PHENOLIC COMPOUND BIOCHEMISTRY
PHENOLIC COMPOUND
BIOCHEMISTRY
By
WILFRED VERMERRIS
Purdue University, West Lafayette, IN, U.S.A.
and
RALPH NICHOLSON
Purdue University, West Lafayette, IN, U.S.A.
123
Authors
Ralph Nicholson
Purdue University
West Lafayette, IN
USA
Dr. Wilfred Vermerris
University of Florida
Genetics Institute Cancer &
Genetics Research
Complex
1376 Mowry Road
Gainesville FL 32610-3610
USA
wev@ufl.edu
ISBN: 978-1-4020-5163-0
e-ISBN: 978-1-4020-5164-7
Library of Congress Control Number: 2008936122
All Rights Reserved
c 2008 Springer Science+Business Media B.V.
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A. Ben-Arieh, I. Frones, Indicators of Children’s Well-Being: Theory and Practice in a Multi-Cultutal
Perspective,
© Springer Science + Business Media B.V. 2009
Contents
v
CONTENTS
PREFACE
xi
CHAPTER 1
Families of Phenolic Compounds and Means of Classification
1. Definitions
2. Classification
3. Classes of phenolic compounds
3.1 Simple phenolics
3.2 Phenolic acids and aldehydes
3.3 Acetophenones and phenylacetic acids
3.4 Cinnamic acids
3.5 Coumarins
3.6 Flavonoids
3.6.1 Chalcones
3.6.2 Aurones
3.6.3 Flavonoids
3.6.3.1 Flavanones
3.6.3.2 Flavanonols
3.6.3.3 Leucoanthocyanidins
3.6.3.4 Flavones
3.6.3.5 Anthocyanidins and deoxyanthocyandins
3.6.3.6 Anthocyanins
3.7 Biflavonyls
3.8 Benzophenones, xanthones and stilbenes
3.9 Benzoquinones, anthraquinones and naphthaquinones
3.10 Betacyanins
3.11 Lignans
3.12 Lignin
3.13 Tannins
3.13.1 Condensed tannins
1
2
3
3
4
4
5
6
7
8
8
9
10
10
11
12
12
15
15
16
17
17
18
20
23
24
vi
Contents
3.13.2 Gallotannins
3.13.3 Ellagitannins
3.13.4 Complex tannins
3.14 Phlobaphenes
4. References
25
26
29
30
32
CHAPTER 2
Chemical Properties of Phenolic Compounds
1
The benzene ring
1.1 Planar versus non-planar bonds
1.2 The acidic nature of the phenolic hydroxyl group
1.2.1 The effects of substituents on acidity
1.2.2 Use of acidity in separations
1.3 Hydrogen bonding and the phenolic hydroxyl group
1.3.1 Intra
- and inter
- molecular hydrogen bond
s
1.3.2 Stability of the hydrogen bond ring
1.4 Metal complexes
1.5 Esterification
1.6 Ethers and glycosides
1.7 Oxidation of the phenolic hydroxyl group
1.7.1 Auto-oxidation of phenolic compounds
1.7.2 Enzymatic oxidation of the phenolic hydroxyl group
1.7.2.1 E.C. 1.10.3
1.7.2.2 E.C. 1.14.18.1
1.7.2.3 E.C. 1.11.1.
1.8 Reactive oxygen species and antioxidants
2. References
35
38
38
39
40
40
41
41
43
45
47
48
48
50
50
53
53
58
61
CHAPTER 3
Biosynthesis of Phenolic Compounds
1. Introduction
2. Protein isolation and purification
3. Gene cloning strategies
3.1 Insertional mutagenesis
3.2 Map-based cloning
3.3 The candidate-gene approach
3.4 QTL mapping
4. Isolation and characterization of recombinant proteins
5. Carbohydrate catabolism
5.1 Glycolysis
5.2 The pentose phosphate pathway
6. The shikimate pathway
63
64
67
70
72
72
73
74
76
77
77
81
Contents
7. The general phenylpropanoid pathway
8. Biosynthesis of phenolic acids
8.1 Salicylic acid
8.2 Gallic acid
9. Biosynthesis of flavonoids and condensed tannins
9.1 Structural genes and enzymes
9.2 Regulatory genes
10. Monolignol biosynthesis
11. Lignan biosynthesis
12. Lignin biosynthesis
12.1 Genetic control of lignification
12.2 Monolignol transport and polymerization
12.3 Modification of lignin for agro-industrial applications
12.3.1 Pulp and paper industry
12.3.2 Forage and silage quality
12.3.3 Ethanol production from ligno-cellulosic biomass
13. Hydroxycinnamic acid biosynthesis
14. Biosynthesis of sinapoyl esters
15. Coumarin biosynthesis
16. Stilbene biosynthesis
17. Biosynthesis of gallotannins and ellagitannins
18. References
vii
84
86
86
88
90
90
96
102
107
112
112
116
120
121
123
124
125
126
129
129
130
134
CHAPTER 4
Isolation and Identification of Phenolic Compounds
1. Introduction
2. Isolation of phenolic compounds
2.1 Total phenolic content: the Folin-Ciocalteu reagent
2.2 Determining the content of condensed tannins
2.2.1 The butanol-HCl assay
2.2.2 The vanillin assay
2.2.3 Precipitation of condensed tannins with formaldehyde
2.3 Determining the content of gallotannins
2.3.1 The potassium iodate assay
2.3.2 The rhodanine assay
2.4 Determining the content of ellagitannins
2.4.1 Nitrous acid oxidation
2.4.2 The NaNO2/HCl assay
2.5 Determining lignin content
2.5.1 Klason lignin
2.5.2 Acid detergent lignin
2.5.3 Thioglycolic acid lignin
2.6 Acetylbromide lignin
151
151
152
154
154
154
155
155
155
156
157
157
158
159
160
161
162
164
viii
Contents
3. Identification and characterization of phenolic compounds
3.1 Thin layer chromatography
3.2 Liquid chromatography: HPLC and LC-MS
3.3 Gas chromatography
3.4 Methods for the identification of lignin subunit composition
3.4.1 The nitrobenzene oxidation
3.4.2 Thioacidolysis
3.4.3 Derivatiation Followed by Reductive Cleavage
3.4.4 Analytical pyrolysis
3.4.5 Nuclear magnetic resonance
3.4.6 Fourier-transform infrared spectroscopy and near
infrared reflectance spectroscopy
4. Visualization of phenolic compounds in planta using
histochemical stains
4.1 An overview of histochemical staining protocols
4.2 Visualizing plant-pathogen interactions involving phenolics
with histochemical stains
5. References
166
166
169
170
171
172
173
176
178
178
181
183
183
185
190
CHAPTER 5
Analysis of Phenolic Compounds with Mass Spectrometry
1. The principles of mass spectrometry
1.1 Ionization
1.2 Mass Analysis
1.3 Detectors
1.4 Sample introduction
2. New developments in mass spectrometry
2.1 Fast atom bombardment
2.2 Plasma desorption ionization
2.3 Electrospray ionization
2.4 Matrix-assisted laser desorption ionization
3. Quantitation
4. References
197
197
198
199
200
202
202
203
204
206
208
210
CHAPTER 6
The Role of Phenols in Plant Defense
1. Preformed antimicrobial and insecticidal metabolites
1.1 Chlorogenic acid
1.2 Phloridzin and phloretin
1.3 Cyanogenic glycosides
1.4 Tuliposides
1.5 Protocatechuic acid
211
213
214
215
216
217
Contents
1.6 Lignin
1.7 C-glycosyl flavones
2. Compounds formed in response to pathogen attack
2.1 3-Deoxyanthocyanidins
2.2 Pisatin
2.3 Stilbenes
2.4 Salicylic acid
2.5 Lignin
3. References
ix
217
217
222
222
224
224
225
227
230
CHAPTER 7
Phenolic Compounds and their Effects on Human Health
1.
2.
3.
4.
5.
Introduction
Antioxidant properties
Disease prevention
Activity against toxins
References
235
237
246
251
253
Appendix
257
Index
267
Contents
xi
PREFACE
Phenolic compounds represent a large group of molecules with a variety
of functions in plant growth, development, and defense. Phenolic
compounds include signaling molecules, pigments and flavors that can
attract or repel, as well as compounds that can protect the plant against
insects, fungi, bacteria, and viruses. Most phenolic compounds are present
as esters or glycosides rather than as free compounds. Tannins and lignin are
phenolic polymers. Tannins are used comercially as dyes and astringents,
and lignin accounts for structural ridgidity of cells and tissues and is
essential to vascular development. From this brief overview it is apparent
that phenolic compounds make up a large and fascinating family.
Our intention is to provide the reader with an overview of this family of
compounds that will show their diversity and provide a basis for continued
investigations. The target audience is intended to include phytopathologists,
botanists, agronomists, entomologists, and people with a general interest in
plant biochemistry. This book will also be a valuable resource when used as
a textbook in a course on phenolic compounds, aimed at advanced
undergraduate or beginning graduate students in the life sciences. While
writing this book we assumed the reader would have basic knowledge of
organic chemistry, biochemistry of DNA, RNA, proteins and lipids, and cell
physiology. At the end of the chapters we have provided some additional
references for further reading, either to obtain background information, or to
obtain more details.
The focus of this book is centered on structure, nomenclature and
occurrence of phenolic compounds (Chapter 1), and their chemical
properties (Chapter 2). Chapter 3 describes the biosynthetic pathways
leading to the major classes of phenolics. This chapter presents an up-to-date
overview of the genetic approaches that have been used to elucidate these
pathways. Chapter 4 presents an overview of methods for the isolation and
identification of plant phenolic compounds. Given that much of the recent
xi
xii
Contents
Preface
advances in the identification of phenolic compounds have been made
possible through the development of mass spectrometry, we have dedicated
an entire chapter to the use of mass spectrometry in the identification of
phenolic compounds (Chapter 5). This chapter was written by Dr. Karl V.
Wood of the Mass Spectrometry Facility in the Department of Chemistry at
Purdue University. We are grateful for his contribution to this book. Chapter
6 highlights the role of phenolic compounds in plant defense. We have
included a number of examples, including phytoanticipins, phytoalexins and
lignin synthesized in response to pathogen attack, to complement the more
chemical nature of the other chapters, and to illustrate the important role
phenolic compounds play in plant survival. The final chapter is an overview
of some of the positive effects phenolics can have on human health.
We would like to thank Dr. Karl Wood for helpful comments on the
manuscript, Drs. William Bourdoncle, Surinder Chopra, Hans Jung and
Michael McMullen for providing details on specific topics, Ana Saballos for
helpful discussions, Shelly Dunk and Flora Mauch for assistance with the
references, and Zuzana Bernhardt and Ineke Ravesloot at Springer
Publishers for their encouragement and flexibility.
This book would not have been possible without our supporters at home,
Heather (R.L.N.) and Lauren and Deirdre (W.V.), and we are grateful for
their patience during the writing of this book, especially when this took
place during evenings and weekends.
West Lafayette, IN
Spring 2006
W. Vermerris
Associate Professor of Agronomy, University of Florida
Genetics Institute, Gainesville, FL
R. L. Nicholson
Professor of Botany and Plant Pathology,
Purdue University, West Lafayette, IN
FAMILIES OF PHENOLIC COMPOUNDS AND MEANS OF
CLASSIFICATION
1
Chapter 1
FAMILIES OF PHENOLIC COMPOUNDS AND
MEANS OF CLASSIFICATION
1.
DEFINITIONS
What are phenolic compounds? They are compounds that have one or
more hydroxyl groups attached directly to an aromatic ring. Phenol (1.1) is
the structure upon which the entire group is based. The aromatic ring in this
case is, of course, benzene.
OH
(1.1)
The phenols are in many ways similar to alcohols of aliphatic structures
where the hydroxyl group is attached to a chain of carbons. The phenolic
hydroxyl group, however, is influenced by the presence of the aromatic ring.
Because of the aromatic ring, the hydrogen of the phenolic hydroxyl is
labile, which makes phenols weak acids. We will deal with their chemical
properties separately in Chapter 2.
1
2
Chapter 1
Polyphenols are compounds that have more than one phenolic hydroxyl
group attached to one or more benzene rings. The term is somewhat
misleading since it tends to make people think of polymers of individual
phenol molecules. Of course such polymers exist. Phenolic compounds are
characteristic of plants and as a group they are usually found as esters or
glycosides rather than as free compounds. It is important to realize this if
you want to extract phenols from plant tissues.
2.
CLASSIFICATION
The term phenolics covers a very large and diverse group of chemical
compounds. These compounds can be classified in a number of ways.
Harborne and Simmonds (1964) classified these compounds into groups
based on the number of carbons in the molecule (Table 1.1).
Table 1-1. Classification of phenolic compounds
Structure
Class
C6
C6 - C1
C6 - C2
C6 - C3
C6 - C3
C15
C15
C15
C15
C15
C15
C15
C30
C6-C1-C6, C6-C2-C6
C6, C10, C14
C18
Lignans, neolignans
Lignin
Tannins
Phlobaphenes
simple phenolics
phenolic acids and related compounds
acetophenones and phenylacetic acids
cinnamic acids, cinnamyl aldehydes, cinnamyl alcohols
coumarins, isocoumarins, and chromones
chalcones, aurones, dihydrochalcones
flavans
flavones
flavanones
flavanonols
anthocyanidins
anthocyanins
biflavonyls
benzophenones, xanthones, stilbenes
quinones
betacyanins
dimers or oligomers
polymers
oligomers or polymers
polymers
An alternative classification has been used by Swain and Bate-Smith
(1962). They grouped the phenols in “common” and “less common”
categories. Ribéreau-Gayon (1972) grouped the phenols into three families
as follows:
Families of phenolic compounds and means of classification
3
1. Widely distributed phenols - ubiquitous to all plants, or of
importance in a specific plant
2. Phenols that are less widely distributed - limited number of
compounds known
3. Phenolic constituents present as polymers.
In this chapter each of the classes will be discussed, and structures of
commonly found or important compounds will be presented. The
compounds will be presented in the order listed in Table 1-1. The main
focus will be on phenolic compounds that are widely distributed.
3.
CLASSES OF PHENOLIC COMPOUNDS
3.1
Simple phenolics
Simple phenolics are substituted phenols. The ortho, meta and para
nomenclature refers to a 1,2-, 1,3- and 1,4-substitution pattern of the
benzene ring, respectively, where in this case one of the functional groups is
the hydroxyl group. With three functional groups, the substitution pattern
can be 1,3,5, which, when all three substituents are identical, is designated
as a meta-tri-substitution pattern, whereas the 1,2,6, substitution pattern is
indicated by the prefix ‘vic’(Figure 1-1).
OH
OH
OH
OH
R
OH
R1
R
R1
R2
R2
R
ortho
meta
para
meta-tri
vic-tri
Figure 1-1. Nomenclature for substitution patterns of phenolic compounds. R, R1
and R2 are generic substituents.
Examples include resorcinol (1,3-dihydroxybenzene; 1.2), a metadihydroxylated
simple
phenolic,
and
phloroglucinol
(1,3,5trihydroxybenzene; 1.3), a meta-trihydroxylated simple phenolic.
4
Chapter 1
OH
HO
OH
HO
(1.2)
3.2
OH
(1.3)
Phenolic acids and aldehydes
Hydroxy-benzoic acids are characterized by the presence of a carboxyl
group substituted on a phenol. Examples include p-hydroxybenzoic acid
(1.4), gallic acid (1.5), protocathechuic acid (1.6), salicylic acid (1.7) and
vanillic acid (1.8). Related are hydroxybenzoic aldehydes, such as vanillin
(1.9), which have an aldehyde group in stead of a carboxyl group.
OH
O
OH
O
O
OH
OH
HO
OH
OH
OH
OH
(1.4)
(1.5)
(1.6)
H
O
OH
O
OH
O
OH
OCH3
OCH3
(1.7)
3.3
OH
OH
(1.8)
(1.9)
Acetophenones and phenylacetic acids
Phenones are C6-C2 compounds that are rarely found in nature.
Examples include 2-hydroxyacetophenone (1.10) and 2-hydroxyphenyl
acetic acid (1.11).
Families of phenolic compounds and means of classification
5
HO
O
O
OH
OH
(1.10)
3.4
(1.11)
Cinnamic acids
There are six common cinnamic acids, which have a C6 - C3 skeleton.
All plants probably contain at least three of them. Shown below are
cinnamic, acid (1.12), p-coumaric acid (1.13), caffeic acid (1.14), ferulic
acid (1.15), 5-hydroxyferulic acid (1.16), and sinapic acid (1.17).
O
OH
O
OH
O
OH
OH
(1.12)
O
OH
OH
OH
(1.13)
(1.14)
O
OH
O
OCH3 H3CO
OCH3 HO
OH
OCH3
OH
OH
OH
(1.15)
(1.16)
(1.17)
Cinnamic acids are commonly found in plants as esters of quinic acid,
shikimic acid, and tartaric acid. For example, chlorogenic acid (1.18) is an
ester of caffeic acid and quinic acid.
6
Chapter 1
O
O
HO
OH
OH
O
OH
OH
OH
(1.18)
Cinnamic esters are also found as sugar esters, or as esters of a variety
of other organic acids. For example, sinapoyl esters represent a class of UVabsorbing compounds in the family of the Brassicaceae. Examples include
sinapoyl malate (1.19) present in leaves, and sinapoyl choline (1.20) present
in roots (Ruegger and Chapple, 2001).
O
O
H3CO
O
CH3
O
HO
O
N+
O
O
H3CO
CH3
CH3
H3CO
(1.19)
H3CO
3.5
O
HO
(1.20)
Coumarins
Coumarins also have a C6 - C3 skeleton, but they possess an oxygen
heterocycle as part of the C3-unit. There are numerous coumarins, many of
which play a role in disease and pest resistance, as well as UV-tolerance.
The coumarin umbelliferone (1.21) is popular in enzyme assays.
Umbelliferone esters can be used as a substrate for non-specific esterase
enzyme assays and in fluorescent immunoassays (Jacks and Kircher, 1967).
In order to quantify the enzyme activity of the popular reporter gene glucuronidase (GUS), plant extracts can be incubated with 4methylumbelliferyl -D-glucuronide (4-MUG; 1.22), which upon hydrolysis
Families of phenolic compounds and means of classification
7
by GUS produces the fluorescent compound 4-methylumbelliferone (4MU), along with glucuronic acid (Gallagher et al., 1992).
Isocoumarins, such as bergenin (1.23) have a structure similar to
coumarins, but the position of the oxygen and carbonyl groups within the
oxygen heterocycle are reversed. Isocoumarins also play a role in defense
responses. For example, bergenin has been shown to inhibit the growth of
the powdery mildew on pea (Prithiviraj et al., 1997).
HO
O
O
O
O
OH
O
(1.21)
O
O
HO
HO
OH
(1.22)
O
HO
O
H
OH
H3CO
H
OH
O
OH
(1.23)
HO
3.6
Flavonoids
Flavonoids are C15 compounds all of which have the structure C6-C3-C6.
Flavonoids may be grouped into three big classes based on their general
structure. In each case, two benzene rings are linked together by a group of
three carbons. It is the arrangement of the C3 group that determines how the
compounds are classified.
8
3.6.1
Chapter 1
Chalcones
Chalcones (1.24) and dihydrochalocones (1.25) have a linear C3-chain
connecting the two rings. The C3-chain of chalcones contains a double bond,
whereas the C3-chain of dihydrochalcones is saturated.
OH
OH
B
B
HO
OH
HO
OH
β
β
A
A
α
α
OH
OH
O
O
(1.25)
(1.24)
Chalcones, such as butein (1.26), are yellow pigments in flowers. An
example of a dihydrochalcone is phloridzin (phloretin-2′-O-D-glucoside)
(1.27), a compound found in apple leaves, and which has been reported to
have anti-tumor activity (Nelson and Falk, 1993).
OH
OH
OH
OH
HO
OH
HO
OH
O
O
O
Glc
(1.26)
3.6.2
(1.27)
Aurones
Aurones (1.28) are formed by cyclization of chalcones, whereby the
meta-hydroxyl group reacts with the α-carbon to form a five-member
heterocycle. Aurones are also yellow pigments present in flowers.
Families of phenolic compounds and means of classification
9
OH
B
HO
O
A
O
OH
3.6.3
(1.28)
Flavonoids
Typical flavonoids, such as flavanone (1.29), have a six-member
heterocycle. Flavonoids have an A-, B-, and C-ring, and are typically
depicted with the A-ring on the left-hand side. The A-ring originates from
the condensation of three malonyl-CoA molecules, and the B-ring originates
from p-coumaroyl-CoA. These origins explain why the A-ring of most
flavonoids is either meta-dihydroxylated or meta-trihydroxylated.
3'
2'
7
9
A
6
B
1
8
HO
1'
O
5'
2
6'
C
3
10
5
OH
4'
4
OH
O
(1.29)
In typical flavonoids one of the meta-hydroxyl groups of the A-ring
contributes the oxygen to the six atom-heterocycle. The six member oxygen
heterocycle of typical flavonoids may be a pyran (1.30), pyrylium (1.31), or
pyrone ring (1.32). The B-ring is typically mono-hydroxylated, orthodihydroxylated, or vic-trihydroxylated. The B-ring may also have methylethers as substituents.
O
O
O
(1.30)
(1.31)
(1.32)
O
10
Chapter 1
Isoflavones, isoflavanones and neoflavonoids are also members of the
flavonoid group. They all have the C6-C3-C6 structure but the B-ring is in a
different position on the oxygen heterocycle. Examples are isoflavone (1.33)
and the neoflavonoid dalbergin (1.34).
H3CO
O
O
O
H3CO
O
(1.34)
(1.33)
3.6.3.1
Flavanones
The heterocycle of flavanones also contains a ketone group, but there is
no unsaturated carbon-carbon bond. The A- and B-ring can be substituted
analogous to the flavones, as in naringenin (1.35).
OH
OH
HO
O
OH
HO
O
OH
OH
O
(1.35)
3.6.3.2
OH
O
(1.36)
Flavanonols
Flavanonols are also known as dihydroflavonols and often occur in
association with tannins in heartwood. An example is taxifolin (1.36), also
known as dihydroquercitin.
Families of phenolic compounds and means of classification
3.6.3.3
11
Leucoanthocyanidins
Leucoanthocyanidins are also referred to as flavan-3,4-cis-diols. They
are synthesized from flavanonols via a reduction of the ketone moiety on
C4. Examples are leucocyanidin (1.37) and leucodelphinidin (1.38). These
compounds are often present in wood and play a role in the formation of
condensed tannins.
OH
OH
OH
OH
HO
HO
O
O
OH
OH
OH
OH
OH
OH
OH
(1.38)
(1.37)
Because of their completely saturated heterocycle, leucoanthocyanidins,
together with flavan-3-ols are referred to as flavans. Examples of flavan-3ols are catechin (1.39) and gallocatechin (1.40). The ‘gallo’ in the latter
compound refers to the vic-tri-hydroxy substitution pattern on the B-ring.
Unlike most other flavonoids, the flavans are present as free aglycones or as
polymers of aglycones, i.e. they are not glycosylated.
OH
OH
OH
HO
O
OH
HO
O
OH
OH
OH
OH
OH
OH
(1.39)
(1.40)
OH
HO
O
O
OH
OH
O
OH
(1.41)
OH
12
Chapter 1
Catechins (1.41) can also be found as gallic acid esters that are esterified at
the 3′ hydroxyl group. Note the difference between the gallic acid ester of
catechin (1.41) and gallocatechin (1.40).
3.6.3.4
Flavones
The heterocycle of flavones contains a ketone group, and has an
unsaturated carbon-carbon bond. Flavones are common in angiosperms. The
most widely distributed flavones in nature are kaemferol (5,7,4′
hydroxyflavone; 1.42), quercetin (5,7,3′,4′ hydroxyflavone; 1.43), and
myricetin (5,7,3′,4′,5′ hydroxyflavone; 1.44).
OH
OH
HO
O
OH
HO
O
OH
OH
OH
O
OH
O
OH
(1.42)
(1.43)
OH
HO
O
OH
OH
OH
O
(1.44)
3.6.3.5
Anthocyanidins and deoxyanthocyandins
The heterocycle of anthocyanidins is a pyrilium kation. Anthocyanidins
are typically not found as free aglycones, with the excepton of the following
widely distributed, colored compounds: Pelargonidin (orange-red; 1.45),
cyanidin (red; 1.46), peonidin (rose-red; 1.47), delphinidin (blue-violet;
1.48), petunidin (blue-purple; 1.49), and malvidin (purple; 1.50). A
convenient mnemonic is: PCP–DPM. The most common anthocyanidin is
cyanidin. These compounds are present in the vacuoles of colored plant
tissues such as leaves or flower petals. The color of the pigment depends on
Families of phenolic compounds and means of classification
13
the pH, metal ions present, and the combination of substituted sugars and
acylesters. Different colors can also result from the presence of
combinations of several anthocyanidins (Figure 1.2).
OH
OH
OH
HO
HO
O
O
OH
OH
OH
OH
(1.45)
(1.46)
OCH3
OH
OH
HO
O
OH
HO
O
OH
OH
OH
OH
OH
(1.47)
(1.48)
OCH3
OCH3
OH
OH
HO
HO
O
O
OCH3
OH
OH
OH
OH
OH
(1.49)
(1.50)
Note that each of the six common anthocyanidins has the basic structure
of the flavylium cation (2-phenyl benzopyrilium; 1.31).
Other anthocyanidins exist, and can be categorized into two groups:
1. Those where either the C5 or C7 position is substituted with a
methoxyl group
2. The deoxyanthocyanidins, which do not contain a hydroxyl
group at the C3 position
14
Chapter 1
There are five deoxyanthocyanidins: Apigeninidin (1.51), luteolinidin
(1.52), 7-methoxyapigeninidin (1.53), 5-methoxy-luteolinidin (1.54), and the
caffeic acid ester of arabinosyl 5-O-apigeninidin (1.55).
Increasing red
OH
OCH3
OH
OH
OH
+
O
HO
OH
OH
OCH3
OH
OH
OH
OH
OH
OCH3
OH
Increasing purple
OCH3
Figure 1-2. Impact of ring substitution on the color of anthocyanidins
OH
OH
HO
O
OH
OH
HO
O
OH
(1.51)
(1.52)
Families of phenolic compounds and means of classification
15
OH
OH
H3CO
O
OH
HO
O
OH
OCH3
(1.53)
(1.54)
OH
O
HO
HO
O
O
O
O
HO
OH
HO
(1.55)
3.6.3.6
Anthocyanins
Anthocyanins are water-soluble glycosides of anthocyanidins. The most
common glycoside is the 3-glycoside. If a second sugar is present, it is
almost always at the 5-hydroxyl position, and almost always a glucose
residue. Such compounds are called 3,5-dimonosides. In addition, there are a
few rare 3,7-substitutions. While glucose is the most common sugar,
substitutions of other sugars, such as arabinose, are sometimes observed.
Anthocyanins can also be acylated. In this case an organic acid - typically pcoumaric acid (1.11), caffeic acid (1.12), or ferulic acid (1.13) - is esterified
to the sugar. An example is petanin (3-[6-O-(4-O-E-p-coumaroyl-O-αrhamnopyranosyl)- -glucopyranoside]-5-O- -glucopyranoside; 1.56), a
compound found in Solanaceae.
3.7
Biflavonyls
Biflavonyls have a C30 skeleton. They are dimers of flavones such as
apigenin or methylated derivatives and are found in gymnosperms. Few
compounds are known. The most familiar is ginkgetin (1.57) from Ginkgo
biloba (fossil tree of Japanese silver apricot).
16
Chapter 1
OCH3
HO
O
O
OH
HO
O
HO
O
O
CH2
CH3
O
O
CH2OH
O
OH
OH
OH
OH
O
OH
OH
OH
(1.56)
OH
H3CO
OH
O
OCH3
O
HO
O
HO
OH
(1.57)
3.8
Benzophenones, xanthones and stilbenes
Benzophenones and xanthones have a C6-C1-C6 structure, whereas
stilbenes have a C6-C2-C6 structure. Xanthones are yellow pigments in
flowers, and stilbenes are associated with heartwood of trees. Shown are the
structures of benzophenone, (1.58), xanthone (1.59), and the stilbenes
resveratrol (1.60) and pinosylvin (1.61).
O
O
O
(1.58)
(1.59)
HO
HO
OH
HO
(1.60)
HO
(1.61)
Families of phenolic compounds and means of classification
3.9
17
Benzoquinones, anthraquinones and
naphthaquinones
Benzoquinones, such as 2,6-dimethoxybenzoquinone (1.62), are present
in root exudates of maize and stimulate parasitic plants to form haustoria
(Matvienko et al., 2001). Ubiquinones, such as ubiquinone(3) (1.63), where
(3) indicates the number of isoprenoid sidechains, is also known as
Coenzyme Q and has a role in electron transport in the mitochondria.
O
O
OCH3
H3CO
OCH3
H3C
O
H3CO
O
O
(1.62)
(1.63)
3
Naphthaquinones are rare. Among the naphthaquinones juglone (1.64) is
relatively common. It is found in walnuts. Anthraquinone is the most widely
distributed of the quinones in higher plants and fungi. There are numerous
compounds. The anthtraquinone emodin (1.65) occurs as a rhamnoside in
rhubarb roots.
OH
OH
O
O
OH
HO
O
(1.64)
3.10
O
(1.65)
Betacyanins
Betacyanins are red pigments and account for the red color of beets
(Beta vulgaris). They are unique compounds to the Centrospermae. They
have absorption spectra that resemble anthocyanins, but they contain
nitrogen. An example is betanidin (1.66). Betacyanins are normally found as
glycosides. Betaxanthins are chemically related to betacyanins, but they are
not phenols. An example is indicaxanthin (1.67). These compounds are
yellow pigments, and are also unique to the Centrospermae.
18
Chapter 1
HO
COOH
COOH
N
HO
N
COOH
COOH
NH
NH
HOOC
HOOC
(1.66)
3.11
(1.67)
Lignans
Lignans are dimers or oligomers that result from the coupling of
monolignols – p-coumaryl alcohol (1.68), coniferyl alcohol (1.69), and
sinapyl alcohol (1.70), with coniferyl alcohol being the most common
monolignol used in lignan biosynthesis. Lignans are present in ferns,
gymnosperms and angiosperms. They are localized in woody stems and in
seeds and play a role as insect deterrents. Some of these compounds have
medicinal properties.
OH
OH
OH
γ
β
α
1
6
5
4
2
3
OCH3
OCH3
H3CO
OH
OH
OH
(1.68)
(1.69)
(1.70)
Lignan biosynthesis results from the reaction of monolignol radicals.
The monolignol radicals (1.71), in this example derived from p-coumaryl
alcohol (1.68), are formed after reaction with hydrogenperoxide radicals
(generated by peroxidases; see also Chapter 2), which eliminate the proton
on the para-hydroxyl-group of the phenol. The radial electron can be
delocalized along the phenol ring, but also along the propane tail, so that the
Families of phenolic compounds and means of classification
19
carbon at the 1, 3 and 5 positions of the ring, as well as the -carbon of the
propane tail, become reactive.
OH
OH
OH
OH
OH
.
.
.
.
.
O
O
O
O
O
(1.71)
The term lignan typically refers to dimers of monolignols that are linked
via an 8-8′ ( - ′) bond, whereas the term neolignan refers to dimers and
oligomers that contain bonds other than the 8-8′ bond. Most lignans are
optically active, and typically only one enantiomer is found in a given
species. Examples of lignans include (+)-pinoresinol (1.72), (+)-sesamin
(1.73), and (–)-plicatic acid (1.74).
H3CO
O
O
OH
O
O
H
H
H
O
H
O
HO
O
OCH3
O
(1.73)
(1.72)
OH
H3CO
OH
OH COOH
HO
H3CO
OH
OH
(1.74)
20
Chapter 1
The stereo-selective formation of certain lignans has been shown to be
mediated by ‘dirigent’ proteins. These proteins hold the monolignols in a
specific orientation, but have no catalytic activity (Davin et al., 1997; see
also Chapter 3).
3.12
Lignin
Lignin is a phenolic polymer. It is the second most abundant biopolymer on Earth (after cellulose), and plays an important role in providing
structural support to plants. Its hydrophobicity also facilitates water
transport through the vascular tissue. Finally, the chemical complexity and
apparent lack of regularity in its structure make lignin extremely suitable as
a physical barrier against insects and fungi.
Like lignans, lignin is synthesized primarily from three monolignol
precursors: p-coumaryl alcohol (1.68), coniferyl alcohol (1.69), and sinapyl
alcohol (1.70). Additional compounds are incorporated into the lignin, but
typically in small quantities. Some of these compounds include:
coniferaldehyde (1.75; Pillonel et al., 1991; Halpin et al., 1994; Ralph et al.,
2001), sinapaldehyde (1.76; Pillonel et al., 1991), dihydroconiferyl alcohol
(1.77; Ralph et al., 1997), 5-hydroxyconiferyl alcohol (1.78; Lapierre et al.,
1988; Ralph et al., 2001; Marita et al., 2003), tyramine ferulate (1.79; Ralph
et al., 1998), p-hydroxy-3-methoxybenzaldehyde (1.80; Kim et al., 2003), phydroxybenzoate (1.81; Landucci et al., 1992), p-coumarate (1.13; Lu and
Ralph, 1999) and acetate (Ralph, 1996).
H
O
H
OCH3 H3CO
O
OH
OCH3
OCH3
OH
OH
OH
(1.75)
(1.76)
(1.77)
The latter three compounds are esterified to the -carbon of the
monolignols. These compounds are found in higher quantities in certain
mutants or genetically engineered plants in which the expression of specific
lignin biosynthetic genes has been altered (Sederoff et al., 1999; Boerjan
Families of phenolic compounds and means of classification
21
et al., 2003; Ralph et al., 2004). The presence of these compounds in lignin
has prompted a broader definition of lignin, based more on the function than
on a narrowly defined chemical composition (Brunow et al., 1999).
O
OH
HN
OCH3
OH
OCH3
HO
OH
OH
(1.78)
(1.79)
O
H
O
O
OCH3
OH
OH
(1.80)
(1.81)
Lignin is formed through a radical-mediated polymerization process, but
lignin is not optically active (Ralph et al., 1999), and the structure of lignin
is believed to be under chemical control, rather than under the control of
dirigent proteins or enzymes (Hatfield and Vermerris, 2001). The lignin
polymer enlarges as additional monolignol radicals react with reactive sites
on the polymer.
After polymerization the different lignin subunits are referred to as phyrdoxyphenyl (H), guaiacyl (G), and syringyl (S) residues, depending on
whether they originated from p-coumaryl alcohol, coniferyl alcohol, or
sinapyl alcohol, respectively.
Different kinds of interunit linkages can be formed depending on the
position of the delocalized radical electron at the time two radicals are
coupled. The most common interunit linkage in lignin is the -O-4 linkage
(1.82). Other coupling modes include: 5-O-4′ (1.83), -1 (1.84), 5-5′ (1.85),
- ′ (1.86), -5 (1.87), and the dibenzodioxocin linkage (1.88; Brunow et al.,
1998). The interunit linkages involving the -carbon are favored. The 5-5′
22
Chapter 1
OH
OH
R4
HO
O
R3
R4
HO
R3
O
HO
R1
O
O
R2
R1
(1.82)
(1.83)
R3
HO
OH
O
HO
R4
R2
OH
R1
R1
O
O
O
R1
R3
(1.84)
OH
(1.85)
O
O
R4
HO
R2
R3
R2
O
O
O
O
R1
R1
(1.86)
HO
OH
(1.87)
OH
O
R3
OCH3
R3
O
O
O
OH
R2
R1
O
(1.88)
R1
O
(1.89)
Families of phenolic compounds and means of classification
23
and the 5-O-4′ linkages are present in only small amounts, and tend to
originate from preformed oligomers, rather than from the addition of new
monolignol radicals to the growing ligninpolymer (Ralph et al., 2004). In
plants that accumulate substantial amounts of 5-hydoxyconiferyl alcohol
(1.78) as a result of reduced activity of the enzyme caffeic acid Omethyltransferase (see Chapter 3), the benzodioxane linkage (1.89) has been
identified. This is a novel linkage between two subunits involving a -O-4′
and an α-O-5′ bond (Ralph et al., 2001; Marita et al., 2003). The substituents
on the phenol ring in structures (1.82) through (1.89) are indicated with an
R. In the case of H-residues both substituents are hydrogens, in the case of
G-residues C3 contains a methoxyl group, and in the case of syringyl
residues both C3 and C5 contain methoxyl groups.
3.13
Tannins
Tannins comprise a group of compounds with a wide diversity in
structure that share their ability to bind and precipitate proteins. The name
tannins refers to the process of tanning animal skin to form leather. This
process has been known since prehistoric times, when animal hides were
treated with animal fat and brain tissue. Chemically this resulted in the
cross-linking of the collagen chains in the hide. Throughout much of history
the tanning process was performed with tannins derived from plants, until
minerals such as aluminum and chromium replaced the use of plant tannins
during the last century. As part of Japanese and Chinese natural medicine
tannins have been used as anti-inflammatory and antiseptic compounds.
They have also been used to treat a wide array of illnesses, including
diarrhea and tumors in the stomach or duodenum (Khanbabaee and Van
Ree, 2001). Another application of tannins is in wine and beer production,
where they are used to precipitate proteins.
Tannins are abundant in many different plant species, in particular oak
(Quercus spp.), chestnut (Castanea spp.), staghorn sumac (Rhus typhina),
and fringe cups (Tellima grandiflora). Tannins can be present in the leaves,
bark, and fruits, and are thought to protect the plant against infection and
herbivory.
Tannins can be classified in three groups: condensed tannins,
hydrolysable tannins, and complex tannins (Khanbabaee and Van Ree,
2001). These groups can then be further divided, as shown in Figure 1-3.
24
Chapter 1
TANNINS
condensed tannins
complex tannins
hydrolyzable tannins
gallotannins
ellagitannins
group A
group B
Figure 1-3. Classification of tannins
3.13.1
Condensed tannins
Condensed tannins are also referred to as proanthocyanidins. They are
oligomeric or polymeric flavonoids consisting of flavan-3-ol (catechin)
units. Hydrolysis under harsh conditions, such as heating in acid, yields
anthocyanidins. An example of a condensed tannin is procyanidin B2
(epicatechin-(4 →8′)-epicatechin; 1.90). In this case the interflavanyl
linkage is between C4 of the ‘lower’ unit, and C8 of the ‘upper’ unit. The
linkage can also be between C4 of one unit and C6 of the second unit.
OH
HO
O
OH
OH
OH
OH
HO
O
OH
OH
OH
(1.90)
Polymers are formed through the action of acids or enzymes. Polymers
made up of between two and ten residues are called flavolans. Polymers
Families of phenolic compounds and means of classification
25
made up of more than 50 catechin units have been identified (Khanbabaee
and Van Ree, 2001).The degree of polymerization affects the ability to
precipitate proteins. This is of importance in wine making, where a high
level of condensed tannins, especially in red wines, can result in the dry
feeling on the inside of the mouth.
3.13.2
Gallotannins
Gallotannins are hydrolysable tannins with a polyol core (referring to a
compound with multiple hydroxyl groups) substituted with 10-12 gallic acid
residues. Gallotannins contain the characteristic meta-depside bonds (1.91)
between gallic acid residues. This bond is more labile than an aliphatic ester
bond, and can be methanolyzed with a weak acid in methanol. In contrast,
methanolysis of an aliphatic ester bond requires methanol with a strong
mineral acid and heat.
OH
OH
O
O
OH
OH
HO
HO
OH
O
O
OH
HO
HO
O
O
O
O
O
O
O
OH
HO
O
O
O
HO
OH
OH
HO
OH
OH
OH
OH
HO
(1.91)
(1.92)
The most commonly found polyol is D-glucose, although some
gallotannins contain catechin and triterpenoid units as the core polyol.
Gallotannins with a D-glucose core are synthesized from 1,2,3,4,6pentagalloylglucose (1,2,3,4,6-penta-O-galloyl- -D-glucopyranose; 1.92; see
also Chapter 3). An example of a gallotannin is the hexagalloylated
compound 2-O-digalloyl-1,3,4,6-tetra-O-galloyl- -D-glucopyranose (1.93),
where the additional gallic acid residue is located on C2 of the
glucopyranose ring.
26
Chapter 1
HO
HO
O
HO HO
OH
O
O
OH
HO
O
O
O
O
O
OH
HO
O
OH
O
O
HO
O
OH
OH
OHO
OH
OH
HO
(1.93)
3.13.3
Ellagitannins
Ellagitannins are also hydrolysable tannins derived from
pentagalloylglucose (1.92), but unlike gallotannins, they contain additional
C-C bonds between adjacent galloyl moieties in the pentagalloylglucose
molecule. This C-C linkage is formed through oxidative coupling between
the two adjacent galloyl residues, and results in the formation of a
hexahydroxydiphenoyl (HHDP) unit, which can have either the S- (1.94) or
the R-configuration (1.95).
HO
HO
O
HO
O
HO
O
O
O
O
HO
HO
O
O
HO
HO
HO
HO
OH
OH
(1.94)
(1.95)
The chirality is the result of the limited free rotation around the axis of
the C-C bond due to the two ester bonds between the galloyl residues and the
Families of phenolic compounds and means of classification
27
polyol (indicated by the wavy bonds in 1.94 and 1.95), combined with the
presence of the ortho-substituents that create steric hindrance.
The name ellagitannins is derived from ellagic acid (1.96), which is
formed spontaneously from hexahydroxydiphenic acid (1.94/1.95) in
aqueous solution via an intra-molecular esterification reaction.
O
HO
HO
O
HO
OH
OH
HO
OH
HO
OH
O
HO
OH
HO
OH
HO
O
HO
O
OH
HO
O
(1.94)
OH
HO
O
OH
O
(1.96)
With the glucopyranose molecule in the 4C1 conformation (a chair
conformation with C4 above the plan, as shown in (1.92)), the most
common linkages are between galloyl residues at the 2- and 3-positions of
the glucopyranose ring, and/or between those at the 4- and 6-positions.
These are referred to as Group A ellagitannins. In addition, ellagitannins
with the less common 3,4-linkage, such as identified in the compound
cercidinin A from the bark of Cercidiphyllum japonicum are included in this
group (Tanaka et al., 2001). Group B ellagitannins have a glucopyranose
molecule in the energetically less favorable 1C4 chair or boat conformations.
In this case 1,6-, 1,3-, 2,4- or 3,6-C-C linkages can be formed between
galloyl residues. Figure 1-4 displays the possible different configurations. It
should be noted that it is possible to have combinations of different linkages,
such as, for example, 3,6- and 2,4-linkages.
The configuration of the HHDP esters (R or S; as shown in Figure 1-4)
varies depending on the position of the HHDP unit, and is in the
28
Chapter 1
energetically most favorable configuration. This is dictated by the
stereochemistry of the sugar molecule (Haslam and Cai, 1993). Aside from
the glucopyranose ring, there are also ellagitannins consisting of an openchain polyol. The open chain ellagitannins identified so far all contain a 2,3linked HHDP unit (Haslam and Cai, 1993).
4
1
C1
C4
OG
OG
OG
6
4
6
5
GO
GO
2
1
(S)
2
OG
3
4
OG
G
O
5
3
1
OG
O
OG
OG
O
O
G
G (R)
4,6
G
O
O
GO
OG
3,6
O
O
OG
OG
OG
OG
OG
2,3
1,6
O
GO
G (S)
G
O
G
O
O
OG
O
(S)
G
OG
O
OG
OG
OG
3,4
O
G
(R)
O
OG
O
G
2,4
GO
OG
OG
OG
O
O
(R)
G
O
G
1,3
GO
G
O
G
O
O
OG
OG
Figure 1-4. Possible linkages between adjacent galloyl residues in D-glucose-based
ellagitannins. Ellagitannins from Group A are shown on the left, from Group B on the right.
The (S) and (R) refer to the conformation of the HHDP-units.
Families of phenolic compounds and means of classification
29
Further modification of the HHDP unit is possible. Adjacent galloyl
residues can undergo further oxidative coupling with participation of the
aromatic hydroxyl groups. The valoneoyl unit (1.97), for example, arises from
the linkage between a galloyl residue with an HHDP-unit. Furthermore, the
meta- and para-hydroxyl groups on one of the galloyl-moieties can be oxidized,
resulting in a dehydro-HHDP unit (1.98).
HO
HO
O
O
HO
HO
OO
O
HO
O
HO
OH
HO
O
O
HO
O
HO
O
O
HO
(1.97)
3.13.4
O
OH
OH
(1.98)
Complex tannins
Complex tannins are defined as tannins in which a catechin unit (1.39) is
bound glycosidically to either a gallotannin or an ellagitannin unit. As the
name implies, the structure of these compounds can be very complex. An
example is Acutissimin A (1.99). This is a flavogallonyl unit bound
glucosidically to C1, with an additional three hydrolyzable ester bonds to a
D-glucose-derived open-chain polyol.
This complex tannin is formed during the aging process of red wine,
whereby the catechin unit originates from the grapes, and the ellagitannin, in
this case vescalagin, originates from the oak barrels.
Acutissimin A has been shown to be a powerful inhibitor of DNA topoisomerase II, an enzyme required for the division of cancer cells, and a
target for chemotherapeutic drugs (Quideau et al., 2003). Based on these
findings, however, it is an overstatement to consider red wine a cancer
preventative. Red wine contains other compounds with medicinal activity
which will be discussed in more detail in Chapter 7
30
Chapter 1
OH
HO
OH
OH
HO
HO
HO
HO
O
O
O
O
OH
O
HO
O
O
O
HO
O
O
OH
O
HO
OH
HO
HO
HO
OH
OH
(1.99)
3.14
Phlobaphenes
Phlobaphenes are phenolic polymers that can be present in floral organs of
maize (Zea mays L.), including the pericarp (the hard, outermost layer of the
kernel, derived from the ovary wall), the cob, the husks (the leaves covering the
ear), the tassel glumes, the cob pith, and the tassel pith. Accumulation of
phlobaphenes results in a red pigmentation (Styles and Ceska, 1989). Certain
lines of sorghum (Sorghum bicolor L. (Moench)) also produce phlobaphenes
(Boddu et al., 2005).
The structure of phlobaphenes is poorly understood. These compounds
are believed to be polymers of flavan-4-ols, notably apiferol (1.100) and
luteoferol (1.101) (Shirley-Winkel, 2001). Both of these monomers are
derived from naringenin (1.35).
The polymerization is thought to be under chemical, rather than
enzymatic control, and give rise to a polymer (1.102) in which the
monomers are linked via a 4-8′ linkage. The C-C bonds between the flavan4-ol monomers would be difficult to break, which could help explain the
difficulties with the structural elucidation of phlobaphenes. The reaction
mechanism whereby the hydroxyl group on C-4 is eliminated and a C-C
Families of phenolic compounds and means of classification
31
bond between two monomers is formed, is however, not easily imagined,
and it is possible that the structure of phlobaphenes is different than is
currently postulated.
OH
OH
HO
O
OH
OH
HO
O
OH
OH
(1.100)
OH
(1.101)
R
OH
HO
O
R
OH
OH
HO
O
OH
(1.102)
32
4.
Chapter 1
REFERENCES
Boddu, J., Svabek, C., Ibraheem, F., Jones, D., and Chopra, S., 2005,
Characterization of a deletion allele of a sorghum Myb gene yellow seed1
showing loss of 3-deoxyflavonoids, Plant Sci. 169: 542-552.
Boerjan, W., Ralph, J., and Baucher, M., 2003, Lignin biosynthesis. Ann.
Rev. Plant Biol. 54: 519-546.
Brunow, G., Lundquist, K. and Gellerstedt, G., 1999, Lignin. In: Sjöström, E. and
Alén R., (eds.), Analytical Methods in Wood Chemistry, Pulping, and
Papermaking, Springer-Verlag, Germany, pp. 77–124.
Gallagher, S. R., 1992, GUS Protocols: Using the GUS Gene as a Reporter
of Gene Expression, Academic Press, San Diego.
Halpin, C., Knight, M. E., Foxon, G. A., Campbell, M. M., Boudet, A. M.,
Boon, J. J., Chabbert, B., Tollier, M.-T., and Schuch, W., 1994,
Manipulation of lignin quality by downregulation of cinnamyl alcohol
dehydrogenase, Plant J. 6, 339-350.
Haslam, E., and Cai, Y., 1994, Plant polyphenols (vegetable tannins) : gallic
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Khanbabaee, K., and Van Ree, T., 2001, Tannins: Classification and
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Families of phenolic compounds and means of classification
33
Matvienko, M., Torres, M. J., and Yoder, J. I., 2001, Transcriptional
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34
Chapter 1
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Grotewold, E., 2005, The Science of Flavonoids, Springer, New York, NY,
274 pp.
CHEMICAL PROPERTIES OF PHENOLIC COMPOUNDS
35
Chapter 2
CHEMICAL PROPERTIES OF PHENOLIC
COMPOUNDS
1.
THE BENZENE RING
The atomic number of carbon is six, which means that the carbon atom
has six protons and six electrons. Electrons are present in orbitals around the
atom’s nucleus. An orbital is defined as the space around the nucleus where
an electron with certain energy is most likely to be found. There are
typically several different orbitals around a nucleus, representing different
energy levels and different spatial distributions.
2
2
2
The electron configuration of carbon is 1s 2s 2p , meaning that the
1s, 2s and 2p orbitals each contain two electrons. Covalent bonds between
atoms result from the sharing of one or more pairs of electrons. This is the
result of overlapping atomic orbitals that form molecular orbitals. Carbon is
able to make four covalent bonds, even though based on its electron
configuration only two unpaired electrons – those in the 2p-orbitals - are
available. The ability to make four instead of two covalent bonds results from
hybridization, whereby one of the 2s electrons is promoted to the 2p orbital
2
1
3
(electron configuration: 1s 2s 2p ), followed by the formation of four
3
equivalent sp orbitals with slightly higher energy than the 2s orbital. The
promotion of one of the 2s-electrons to the 2p-orbital can also result in the
2
2
formation of three sp and one 2p orbitals (sp hybridization), or in two sp
orbitals and two 2p orbitals (sp hybridization). The angle between these
different orbitals is always such that the distance between the orbitals is
3
maximal. Thus, the four sp orbitals make an angle of 109.5°. This can be
visualized as a regular tetrahedron, with the C-atom in the center and the
35
36
Chapter 2
2
orbitals pointing into the corners. The three sp orbitals make an angle of
120° with each other, and lie in a plane. The two sp orbitals make an angle
of 180° with each other.
If the bond formed between two adjacent atoms is symmetric along the axis
between the two nuclei, we refer to the bond as a σ-bond. In contrast, the
bond that is formed between two electrons in the p-orbitals is called a πbond. These molecular orbitals have a very different shape. The single bond
between two carbon atoms (-C-C-) is formed between electrons in the sp3orbitals (σ-bond) of two adjacent carbon atoms (Figure 1a). A double bond
between two C-atoms (-C=C-) consists of a σ-bond formed between
electrons in the sp2 orbitals of two adjacent C-atoms, and a π-bond formed
between two 2p-orbitals (Figure 2-1).
Figure 2-1. Formation of a σ and a π-bond between two sp2 hybridized carbon atoms. The porbital has two lobes, one above and one below the C-atom.
A single bond has a length of 1.54 Å (1Å = 1Ångstrom = 10-10 m). A
double bond is 1.34 Å. In conjugated molecules there is an alternation
between single and double bonds, such that the π-electrons can be shared by
all C-atoms that are part of the conjugated system. These electrons are
referred to as delocalized electrons, because they are less confined to the
axis of the bond. A conjugated molecule is considered aromatic if it contains
a cyclic π-system with (4n+2) π-electrons (n = 1, 2, 3, …). Hence, benzene
is the simplest aromatic compound (n = 1). The π-electrons of benzene are
present in a molecular orbital that lies above and below the plane formed by
the C-atoms (Figure 2-1). The bond between the C-atoms is 1.39 Å, which
lies in between the length of the single and double bond.
Chemical properties of phenolic compounds
37
Figure 2-2. Spatial representation (ball-and-stick model) of benzene, with C-atoms in grey
and H-atoms in white. The dotted lines between the C-atoms represent the delocalized
electrons. The image on the right shows the surface area of the highest occupied molecular
orbital (HOMO). Note how the π-electrons are above and below the benzene ring.
The benzene ring is usually depicted as one of two mesomeric structures
(2.1). The double arrow indicates that the true structure of the molecule lies
somewhere in between the two drawn structures. It is therefore more
accurate to use structure 2.2, since the six C-C bonds of the ring are
identical, with the π-electrons delocalized over the entire ring. The
configuration shown in 2.2 is, however, less convenient for drawing reaction
mechanisms.
(2.1)
(2.2)
The delocalization of the π-electrons is energetically favorable, and this
affects the reactivity of aromatic compounds: There is a tendency towards
restoring aromaticity. This is why aromatic compounds, in contrast to
regular alkenes (linear chains of carbon atoms containing at least one double
bond), do not easily undergo addition reactions, whereby a double bond is
replaced by two single bonds. Aromatic compounds show a preference for
substitution reactions, which means that atoms are replaced.
38
1.1
Chapter 2
Planar versus non-planar bonds
The three-dimensional structure of molecules is important, because it
affects both chemical reactivity and biological activity. The number of bonds
the carbon atom is involved in is important in this respect, because it affects
whether the molecule is planar (i.e. flat) or non-planar. Since the angle between
the sp3 orbitals is 109.5°, alkanes (a chain of carbon atoms connected via single
bonds) cannot lie in a flat plane. A double bond, however, confines the
neighboring single bonds to a plane. Hence, because of the conjugated structure,
the benzene ring is planar.
B
B
O
A
O
A
C
C
OH
O
O
(2.3)
(2.4)
The spatial structure of flavone (2.3) and flavanonol (2.4) is very
different. The presence of the double bond in the C-ring of flavone results in
the A- and C-rings being planar, whereas the C-ring of flavanonol is not
planar.
1.2
The acidic nature of the phenolic hydroxyl group
Since phenol is benzene with a hydroxyl group, the reactivity of phenol and
phenolic compounds is in many ways dictated by the chemical properties of the
benzene ring. The first property to consider is acidity. A compound is considered
an acid when it can release a proton (H+) while in solution. The acid constant Ka
of a compound defines to what extent the proton is released. Strong acids will
completely dissociate, whereas weak acids (HA) are at equilibrium with their
dissociated state:
A - + H+
HA
The Ka is defined as:
Ka =
The pKa is the negative
convenient numbers.
10
[H+] [A-]
[HA]
log of Ka, which tends to result in more
Chemical properties of phenolic compounds
39
Phenolic compounds are, in general, weak acids. Compared to the hydroxyl
group of unsubstituted aliphatic alcohols, however, the phenolic OH-group is
more acidic. The reason for this is that the anion formed after abstracting the
proton from the hydroxyl group is relatively stable because of the existence of
several mesomeric structures. The anion is referred to as the phenolate anion.
Hence, phenol (2.5) is a weak acid, with a pKa value of 10. This places phenol
in between carboxylic acids (pKa = 4-5) and aliphatic alcohols (pKa = 16-19).
H
..
..
..
..
..
..
..
O
O
O
O
..
..
..
..
O
OH
(2.5)
1.2.1
The effects of substituents on acidity
The actual pKa of phenolic compounds depends upon the overall
structure of the molecule and the nature of the substituents on the aromatic
ring(s). The compound 2,4,6-trinitrophenol (2.6) has a pKa value 0.71,
equivalent to that of a strong acid. This low pKa-value is the result of the
electron-pulling NO2 substituents. The presence of these groups stabilizes
the anion that results after the proton is removed.
O2 N
O
O
OH
NO2
O2N
NO2
O2 N
O
N
O
OH
....
NO2
(2.6)
NO2
NO2
40
1.2.2
Chapter 2
Use of acidity in separations
The pKa is important since it affects the way certain phenolic
compounds are extracted. If we consider having a mixture of phenols that
range from weak acids to strong acids, the addition of sodium carbonate
(Na2CO3) or sodium bicarbonate (NaHCO3) to the mixture will allow
separation of these phenols. The weak base picks up the H+ from the strong
acids or the more acidic phenols. This results in formation of phenolate salts
of the phenols that are soluble in water.
The less acidic phenols are not neutralized or do not lose a H+ and tend
to remain as “free phenols” and will not be as soluble in water. These
compounds can then be extracted with organic solvents.
1.3
Hydrogen bonding and the phenolic hydroxyl group
The hydrogen bond is an electrostatic interaction between a hydrogen atom
bound to an electronegative atom such as oxygen, fluorine or nitrogen, and the
free electrons of other atoms. An actual covalent bond is not possible, because
that would result in the presence of more than two electrons in the orbital
around the hydrogen atom.
The proton of the hydroxyl group of phenol is an ideal candidate for
hydrogen bonding. Shown below (2.7) are three phenol molecules, with the
hydrogen bonds indicated by dotted lines.
O
O
O
H
H
H
(2.7)
The hydrogen bond is weaker than a shared covalent bond, and the
distance between the hydrogen nucleus and the oxygen nucleus is
approximately 1.5 times larger than that of the covalent bond. The presence
of hydrogen bonds raises the melting and boiling points of compounds,
because more energy is required to break intermolecular bonds. The
presence of hydrogen bonds can alter the UV and IR spectra of a given
compound.
Chemical properties of phenolic compounds
1.3.1
41
Intra- and inter-molecular hydrogen bonds
Phenolic compounds may form both inter- and intra-molecular hydrogen
bonds, referring to bonds that are formed between or within molecules,
respectively. Intra-molecular hydrogen bonds are common between adjacent
hydroxyl groups (ortho-substitution), or groups in the ortho-position relative
to a carbonyl group. An example is quercetin (2.8). The B-ring of flavonoids
is more stable with respect to hydroxyl groups. The hydroxyl groups on the
B-ring are placed in either the ortho- or the tri-vic configuration, both of
which allow hydrogen bonding. The hydroxyl groups on the A-ring are
typically in the meta-position, which precludes hydrogen bonds from
forming. Catechin (2.9) may form a hydrogen bond between the hydroxyl
group and the oxygen of the heterocycle.
H
O
OH
HO
O
OH
OH
O
O
OH
H
(2.8)
HO
O
H
O
OH
1.3.2
(2.9)
Stability of the hydrogen bond ring
Intramolecular hydrogen bonding is considered to reduce the reactivity
of the phenolic hydroxyl group. Thus, it reduces solubility in alcohol, and
may reduce the ability to form esters and ethers. Note that the hydrogen
bond results in the formation of a ring. This ring also has a level of stability.
The six-member rings are more stable and stronger than the five-member
rings. Compare for example O-hydroxyacetophenone (six-member ring;
2.10) to catechol (a five-member ring; 2.11).
42
Chapter 2
O
H
H
H
O
O
O
(2.10)
(2.11)
Intermolecular hydrogen bonds raise melting points and solubility.
Table 2.1 lists physical properties of phenol (2.5), resorcinol (2.12) and
phloroglucinol (2.13) that are influenced by hydrogen bonds.
OH
HO
OH
(2.12)
HO
OH
(2.13)
Table 2-1. Impact of substituents on solubility
phenol
resorcinol
phloroglucinol
Melting point (°C)
Solubility in ethanol
Solubility in water
118
1 g/0.9 ml
1 g/0.9 ml
218
1 g/12 ml
1 g/100 ml
41
very good
1 g/15 ml
Intermolecular hydrogen bonds make it difficult to purify phenolic
compounds from mixtures, because of the interactions between different
molecules, including the solvent.
Hydrogen bond formation in phenolic compounds can be summarized
with the following general rules:
1. Unless they are sterically hindered, all phenolic compounds take
part in hydrogen bonding.
2. Intramolecular hydrogen bonds are less stable than
intermolecular hydrogen bonds. The formation of intramolecular
hydrogen bonds diminishes reactivity, whereas the formation of
intermolecular hydrogen bonds can complicate purification.
Chemical properties of phenolic compounds
43
3. Phenolic compounds that form intermolecular hydrogen bonds
are typically solid at room temperature.
4. Ring structures can be formed as a result of hydrogen bonds.
Six-member rings are more stable than five-member rings.
5. Some phenolic compounds can form flat hexagonal structures
with the aromatic rings facing outward, and linked by hydrogen
bonds. The internal space thus formed contains solvent. Such
compounds are called inclusion compounds or clathrates. An
example is dianin (2.14), which can form clathrates in more than
50 different solvents.
O
(2.14)
1.4
OH
Metal complexes
Metal complexes of phenols are important in nature and useful in the
laboratory. The metals involved usually include iron, aluminum and
magnesium. In nature the flavonoids account for most red, blue, and violet and to some extent yellow - colors. The majority of yellow colors are the
result of the presence of carotenoids and aurones.
The precise color of anthocyanins depends on the substitution pattern,
the pH (red in acid, blue in base), but also on the formation of complexes
formed with iron, aluminum and magnesium ions. Such complexes can be
rather large, and chemically diverse. An example is the blue pigment
protocyanin (2.15) from cornflower. This is a cyanidin 3,5-diglucoside
complex of two molecules of cyanidin linked through their o-diphenol
groups with one Al3+ or Fe3+ ion. Cyanocentaurin is even more complex:
four molecules of cyanidin 3,5-diglucoside, iron, and three molecules of
biflavone glycoside.
Several structures are capable of forming metal complexes: o-dihydroxy
phenols (2.16), 3-hydroxychromones (2.17), 5-hydroxychromones (2.18),
and o-hydroxycarbonyls (2.19).
44
Chapter 2
M
O
O
HO
O
O
O
O
OH
RO
OR
OH
OH
(2.15)
O
OH
M
OH
O
O
O
(2.16)
O
OH
O
O
O
O
M
(2.17)
OH
O
O
O
M
(2.18)
O
OH
M
O
O
R
R
(2.19)
The overall structure of the molecule determines the reactivity of the
molecule with the metal, and the presence of the metal ion will impact the
chemical properties of the complex. The degree to which the chemical
Chemical properties of phenolic compounds
45
properties are altered as a result of complex formation depends on the
structure of the phenolic compound. For example, aluminum chloride has
less effect on the absorption spectrum of catechol (2.20) than on that of 3,4dihydroxychalcone (2.21).
OH
OH
OH
OH
O
(2.20)
(2.21)
Metal complexes are used for compound identification. They can shift
or change absorption spectra, change the Rf of compounds in thin layer
chromatography, and change visual colors used in chromatography.
1.5
Esterification
Esters (RCOOR) are formed by reaction of a carboxylic acid with the
hydroxyl group of an alcohol. The hydroxyl group of phenolic compounds
can participate in ester formation.
Esters of two phenols are not
particularly common in nature. The most familiar is the diester of gallic acid
(2.22), which is ellagic acid (2.23), along with other gallotannin compounds.
O
OH
O
HO
O
OH
HO
HO
OH
HO
HO
O
O
(2.22)
(2.23)
Generally the esters of phenols found naturally are compounds where
the phenol contributes the carboxyl group, and another compound
contributes the alcoholic hydroxyl group. The hydroxycinnamic acids do not
seem to undergo intermolecular condensation, but esters with quinic acid
and other acids do occur. For example, chlorogenic acid is an ester of caffeic
acid and quinic acid (3-caffeoylquinic acid; 2.24).
46
Chapter 2
O
OH
O
OH
OH
HO
OH
O
OH
(2.24)
o-Hydroxycinnamic acids undergo intramolecular esterification to yield
lactones that are called coumarins (see also Chapter 1, Section 3.5). Shown
below is the formation of coumarin (2.28) from coumaric acid (2.25; by
definition coumaric acid has the hydroxyl group in the ortho–position),
which involves glycoslyation (2.26; see also Section 1.7), isomerization
from the trans- to the cis-form (2.27), and intramolecular esterification.
O
OH
OH
O
O
O
OH
O-Glc
O
+Glc
(2.25)
OH
O
Glc -Glc
(2.26)
(2.27)
(2.28)
Of interest to mycologists, the basidiomycete Polyporus leucomelas
produces the phenolic ester protoleucomelone (2.29).
OAc
OAc
OAc
AcO
OAc
OAc
AcO
(2.29)
Ac = CH3CO
Chemical properties of phenolic compounds
1.6
47
Ethers and glycosides
Ethers (R-O-R) are frequently found as natural products in nature. The
most common ether is that of methanol and the phenolic hydroxyl group.
The methyl ether (methoxyl group) is very stable and therefore not reactive.
Shown below is the formation of methoxybenzene (2.30) from phenol and
methanol.
H+
H3 C
OH
H 2O
O
OH
CH3
(2.5)
(2.30)
Glycosides – formed between a sugar molecule and an alcohol – are in
some sense similar to ethers. Glycosides are formed between the sugar
molecule in a ring conformation (pyranose or furanose form) and an alcohol.
The example below shows D-glucose (2.31), in equilibrium with -Dglucopyranose (2.32).
CHO
H
H OH
OH
HO
H O
H
HO
H
OH
H
OH
HO
H
OH
OH
(2.32)
H
CH2OH
H
+ H+, -H2O
(2.31)
H OH
H
O
HO
H
HO
H
HO
OH
(2.5)
(2.33)
H
- H+
H OH
H OH
H O
H O
HO
HO
H
HO
H
O
HO
H
OH
H
O
(2.34)
OH
H
H
(2.35)
Figure 2-3. Formation of phenolic glycosides
48
Chapter 2
In presence of acid, cation 2.33 reacts with phenol (2.5) to result in a
mixture of the α- and -glucoside (2.34 and 2.35, respectively). Chemically
this reaction involves the formation of an acetal (2.34 and 2.35) from a semiacetal (the pyranose 2.32; the furanose could also react). Unlike typical ether
bonds, the glycoside bond is susceptible to acid hydrolysis.
1.7
Oxidation of the phenolic hydroxyl group
Oxidation of phenols is one of the most important aspects of these
compounds to the biologist. Oxidation of phenolic compounds can result in
the browning of tissues. Well-known examples are the browning of fruits
after they have been cut. Oxidation can also result in the formation of
metabolites that are toxic to animals and plants, and that can account for
spoilage of foods in processing. On the other hand, toxic compounds formed
from the oxidation of phenolics can inhibit pathogenic microorganisms.
Certain phenols are used as retardants or antioxidants to prevent the
oxidation of fatty acids.
1.7.1
Auto-oxidation of phenolic compounds
Auto-oxidation refers to the formation of cross-linked structures as a
result of exposure to light and oxygen. Under the influence of light, oxygen
can abstract a proton, thereby generating a radical. This is particularly likely
to occur if the proton is adjacent to a double bond, because the radical
electron can be delocalized, thus lowering the energy.
Given their aromatic nature, phenolic compounds are easily autooxidized. The radical that is generated can subsequently react with other
radicals to form a dimer. Since the radical electron is delocalized, several
structures can be formed depending on the precise location of the radical
electrons at the time of the reaction.
Figure 2-4 shows how radicals of catechol (2.11) can react to form
mixtures of tetrahydroxy-biphenyls (2.36) and quinines (2.37). Another
example, shown in Figure 2-5, shows the formation of dimers of p-cresol
(2.38).
A variety of complex compounds can arise through these mechanisms,
including biflavonyls and bianthraquinones. An example of the latter is the
compound iridoskyrim (2.39) formed by the fungus Penicillium islandicum.
Chemical properties of phenolic compounds
.
OH
O
OH
OH
49
O
.
OH
(2.11)
O
OH
HO
OH
OH
OH
HO
OH
(2.36)
O
OH
O
OH
(2.37)
Figure 2-4. Auto-oxidation of catechol can result in the formation of different dimers
OH
H2O
.
O
OH
OH
O
O
.
O2
.
(2.38)
H2O
O
O
O
O
Figure 2-5. Auto-oxidation of p-cresol can result in the formation of different dimers
50
Chapter 2
OH
O
HO
O
H3C
OH
(2.39)
1.7.2
OH
CH3
O
OH
O
OH
Enzymatic oxidation of the phenolic hydroxyl group
An alternative mechanism for the oxidation of phenolic compounds is
enzyme-catalyzed oxidation. Several classes of enzymes can catalyze this
reaction. According to the Nomenclature Committee of the International
Union of Biochemistry and Molecular Biology (NC-IUBMB), these
enzymes are part of the E.C. 1 class of oxidoreductases (see the Internet web
site: http://www.chem.qmul.ac.uk/iubmb/enzyme/EC1). The three main
classes of enzymes that catalyze the oxidation of phenolic compounds are
the oxidoreductases that use oxygen as electron acceptor (E.C. 1.10.3), the
peroxidases (E.C. 1.11.1), and monophenol monooxygenase (E.C.
1.14.18.1).
1.7.2.1
E.C. 1.10.3
This class includes enzymes that use diphenols or related compounds as
electron donors and oxygen as the acceptor, thereby forming the oxidized
donor and water. Members include catechol oxidase (E.C. 1.10.3.1), laccase
(E.C. 1.10.3.2), and o-aminophenol oxidase (E.C. 1.10.3.4). Laccase is also
known as p-diphenoloxidase, whereas catechol oxidase is also known as
diphenoloxidase, phenoloxidase, polyphenoloxidase, o-diphenolase,
phenolase and tyrosinase. Many of these names are also used in reference to
a different enzyme, monophenol monooxygenase (E.C. 1.14.18.1). This
enzyme will be discussed further in Section 1.8.2.2.
The phenol-oxidizing enzyme tyrosinase has two types of activity: (1)
phenol o-hydroxylase (cresolase) activity, whereby a monophenol is
converted into an o-diphenol via the incorporation of oxygen, and (2)
cathecholase activity, whereby the diphenol is oxidized. The two reactions
are illustrated in Figure 2-6, in the conversion of tyrosine (2.40) to L-DOPA
(3,4-dihydroxyphenylalanine; (2.41), dopaquinone (2.42), and indole-5,6quinone carboxylate (2.43), which is further converted to the brown pigment
Chemical properties of phenolic compounds
51
melanin via enzyme-mediated oxidation (reviewed by Sánchez-Ferrer et al.
(1995)).
Melanin is the major determinant of skin color in humans
(Sturm, 1998), and is formed when the cut surfaces of fruits, such as apples,
bananas and avocados, are exposed to air.
O
O
O
O
O
O
O
O
NH3
NH3
NH3
NH
1
H+
1
/2 O 2
/ 2 O2
HO
O
O
OH
OH
O
O
(2.40)
(2.41)
(2.42)
(2.43)
Figure 2-6. Tyrosinase-catalyzed oxidation of tyrosine results in precursors of melanin
Laccase catalyzes the oxidation of p-diphenols to p-quinones. Shown in
Figure 2-7 is the oxidation of 1,4-dihydroxybenzene (2.44) to p-quinone
(2.45).
.
OH
O
O
laccase
1
/2 O2
OH
(2.44)
H2O
.
O
O
(2.45)
Figure 2-7. The oxidation of 1,4-dihydroxybenzene to p-quinone
This enzyme exhibits no hydroxylase activity and is involved in the final
synthesis of many naturally occurring p-quinones, e.g. the naphthaquinone
juglone in walnut (1.58) and the benzoquinone arbutin (hydroquinone- -Dglucopyranoside; 2.46). Arbutin is a plant cryo-protectant that stabilizes
membranes (Hincha et al., 1999). This compound has medicinal properties
and has, for example, been used to treat urinary tract infections in humans. It
is also used to lighten skin color, because it inhibits tyrosinase and hence the
formation of melanin. The derivative deoxyarbutin (2.47; note the difference
in the sugar molecule) was recently reported to be considerably more
effective as a skin-lightening compound (Boissy et al., 2005).
52
Chapter 2
OH
OH
H OH
H
H
H
O
HO
H
O
H
O
HO
H
O
H
H
OH
H
H
H
H
H
(2.46)
(2.47)
While initially controversial, there is evidence that laccases play a role
in the polymerization of the cell wall polymer lignin (see Chapter 1, section
3.12), which occurs via the oxidative coupling of monolignol radicals with
reactive (oxidized) sites on the lignin polymer. The evidence for their
involvement comes from a number of studies in which laccases were
localized to lignifying tissues in woody species through the use of
histochemical stains. Furthermore, the expression of the laccase genes was
shown to be specific for lignifying tissues. In addition, when laccases
purified from these tissues were mixed with monolignols under aerobic
conditions, a dehydrogenation polymer (DHP) with lignin-like
characteristics was formed (Sterjiades et al., 1992; Driouich et al., 1992; Boa
et al., 1993; Ranocha et al., 1999). Down-regulation of laccase genes in
poplar through the introduction of antisense constructs did not, however,
impact lignin content nor subunit composition, but did have an effect on cell
wall structure (Ranocha et al., 2002).
Figure 2-8 shows laccase-mediated generation of radicals of coniferyl
alcohol (2.48), with the stoichiometry of the reaction adjusted for coniferyl
alcohol. The radical electron is delocalized, enabling the formation of
various interunit linkages, as discussed in Chapter 1.
CH2OH
CH2OH
CH2OH
.
laccase
1
/4 O2
OH
1
/2 H2O
O
.
O
(2.48)
Figure 2-8. Laccase-catalyzed formation of coniferyl alcohol radicals
Chemical properties of phenolic compounds
53
1.7.2.2
E.C. 1.14.18.1
The E.C. 1.14 class of monooxygenases contains enzymes acting on
paired donors, with the incorporation or reduction of molecular oxygen.
Monophenol monooxygenase (E.C. 1.14.18.1) catalyzes the same reactions
as catechol oxidase (E.C. 1.10.3.1; see Section 1.8.2.1) if only 1,2benzenediols are available as substrate. In this case one of the monophenols
acts as a donor for the oxidation of the other monophenol, and one atom of
oxygen is incorporated. Common names like tyrosinase and phenolase can
refer to both catechol oxidase and monophenol monooxygenase, but they
can be distinguished based on the fact that E.C. 1.14 uses paired donors. In
addition, the E.C. number is generally indicated in the text to further clarify
this.
1.7.2.3
E.C. 1.11.1.
The E.C. 1.11.1 subclass contains the peroxidases, which use hydrogen
peroxide (H2O2) as electron acceptor to oxidize the donor, thereby forming
the oxidized donor and water. Members include horseradish peroxidase
(E.C. 1.11.1.7; also known as guaiacol peroxidase and scopoletin
peroxidase), manganese peroxidase (E.C. 1.11.1.13) and diarylpropane
peroxidase (E.C. 1.11.1.14). All three classes are hemoproteins. Horseradish
peroxidase and related peroxidases are involved in the oxidative coupling of
lignans, lignin, and tannins. Mechanistically, hydrogen peroxide oxidizes the
active site of the peroxidase enzyme, and upon binding of the substrate in
the active site, the substrate becomes oxidized and the enzyme returns to its
reduced state.
Peroxidases are encoded by large multi-gene families, which has
complicated the study of individual peroxidase enzymes (cf. Christensen et
al., 1998). Manganese and diarylpropane peroxidases are used by white rot
fungi (basiodiomycetes) to degrade lignin via oxidation.
The origin of the H2O2 that peroxidases use is not entirely clear. The
best studied peroxidases are the ones involved in cell wall lignification, and
several mechanisms that describe the generation of H2O2 have been
identified in different plant species, as will be discussed below.
Ogawa et al. (1997) investigated the formation of H2O2 and the
superoxide radical (O2.-) in spinach (Spinacia oleracea) hypocotyls with the
use of histochemical stains. Nitroblue tetrazolium (NBT) is used to detect
O2.- radicals. The colored reaction product formazan was only detected in the
vascular tissue of developing spinach hypocotyls if CuZn-superoxide
dismutase (CuZn-SOD; E.C. 1.15.1.1) was inhibited by DDC (N,N-
54
Chapter 2
diethyldithiocarbamate), suggesting that CuZn-SOD effectively catalyzes
the elimination of the O2.- radicals. The mechanism for the elimination of
these radicals is through dismutation of superoxide into oxygen (O2) via
oxidation,
and
H2O2
via
reduction.
Imidazole
and
DPI
(diphenyleneiodonium), inhibitors of NAD(P)H-oxidase (E.C. 1.6.99.6),
were shown to suppress the formation of formazan, indicating the
involvement of NAD(P)H-oxidase in superoxide radical formation. NADPH
is nicotinamide dinucleotide phosphate, a compound that is used as an
electron donor throughout cellular metabolism. Based on these results they
proposed a mechanism for the generation of H2O2, shown below in scheme
2.1, that involves the concerted action of a membrane-bound NADPH
oxidase and a CuZn-superoxide dismutase The H2O2 that is generated by the
combined action of these two enzymes is then used to activate a peroxidase
that oxidizes monolignols.
NADPH-oxidase
NAD(P)H + 2 O2
2 O2.-
+
2 H+
NAD(P)+ + 2 O2.-
CuZn SOD
H2O2 + O2
Figure 2-9. Formation of H2O2 through the concerted action of NAD(P)H oxidase and CuZnsuperoxide dismutase, as proposed by Ogawa et al. (1997).
There are cases where the peroxidase is not activated by H2O2, but by a
reaction product instead. Ferrer et al. (1990) described the oxidation of the auxin
indole-3-acetic acid (IAA; 2.49) and molecular oxygen by a cell wall peroxidase
that was able to oxidize coniferyl alcohol (2.48) in the absence of H2O2.
Auxins are plant hormones involved in a number of developmental
processes in plants, including embryo development, leaf formation, and
apical dominance (reviewed by Leyser (2005), and Woodward and Bartel
(2005)). IAA is transported through the extracellular space, and would thus
be readily available as a reductor to cell-wall bound peroxidases. The cellwall bound peroxidases in this study were isolated from lupin (Lupinus
alba), and the oxidation of coniferyl alcohol at the expense of IAA was
monitored spectrophotometrically in the UV range of the spectrum. The IAA
was converted to oxindoles. Previous studies showed that 3-methylene 2oxindole (2.54) is the predominant oxindole formed (Ricard and Job, 1974).
Ferrer et al. (1990) showed that the oxidation of coniferyl alcohol was
dependent on the concentration of IAA, whereby high concentrations
inhibited the reaction. This makes sense physiologically, since lignification
is associated with a terminal developmental process, whereas high levels of
Chemical properties of phenolic compounds
55
auxin are correlated with growth and differentiation. Folkes et al. (2002)
proposed a reaction mechanism for the peroxidase-mediated oxidation of
IAA without the involvement of H2O2, which is shown in Figure 2-10.
Oxidation of IAA (2.49) results in cation 2.50, which undergoes
decarboxylation and results in the skatolyl radical (2.51). This compound
reacts with molecular oxygen to form peroxyl radical 2.52. With IAA or
another cellular redactor, the hydroperoxide 2.53 is formed. It is this
compound that activates the peroxidase, and thus allows the oxidation of
other substrates, such as coniferyl alcohol. Among the degradation products
of 2.53, 3-methylene 2-oxindole (2.54) is the most abundant.
OH
OH
O
O
.N
N
H
(2.49)
(2.50)
O
H2C
.
O
H
.
CH2
O2
N
N
H
(2.52)
H
(2.51)
O
H2C
OH
CH2
O
N
N
(2.53)
H
(2.54)
H
Figure 2-10. Oxidation of IAA
An alternative mechanism for the generation of H2O2 was described by
Caliskan and Cuming (1998), who studied the wheat protein germin. This
protein is synthesized de novo when wheat embryos germinate. It was
shown to be highly resistant to proteolytic degradation, and to have oxalate
oxidase (E.C.. 1.2.3.4) activity. This enzyme catalyzes the oxidation of
oxalate (2.55), and the formation of H2O2 as shown in Figure 2-11. Given
that in 9-day old seedlings both the oxalate oxidase mRNA and protein were
localized to the vascular tissue, the authors speculated that wheat germin
56
Chapter 2
plays a role in providing H2O2 in those tissues of the seedling where the cell
wall needs to be cross-linked to restrict cell growth.
O
O
oxalate oxidase
2 H+ + O2
O
2 CO2 + H2O2
O
(2.55)
Figure 2-11. Formation of H2O2 via oxidation of oxalate by oxalate oxidase
A fourth possibility is the generation of H2O2 via oxidation of putrescine
(butane-1,4-diamine; 2.56). This reaction is catalyzed by copper amine
oxidase (E.C. 1.4.3.6). Copper amine oxidases are homodimers in which
each unit contains a copper ion and a 1,3,5-trihydroxyphenylalanine quinine
co-factor. In plants copper amine oxidases generally oxidize putrescine to 4aminobutanal (2.57). This latter compound undergoes spontaneous
cyclization to ∆1 pyrroline (2.58), ammonia, and H2O2, as shown in Figure
2-12 (Medda et al., 1995).
NH2
O
Cu-amine oxidase
H
NH
NH3 + H2O2
O2
H2O
H2O
NH2
(2.56)
NH2
(2.57)
(2.58)
Figure 2-12. Formation of H2O2 via oxidation of putrescine by copper amine oxidase
The copper amine oxidase in the model plant Arabidopsis thaliana is
encoded by the ATAO1 gene (Møller and McPherson, 1999). In situ
hybridizations and analyses of transgenic plants expressing a reporter gene
under control of the ATAO1 promoter revealed expression of the ATAO1
gene in the root cap and the vascular tissue. This would make it feasible that
H2O2 generated via this mechanism could be used by a peroxidase involved
in lignification and cross-linking of cell wall proteins in Arabidopsis.
Ros-Barceló et al. (2002) analyzed lignifying xylem of Zinnia elegans.
Based on the inhibition of H2O2 production as a result of treatment with
Chemical properties of phenolic compounds
57
imidazole, an involvement of NADPH-oxidase (E.C. 1.6.3.1) was
hypothesized. This enzyme catalyzes the formation of H2O2 from the
oxidation of NADPH with molecular oxygen, as shown in Figure 2-13.
NADPH + H+ + O2
NADPH-oxidase
NADP+
+ H2O2
Figure 2-13. Formation of H2O2 via oxidation of NADPH by NADPH oxidase
Önnerud et al. (2004) recently proposed a mechanism involving a redox
shuttle for the oxidative coupling of coniferyl alcohol (2.48), as it may occur
during lignification. Given that lignin is very compact, the authors
speculated that enzymes such as peroxidases may be too large to be
effective. They investigated whether manganese (II) oxalate (2.55; see
Figure 2-11) could function as a redox shuttle. Figure 2-14 depicts how
Mn(II) oxalate is reduced to Mn(III) oxalate by a membrane or cell-wall
bound manganese peroxidase (E.C. 1.11.1.13). The Mn(III) diffuses into the
cell wall, oxidizes monolignols and the lignin polymer, and returns to the
manganese peroxidase to get oxidized again.
H2O2
Mn2+
Mn2+
monolignol radical
oxidized lignin residue
Mn3+
Mn3+
monolignol
lignin
Mn peroxidase
H2O
lignifying cell wall
Figure 2-14. Oxidation of monolignols via a manganese oxalate redox shuttle as proposed by
Önnerud et al. (2004)
Based on this overview, it is clear that there are many different
mechanisms by which H2O2 can be generated, and it is possible that even
more mechanisms exist. Further research is needed to determine to what
extent these mechanisms are unique to particular plant species, tissues,
metabolic processes, or developmental stages. Given that the availability and
concentration of enzyme substrates is likely to fluctuate as a function of both
the developmental stage and the environmental conditions, the availability
of multiple mechanisms to generate H2O2 offers a high degree of flexibility.
58
1.8
Chapter 2
Reactive oxygen species and antioxidants
Radicals are molecules with a free (unpaired) electron that are highly
reactive. Radicals are formed in all living organisms during oxidation
reactions that occur as part of normal metabolism. Under certain
circumstances such as environmental stress, wounding, and pathogen attack,
the concentration of free radicals is increased beyond the normal levels.
Radicals can do considerable damage to living organisms when left
unchecked. This results in part from their reactivity, particularly towards
DNA and membranes (lipids and proteins), and in part from the chain
reactions they can initiate. Chain reactions occur when a radical reacts with
another molecule, abstracts an electron, and thereby creates a new radical
that can react with other molecules.
Reactive oxygen species (ROS) are molecules that contain an oxygen
atom and that are highly reactive as a result of the presence of a free radical,
or a configuration of the oxygen atom whereby there are more electrons than
usual. Examples of the first class include the hydroxyl radical (.OH), and the
superoxide radical (O2. —), whereas the peroxide (O22—) and hypochlorite
(ClO. —) ions belong to the second class. Hydrogen peroxide (H2O2) is also
considered as a ROS because of its reactivity (Halliwell, 1991), as we have
seen in Section 1.8.2.3. The hydroxyl radical is the most reactive. In fact, it
is considered the most reactive radical known, with an ability to react with a
very wide range of (bio-)molecules. It can be produced via the Fenton
reaction, first described in 1894 (Figure 2-15).
Fe2+ + H2O2
Fe3+ + OH- + OH.-
Figure 2-15. Production of the hydroxyl radical via the Fenton reaction
Because of the reactivity of the hydroxyl radical, and the fact that the
ingredients are inexpensive, the Fenton reaction is used on a commercial
scale to treat waste water. The Fenton reaction can also occur with copper as
the transition metal. Given that Fe2+, Cu+, and H2O2 are abundantly present
in biological systems, hydroxyl radicals can be generated via the Fenton
reaction in vivo. Reviews by Schützendübel and Polle (2002) and by Valko
et al. (2005) describe the impact of ROS in plants and humans, respectively.
Chemical properties of phenolic compounds
59
A particularly damaging reaction is the reaction between the hydroxyl
radical and unsaturated fatty acid side chains of phospholipids in the cell
membrane, a reaction referred to as lipid peroxidation (Figure 2-17).
.OH
.
COOR
COOR
.
COOR
O2
COOR
.
O
COOR
O
COOR
O
HO
+
.
COOR
Figure 2-17. Lipid peroxidation. A hydroxyl radical abstracts a hydrogen from a fatty acid or
lipid molecule. After rearrangement to a conjugated structure, the radical reacts with oxygen
to form a peroxyl radical. The newly formed peroxyl radical can initiate a chain reaction
whereby new peroxyl radicals are formed.
The hydroxyl radical will abstract a hydrogen atom from the fatty acid,
creating a fatty acid radical with the free electron on a carbon atom in the
chain. The radical will typically undergo a rearrangement resulting in a more
stable conjugated structure. This newly generated radical can crosslink with
a nearby fatty acid radical. Alternatively, the fatty acid radical can react with
molecular oxygen to produce a peroxyl radical. This then sets in motion a
chain reaction, whereby many new peroxyl radicals are generated. As a
consequence of lipid peroxidation, the fluidity of the membrane can be
affected, and membrane proteins, especially receptors, can become part of
the radical reactions, affecting their function. Ultimately, the membrane can
collapse.
60
Chapter 2
More recently, the impact of excess iron on carcinogenesis has been
studied. Toyokuni (2002) reported that in kidney cells of rats an overload of
iron can result in carcinogenesis because of Fenton-reaction induced damage
to a tumor suppressor gene. This is currently an active area of research.
Living organisms have developed various ways to deal with ROS. One
mechanism is enzymatic inactivation. The enzyme superoxide dismutase,
(E.C. 1.15.1.1) catalyzes the dismutation of superoxide into oxygen (O2) via
oxidation, and H2O2 via reduction (see also Section 1.8.2.3). The H2O2,
which is reactive itself, is removed through the action of the enzymes
catalase (E.C. 1. 11.1.6) and glutathione peroxidase (E.C. 1.11.1.9). Catalase
catalyzes the conversion of H2O2 to water and oxygen, whereas glutathione
peroxidase catalyzes the formation of oxidized glutathione (G-S-S-G) from
reduced glutathione (G-SH), at the expense of H2O2 (Halliwell, 1991).
The other commonly used mechanism to inactivate ROS is through the
use of antioxidants. Antioxidants can react with the radical, but rather than
turning into another reactive molecule, these compounds are relatively stable
in the presence of the radical electron. As a consequence, they scavenge the
radical electrons, quench the chain reaction, and avoid further damage. The
relative stability of antioxidants containing a radical electron is generally the
result of the presence of conjugated bonds, so that the radical electron can be
delocalized. As a consequence, aromatic compounds in general, and
phenolic compounds in particular are very effective antioxidants. Examples
of delocalized radical electrons in phenolic compounds are given in
structures 2.11 and 2.38. The antioxidant properties of phenolics will be
discussed in more detail in Chapter 7.
Chemical properties of phenolic compounds
2.
61
REFERENCES
Bao, W., O'Malley, D. M., Whetten, R., and Sederoff, R.R., 1993, A laccase
associated with lignification in loblolly pine xylem. Science 260: 672674.
Boissy, R. E., Visscher, M., deLong, M. A., 2005, Deoxyarbutin: a novel
reversible tyrosinase inhibitor with effective in vivo skin lightening
potency, Exp. Dermatol. 14: 601-608.
Christensen, J. H., Bauw, G., Gjesing Welinder, K., Van Montagu, M., and
Boerjan, W., 1998, Purification and characterization of peroxidases
correlated with lignification in poplar xylem, Plant Physiol. 118: 125135.
Driouich, A., Laine, A. C., Vian, B., and Faye, L., 1992, Characterization
and localization of laccase forms in stem and cell cultures of sycamore,
Plant J. 2: 13-24.
Ferrer, M. A., Pedreño, M. A., Muñoz, R., and Ros Barceló, A., 1990,
Oxidation of coniferyl alcohol by cell wall peroxidases at the expense of
indole-3-acetic acid and O2, FEBS Lett. 276: 127-130.
Folkes, L. K., Rossiter, S., and Wardman, P., 2002, Reactivity toward thiols
and cytotoxicity of 3-methylene-2-oxindoles, cytotoxins from indole-3acetic acids, on activation by peroxidases, Chem. Res. Toxicol. 15: 877882.
Halliwell, B., 1991, Reactive oxygen species in living systems, Am. J. Med.
91: 14S
Hincha, D. K., Oliver, A. E., and Crowe, J. H., 1999, Lipid composition
determines the effects of arbutin on the stability of membranes, Biophys.
J. 77: 224-234.
Leyser, O., 2005, Auxin distribution and plant pattern formation: how many
angels can dance on the point of PIN?, Cell 121: 819-822.
Medda, R., Padiglia, A., Pedersen, J. Z., Rotilio, G., Finazzi Agró, and
Floris, G, 1995, The reaction mechanism of copper amine oxidase:
detection of intermediates by the use of substrates and inhibitors,
Biochem. 34: 16375-16381.
Møller, S. G., and McPherson, M. J., 1998, Developmental expression and
biochemical analysis of the Arabidopsis atao1 gene encoding an H2O2generating diamine oxidase, Plant J. 13: 781-791.
Ogawa, K., Kanematsu, S., and Asada, K., 1997, Generation of superoxide
anion and localization of CuZn-superoxide dismutase in the vascular
tissue of spinach hypocotyls: their association with lignification, Plant
Cell Physiol. 38: 1118-1126.
Önnerud, H., Zhang, L., Gellerstedt, G., and Henriksson, G., 2004,
Polymerization of monolignols by redox shuttle–mediated enzymatic
62
Chapter 2
oxidation: a new model in lignin biosynthesis I, Plant Cell 14: 19521963.
Ranocha P., Chabannes, M., Chamayou, S., Danoun, S., Jauneau, A.,
Boudet, A.-M., and Goffner, D., 2002, Laccase down-regulation causes
alterations in phenolic metabolism and cell wall structure in poplar, Plant
Physiol. 129: 145-155.
Ranocha, P., McDougall, G., Hawkins, S., Sterjiades, R., Borderies, G.,
Stewart, D.,, Cabanes-Macheteau, M., Boudet, A.-M., and Goffner, D.,
1999, Biochemical characterization, molecular cloning and expression of
laccases – a divergent gene family – in poplar, Eur. J. Biochem. 259:
485-495.
Ricard, J. and Job, D., 1974, Reaction mechanisms of indole-3-acetate
degradation by peroxidases. A stopped-flow and low-temperature
spectroscopic study, Eur. J. Biochem. 44: 359-374.
Ros-Barceló, A., Pomar, F., López-Serrano, M., Martínez, P., Pedreño, M.
A., 2002, Developmental regulation of the H2O2-producing system and of
a basic peroxidase isoenzyme in the Zinnia elegans lignifying xylem,
Plant Physiol. Biochem. 40: 325-332.
Sánchez-Ferrer, A., Rodríguez-López, J. N., García-Cánovas, F., and
García-Carmona, F., 1995, Tyrosinase: a comprehensive review of its
mechanism, Biochim. Biophys. Acta 1247: 1-11.
Schützendübel, A., and Polle, A., 2002, Plant responses to abiotic stresses:
heavy metal-induced oxidative stress and protection by mycorrhization,
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Sterjiades, R., Dean, J. F. D., and Eriksson, K.-E. L., 1992, Laccase from
sycamore maple (Acer pseudoplatanus) polymerizes monolignols, Plant
Physiol. 99: 1162-1168.
Sturm, R. A., 1998, Human pigmentation genes and their response to UV
radiation, Mutat. Res. Rev. 422: 69-76.
Toyokuni, S., 2002, Iron and carcinogenesis: from Fenton reaction to target
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Valko, M., Morris, H., and Cronin, M. T., 2005, Metals, toxicity and
oxidative stress, Curr. Med. Chem. 12: 1161-1208.
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For further reading:
Denisov, E., and Afanas’ev, I. B., 2005, Oxidation and Antioxidants in
Organic Chemistry and Biology, CRC Press, Boca Raton, 1024 pp.
Smirnoff, N., Antioxidants and Reactive Oxygen Species in Plants,
Blackwell Publishing, Oxford, 302 pp.
BIOSYNTHESIS OF PHENOLIC COMPOUNDS
63
Chapter 3
BIOSYNTHESIS OF PHENOLIC COMPOUNDS
1.
INTRODUCTION
This chapter provides an overview of the biosynthetic pathways leading
to the major classes of phenolic compounds as outlined in Chapter 1. This
overview is intended to show the origin of the different families of phenolic
compounds, many of which share precursors. In addition to an overview of
established pathways, newly elucidated steps in the biosynthesis of several
classes of compounds will be highlighted.
Detailed studies of biosynthetic pathways have recently become much
more feasible due to the availability of mutants in which genes that affect
phenolic metabolism are defective. Genes consist of coding regions and
regulatory sequences. The coding region is transcribed into messenger RNA
(mRNA) by (generally) RNA polymerase II and stipulates the amino acid
sequence of the protein encoded by the gene. The regulatory sequences are
located at the upstream (5´) and sometimes also the downstream (3´) end of
the coding region and control the spatio-temporal expression of the gene.
‘Spatio’ refers to cells, tissues or organs where a gene is expressed, whereas
‘temporal’ refers to the developmental stage at which a gene is expressed.
Mutations are genetic alterations in the DNA that may affect gene
expression. This can be the result of changes in regulatory elements of the
gene, so that the gene is no longer expressed, expressed at lower levels, or
has a different spatio-temporal pattern of expression. Alternatively,
mutations can affect the part of the gene that encodes the protein. In this
case truncated proteins are synthesized, or proteins in which a critical amino
63
64
Chapter 3
acid has been substituted, affecting substrate binding ability, substrate
specificity, or the ability to catalyze the substrate conversion. In these cases
the protein can no longer function in its normal way, which oftentimes leads
to an altered phenotype, such as altered growth characteristics, plant
architecture, or color.
Below follows a brief overview of commonly used strategies for protein
isolation, gene cloning, and protein characterization to describe the
principles of using biochemistry and genetics for the elucidation of
biosynthetic pathways. Next, biochemical pathways leading to the different
classes of phenolic compounds will be presented. Each compound will be
referred to with one number (e.g. 3.12), which will be used throughout the
chapter, even if the compound is an intermediate of several different
pathways and is thus included in several sections of this chapter. Enzymes in
this chapter are referred to by their name and E.C. number whenever
possible. The E.C. numbers were designated by the Enzyme Commission of
the Nomenclature Committee of the International Union of Biochemistry
and Molecular Biology (IUBMB). This information can be accessed via the
Internet at www.expasy.org.
2.
PROTEIN ISOLATION AND PURIFICATION
The traditional methodology for the isolation of proteins involves
biochemical separation techniques, whereby the protein of interest is
isolated from all the other proteins based on its unique physical and
chemical properties. This includes molecular weight (and hence size), shape
(for example, globular versus rod), hydrophobicity, and net electric charge.
In the case of enzymes involved in biochemical pathways, the isolation
is often based on activity assays. The nature of the activity assay depends on
the enzymatic reaction and can involve, for example, the detection of a
product on a thin-layer chromatography (TLC) plate (see Chapter 4, Section
1.2.1), the appearance or disappearance of a specific absorbance in a
spectrophotometric assay, or a coupled assay involving the oxidation or
reduction of a co-factor such as nicotinamide dinucleotide (NAD(H)), which
can be measured by changes in fluorescence.
The isolation procedure starts with the preparation of a cell extract
in which the enzyme activity can be detected. This typically involves
grinding the tissue in an extraction buffer so that the cell contents, including
the proteins, become accessible. Protease inhibitors, such as phenylmethyl
Biosynthesis of phenolic compounds
65
sulphonylfluoride (PMSF), are added to the extraction buffer to avoid
proteolytic degradation of the enzyme. The first fractionation is generally a
centrifugation step, whereby the enzyme is precipitated if it is bound to the
cell wall or the cell membrane, and otherwise ends up in the supernatant.
The supernatant will also contain membrane fractions derived from the
organelles (mitochondria, chloroplasts, endoplasmic reticulum, Golgi
complex), which can be precipitated by ultracentrifugation if desired.
Soluble proteins can be separated from each other based on variation in
their solubility in high-salt solutions. Most proteins will precipitate if the salt
concentration exceeds a certain level, a process referred to as ‘salting out’.
The salt concentration at which proteins precipitate varies depending on the
size, shape, and the proportion and distribution of polar, apolar and charged
amino acids. A successful strategy to separate proteins from each other,
therefore, is to add a salt, followed by centrifugation to remove the
precipitated proteins. Additional salt can be added to the supernatant, so that
different protein fractions representing different solubility values can be
generated. The precipitated proteins can be redissolved by diluting the
precipitate in a low-salt buffer. Ammonium sulfate is commonly used to
precipitate proteins, because most proteins will precipitate in a saturated (4
Molar) solution of this salt. Furthermore, little heat is generated when
ammonium sulfate is dissolved, thus preventing heat-induced denaturation
of the proteins. The ammonium sulfate can be added as a powder, or as a
saturated solution. Enzyme activity assays are performed on the different
fractions that are generated, so that the amount of ammonium sulfate that
needs to be added in order to remove a subset of the proteins in the extract
can be determined. Alternatively, other salts, polyethylene glycol, apolar
solvents, protamine sulfate, or trifluoroacetic acid can be used to precipitate
proteins. After this initial enrichment step, chromatography is typically used
to further purify the enzyme. The main types of chromatography will be
discussed below.
Ion exchange chromatography relies on variation in charge between
different proteins. In this case the chromatography column is filled with a
resin harboring fixed charged groups, and counter ions of the opposite
charge. The protein mixture is loaded onto the column, and proteins with a
charge opposite of the charge of the resin will replace the counter ions and
adsorb to the column, whereas proteins with the same charge as the resin
and uncharged proteins can be removed by flushing the column with a lowsalt buffer. An elution buffer of increasing ionic strength (increasing salt
concentration) or changing pH is pumped through the column. Depending
on the strength of the electrostatic interaction between the protein and the
66
Chapter 3
matrix, the proteins elute from the column at different ionic strengths or pHvalues of the elution buffer. The column can be recharged afterwards.
Hydrophobic interaction chromatography relies on hydrophobic
interactions between apolar amino acid residues in the proteins and a resin
containing hydrophobic groups, such as n-octyl or phenyl groups. After the
protein mixture is applied to the column, an elution buffer with decreasing
ionic strength is used. Hydrophilic proteins will elute first, whereas
hydrophobic proteins elute last.
Bio-affinity chromatography is based on adsorption of the target enzyme
to a resin to which the substrate of the target enzyme has been covalently
linked. An example is the purification of a cellulose-degrading enzyme
through the use of a column containing a cellulose-based resin. Proteins that
have no affinity for the substrate are removed from the column with a wash
buffer, and the target enzyme is eluted through the application of a buffer
containing unbound competitor molecules, or through the use of a buffer
with a high ionic strength or a pH that reduces the affinity of the enzyme for
the resin-bound substrate.
Gel filtration or size exclusion chromatography relies on separation of
proteins by size through a matrix made of small beads in a chromatography
column. The beads contain pores of a fixed size. Large proteins will move
faster through the column than small proteins, because the large proteins
move in between the beads of the matrix, whereas the small proteins move
through the pores. The size of the beads and the size of the pores within the
beads determine the fractionation range, i.e. the range in molecular weight
that can be effectively separated on the column. The dimensions of the
column and the flow rate of the elution buffer determine the resolution.
While there are no set protocols for the specific order in which the
different chromatographic separations are carried out, ion exchange and
hydrophobic interaction chromatography typically precede gel filtration and
bio-affinity chromatography. The latter can involve expensive resins and is
often performed after various contaminants have been removed during
earlier purification steps.
High-performance liquid chromatography (HPLC) and fast protein
liquid chromatography (FPLC) rely on the same separation principles as the
traditional chromatography columns, but tend to be much faster because of
high flow rates that are possible due to the uniform bead size and the
mechanical strength of the beads. See also Chapter 4, section 1.2.2.
Biosynthesis of phenolic compounds
67
Regardless of the chromatography method that is used, fractions
containing subsets of the proteins in the sample are collected at the bottom
of the column. Each of the fractions is assayed for enzyme activity. In
addition, the complexity of the fraction is evaluated by separating the
proteins in a small sample of the fraction using sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE). This method separates
proteins in mixtures based on their size.
The purification is complete when the protein of interest has been
purified to (apparent) homogeneity, meaning that a fraction has been
obtained in which the protein of interest is the only protein, or in which any
remaining contaminants are below the level of detection.
3.
GENE CLONING STRATEGIES
The availability of mutants with altered phenolic content or composition
enables cloning of the mutated gene, and subsequently cloning of the wildtype (normal) version of the gene. It is important to keep in mind that if a
plant lacks a certain phenolic compound as a result of a mutation, the wildtype version of the gene – referred to as the wild-type allele – plays a role in
the biosynthesis of this compound. Mutations occur spontaneously as a
result of errors during DNA replication, but can also be induced by the use
of alkylating agents such as ethyl methanesulfonate (EMS) and
diethylsulfate (DES), radiation with fast neutrons, and insertional elements
(see Section 3.1). The gene name is written in italics, and the protein
encoded by the gene is written in regular font style. Depending on the
species, the whole name or just the first letter of the wild-type allele is
capitalized, whereas the mutant allele is referred to in lower case letters.
The sequence of the gene can be used to deduce the amino acid
sequence of the protein encoded by the gene. The DNA and amino acid
sequences can then be used to identify similar sequences in the large
sequence databases such as GenBank (www.ncbi.nlm.nh.gov) or
SWISSPROT (www.expasy.org). The chemical data obtained form the
mutant combined with the sequence data from the gene that is defective in
the mutant can then provide information on the function of the gene in the
biosynthesis of a certain class of phenolic compounds.
Several methods to clone genes will be discussed in the next three
sections. The ability to clone genes was revolutionized by the development
68
Chapter 3
of the polymerase chain reaction (PCR). This is an in vitro method for the
amplification of specific regions of DNA or cDNA. cDNA (complementary
DNA) is a DNA copy of an mRNA molecule and which is synthesized in
vitro by a retroviral reverse transcriptase. cDNA is more stable than mRNA,
and more compact than the corresponding genomic DNA region, because
cDNA does not contain introns.
PCR was developed in 1984 by Dr. Kary Mullis and co-workers (Saiki
et al., 1985), who subsequently won (half of) the 1993 Nobel Prize in
Chemistry for his work. The breakthrough that made PCR possible was the
use of a heat-stable DNA polymerase for the synthesis of DNA. The
polymerase is typically isolated from a thermophilic archaebacterium, such
as Thermus aquaticus, Thermococcus gorgonarius, or Thermus ubiquitus.
The name of the polymerase reflects its origin. In the examples above, the
polymerase is referred to as Taq, Tgo and Tub polymerase, respectively.
These polymerases are primer- and template-dependent, i.e. they require a
nucleotide with a free 3´ OH group that is part of a short double stranded
DNA structure, to which they will add a new nucleotide that complements
the nucleotide on the template strand. The primers are designed by the
researcher and are typically oligonucleotides (‘oligo’s’) of 18-25 residues
long. This length results in sequences that in most cases will define a unique
site in the genome of the species of interest. Exceptions may be oligo’s that
bind to repetitive sequences or that bind to a conserved sequence in a gene
that is part of a multigene family.
PCR involves three steps: 1) denaturation, performed at 94°C, to melt
(separate) the two DNA strands of the template, 2) primer annealing,
performed at 45-70°C depending on the length and GC-content of the
primer, and 3) extension, performed at 72°C (or a specific temperature
recommended by the manufacturer of the enzyme), during which the DNA
delineated by the primers is being synthesized by the polymerase. This
process is repeated 20-40 times, and during each cycle each template strand
is being duplicated. Consequently, there is an exponential amplification of
the target DNA, so that PCR with pico- or nanogram quantities of template
DNA will result in enough product to perform further manipulations
(cloning, sequencing, transfection). Given the expense of the enzyme, PCR
is performed in small volumes (10-50 µl) in a thermal cycler, which is a
machine specifically designed for this process. PCR conditions typically
need to be optimized empirically in order to obtain highly specific products
and a reasonable yield. Varying the annealing temperature of the primers,
the total number of cycles, and the concentrations of primers, nucleotides,
and the cofactor Mg2+ will generally allow the identification of suitable
Biosynthesis of phenolic compounds
69
amplification conditions. PCR has become a routine technique in plant,
animal and microbial biology as well as medicine, and is used for cloning of
genes and cDNA’s, as well as for genotyping using so-called molecular
markers (Innis et al., 1999).
5’
3’
3’
5’
Step1: denaturation (94°C)
5’
3’
3’
5’
Step 2: primer annealing (50-70°C)
5’
3’
5’
3’
5’
3’
3’
5’
Step 3: primer extension (72°C)
5’
3’
5’
3’
3’
5’
5’
3’
Next round…..
5’
3’
5’
5’
3’
3’
Figure 3-1. Schematic representation of the polymerase chain reaction. The two strands of the
template DNA are represented by the white and black bars. The upper (forward) and lower
(reverse) primers are indicated by a hatched and solid white arrow, respectively. The 5´ and
3´ refer to the corresponding hydroxyl groups on the ribose residue in the DNA backbone,
and indicate the directionality of the DNA. DNA polymerases synthesize DNA in the 5´ to 3´
direction.
70
3.1
Chapter 3
Insertional mutagenesis
Insertional mutagenesis is based on the insertion of a known sequence,
referred to as a tag, in the gene of interest. Depending on whether the tag is
a transposable element or T-DNA, the method of cloning genes is referred to
as transposon tagging or T-DNA tagging (Walbot, 1992). Transposable
elements, or transposons, are mobile genetic elements that were first
discovered in maize (Zea mays L.) by the late Dr. Barbara McClintock
(McClintock, 1947), who won the 1983 Nobel Prize in Medicine/Physiology
for her pioneering research. The three major transposable element systems in
maize are Activator (Ac)/Dissociation (Ds), Supressor mutator
(Spm)/defective Suppressor mutator (dSpm) and Robertson’s Mutator
(MuDR)/Mu. The Spm/dSpm system is also known as Enhancer
(En)/Inhibitor (I). All of these transposon systems are two-element systems:
the first element (Ac, Spm (En), MuDR) is the autonomous element which
encodes the transposase enzyme necessary for transposition, and the second
element is the non-autonomous element, which can only transpose in the
presence of an active autonomous element. In addition to these transposable
element systems, the maize genome contains many other transposable
elements, as well as retrotransposons. The latter class of mobile elements
replicate through an RNA intermediate. The other species in which
transposable elements are commonly used for cloning purposes is
snapdragon (Antirrhinum majus). The elements in this species are called
Tam, where Tam1 is the autonomous element and Tam2 is the nonautonomous element. Several additional Tam elements have been identified
(reviewed by Schwarz-Sommer et al., 2003). The Ac and Spm (En) systems
have also been used in species that do normally not harbor transposable
elements themselves, including the model plant Arabidopsis thaliana,
tomato (Lycospersicon esculentum) and tobacco (Nicotiana tabacum).
T-DNA is DNA transferred by the soil borne pathogen Agrobacterium
tumefaciens. This bacterium transfers T-DNA, which is harbored on the
bacterium’s tumor-inducing (Ti) plasmid, to cells of the host plant via
wound sites. The T-DNA then integrates in the genome of the plant, in a
more or less random fashion. If the T-DNA inserts into a gene, the gene will
likely loose its normal function. The principle of Agrobacterium-mediated
transformation of plants has recently been reviewed (Gelvin, 2000; 2003).
There are two strategies to use insertional mutagenesis for cloning
purposes. One is the direct tagging approach. In this case a mutant, for
example a spontaneous or chemically induced mutant, has already been
identified, but no information on the nature of the mutated gene is available.
Biosynthesis of phenolic compounds
71
The mutant is then crossed with a wild-type line carrying an active
transposable element, and the F1 progeny is screened for the presence of a
plant with the mutant phenotype. Such a mutant will only be identified if the
wild-type allele of the gene of interest has been mutated, presumably as the
result of an insertion.
The second approach that can be taken is the random tagging method.
This method is based on the principle that the insertion elements can insert
in any gene, so that all of the genes controlling the trait of interest can be
uncovered, as long as the mutation is not lethal. Since most mutations are
recessive, meaning that both the maternal and paternal copy of the gene need
to be defective in order to see a mutant phenotype, the screening is typically
performed using F2 families in which mutations will segregate. Given the
relative low mutation rate (1:10,000 to 1:1,000,000), large populations of
plants need to be screened. Therefore, an efficient method of screening has
to be available in order to identify mutants of interest.
Once an insertional mutant with the desirable phenotype has been
obtained, the cloning strategy is similar regardless whether transposons or TDNA were used. In the direct tagging strategy the mutant is crossed with a
wild-type plant to produce F1 progeny. Since most mutations are recessive,
the F1 progeny is self pollinated to produce F2 progeny. The plants of the F2
population are scored for the presence of the mutation. DNA is isolated from
these plants, as well as from the wild-type siblings. In the case of the
random tagging strategy, DNA is isolated from the mutant and its wild-type
siblings from the same family. Under both scenarios the isolated DNA is
then used to identify the insertion element that is likely the cause of the
mutation. There are several methods available to do this, including the use
of the traditional Southern blot hybridized with a radioactive or chemically
labeled probe that will hybridize to the insertion element (Federoff et al,
1984; Tan et al., 1997), methods based on the polymerase chain reaction
(PCR) (Liu et al., 1995; Frey et al., 1997; Ribot, 1998), or plasmid rescue
(Behringer and Medford, 1992; Meyer et al., 1996). All of these methods
will ultimately result in the isolation of the DNA adjacent to the insertion
element, which is typically the gene of interest. Sequencing of this DNA
followed by homology searches in the large public sequence databases can
then provide information on the identity and function of the gene of interest.
The function will ultimately need to be tested with additional experiments.
72
3.2
Chapter 3
Map-based cloning
Map-based cloning strategies work best with species with completely
sequence genomes. At this time plantgenomes that have been sequenced
include the model plant Arabidopsis thaliana (The Arabidopsis Genome
Initiative, 2000), rice (Oryza sativa; International Genome Sequencing
Project, 2005) and the poplar tree (Populus trichocarpa; Joint Genome
Institute, http://genome.jgi-psf.org/Poptr1/Poptr1.home.html). In this case
the first step after identifying a mutant of interest is placing the mutation on
the genetic map. This is done by crossing the mutant with a wild-type plant
that has a different genetic origin. This will generate F1 progeny, which, for
the same reason as described in Section 3.1, is self pollinated to produce an
F2 population. The mutants and wild-type plants are identified in this F2
population, DNA is isolated, followed by the determination of the genotype
of these plants at a large number of genetic loci across the genome.
Genotyping is generally performed with so-called molecular markers,
which detect genetic variation across the genome. Mapping the mutation
involves identifying the molecular marker(s) that are associated with the
presence of the mutation. If the mutation is closely linked to a particular
marker, the mutant plants will show predominantly the marker allele from
the mutant parent, whereas the wild-type plants will show the marker allele
from the wild-type parent. Once the precise map location has been
determined, the gene sequence can be obtained based on the available
genome sequence. Oftentimes several genes are located in the region where
the mutation maps, but typically some of these genes can be discarded based
on their known or deduced function. Introduction of a wild-type copy of the
gene into a mutant plant via transformation and showing that the
transformed plants have a wild-type phenotype is proof that the right gene
has been identified. An example of the map-based cloning of a gene
involved in the biosynthesis of a phenolic compound can be found in Franke
et al. (2002a).
3.3
The candidate-gene approach
The candidate-gene approach (Pflieger et al., 2001) became possible
after the establishment of large DNA and protein databases. When using this
approach, a mutant of interest is characterized chemically, and based on the
knowledge of the pathway most likely affected by the mutation, a candidate
gene is proposed. Thus, the candidate gene is defined as the gene that, if it
were defective, would cause the observed mutant phenotype. Once such a
candidate gene has been proposed, the sequence databases can be searched
in an attempt to identify DNA or protein sequences from the candidate gene.
Biosynthesis of phenolic compounds
73
If the DNA sequence is available in a database, PCR can be used to obtain
the gene from the mutant. After sequencing the gene, sequence comparison
between the gene from the mutant and the sequence in the database (or
better yet, the sequence obtained from the wild-type progenitor of the
mutant) can reveal whether or not the candidate gene is the gene that is
responsible for the mutation. If no mutations are identified during the
sequence comparison, additional sequence, especially from the upstream
regulatory elements, needs to be obtained in order to exclude the candidate
gene from further consideration. If there is no evidence for mutations, a new
candidate gene needs to be considered. This approach will also work if the
sequence of the candidate gene is available from a different but related
species, because the degree of sequence homology is typically high between
related species.
The efficiency of the candidate-gene approach can be improved
considerably if comparisons between the mutant and a wild-type
(progenitor) show differences in gene expression (observed via northern
blots or reverse-transcription PCR), differences in enzyme activity, or
differences in the amount of protein (observed via western blotting) (Bout
and Vermerris, 2003). If differences are observed prior to the cloning of the
candidate gene, the evidence in support of the candidate gene being mutated
and responsible for the mutant phenotype is much stronger. It is, however,
possible that the mutation has no impact on gene expression levels, and that
the mutation results in reduced enzyme activity. Enzyme assays performed
with crude protein extracts are not always specific for one enzyme.
Therefore, even if the enzyme encoded by the candidate gene is less active, a
different protein with similar activity may obscure differences in enzyme
activity between the mutant and the wild-type control.
3.4
QTL mapping
Quantitative traits such as, for example, weight, height and yield, show a
continuum in values because they tend to be controlled by multiple
independent genetic loci. In addition, they are influenced by the
environment. In the case of plant height, for example, rainfall and soil
mineral concentrations will play a role. The identification of genes
controlling quantitative traits is more complex than the identification of
genes controlling traits that are inherited in a qualitative or discrete manner,
such as, for example, flower color. A quantitative trait locus (QTL; this is
also the abbreviation for the plural, quantitative trait loci), defined as a
genetic locus delineated by two molecular markers on a genetic map of the
species of interest and affecting a quantitative trait of interest, can be
identified in an F2 population generated from two parental lines that differ as
74
Chapter 3
much as possible from each other with respect to the trait of interest. By
evaluating a large F2 population (at least several hundred individuals) one
ensures that so many recombination events between the two parental
genomes are represented that the two parental genomes have essentially
been shuffled. The individual plants in the F2 population are evaluated for
the trait of interest, and their genotypes are established with the use of
genetic and/or molecular markers. One of several possible statistical
analyses is performed to identify associations between the trait of interest
and specific alleles at various genetic loci. In the simplest scenario, all
individuals with high values for the trait share a particular allele at a given
locus, and the individuals with low values for the trait share the other allele
at that locus.
The F2 population should be evaluated in several locations and/or years
to separate the genetic and environmental effects on the trait. The QTL is
then mapped to a region of the chromosome. Oftentimes, several QTL are
identified for a given quantitative trait, each representing a portion of the
variance for the trait. A relatively dense marker map will help narrow down
any chromosomal regions of interest as much as possible. The identification
of a QTL will often lead to the identification of a candidate gene at that
position. When the genome sequence is available, one or more candidate
genes can be identified relatively easily and evaluated further. When the
genome sequence is not (yet) available, map based cloning using, for
example, bacterial artificial chromosomes (BACs) can be considered, as
long as the map interval is small enough and sufficient resources are
available. Further background on the theory and application of this
approach, referred to as QTL mapping, can be found in the review by
Doerge (2002).
4.
ISOLATION AND CHARACTERIZATION OF
RECOMBINANT PROTEINS
Knowing the sequence of the gene allows the isolation of the cDNA.
Unlike the genomic DNA, cDNA’s do not contain introns, which are
stretches of non-coding sequences interspersed between the exonic (coding)
DNA regions. The introns are removed after transcription through a process
called splicing. Intron-exon boundaries and splicing mechanisms vary
between different species, so that introduction of foreign genomic sequences
into a particular species will not automatically result in the synthesis of a
functional protein. Introduction and expression of cDNA sequences,
however, generally does result in the synthesis of functional proteins. Such
proteins are called recombinant proteins.
Biosynthesis of phenolic compounds
75
Recombinant protein can be obtained in large amounts via
overexpression of the cDNA of interest in, for example, the bacterium
Escherichia coli, the yeasts Saccharomyces cerevisiae and Pichia pastoris,
or the Sf9 insect cell line of Spodoptera frugiperda. This is typically
achieved by the introduction of a plasmid vector containing the cDNA. He
plasmid is a circular DNA replicon that is maintained because it confers a
selective advantage, such as an antibiotic resistance. The use of an
appropriate promoter results in high expression levels of the cDNA. As a
consequence, the abundance of recombinant proteins in these systems is
typically much higher than that of the native protein in a crude plant extract.
The higher abundance facilitates the purification of the protein to apparent
homogeneity. Purification of the recombinant protein can be achieved
through traditional biochemical separation techniques, including various
types of chromatography (see Section 2). Many of the expression systems,
however, are based on so called tags at the N- or C-terminus of the protein.
These tags allow binding of the recombinant protein to a specially
formulated resin. The crude cell extract containing the recombinant protein
is loaded on a column containing the resin, and the recombinant protein
becomes bound to the resin, typically via an electrostatic interaction.
Undesirable proteins are subsequently removed by washing the column with
a buffer that does not disrupt the binding of the recombinant protein. The
recombinant protein is eventually eluted with a buffer that disrupts the
interaction between the recombinant protein and the resin.
An example of the use of tags to purify recombinant proteins is the
addition of six histidine (His) residues to a recombinant protein. The Hisresidues are encoded by the DNA of the expression vector and will be added
to the protein during translation. The purification of the recombinant protein
is achieved through immobilized metal affinity chromatography (IMAC).
Since His can chelate transition metal ions such as Ni2+, Zn2+, and Cu2+, the
His-tag will allow the recombinant protein to bind to a resin containing Ni2+
ions, such as Ni2+-Sepharose. Use of Ni2+ appears to be more successful than
the other ions. The recombinant protein is eluted from the column by
washing with imidazole, which competes for the Ni2+ binding sites.
Alternatively, a low-pH buffer can be used to elute the recombinant protein.
The low pH disrupts the electrostatic interactions between the recombinant
protein and the Ni2+ on the column, but has the risk of denaturing the protein
of interest. In general the presence of the six His-residues has no impact on
protein function, but the tag can be removed by endoproteolytic cleavage.
An alternative method to purify recombinant proteins, similar in principle
to the His-tag described above, is the fusion with glutathione S-transferase
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Chapter 3
(GST). This is an enzyme that has high affinity for glutathione. The crude
cell extract containing the recombinant GST-fusion protein is loaded on a
column of a resin containing glutathione (such as glutathione Sepharose).
Undesirable proteins are removed by washing, and the recombinant GSTfusion protein is eluted by washing the resin with reduced gluthathione,
which competes with the recombinant protein. The enzyme thrombin is used
to specifically remove the GST-tag from the purified recombinant protein.
Plasmid vectors to synthesize recombinant proteins with His-tags or GSTfusion proteins, as well as various other types of tags are available from
various biochemical supply companies.
Data from in vitro activity assays with these purified recombinant
proteins can typically be interpreted much more easily than data obtained
from experiments with crude or partially purified protein extracts, because
(1) there will be no competing proteins with similar activity present in the
assay, and (2) there will no enzymes present that convert the product
generated by the enzyme of interest, and hence reduce the effective product
concentration. A potential downside of the use of recombinant protein over
crude extracts is the fact that critical co-factors that will ensure proper
activity may not be present in the purified protein fraction. If that is the case,
the researcher will have to empirically determine which co-factor and at
what concentration needs to be included in the assay. Another consideration
is that the native protein may have undergone post-translational processing,
such as acetylation, glycosylation, myristoylation, etc. These modifications
may not occur or may not occur properly when the protein is expressed in
bacterial, fungal or insect cells. Assuming that these potential problems do
not occur or can be dealt with, the availability of pure recombinant protein
will enable the determination of substrate specificity, as well as kinetic
experiments in which the rate of conversion is measured as a function of
time and/or substrate concentration.
5.
CARBOHYDRATE CATABOLISM
All plant carbohydrates are derived from photosynthesis, the process
during which CO2 from the atmosphere is fixed and converted to
carbohydrates with energy from light. The carbohydrates that are generated
during photosynthesis form the building blocks for all other carbon-based
compounds in the cell, including the phenolic compounds. The precursors
for plant phenolic compounds are derived from two catabolic processes in
the plant cell: glycolysis and the pentose phosphate pathway. For detailed
Biosynthesis of phenolic compounds
77
background on carbohydrate metabolism, please refer to Dennis and
Blakeley (2000) or Plaxton and McManus (2006).
5.1
Glycolysis
Glycolysis, also known as the Embden-Meyerhof-Parnas pathway, is the
catabolic process during which carbohydrates generated during
photosynthesis are broken down to pyruvate, and ultimately CO2 (Figure 31). This process fulfils two fundamental roles: It oxidizes hexoses to
generate ATP, reductant, and pyruvate, and it produces building blocks for
anabolism. Glycolysis in plants has been reviewed extensively by Plaxton
(1996).
Glycolysis starts with the conversion of glucose-6-phosphate (3.1) to
fructose-6-phosphate (3.2) by the enzyme hexose phosphate isomerase.
Glucose-6-phosphate (3.1) can be generated from glucose via hexokinase, or
from glucose-1-phosphate resulting from hydrolysis of sucrose or starch. In
the latter case, phosphoglucomutase converts glucose-1-phosphate to
glucose-6-phosphate. The pentose phosphate pathway (see Section 5.2)
generates fructose-6-phosphate (3.2), which can enter glycolysis at this
point.
Fructose-6-phosphate (3.2) is converted to fructose-1,6-bisphosphate
(3.3) by phosphofructokinase. This compound is subsequently broken down
by fructose bisphosphate aldolase to dihydroxyacetone-phosphate (DHAP;
3.4) and glyceraldehyde-3-phosphate (GA3P; 3.5). DHAP is converted to
GA3P by triose-phosphate isomerase. In the subsequent reaction GA3P is
converted to glycerate-1,3-bisphosphate (3.6) by GAP-dehydrogenase.
Glycerate-1,3-bisphosphate is converted by glycerate-3-phosphate kinase to
glycerate-3-phosphate (3.7), which is subsequently converted to glycerate-2phosphate (3.8) by glycerate phosphate mutase. An enolase,
phosphopyruvate
hydratase,
converts
glycerate-2-phosphate
to
phosphoenolpyruvate (PEP; 3.9). As a final step, PEP is converted to
pyruvate (3.10) by pyruvate kinase. Alternatively, PEP can enter the
shikimate pathway (see Section 6).
5.2
The pentose phosphate pathway
The plant can also use the pentose phosphate pathway to break down
glucose, but the main purpose of this pathway is the generation of reducing
power in the form of NADPH (nicotinamide adenine dinucleotide
78
Chapter 3
phosphate). In addition, this pathway provides sugar intermediates that can
serve as building blocks for aromatic amino acids and nucleic acids.
H O-P
CH2OH
CH2O-P
H O
O
a
b
OH
HO
H
HO
H
OH
H
OH
OH
ATP
OH
(3.1)
(3.2)
O
CH2O-P
CH2O-P
P-O
OH
(3.4)
O-P
(3.5)
O
c
OH
OH
d
O
OH
OH
(3.3)
NAD+, Pi
O-P
e
NADH, H+
O
O-P
(3.6)
O-P
(3.7)
OH
(3.8)
OH
ADP
O
f
ATP
O
OH
g
O
O
O-P
h
O
H 2O
(3.9)
O
O-P
ADP
i
O
ATP
(3.10)
O
O
ADP
Biosynthesis of phenolic compounds
79
The pentose phosphate pathway can be divided in two phases: an
oxidative phase during which glucose-6-phosphate is converted to ribulose5-phosphate, and a non-oxidative phase constituting of a series of reversible
reactions in which two pentose-phosphate residues are converted to a series
of sugar-phosphate molecules of differing lengths (Figure 3-2).
The oxidative part of the pentose phosphate pathway starts with the
oxidation of glucose-6-phosphate (3.1) to gluconolactone-6-phosphate
(3.11) by glucose-6-phosphate 1-dehydrogenase with the reduction of
NADP+ to NADPH. Gluconolactone-6-phosphate (3.11) is converted to
gluconate-6-phosphate (3.12) by gluconate-6-phospate lactonase.
Irreversible oxidative decarboxylation of (3.12) by gluconate-6-phosphate
reductase results in ribulose-5-phosphate (3.13), with the generation of
another NADPH molecule.
Ribulose-5-phosphate (3.13) can be converted to ribose-5-phosphate
(3.14) and xylulose 5-phosphate (3.15), by the enzymes ribose-5-phosphate
isomerase and ribulose 5-phosphate 3-epimerase, respectively. The two
pentose-phosphate molecules, 3.14 and 3.15, are converted to a C3 and a C7
sugar-phosphate, glyceraldehyde 3-phosphate (3.4) and sedoheptulose-7phosphate (3.16), respectively, via the action of a transketolase.
Transketolases are characterized by their ability to transfer a two-carbon
unit from a ketose to an aldehyde. The C3 and C7 sugar-phosphates can
subsequently be converted to a C4 and a C6 sugar-phosphate, erythrose 4phosphate (3.17) and fructose 6-phosphate (3.2), respectively. This reaction
is catalyzed by a transaldolase, which transfers a three-carbon
glyceraldehyde unit from an aldose to a ketose. Erythrose-4-phosphate
(3.17) can be used in the shikimate pathway (see Section 6). A second
transketolase reaction can generate a second fructose-6-phosphate (3.2) and
glyceraldehyde-3-phosphate (3.4) residue from erythrose-4-phosphate (3.17)
and xylulose-5-phosphate (3.15). Hexose-phosphate isomerase converts the
Figure 3-2 Glycolysis. The enzymes involved in this pathway are: (a) hexose phosphate
isomerase (E.C. 5.3.1.9), (b) phosphofructokinase (E.C. 2.7.1.1), (c) fructose bisphosphate
aldolase (E.C. 4.1.2.13), (d) triose-phosphate isomerase (E.C. 5.3.1.1), (e) GAPdehydrogenase (E.C. 1.2.1.12), (f) glycerate-3-phosphate kinase (E.C. 2.7.2.3), (g) glycerate
phosphate mutase (E.C. 5.4.2.1), (h) enolase (phosphopyruvate hydratase; E.C. 4.2.1.11), and
(i) pyruvate kinase (E.C. 2.7.1.40).
80
Chapter 3
H O-P
H O
H O
a
HO
H
H
HO
H
OH
(3.1)
b
HO
OH
HO
H O-P
NADP+ NADPH
H
H
OH
O
(3.11)
e
COO
H
NADP
OH
HO
H
H
OH
H
OH
CH2O-P
c
+
NADPH
O
CO2
CH2OH
CHO
CH2OH
d
H
OH
O
H
OH
H
OH
OH
H
OH
H
OH
H
(3.13)
f
(3.14)
OH
CH2O-P
CH2O-P
CH2O-P
H
(3.15)
CH2OH
(3.12)
O
CHO
H
HO
H
OH
H
OH
H
OH
H
OH
CH2O-P
(3.4)
i
CH2O-P
(3.16)
g
CH2OH
CHO
O
HO
H
OH
H
OH
H
H
OH
H
OH
(3.2)
CH2O-P
(3.17)
CH2O-P
CH2OH
h
O
HO
H
H
OH
H
OH
(3.2)
CH2O-P
CHO
H
OH
CH2O-P
(3.4)
Biosynthesis of phenolic compounds
81
two fructose-6-phosphate molecules to glucose-6-phosphate (3.1), which can
enter the pentose phosphate pathway again to generate additional NADPH.
So in summary, three glucose-6-phosphate (3.1) molecules (3 x C6) are
oxidized to three ribulose-5-phosphate (3.13) residues (3 x C5) and three
molecules of CO2 (3 x C1) under generation of six molecules of NADPH.
The three ribulose-5-phosphate residues are then converted to one
glyceraldehyde-3-phosphate (3.14) molecule (1 x C3) and two fructose-6phosphate (3.2) molecules (2 x C6). Fructose-6-phosphate can be converted
to glucose-6-phosphate and reenter the oxidative part of the pentose
phosphate pathway. Fructose-6-phosphate and glyceraldehydes can also
serve as intermediates in glycolysis (Section 5.1), which offers the cell
considerable flexibility in terms of its metabolic flux.
The availability of the Arabidopsis genome sequence revealed that there
are multiple genes encoding the different enzymes in the oxidative pentose
phosphate pathway (reviewed by Kruger and von Schaewen, 2003). Studies
across a range of species indicate that genes encoding individual isozymes
may be differentially expressed in different tissues, at different
developmental stages, and in response to different growth conditions,
especially those that alter demand for NADPH or intermediates of the
oxidative pentose phosphate pathway for biosynthesis. The ability to use
specific isoforms that allow optimal performance under certain conditions
offers the plant a greater degree of metabolic flexibility.
6.
THE SHIKIMATE PATHWAY
The shikimate pathway results in the biosynthesis of chorismate, which
can subsequently serve as a recursor for the biosynthesis of the aromatic
amino acids tryptophan, phenylalanine and tyrosine. The biochemistry of
Figure 3-3 The pentose phosphate pathway. The enzymes involved in this pathway are: (a)
glucose-6-phosphate dehydrogenase (E.C. 1.1.1.49), (b) gluconate-6-phospate lactonase (E.C.
3.1.1.31), (c) gluconate-6-phosphate reductase (E.C. 1.1.1.44), (d) ribose-5-phosphate
isomerase (E.C. 5.3.1.6), (e) ribulose5-phosphate 3-epimerase (E.C. 5.1.3.1.), (f)
transketolase (E.C. 2.2.1.1), (g) transaldolase (E.C. 2.2.1.2), (h) transketolase, and (i) hexosephosphate isomerase (E.C. 5.3.1.9).
82
Chapter 3
the shikimate pathway has been extensively reviewed by Weaver and
Herrmann (1997) and Hermann and Weaver (1999).
The shikimate pathway is common to both plants and microorganisms
(Figure 3-3). Shikimate is synthesized from the substrates
phosphoenolpyruvate (3.9) and erythrose 4-phosphate (3.17). These two
precursors are derived from glycolysis and the pentose phosphate pathway,
respectively, and are condensed to 3-deoxy-D-arabino-heptulosonate 7phosphate (DAHP; 3.18) by the enzyme DAHP synthase. The subsequent
steps result in the formation of 3-dehydroquinate (3.19) by the enzyme 3dehydroquinate synthase, 3-dehydroshikimate (3.20) by the enzyme 3dehydroquinate dehydratase, and finally shikimate (3.21) by the enzyme
shikimate dehydrogenase.
Shikimate is further converted to shikimate 3-phosphate (3.22) by
shikimate kinase, and subsequently to 5-enolpyruvylshikimate 3-phosphate
(EPSP; 3.23) by 5-enolpyruvylshikimate 3-phosphate synthase. EPSP is then
converted to chorismate (3.24) by chorismate synthase.
Chorismate is at a branch point for the biosynthesis of aromatic amino
acids: tryptophan on the one hand, and phenylalanine (3.27) and tyrosine
(3.28) on the other hand. While this is strictly speaking no longer part of the
shikimate pathway, the biosynthesis of phenylalanine and tyrosine is
included in Figure 3-3, because they are the precursors of the important class
of phenolic compounds, the phenylpropanoids, as well as several other
classes of phenolic compounds. This requires the conversion of chorismate
to prephenate (3.25), catalyzed by chorismate mutase, and arogenate (3.26),
catalyzed by prephenate aminotransferase. The enzyme arogenate
dehydratase converts arogenate to phenylalanine (3.27), whereas the enzyme
arogenate dehydrogenase generates tyrosine (3.28).
Figure 3-3 The shikimate pathway. The enzymes involved in this pathway are: (a) DAHP
synthase (E.C. 2.5.1.54), (b) 3-dehydroquinate synthase (E.C. 4.2.3.4), (c) 3-dehydroquinate
dehydratase (E.C. 4.2.1.10), (d) shikimate dehydrogenase (E.C. 1.1.1.25), (e) shikimate
kinase (E.C 2.7.1.71), (f) 5-enolpyruvylshikimate 3-phosphate synthase (E.C. 2.5.1.19), (g)
chorismate synthase (E.C. 4.2.3.5), (h) chorismate mutase (E.C. 5.4.99.5), (i) prephenate
aminotransferase (E.C. 2.6.1.78 and E.C. 2.6.1.79), (j) arogenate dehydratase (E.C. 4.2.1.91),
and (k) arogenate dehydrogenase (E.C. 1.3.1.43, E.C. 1.3.1.78, E.C. 1.3.1.79).
Biosynthesis of phenolic compounds
83
H
O
OH
(3.17)
O
O
P
C
O
HO
OH O
(3.25)
(3.9)
P
O
O
O
O
O
O
C
C
O
OH
OH
O
P
HO
C
OH
O
O
O
O
C
OH
(3.26)
C
H3N
O
O
(3.18)
O
HO
g
O
(3.24)
O
O
O
O
C
O
O
C
C
O
OH
NH3
NH3
(3.19)
O
O
c
O
HO
HO
O
C
(3.23)
O
O
d
OH
O
C
HO
P
(3.20)
HO
f
HO
O
e
C
O
HO
C
O
HO
k
O
C
HO
j
C
b
HO
O
O
C
O
O
HO
O
HO
O
O
HO
P
O
C
h
a
HO
i
O
O
O
(3.21)
O
O
(3.22)
P
(3.27)
(3.28)
84
7.
Chapter 3
THE GENERAL PHENYLPROPANOID
PATHWAY
The general phenylpropanoid pathway (Figure 3-4), as the name
implies, generates a substrate common to a number of phenylpropanoid
compounds, including flavonoids, monolignols, hydroxycinnamic acids,
sinapoyl esters, coumarins and stilbenes. The general phenylpropanoid
pathway starts with phenylalanine (3.27) generated via the shikimate
pathway (see Section 6). Deamination of phenylalanine is catalyzed by the
enzyme phenylalanine ammonia lyase (PAL) and results in cinnamic acid
(3.29). Cinnamic acid is subsequently hydroxylated by cinnamic acid 4hydroxylase (C4H) to give p-coumaric acid (3.30). In graminaceous species,
such as maize, this compound can also result from the deamination of
tyrosine (3.28). In vitro assays with recombinant enzyme demonstrated that
the catalytic activity towards tyrosine resides in the same enzyme, in other
words, in grasses PAL has activity against both phenylalanine and tyrosine
(Roesler et al., 1997). p-Coumaric acid (3.30) is converted to p-coumaroyl
Coenzyme A (3.31) by the enzyme 4-coumaric acid:CoA ligase (4CL). The
general phenylpropanoid pathway ends with p-coumaroyl Coenzyme A
(3.31), as further reactions lead to the biosynthesis of specific classes of
compounds.
Older text books and articles typically describe the general
phenylpropanoid pathway as the pathway leading to the full set of
hydroxycinnamic acids (p-coumaric acid (3.30), caffeic acid (3.32), ferulic
acid (3.33), 5-hydroxyferulic acid (3.34) and sinapic acid (3.35), as well as
their corresponding CoA-esters (3.31, 3.36-3.39). This is indicated by the
grey structures in Figure 3-4. Recent advances in the cloning and
characterization of genes encoding enzymes involved in phenylpropanoid
metabolism have made it possible to determine substrate specificity and
catalytic activity of a number of enzymes. This provided evidence
Figure 3-4 The general phenylpropanoid pathway. The enzymes involved in this pathway are:
(a) phenylalanine ammonia lyase (PAL; E.C. 4.3.1.5), (b) cinnamic acid 4-hydroxylase (C4H;
E.C. 1.14.13.11), and (f) 4-coumaric acid:CoA ligase (4CL; E.C. 6.2.1.12). (a′) depicts
tyrosine ammonia lyase activity in PAL of graminaceous species. The grey structures in the
box represent an older version of the phenylpropanoid pathway in which the ring substitution
reactions were thought to occur at the level of the hydroxycinnamic acids and/or
hydroxycinnamoyl esters. The enzymes involved in these conversions are (c) coumarate 3hydroxylase (C3H; E.C. 1.14.14.1), (d) caffeate O-methyltransferase (COMT; EC 2.1.1.68),
(e) ferulate 5-hydroxylase (F5H; EC 1.14.13), and (g) caffeoyl-CoA O-methyltransferase
(CCoA-OMT; EC 2.1.1.104). These enzymes are discussed in more detail in Section 10.
Biosynthesis of phenolic compounds
O
HO
O
(3.27)
a
H 3N
(3.28)
O
H3 N
(3.29)
HO
O
O
(3.30)
HO
f
S-CoA
(3.31)
O
c
O
(3.32)
d
HO
(3.33)
f
HO
O
O
H3CO
S-CoA
HO
f
(3.36)
g
HO
(3.37)
H3CO
O
O
S-CoA
O
e
e
HO
HO
O
HO
(3.34)
H3CO
f
HO
S-CoA
(3.38)
H3CO
O
O
g
d
H3CO
H3CO
HO
H3CO
O
c
HO
HO
O
a'
O
b
85
O
(3.35)
O
f?
S-CoA
HO
H3CO
(3.39)
O
86
Chapter 3
against the hydroxylation and methylation reactions occurring at the level of
the hydroxycinnamic acids. Rather, these reactions appear to occur at the
level of the hydroxycinnamoyl esters, aldehydes, and alcohols (reviewed by
Humphreys and Chapple, 2002). The hydroxycinnamic acids are thought to
be generated from cinnamaldehydes, as will be discussed in Section 12. As a
consequence, the current model of the general phenylpropanoid pathway is
more streamlined, as shown in black in Figure 3-4. There is some evidence,
however, that in certain plant species, specifically poplar (Meyermans et al.,
2000), variations of the general phenylpropanoid pathway exist that are
different from the general model depicted here.
8.
BIOSYNTHESIS OF PHENOLIC ACIDS
Phenolic acids are generally not abundant in most plants. There are a
few exceptions: gallic acid (1.5) and salicylic acid (SA; 1.8). Gallic acid is a
precursor for the ellagitannins and gallotannins, which will be discussed in
more detail in section 15 of this chapter. Salicylic acid is an important
defense compound because it mediates systemic acquired resistance (SAR),
a resistance mechanism whereby SA is used as a signaling molecule to relay
information on pathogen attack to other parts of the plant. Upon receiving
the SA signal, a general defense response is activated that includes the
biosynthesis of pathogenesis-related (PR) proteins. This will be discussed in
more detail in Chapter 6, Section 2.4.
8.1
Salicylic acid
There are two possible biosynthetic routes to SA, one plant-specific, and
the other more similar to the one found in bacteria (Figure 3-5). The ‘plantspecific’ biosynthetic route to SA starts with phenylalanine (3.27), which, as
part of the general phenylpropanoid pathway (see Section 7), is converted to
cinnamic acid (3.29) by the enzyme phenylalanine ammonia lyase (PAL).
Cinnamic acid is converted to benzoic acid (3.40), probably through a
process similar to –oxidation of fatty acids. The hydroxylation of C2,
catalyzed by the enzyme benzoic acid 2-hydroxylase (BA2H), results in SA
(3.41). Characterization of purified tobacco BA2H showed that this enzyme
is a cytochrome P450-dependent oxygenase (Léon et al., 1995).
Alternatively, BA2H may hydroxylate cinnamic acid (3.29) on C2 to
produce coumaric acid (3.42), which, after oxidation of the propane side
chain, results in SA (3.41).
Biosynthesis of phenolic compounds
87
O
O
O
O
C
a
b
c
NH2
O
C
O
O
OH
(3.24)
(3.27)
g
d
O
O
O
O
C
OH
O
O
C
O
O
O
f
(3.43)
h
(3.29)
e
OH
e
O
O
O
O
O
C
O
OH
C
(3.42)
f
O
(3.10)
(3.41)
(3.40)
Figure 3-5. Biosynthesis of salicylic acid. The enzymes involved in this pathway are: (a)
chorismate mutase (E.C. 5.4.99.5), (b) prephenate aminotransferase (E.C. 2.6.1.78 and E.C.
2.6.1.79), (c) arogenate dehydratase (E.C. 4.2.1.91), (d) phenylalanine ammonia lyase (E.C.
4.3.1.5), (e) presumed –oxidation by a yet to be identified enzyme, (f) benzoic acid 2hydroxylase, (g) isochorismate synthase (E. C. 5.4.4.2), and (h) a putative plant pyruvate
lyase.
An alternative biosynthetic pathway towards SA was hypothesized to
exist after experiments with labeled benzaldehyde, benzyl alcohol, and
phenylalanine resulted in lower incorporation of the label in SA than
88
Chapter 3
expected (Ribnicky et al., 1998). Sequence analysis of the Arabidopsis
genome revealed two genes with homology to the bacterial gene encoding
isochorismate synthase, an enzyme involved in SA production in bacteria
such as Pseudomonas aeruginosa. One of these genes, ICS1 was shown to
be up-regulated when Arabidopsis leaves were infected with bacterial
pathogens (Wildermuth et al., 2001). In addition, two mutants that were
lacking a functional copy of the ICS1 gene produced less SA, exhibited a
reduced pathogenesis-related gene expression, and were more susceptible to
pathogens. The ICS1 protein contains a putative plastid transit sequence and
cleavage site that would be consistent with biosynthesis of SA from the
plastid-synthesized pool of chorismate (3.24). In bacteria isochorismate
(3.43) is converted to SA and pyruvate (3.10) by the enzyme pyruvate lyase.
It is yet unclear whether plants would use a similar enzyme. Wildermuth et
al. (2001) speculated that the SA synthesized by isochorismate synthase is
crucial for SAR, whereas SA synthesized from phenylalanine may be
important in mediating cell necrosis in response to certain pathogens or
fungal elicitors.
8.2
Gallic acid
The biosynthesis of gallic acid (3.47) has been under investigation for
more than 50 years. Different biosynthetic routes have been proposed, as
depicted in Figure 3-6: (1) direct biosynthesis from an intermediate of the
shikimate pathway, (2) biosynthesis via phenylalanine (3.27), cinnamic acid
(3.29), p-coumaric acid (3.30), caffeic acid (3.32), and 3,4,5trihydroxycinnamic acid (3.44), or (3) biosynthesis via caffeic acid (3.32)
and protocatechuic acid (3.45). The possibility that different pathways coexisted in different species or even within one species was also considered.
Werner et al. (1997) used 13C-labeled glucose in feeding experiments
with the fungus Phycomyces blakesleeanus and in leaves of sumac (Rhus
typhina). After incubation with the labeled glucose, gallic acid and aromatic
amino acids were isolated and subjected to NMR analyses. The NMR data
indicated that in both the fungus and the plant the carbon atoms in gallic
acid were biosynthetically similar to those in shikimate (3.21), and different
from those in phenylalanine (3.27) and tyrosine (3.28). Based on these data
5-dehydroshikimate (3.46) was proposed as the most likely precursor of
gallic acid. Ossipov et al. (2003) indeed isolated an enzyme from birch
(Betula pubescence) that was able to reduce 5-dehydroshikimate to gallic
acid.
Biosynthesis of phenolic compounds
O
OH
O
OH
89
O
OH
OH
P
O
O
(3.17)
O
b
a
c
P
OH
OH HO
O
OH HO
O
OH
OH
OH
(3.9)
(3.21)
(3.46)
OH
OH
(3.47)
h
O
OH
i
d
(3.45)
OH
g
O
OH
O
OH
O
OH
O
OH
OH
NH2
e
f
e
OH
(3.27)
OH
OH
(3.30)
(3.32)
OH
HO
OH
(3.44)
Figure 3-6. Biosynthesis of gallic acid in sumac (Rhus typhina). The enzymes involved in
this pathway are: (a) enzymes involved in the shikimate pathway (see Figure 3-3), (b)
proposed shikimate dehydrogenase, (c) proposed 5-dehydroshikimate dehydrogenase
identified in birch by Ossipov et al. (2003), (d) enzymes involved in the biosynthesis of
phenylalanine (see Figure 3-3), (e) enzymes involved in the general phenylpropanoid
pathway (see Figure 3-4), (f) proposed caffeic acid 5-hydroxylase, (g) and (i) oxidizing
enzymes that reduce the propane side chain from C3 to C1, (h) proposed protocatechuic acid
5-hydroxylase. The structures in black represent the pathway based on the experiments by
Werner et al. (2004), which is responsible for > 90% of the gallic acid synthesis. The grey
structures in the box represent alternative pathways that had been hypothesized to exist, but
that do not contribute (significantly) to the biosynthesis of gallic acid.
90
Chapter 3
Further evidence in favor of 5-dehydroshikimate (3.46) as a precursor of
gallic acid came from a recent study by Werner et al. (2004) in which the
ratio of oxygen isotopes (16O/18O) in gallic acid was measured. The 16O
isotope of oxygen is by far the most common (99.8 atom%), with the 17O
and 18O isotopes representing 0.04 and 0.2 atom%, respectively. The isotope
abundance ( ) in a biological compound is typically expressed as the relative
difference of the isotope ratio of the compound to that of an international
standard, and is expressed in ‰. In the case of 18O, ocean water is used as
the standard. Interestingly, the isotope abundance of 18O varies among the
main sources of oxygen in plant-based compounds. The 18O values of CO2
and O2 are approximately +40 and +24‰, whereas the value for
groundwater varies between -10 and +2‰. In this case positive numbers are
indicative of 18O/16O ratios higher than ocean water. Hence, 18O of a
particular compound varies depending on its biosynthetic origin (Schmidt et
al., 2001).
If gallic acid was synthesized from an intermediate of the shikimate
pathway, the three phenolic oxygen atoms would originate from the
carbohydrate erythrose 4-phosphate (3.17). In the alternative route via
phenylalanine, the phenolic oxygen atoms would have to be introduced by a
monooxygenase using atmospheric oxygen. Since carbohydrates and
molecular oxygen have different 18O values, the 18O value for gallic acid
will reflect the origin of its oxygen atoms. Werner et al. (2004) measured
18
O of gallic acid and water isolated from sumac leaves using an isotope
ratio mass spectrometer and calculated that the biosynthesis of gallic acid
from 5-dehydroshikimate (3.46) was consistent with the experimental value,
whereas biosynthesis from phenylalanine (3.27) was not.
9.
BIOSYNTHESIS OF FLAVONOIDS AND
CONDENSED TANNINS
9.1
Structural genes and enzymes
The identification and isolation of genes involved in flavonoid
biosynthesis has benefited from the fact that many of the flavonoids are
colored compounds. Mutant phenotypes are therefore often easily
identifiable based on variation in color. In Arabidopsis many of the genes
involved in flavonoid bioysnthesis have been uncovered based on the change
in seed coat (testa) color. Wild-type Arabidopsis seeds have a brown color,
and mutations in flavonoid biosynthetic genes result in yellow or pale brown
color because the underlying cotyledons are visible. These mutants are
Biosynthesis of phenolic compounds
91
referred to as transparent testa (tt) mutants. A total of 21 of these mutants
have been identified, resulting from either chemical mutagenesis with EMS,
or ionizing radiation (X-ray or fast neutrons). This includes 19 tt mutants,
and two transparent testa glabra (ttg) mutants, which have pale seeds but
also lack trichomes (leaf hairs) (reviewed by Winkel-Shirley, 2001). The
absence of flavonoids in the seed coat reduces seed dormancy, and some of
the tt mutants were actually identified based on their reduced dormancy, as
opposed to the seed coat color.
Maize mutants with altered flavonoid metabolism can also be identified
based on variation in color, either of the seeds, the vegetative parts of the
plant, or the floral structures (anthers and silks). Petunia (Petunia hybrida)
and snapdragon (Antirrhinum majus) have also been widely used as model
species for the elucidation of flavonoid biosynthesis (reviewed by WinkelShirley, 2001). In the description of genes involved in flavonoid
biosynthesis presented in this section, the emphasis will be on maize and
Arabidopsis.
Flavonoid biosynthesis (Figure 3-7) is initiated from the condensation of
p-coumaroyl-CoA (3.31) with three molecules malonyl-CoA (3.48), which
is catalyzed by the enzyme chalcone synthase (CHS), and gives rise to
4,2′,4′,6′ tetrahydroxychalcone (3.49). This compound can undergo a
number of reactions that give rise to the different classes of compounds
described in Section 3.6 of Chapter 1.
CHS in maize is encoded by the Colorless2 (C2) gene. Mutations in this
gene result in yellow kernels as a result of a colorless aleurone (Reddy and
Coe, 1962). The aleurone is the cell layer under the pericarp (the hard outer
cell layer of the maize kernel). The C2 gene was cloned via transposon
tagging with the Spm transposon by Wienand et al. (1986). NiesbackKlösgen et al. (1987) further characterized the gene. In Arabidopsis CHS is
encoded by the TT4 locus (Feinbaum and Ausubel, 1988).
The product generated by CHS, 4,2′,4′,6′ tetrahydroxychalcone (3.49) is
the substrate for aurones, yellow pigments common in the petals of flowers,
that contain a five-member ring and additional hydroxyl groups on the Bring. An example is aureusidin (4,6,3′,4′-tetrahydroxyaurone; 3.50).
Aureusidin synthase, the enzyme responsible for the formation of aureusidin
(3.50) was isolated from 32 kg of yellow snapdragon buds via a series of
biochemical separations (Nakayama et al., 2000). Oligonucleotide primers
based on partial amino acid sequence obtained from the isolated protein
enabled the isolation of the corresponding cDNA clone. The cDNA was
shown to encode a 64 kDa protein with similarity to polyphenoloxidases
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Chapter 3
from various plant species. The mature aureusidin synthase can be produced
after cleavage of a 10 kDa N-terminal transit peptide, and a 15 kDa Cterminal peptide of unknown function. The mature aureusidin synthase is a
39 kDa copper-containing glycoprotein that catalyzes both the hydroxylation
of the B-ring and the oxidative cyclization of the 5-member ring
characteristic for aurones. The compound 3,4,2′,4′,6′ pentahydroxychalcone
was shown to be a better substrate for the production of aureusidin (3.50)
and the aurone bracteatin (4,6,3′,4′,5′-pentahydroxyaurone). Given the
similarity of aureusidin synthase to polyphenol oxidases (PPO’s), Nakayama
et al. (2001) investigated the specificity of the enzyme, and concluded it was
a highly specific PPO with substrate specificity for chalcones with a 4-mono
or 3,4-dihydroxy substitution pattern.
The formation of a six-member ring from 4,2′,4′,6′ tetrahydroxychalcone
(3.49), catalyzed by chalcone isomerase (CHI), results in the flavanone
naringenin (3.51). In Arabidopsis CHI is encoded by the TT5 locus (Shirley
et al., 1992) Naringenin is subsequently converted by flavanone 3hydroxylase (F3H) to yield the flavanonol dihydrokaempferol (3.52). This
compound can be converted by flavone synthase (FLS) to the flavone
kaempferol (3.53). Alternatively, the B-ring of dihydrokaempferol can be
substituted with additional hydroxyl groups by flavonoid 3′-hydroxylase
(F3′H) or flavonoid 3′,5′-hydroxylase (F3′5′H) to produce dihydroquercetin
(3.54) and dihydromyricetin (3.55), respectively. These latter two
compounds can subsequently be converted by FLS to their corresponding
flavones, quercetin (3.56) and myricetin (3.57). F3H and F3′H are encoded
by the Arabidopsis TT6 and TT7 genes, respectively (Pelletier and Shirley,
1996; Schoenbohm et al., 2000).
The flavanonols can also give rise to anthocyanins. This involves a
multi-step process whereby they are first reduced to leucoanthocyanidins –
leucopelargonidin (3.58), leucocyanidin (3.59) and leucodelphinidin (3.60) –
by the enzyme dihydroflavonol 4-reductase (DFR) (Figure 3-7), followed by
dehydration and glycosylation (Figure 3-8). The enzyme DFR is encoded by
the maize Anthocyaninless1 (A1) gene, based on the colorless aleurone layer
in the seed, and lack of pigmentation of the green tissues of the plant in the
a1 mutant. This gene was cloned by O’Reilly et al. (1985) using transposon
tagging, and its sequence was analyzed by Schwarz-Sommer et al. (1987). In
Arabidopsis DFR is encoded by the TT3 locus (Shirley et al., 1992).
Anthocyanidin synthase (ANS) is the enzyme that dehydrates the
leucoanthocyanidins (Figure 3-8). While this enzyme had been postulated to
exist, and cDNA’s encoding the putative ANS had been identified (e.g. A2 in
Biosynthesis of phenolic compounds
93
maize; Messen et al., 1990), in vitro ANS activity was only recently
demonstrated in extracts from the beefsteak plant (Perilla frutescens (L.)
Britt; Saito et al., 1999).
ANS is a 2-oxoglutarate-dependent oxygenase that is thought to abstract
a hydrogen radical from C2 of leucoanthocyanidin (3.61) to yield the radical
(3.62) (Figure 3-8). Following a second hydrogen abstraction at C3, the 2flaven-3,4-diol (3.63) is formed. The reaction can also occur in the reverse
order, i.e. abstraction of the hydrogen at C3 followed by the one on C2. The
colorless 2-flavene-3,4-diol is hydrated to 3-flavene-2,3-diol (3.64), which
under acid conditions can give rise to anthocyanidin (3.65). The
glycosylation of anthocyanidins results in the formation of anthocyanins
(3.66) and is catalyzed by UDP-glucose:flavonoid 3-O-glucosyltransferase,
also referred to as anthocyanidin 3-glycosyl transferase (3GT). In maize this
enzyme is encoded by the Bronze1 (Bz1) gene (Dooner and Nelson, 1977;
Larson and Coe, 1977), and mutations in this gene result in bronze aleurone
in the seed and brown vegetative tissues. The Bz1 gene was cloned via
transposon-tagging with Ac by Federoff et al. (1984). The maize bronze2
(bz2) mutant looks very similar to the bz1 mutant. The Bz2 gene encodes a
glutathione S-transferase required for tagging anthocyanins, synthesized as a
result of Bz1 activity, with glutathione. This modification appears necessary
for transfer of anthocyanins into the vacuole. The Bz2 gene was cloned via
transposon-tagging with Mu and Ds by McLaughlin and Walbot (1987) and
Theres et al. (1987), respectively.
Condensed tannins (3.68) arise from polymerization of flavonoids.
Polymerization starts with the condensation of a 2,3-cis-flavonol residue
(3.67) onto a 2,3-trans-flavonol ‘starter’ residue, after which additional 2,3cis-flavonol residues polymerize. The biosynthesis of the monomers of
condensed tannins was poorly understood until the discovery of the
Arabidopsis banyuls mutant. This mutant, named after a French wine,
displays transparent testa as a result of accumulation of red anthocyanins,
and loss of condensed tannins in the seed coat (Albert et al., 1997). The
BANYULS gene was cloned and shown to encode an enzyme with similarity
to DFR (Devic et al., 1999). Thus, it was initially proposed that the
BANYULS gene encoded leucoanthocyanidin reductase (LAR), which
reduces leucoanthocyanidins to the 2,3-trans-flavonol ‘starter’ residue. This
was subsequently disproved, as the product of the BANYULS gene did not
show any activity towards leucoanthocyanidins, but instead was shown to
use anthocyanidins as a substrate (Xie et al., 2003). The resulting products
are 2,3-cis-flavonols (3.67). The biosynthetic origin of the 2,3-transflavonols remains to be elucidated, but several uncharacterized DFR-like
94
Chapter 3
OH
OH
O
OH
HO
S-CoA
(3.31)
a
O
(3.49)
O
OH
O
CoA-S
c
OH
O
(3.48)
O
HO
(3.51)
OH
O
d
OH
O
HO
O
HO
e
(3.53)
(3.52)
OH
OH
OH
OH
O
OH
O
OH
OH
f
OH
OH
g
O
HO
O
HO
e
(3.56)
(3.54)
OH
OH
OH
O
OH
O
OH
OH
OH
OH
O
HO
O
HO
OH
(3.57)
OH
e
(3.55)
OH
OH
OH
O
OH
O
Figure 3-7. Flavonoid biosynthesis (this page and nexts page). The enzymes involved in this
pathway are: (a) chalcone synthase (E.C. 2.3.1.73), (b) aureusidin synthase (E.C. 1.21.3.6),
(c) chalcone isomerase (E.C. 5.5.1.6), (d) flavanone 3-hydroxylase (E.C. 1.14.11.9), (e)
flavone synthase (E.C. 1.14.11.22), (f) flavonoid 3′-hydroxylase (E.C. 1.14.13.21),
Biosynthesis of phenolic compounds
95
OH
OH
HO
O
b
(3.50)
O
OH
OH
O
HO
h
(3.58)
OH
OH
OH
OH
OH
O
HO
h
(3.59)
OH
OH
OH
OH
OH
O
HO
OH
h
(3.60)
OH
OH
OH
(g) flavonoid 3′,5′-hydroxylase (E.C. 1.14.13.88), and (h) dihydroflavonol 4-reductase (E.C.
1.1.1.219).
96
Chapter 3
genes have been identified in the Arabidopsis genome, one or more of which
may encode LAR.
A detailed analysis of the Arabidopsis tt12 mutant revealed that the
vacuole in the endothelial cells (the innermost layer of the testa)
accumulated lower levels of condensed tannins, and that instead the cytosol
contained higher levels of these compounds. The TT12 gene was cloned
using T-DNA tagging. Sequence analysis revealed that the TT12 protein
shows similarity to a multidrug secondary transporter of the MATE
(multidrug and toxic compound extrusion) family. Based on the phenotype
and the sequence similarity, the most likely role of the TT12 protein is that it
functions as a vacuolar transporter for the precursors of condensed tannins
(leucocyanidin and catechin), as it is very unlikely that the polymeric
procyanidin itself may be handled by a transporter. Consequently, the pale
brown color of the tt12 seeds may be the result of the accumulation of
precursors of condensed tannins in the cytoplasm (Debeaujon et al., 2001).
9.2
Regulatory genes
Efforts to elucidate the genetic basis of biosynthetic pathways tend to
result in the identification of structural genes, i.e. genes encoding enzymes
that catalyze the conversion of intermediates in a specific pathway. This is in
part due to the relatively high expression of the structural genes, which
results in abundant representation in expressed sequence tag (EST) libraries,
or high probability of identification when cDNA subtraction methods or
other methods that rely on differential expression are used.
In order to fully understand the biochemical pathway, it is also
important to identify the regulatory mechanisms that control both the timing
(developmental stage, environmental cues) and the location (cells, tissues or
organs) of the biosynthesis of a particular compound or class of compounds.
The regulation of biosynthetic pathways is mediated by regulatory genes
that encode transcription factors and repressors that can enhance and inhibit
the expression of the structural genes, and that are under control of internal
(e.g. developmental) and/or external (e.g. environmental) cues. The
identification of regulatory genes is generally much more difficult, in part
because the expression of regulatory genes tends to be much lower than the
expression of the structural genes.
Biosynthesis of phenolic compounds
97
R1
R1
OH
OH
H
O
HO
.
O
HO
R2
R2
a
OH
OH
OH
OH
OH
OH
(3.62)
(3.61)
R1
R1
OH
OH
OH
O
HO
O
HO
R2
a
R2
H2O
OH
OH
OH
OH
H
OH
(3.64)
(3.63)
R1
R1
OH
O
HO
O
HO
R2
H+
OH
R2
b
OH
O-Glc
H2O
OH
H
OH
H
(3.66)
(3.65)
R1
c
OH
R1
O
HO
R2
2
OH
3
OH
OH
O
HO
R2
R1
H
(3.67)
OH
n
OH
OH
O
HO
R2
OH
OH
H
(3.68)
Figure 3-8. Biosynthesis of anthocyanins and condensed tannins. The enzymes involved in
this pathway are: (a) anthocyanidin synthase (E.C. 1.14.11.19), (b) anthocyanin 3-glycosyl
transferase, and (c) BANYULS.
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Chapter 3
Furthermore, there is considerable sequence similarity between
transcription factors, even though they may operate in different biosynthetic
pathways. Hence, based on sequence similarity it is often difficult to identify
the target genes of a given transcription factor.
Interestingly, regulatory genes involved in the biosynthesis of
anthocyanins were identified relatively early on. This was possible because
of a number of mutants that accumulated high levels of anthocyanins, or that
accumulated anthocyanins in tissues where they were normally not found, in
combination with the easily scorable phenotype of these mutants.
The Red color (R) and Booster (B) genes are regulatory genes that
control the tissue-specific deposition of anthocyanins in maize. These two
genes were shown to independently activate the same target gene Bz1
(Dooner and Nelson, 1977). In most maize lines the R gene is actually
represented by a small family of homologous genes that map closely
together and that are thought to have arisen through gene duplication and
divergence. A functional R gene is required for the pigmentation of all plant
tissues. More than 50 naturally occurring R alleles have been identified,
which differ from each other in the spatial and temporal patterns of
anthocyanin accumulation. The particular pattern of pigmentation displayed
by a given plant is the result of the combined expression of all R family
members that it contains. The standard R locus is responsible for
pigmentation of the aleurone, anthers, and coleoptile. This phenotype is due
to the expression of two tightly linked members of the R gene family, S and
P. The S gene controls pigmentation of the aleurone of the kernel, whereas
the P gene controls pigmentation of the anthers and coleoptile of the plant.
Another member of the R gene family is the Lc gene, which conditions the
pigmentation of the leaf midrib, the ligule and auricle (both of these tissue
are at the boundary of the leaf blade and leaf sheath), several tissues in the
male inflorescence (glume, lemma, palea), and the seed pericarp. The R-nj
gene was cloned by transposon tagging (Dellaporta et al., 1988) and used to
isolate genomic and cDNA clones of the Lc member of the R gene family.
The Lc gene was shown to encode a transcriptional activator of the myc
class. This transcription factor is required for the accumulation of transcripts
of the chalcone synthase (C2) and dihydroflavonol4-reductase (A1) genes in
the anthocyanin biosynthetic pathway (Ludwig et al., 1989). The R-r gene
complex was characterized in detail at the molecular level by Robbins et al.
(1991). It was shown to contain three complete R transcription units P, S1,
S2, and one incomplete unit, Q. The P gene controls plant pigmentation,
whereas the S genes control the pigmentation of the aleurone layer in the
seed,
Biosynthesis of phenolic compounds
99
The B gene was cloned by Chandler et al. (1989) based on postulated
sequence homology to the R1 gene. A probe derived from the cloned R1
gene was used to screen Southern blots obtained from a population of
recombinant inbred lines in which two B alleles (b and B) were segregating.
In addition to a strong hybridization signal corresponding to the R1 gene
itself, a weak hybridization signal was detected, the size of which varied
depending on whether the b or the B allele was present. The B-Peru allele,
conferring a blotched pigmentation pattern to the leaf sheaths, was
subsequently cloned from a sub-genomic library. The coding sequence and
the direction of transcription were determined. Chandler et al. (1989) also
showed a direct correlation between the expression level of the B gene and
the expression of the target genes A1 and Bz1. This was based on expression
analyses of maize plants homozygous for the b null allele, the weakly
expressed B-Peru allele, and the highly expressed B-I allele. The B-Peru
gene and the corresponding cDNA were subsequently sequenced (Radicella
et al., 1991) and shown to encode a protein with similarity to the myc
transcription factors. Comparison of the B-Peru allele with the sequence of
the B-I allele indicated that the variation in expression pattern was the result
of sequence variation in the promoter and the 5´ part of the gene (Radicella
et al., 1992).
The maize C1 (Colorless1) gene regulates the expression of the
structural genes C2, A1, Bz1, Bz2, and A2 in the seed. Presence of a
dominant C1 allele, in combination with functional copies of the
abovementioned target genes results in seeds with colored (purple) aleurone.
The C1 gene was cloned independently by Cone et al. (1986) and Paz-Arez
et al. (1986) using a transposon-tagging strategy, and shown to encode a
transcription factor of the myb class (Paz-Ares et al., 1987). Aside from a
number of mutant c1 alleles that reduce or abolish the expression of the
gene, a dominant mutant allele C1-I was identified. The protein encoded
by the C1-I allele lacks 21 amino acid residues at the carboxy terminus
of the protein and contains a mutation that results in an amino acid
substitution (Paz-Ares et al., 1990). As a result of these two changes, the
mutant C1-I protein can bind to the promoter regions of its target genes,
but not activate transcription, and hence acts as a dominant inhibitor of
the functional C1 protein. The similarity between the R and B proteins,
both at the protein sequence level, and in molecular complementation
studies, prompted Cone et al. (1993a) to investigate whether a similar
relationship existed between the C1 and Purple plant (Pl) genes. The C1
gene determines the color of the aleurone, whereas the Pl gene determines
the color of the vegetative and reproductive parts of the plant. Evidence
in support of this hypothesis came from the existence of alleles for
both genes that were only expressed after exposure to light. Cone
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Chapter 3
et al. (1993a) were thus able to clone the maize Pl gene with a DNA
fragment from the C1 locus as a probe to screen genomic and cDNA
libraries. Based on sequence comparison of the Pl and C1 cDNA’s, more
than 90% of the amino acids at the amino and carboxyl terminal domains
that are important for the regulatory function of the C1 protein were shown
to be identical. The difference between the light-dependent expression of the
pl allele and the essentially constitutive expression of the Pl allele was
subsequently shown to reside in the promoter (Cone et al., 1993b). The pl
allele was expressed at very low levels, but was not a null allele. Rather,
Cone et al. (1993b) proposed a threshold model, whereby the amount or
concentration of the Pl protein had to be above a minimum in order for the
structural anthocyanin biosynthetic genes to be activated.
In summary, anthocyanin biosynthesis in maize requires a combination
of R1 and C1 or B and Pl, in addition to functional structural genes. The R1
and C1 combination is necessary for anthocyanin biosynthesis in the seeds,
whereas the B and Pl combination stipulates anthocyanin synthesis in the
vegetative parts of the plant. A range of modifications to this general model,
such as tissue-specific deposition or light-dependent deposition, exists as a
consequence of the availability of numerous mutant alleles.
In Arabidopsis the TT2, TT8, TTG1 and TTG2 genes have been shown to
be regulatory genes controlling the biosynthesis of flavonoids. Walker et al.
(1999) cloned the TTG1 gene using map-based cloning and showed that this
gene encodes a protein with four ‘WD40’ repeats. WD40 proteins have
diverse roles in intracellular signaling, including control of the cell cycle and
vesicular trafficking. The TTG1 protein is thought to act as a signaling
molecule that activates transcription factors of the myc class. Nesi et al.
(2000) cloned the TT8 gene using T-DNA tagging. Sequence analysis
revealed that this gene encodes a protein containing a basic helix-loop-helix
at its C-terminus, with similarity to the maize R protein and other
transcription factors of the myc class. Based on expression data obtained
with quantitative PCR, Nesi et al. (2000) concluded that the TT8 protein is
important for the expression of the structural genes DFR and BAN. They
also provided data that TTG1 and TT2 are important regulators of these two
genes.
The cloning of the TT2 gene by T-DNA tagging was reported by Nesi et
al. (2001). This gene encodes an R2R3 myb protein that shows similarity to
the maize C1 protein. In order to test whether the TT2 protein functions as a
transcriptional activator, Nesi et al. (2001) were able to show that this
protein is localized in the nucleus, that the spatio-temporal expression
Biosynthesis of phenolic compounds
101
pattern is consistent with the production of condensed tannins in the seed
coat, and that over-expression of TT2 in the presence of a functional TT8
protein results in ectopic expression (i.e. throughout the plant) of the BAN
gene. Hence, TT2 and TT8 appear to work together to regulate the
expression of BAN.
The TTG2 gene encodes a WRKY transcription factor (Johnson et al.,
2002). This class of transcription factors is characterized by the presence of
a WRKYGQK amino acid sequence (one-letter amino acid code) near the Nterminal region and a conserved C-X4-5-C-X22-23-H-X1-H sequence that
resembles zinc finger motifs. Together, these motifs are referred to as the
WRKY domain. WRKY transcription factors were initially implicated in the
response to wounding or pathogen attack, but based on the phenotype of the
ttg2 mutant, which includes developmental defects to the trichomes, it is
clear that they can also function in plant development. Based on the lack of
condensed tannins in the endothelial cells, the TTG2 gene is thought to play
a role in the regulation of structural genes, possibly BAN, involved in the
synthesis of condensed tannins.
The TT1 gene was cloned from a transparent testa mutant that was
allelic to the original tt1 mutant. The new mutant originated from the
insertion of the maize En transposon (see Section 3.2) that had been
introduced in Arabidopsis via transformation. The insertion of the En
element enabled the cloning of the TT1 gene (Sagasser et al., 2002). The
deduced amino acid sequence of the TT1 protein revealed the presence of
two zinc fingers, one near the N-terminus and one near the C-terminus of the
protein, and an additional two zinc fingers in the C-terminal part of the
protein. The presence of zinc fingers is indicative of a role as transcription
factor. The TT1 sequence, however, revealed very limited homology with
known proteins. TT1 and a small number of other plant proteins were
classified as a novel group of transcriptional activators, the WIP subfamily
of zinc finger proteins, where WIP refers to the first three conserved amino
acids. The TT1 gene was shown to be expressed in the endothelial cells of
the seed coat using a reporter gene construct with the GUS gene (see
Chapter 1, Section 3.5). The expression of BAN was reduced, but not
completely eliminated in the tt1 mutant, suggesting that TT1 is not as
specific as TT8 in its regulation of BAN expression. TT1 may instead play a
more general role in the differentiation of the endothelial tissue.
The maize P gene has been implicated in the regulation of phlobaphene
synthesis. Phlobaphenes are red pigments that accumulate in the pericarp of
the maize kernel, as well as various other parts of the plant, including the
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Chapter 3
cob, husks, and tassel glumes. Phlobaphenes arise from the polymerization
of flavan-4-ols, although the exact structure is not known (see Chapter 1,
Section 3.14). Alleles of the P gene are typically referred to by a two-letter
suffix that reflects their expression in the pericarp ad the cob. For example,
the P-rr allele results in red pericarp and red cobs, whereas the P-rw allele
results in red pericarp and white cobs. The P gene was isolated using a P-vv
allele that resulted in variegated patterns of phlobaphene deposition in both
pericarp and cob as a result of an Ac insertion in a P-rr allele (Lechelt et al.,
1989). The P gene encodes two different transcripts that have the 5´ exon
and first intron in common, but that differ from each other at the 3´ end
(Grotewold et al., 1991). The proteins encoded by the P gene contain a DNA
binding domain resembling that of the myb-class of transcriptional
activators, and this domain of the P protein is similar to that of the R and Pl
proteins that regulate anthocyanin biosynthesis. The functional P protein
regulates the expression of the A1 (DFR) and the C2 (CHS) genes
(Grotewold et al., 1991; 1994).
10.
MONOLIGNOL BIOSYNTHESIS
The monolignols are the building blocks of lignans (Section 11) and
lignin (Section 12), whereas some of the intermediates of monolignol
biosynthesis serve as precursors for hydroxycinnamic acids (Section 13) and
sinapoyl esters (Section 14). Monolignols are synthesized from pcoumaroyl-CoA (3.31) generated via the shikimate and general
phenylpropanoid pathways (see Sections 6 and 7). As part of monolignol
biosynthesis p-coumaroyl-CoA (3.31) can undergo two types of
modifications: reduction of the carboxyl group on the propane side chain to
an alcohol, and substitution of the phenyl ring (Figure 3-9). The two
predominant monolignols are coniferyl alcohol (3.79) and sinapyl alcohol
(3.81). p-Coumaryl alcohol (3.70) and 5-hydroxyconiferyl alcohol (3.80) are
generally much less abundant, and are found only in trace amounts in some
species or tissues.
The reduction of p-coumaroyl-CoA (3.31) to p-coumaryl aldehyde (3.69)
is catalyzed by the enzyme cinnamoyl-CoA : NADP oxidoreductase (CCR).
This enzyme was initially purified from soybean cultures (Wegenmayer et
al., 1976), and was later on efficiently isolated from lignifying cambium of
eucalyps (Eucalyptus gunnii) (Goffner et al., 1994). A CCR cDNA was
identified in a cDNA library that was screened with oligonucleotiede derived
from the peptide sequence of the CCR protein. CCR is considered the first
enzyme committed towards the biosynthesis of monolignols and shows
Biosynthesis of phenolic compounds
103
homology to the flavonoid biosynthetic gene flavonol 4-reductase (Lacombe
et al., 1997).
The substitution of the phenyl ring necessary for the biosynthesis of
coniferyl alcohol (3.79) and sinapyl alcohol (3.81) begins with the
hydroxylation of C3. This is a conversion that requires the formation of the
ester of p-coumaroyl-CoA with D-quinate (3.73) or shikimate (3.74)
catalyzed by the enzyme hydroxycinnamoyl-CoA shikimate/quinate
hydroxy-cinnamoyl transferase (HCT; Hoffmann et al., 2003). The
hydroxylation of this ester intermediate is catalyzed by the enzyme pcoumaroyl-CoA 3′-hydroxylase (C3′H; Schoch et al., 2001; Franke et al.,
2002a,b). The resulting shikimate or quinate ester (3.75; 3.76) is
subsequently hydrolyzed by the same HCT, resulting in caffeoyl-CoA
(3.36).
The enzyme responsible for the hydroxylation of C3 was extremely difficult
to identify. It had been postulated to be a phenol oxidase, dioxygenase, or a
cytochrome P450 monooxygenase, but biochemical approaches aimed at
isolating a protein displaying activity toward p-coumaric acid (3.30) were
unsuccessful. With the availability of the Arabidopsis thaliana genome
sequence, Schoch et al. (2001) performed a phylogenetic analysis of the
genes encoding cytochrome P450 enzymes (for a review on this class of
enzymes, see Chapple (1998)). This analysis resulted in the identification of
CYP98A3 as a putative C3H. Expression analyses confirmed that the gene
encoding CYP98A3 was expressed in tissues that would be expected to
synthesize caffeic acid derivatives, including lignified tissues, and wounded
tissues that produced chlorogenic acid (1.18). After cloning of the
corresponding cDNA and expression of the recombinant enzyme in yeast,
substrate specificity could be investigated. The enzyme did not show activity
towards p-coumaric acid (3.30), p-coumaroyl-CoA (3.31), the p-coumaroyl
glucose ester, nor the p-coumaroyl 4-glucoside. Based on experiments by
Heller and Kühnl (1985) and Kühnl et al. (1987) with parsley cell
suspension cultures, Schoch et al. (2001) were able to show activity of the
recombinant CYP98A3 enzyme towards the shikimate and D-quinate esters
of p-coumaroyl-CoA (3.71; 3.72), which resulted in the shikimate and Dquinate esters of p-caffeoyl CoA (3.73; 3.74), respectively. When the
numbering of carbon atoms in the p-coumaroyl-CoA esters is taken into
consideration, the carbon at the 3´ position - not the 3 position - is
hydroxylated. Consequently, the enzyme is now referred to as C3´H. Franke
et al. (2002a) showed that the Arabidopisis reduced epidermal fluorescence8
(ref8) mutant was unable to synthesize caffeic acid (3.32) as a result of a
defective copy of the C3´H gene.
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Chapter 3
OH
HO
OH
OH HO
O
(3.72)
O
b
CoA-S
(3.74)
HO
O
O
OH
O
O
O
OH
HO
OH
OH HO
O
O
OH
b
(3.71)
a
(3.73)
HO
O
O
O
a
O
OH
(3.36)
O
O
OH
OH
OH
O
O
c
CoA-S
O
CoA-S
O
a
OCH3
OH
OH
(3.31)
d
(3.75)
d
O
O
O
O
g
f
OCH3 H3CO
OCH3 HO
OCH3
OH
OH
OH
OH
(3.69)
(3.76)
(3.77)
(3.78)
e
OH
e
e
OH
f
(3.70)
OH
g
OCH3 HO
OH
e
OH
OCH3 H3CO
OCH3
OH
OH
OH
(3.79)
(3.80)
(3.81)
Figure 3-9. Biosynthesis of monolignols. The enzymes involved in this pathway are: (a)
hydroxycinnamoyl-CoA shikimate/quinate hydroxy-cinnamoyl transferase, (b) p-coumaroylCoA 3′-hydroxylase (E.C. 1.14.14.1), (c) caffeoyl-CoA O-methyltransferase (E.C. 2.1.1.104),
(d) cinnamoyl-CoA reductase (E.C. 1.2.1.44) (e) cinnamyl alcohol dehydrogenase (E.C.
1.1.1.195), (f) coniferyl aldehyde/coniferyl alcohol 5-hydroxylase (E.C. 1.14.13), (g)
coniferaldehyde/coniferyl alcohol O-methyltransferase (E.C. 2.1.1.68).
Biosynthesis of phenolic compounds
105
HCT, the enzyme responsible for the formation of the D-quinate and
shikimate esters of p-coumaroyl CoA (3.71; 3.72), was identified by
Hoffmann et al. (2003) in stem extracts of tobacco. Separation of the
proteins with HPLC resulted in a fraction containing HCT activity. The
protein was partially sequenced, and degenerate primers were synthesized to
amplify the corresponding cDNA. Purification of the recombinant protein
from E. coli enabled more detailed studies on substrate specificity and
catalytic properties. These studies showed that HCT was able to catalyze the
esterification reaction of D-quinate and shikimate with both p-coumaroylCoA (3.31) and caffeoyl-CoA (3.36). The enzyme could also catalyze the
reverse reaction, i.e. the hydrolysis of the ester, thus producing caffeoyl CoA
and either D-quinate or shikimate. The role of this enzyme in
phenylpropanoid metabolism was further demonstrated via down-regulation
of the HCT gene in Arabidopsis and tobacco (Nicotiana benthamiana). The
transgenic plants were dwarfed, accumulated caffeoylquinate esters in their
leaves, and showed different lignin subunit composition (Hoffmann et al.,
2004).
Caffeoyl-CoA (3.36) is methylated to feruoyl-CoA (3.75) by the enzyme
caffeoyl-CoA O-methyltransferase (CCoA-OMT). CCoA-OMT had been
implicated in disease responses based on its induction in carrot (Daucus
carota) cell suspension cultures that were treated with elicitors (Kühnl et al.,
1989). A more general role of CCoA-OMT in phenylpropanoid metabolism
was proposed after Ye et al. (1994) showed that the CCoA-OMT gene was
up-regulated during the in vitro development of lignified tracheary elements
derived from Zinnia elegans mesophyl cells. Feruoyl-CoA (3.75) is
subsequently reduced to coniferaldehyde (3.76) by CCR, analogous to the
reduction of p-coumaroyl CoA (3.31) to p-coumaryl aldehyde (3.69).
Coniferaldehyde (3.76) can undergo several fates, some of which can
ultimately lead to the same end product. It can be reduced to coniferyl
alcohol (3.79) by the enzyme cinnamyl alcohol dehydrogenase (CAD).
Alternatively, the enzyme coniferyl aldehyde/coniferyl alcohol 5hydroxylase (C5H), also known by its less accurate name ferulic acid 5hydroxylase (F5H; Humphreys et al., 1999) can catalyze the hydroxylation
of C5 to result in 5-hydroxyconiferyl aldehyde (3.77). C5H is also able to
form 5-hydroxyconiferyl alcohol (3.80) from coniferyl alcohol (3.79). This
enzyme was initially identified as F5H, after analysis of the Arabidopsis
ferulic acid hydroxylase1 (fah1) mutant, which was isolated in a mutant
screen based on reduced levels of the UV-fluorescent sinapoyl esters
(Section 13; Chapple et al., 1992). The FAH1 gene was cloned using a TDNA tagged mutant allele (Meyer et al., 1996), which revealed that the gene
106
Chapter 3
encoded a cytochrome P450 monooxygenase with homology to flavonoid
3´, 5´ hydroxylases. Substrate specificity of recombinant F5H was evaluated
by Humphreys et al. (1999) and Osakabe et al. (1999). Their analyses
revealed that F5H had much higher activity towards coniferaldehyde (3.76)
and coniferyl alcohol (3.79) than against ferulic acid (3.33).
Methylation of 5-hydroxyconiferyl aldehyde (3.77) and 5-hydroxy
coniferyl alcohol (3.80) by the enzyme 5-hydroxyconiferaldehyde/5hydroxyconiferyl alcohol O-methyltransferase results in sinapaldehyde
(3.78) and sinapyl alcohol (3.81), respectively. The enzyme catalyzing this
step is known by the historic but inaccurate name caffeic acid O-methyl
transferase (COMT). So COMT is now thought to be responsible for the
methylation of the hydroxyl group on C5, whereas CCoA-OMT is
responsible for methylation of the hydroxyl group on C3. This explains why
mutations in the COMT gene, such as in the maize brown midrib3 mutant
(Vignols et al., 1995) and the sorghum brown midrib26 mutant (Bout and
Vermerris, 2003), result in reductions in lignin units derived from sinapyl
alcohol, and not in lignin subunits derived from coniferyl alcohol (see also
Section 12).
As described above, sinapyl alcohol (3.81) can be synthezed via
methylation of 5-hydroxyconiferyl alcohol (3.80) by COMT. An alternative
route is via the reduction of sinapaldehyde (3.78) by CAD or, in the case of
aspen (Populus tremuloides) and several other angiosperm trees, sinapyl
alcohol dehydrogenase (SAD; Li et al, 2001). SAD cDNA’s were identified
as a distinct class of hybridizing fragments during the screening of an aspen
cDNA library derived from lignifying xylem tissue with a probe derived
from an aspen CAD cDNA. Analysis of the substrate specificity of the
recombinant protein generated by expression of a SAD cDNA in E.coli
indicated that SAD had a 60-fold higher affinity for sinapaldehyde than
coniferaldehyde.
CAD is encoded by a multigene family in Arabidopsis (Raes et al.,
2003; Goujon et al., 2003) and rice (Oryza sativa; Tobias and Chow, 2005),
and probably in many other species. Mutants of Arabidopsis in which the
genes encoding two distinct isoforms of CAD, CAD-C and CAD-D, were
down-regulated as a result of T-DNA insertions were analyzed by Sibout et
al. (2003). The reduction in CAD-C activity resulted in minor changes in
lignin composition, whereas reduction CAD-D activity resulted in a 45%
and 24% reduction in lignin residues derived from sinapyl alcohol in stem
and root tissue, respectively. Taken together with the fact that both isoforms
display activity towards both coniferaldehyde and sinapaldehyde, these data
suggest
Biosynthesis of phenolic compounds
107
that the CAD enzymes in Arabidopsis do not display the same substrate
specificity for either sinapaldehyde or coniferaldehyde as was observed in
aspen. Rather, in Arabidopsis, and possibly many other species, the
biosynthesis of coniferyl alcohol and sinapyl alcohol appears to be catalyzed
by a combination of isoforms, some of which have a preference towards one
of the substrates. The combination of isoforms varies depending on the
developmental stage and the tissue (Sibout et al., 2003).
11.
LIGNAN BIOSYNTHESIS
Lignans are synthesized from the oxidative coupling of monolignol
radicals (see Section 10 in this chapter, and Chapter 1, Section 3.11). The
monolignol radicals are generated through the action of laccases (E.C.
1.10.3.2) or peroxidases (E.C. 1.11.1.7; see also Chapter 2, section 1.8).
Unlike lignin, however, lignans are optically active, and typically only one
enantiomer is present in a given species. This means that the coupling
between the monolignols is under regio-chemical control, whereby both the
coupling site and the orientation of the two monomers are controlled.
Analysis of Forsythia intermedia stem extracts, which accumulate high
levels of the lignan (+)-pinoresinol (3.82) resulted in the purification of a
protein that has no catalytic activity, but that is able to stipulate the
formation of (+)-pinoresinol from two coniferyl alcohol (3.79) radicals
(Davin et al., 1997). This protein was referred to as ‘dirigent protein’. It is
thought to hold the two coniferyl alcohol radicals in a specific configuration
during bond formation so that both the position of the bond (8-8´) and the
conformation are controlled (Figure 3-10). Once the bond is formed,
intramolecular cyclization results in (+)-pinoresinol (3.82). This particular
dirigent protein was only able to recognize coniferyl alcohol radicals and
none of the other monolignol radicals. Since the initial discovery of this
protein from Forsythia intermedia, homology searches in sequence
databases have revealed the existence of additional genes encoding putative
dirigent proteins, from a variety of species (Gang et al., 1999; Davin and
Lewis, 2000). Recently, evidence for the existence of dirigent proteins able
to direct the formation of 8-O-4´ linkages between coniferyl alcohol and
sinapyl alcohol was reported (Lourith et al., 2005).
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Chapter 3
HO
.
H
H
H
OH
HO
H
H
.
H3CO
H3CO
OH
O
H
H3CO
H3CO
O
H
H
O
O
dirigent protein
(3.79)
H3CO
OH
O
H
H
O
HO
OCH3
(3.82)
Figure 3-10. Dirigent-protein mediated formation of (+)-pinoresinol from two coniferyl
alcohol radicals. The formation of the ring results from intramolecular cyclization.
Lignans represent an extremely diverse group of compounds. This is the
result of both structural diversity and stereo-selective biosynthesis. One
particular plant species generally makes only one enantiomer of a particular
compound. The other enantiomer may be synthesized by a different species.
As a consequence, it is virtually impossible to summarize the biosynthesis of
lignans in general. Instead, the focus here will be on the biosynthesis of the
lignan podophyllotoxin in a number of different plant species, as an
illustration of the different biosynthetic routes that can be used to synthesize
the same compound.
Podophyllotoxin (3.86) is used as an ectopic antiviral agent in the
treatment of venereal warts. It is too cytotoxic to be ingested, but after
chemical modifications podophyllotoxin can be used as a powerful anti-
Biosynthesis of phenolic compounds
109
cancer drug in the treatment of small cell lung cancer, acute leukemia, and
non-Hodgkin’s lymphoma (Canel et al., 2000). Podophyllotoxin is isolated
from the plant Podophyllum peltatum, commonly known as Mayapple (also
referred to as Devils’s apple or hog apple), or from the related species
Podophyllum hexandrum (Himalayan mayapple). In order to be able to
produce podophyllotoxin without depleting the natural populations of these
wild species, attempts have been made to produce podophyllotoxin in tissue
culture. In this context it is important to know the biosynthetic route to
podophyllotoxin production, so that lines that produce high levels of the
relevant biosynthetic enzymes can be selected. Broomhead et al. (1991)
investigated the biosynthesis of podophyllotoxin in Podophyllum
hexandrum using radioactive feeding experiments in whole plants. They
proposed the biosynthetic route displayed in Figure 3-11. This shows how
two coniferyl alcohol (3.79) radicals are coupled in a stereo-selective
manner to form matairesinol (3.83). Subsequent hydroxylation and
methylation of C5´ and lactone ring formation at C3 and C4 gives yatein
(3.84). Ring closure results in the formation of deoxypodophyllotoxin
(3.85), which is hydroxylated to yield podophyllotoxin (3.86).
In addition to podophyllotoxin itself, closely related compounds, such as
5-methoxypodophyllotoxin (3.89), can be used as a precursor for anti-cancer
drugs. Linum species (flax, linseed) can produce podophyllotoxin or
substituted podophyllotoxins in tissue culture with yields of up to 0.35% of
dry weight. The biosynthesis of 5-methoxypodophyllotoxin in Linum flavum
was investigated by Xia et al. (2000), whereas Seidel et al. (2002)
investigated podophyllotoxin (3.86) production in cell cultures of Linum
album.
Xia et al (2000) proposed a biosynthetic route starting coniferyl alcohol
(3.79) and subsequent formation of (+)-pinoresinol (3.82), as shown in
Figure 3-12. The enzyme pinoresinol/lariciresionol reductase converts this
compound to (+)-lariciresinol (3.87), and then to (–)-secoisolariciresionol
(3.88). The enzyme secoisolariciresinol dehydrogenase converts (3.88) into
(–)-matairesinol (3.83). The conversion from (–)-matairesinol (3.83) to
podophyllotoxin (3.86) is likely to be similar to the route shown in Figure 311.
110
Chapter 3
H3CO
.
.
OH
H3CO
O
OH
O
HO
O
OCH3
OCH3
O
OH
(3.83)
(3.79)
O
O
O
O
O
O
O
H3CO
OCH3
OCH3
O
OCH3
H3CO
OCH3
(3.85)
(3.84)
HO
O
O
O
O
H3CO
OCH3
OCH3
(3.86)
Figure 3-11. Biosynthesis of podophyllotoxinin in Podophyllum hexandrum, according to
Broomhead et al. (1991). The dotted arrow refers to a series of reactions that have not yet
been fully elucidated: formation of a methylene dioxybridge, and hydroxylation and
methylation reactions.
Biosynthesis of phenolic compounds
111
OCH3
OCH3
OH
OH
O
O
a
a
NADP+
NADPH
NADP+
NADPH
O
HO
HO
HO
OCH3
OCH3
(3.82)
(3.87)
H3CO
H3CO
OH
O
OH
HO
b
c
HO
O
NAD+
NADH
OCH3
OCH3
OH
OH
(3.88)
(3.83)
OH
H3CO
H3CO
HO
O
O
O
O
HO
O
O
d
OCH3
OH
(3.86)
e
f
g
H3CO
OCH3
OCH3
(3.89)
Figure 3-12. Biosynthesis of 5-methoxypodophyllotoxinin in Linum flavum, according to Xia
et al. (2000). (a) pinoresinol/lariciresionol reductase, and (b) secoisolariciresinol
dehydrogenase. The enzymes catalyzing steps c-g have not yet been elucidated, but involve
(c) hydroxylation of C7, (d) ring closure, (e) hydroxylation of C5, (f) methylation of the OHgroup on C5, and (g) formation of a methylene dioxy bridge. The exact order of these
reactions is also still unknown.
112
Chapter 3
Synthesis of podophyllotoxin (3.86) in cell culture of Linum album
results in yields comparable to those of the most efficient tissue cultures of
Podophyllum hexandrum. In order to further improve L. album cultures,
Seidel et al. (2002) investigated the biosynthesis of podophyllotoxin (3.86).
They fed a number of labeled compounds that to L. album cell cultures to
identify which of these compounds could be used as precursors to
podophyllotoxin. They determined that the substitution pattern on the
benzene ring is critical. The substitution has to be either 3-methoxy, 4hydroxy, as in ferulic acid (3.33), or, alternatively, 3,4methylenedioxycinnamic acid (3.90) can serve as precursor. The precursor
of podophyllotoxin in L. album appears to be deoxypodophyllotoxin (3.83),
based on the higher level of isotope incorporation in the latter compound.
This means that 7-hydroxymatairesinol, the precursors of 5methoxypodophyllotoxin in L. flavum (Xia et al., 2000), is not a precursor of
podophyllotoxin in L. album.
O
OH
O
OCH3
OH
O
OH
O
(3.33)
(3.90)
12.
LIGNIN BIOSYNTHESIS
12.1
Genetic control of lignification
All of the enzymes involved in the biosynthesis of monolignols have
been identified and characterized, and the genes encoding these enzymes
have been cloned. As discussed in Section 10, the specific role of individual
genes that are part of a multigene family needs further investigation.
Another aspect that is relatively poorly understood is the regulation of lignin
biosynthesis. Given that the biosynthesis of lignin involves a large number
of enzymes, the genes encoding these enzymes need to be coordinately
expressed. One mechanism to achieve this would be to have one or a few
Biosynthesis of phenolic compounds
113
transcriptional activators that can bind to a common motif present in the
promoters of several monolignol biosynthetic genes. Comparison of the
promoter sequences of structural genes involved in phenylpropanoid
metabolism revealed a motif conserved among several plant species referred
to as the PAL-box: CCA (C/A) (A/T) A (A/C) C (C/T) CC; the nucleotides
in parentheses are degenerate, meaning that the exact nucleotide that is
present varies among promoters. This motif is also referred to as ACelements and H-box.
Kawaoka et al. (2000) used the PAL-box sequence to isolate a cDNA
encoding a transcription factor that could bind to the PAL box. They
screened an expression library generated from tobacco (Nicotiana tabacum)
stem cDNA’s with a radio-labeled oligonucleotide comprising of three
repeats of a PAL-box. This so-called southwestern screening resulted in a
partial cDNA clone corresponding to a LIM transcription factor. This class
of transcription factors was first identified in the nematode Caenorhabditis
elegans. The LIM domain is cysteine rich and contains two DNA-binding
zinc fingers separated from each other by two amino acids. Sequence
analysis of the full-length cDNA sequence obtained by Kawaoka et al.
(2000), referred to as Ntlim1, revealed the presence of two LIM domains and
one acidic domain. All three domains had to be present to effect
transcriptional activation of a target gene based on transient expression
studies with tobacco protoplasts. In vivo transcriptional activity of the
NtLIM1 protein was demonstrated by studies with transgenic plants
expressing the Ntlim1 gene in either the sense of antisense orientation under
the control of the strong cauliflower mosaic virus (CaMV) 35S promoter.
Introduction of the sense constructs did not enhance expression of the genes
encoding PAL, 4CL, and CAD, nor did it result in increased enzyme
activity. The antisense constructs, however, resulted in a coordinate downregulation of the PAL, 4CL and CAD genes, along with a reduction in the
activities of the enzymes encoded by these genes. Depending on the
transgenic line, variations in lignin content and subunit composition was
observed.
The myb transcription factors had been shown to be involved in the
regulation of phenylpropanoid metabolism (cf. Grotewold et al., 1994; see
Section 9.2). They were first shown to be involved in the regulation of lignin
biosynthesis by showing that lignin content and phenolic ester content were
reduced in transgenic tobacco in which the snapdragon AmMYB308 or
AmMYB330 cDNA’s were overexpressed (Tamagnone et al., 1998). The
reduction rather than increase in phenolic metabolism suggests that these
two myb transcription factors are normally weak activators, but that their
114
Chapter 3
over-expression out-competes the binding of stronger transcriptional
activators that are present. Based on these results Patzlaff et al. (2003)
screened a cDNA library made from differentiating loblolly pine (Pinus
taeda) xylem with a pool of radio-labeled DNA fragments representing
conserved sequences of R2R3 myb genes. R2R3 myb proteins are
characterized by two 50- to 52-residue long imperfect repeats. Each of the
repeats contains three α-helices, with the second and third helices forming a
helix-turn-helix structure when bound to DNA (Sablowski et al., 1994; Dias
and Grotewold, 2003). The PtMYB4 cDNA they isolated was shown to
encode an R2R3 myb protein able to bind to AC-elements in an
electrophoretic mobility shift assay (EMSA) with recombinant PtMYB4
protein and radio-labeled AC-elements based on the bean PAL2 promoter
sequence. Binding of PtMYB4 protein to these targets would reduce their
electrophoretic mobility through polyacrylamide gels, as was indeed the
case. Analysis of transgenic tobacco over-expressing PtMYB4 cDNA
showed that the expression of the PAL gene was reduced, the expression of
the C4H and 4CL genes was not changed, and that the expression of the
genes encoding C3′H, CCoA-OMT, CCR, COMT and CAD (see Section 10)
was increased. Furthermore, the transgenic plants showed ectopic
lignification, which means that tissues that are normally not lignified were
producing lignin. Given that PAL, C4H and 4CL are part of the general
phenylpropanoid pathway (Section 7), whereas the other genes are part of
the more specific monolignol biosynthetic pathway (Section 10), these data
indicate that the PtMYB4 protein appears to be specifically involved in
lignification.
Very similar results were obtained with an R2R3 myb cDNA isolated
from eucalypt (Eucalyptus grandis) xylem (Goicoechea et al., 2005). The
EgMYB2 protein was able to bind to radio-labeled promoter fragments from
the eucalypt CAD2 and CCR genes. As was the case with the PtMYB4
protein, analysis of transgenic tobacco plants over-expressing the EgMYB2
gene showed limited effects on the expression of the PAL, C4H and 4CL
genes, but a 5- to 40-fold increase in the expression levels of the genes
encoding HCT, C3′H, CCR, CCoA-OMT, F5H, COMT and CAD.
The Arabidopsis AtMYB61 gene is homologous to the PtMYB4 gene and
was shown to be mis-expressed in the Arabidopsis de-etiolated3 (det3)
mutant, which displays ectopic lignification and is dark photomorphogenic,
meaning that the mutant will grow and develop in the dark as if it was
growing in the light (Newman et al., 2004). Overexpression of AtMYB61 in
Arabidopsis resulted in a det3 phenotype, whereas down-regulation of
ATMYB61 in det3 mutants resulted in a loss of the mutant phenotype. These
Biosynthesis of phenolic compounds
115
data showed that the mis-expression of AtMYB61 was necessary and
sufficient to produce the det3 mutant phenotype.
Mutants in which lignin deposition is disturbed are valuable sources to
obtain information on the mechanisms involved in the spatio-temporal
control of lignin deposition. The maize brown midrib2 (bm2) mutant has
brown vascular tissue in the leaf blade and leaf sheath. Chemical analysis of
dissected vascular tissue obtained from this mutant and the wild-type control
indicated an overall reduction in lignin residues derived from both coniferyl
alcohol (3.79) and sinapyl alcohol (3.81), but the most striking difference ws
that the developmental-specific gradient in lignin content, whereby older
tissues are more heavily lignified than younger tissues, was disrupted as a
result of the bm2 mutation (Vermerris and Boon, 2001). The functional Bm2
gene has been cloned, but its molecular function remains to be elucidated.
Rogers and Campbell (2004) provided an overview of Arabidopsis
mutants in which normal patterns of lignin deposition were altered. This set
of mutants includes the det3, ectopic lignification1 (eli1) and pom-pom1
(pom1) mutants, all of which synthesize lignin ectopically. These mutants
were the subject of study by Rogers et al. (2005) in an attempt to identify
whether a common regulatory circuit was disturbed Chemical and
histochemical analyses revealed variation in cell wall composition among
the three mutants. A comparison of the expression of structural genes
involved in monolignol biosynthesis revealed that the expression of some
genes (e.g. PAL1, C4H, F5H and CCoA-OMT) was increased in all mutants,
whereas the expression of the other genes changed only in a subset of the
mutants. A microarray analysis was performed to identify genes that were
coordinately expressed with the monolignol biosynthetic genes. Microarray
experiments are performed with so-called gene chips on which several
thousand genes are represented by short gene-specific DNA molecules
termed oligonucleotides. The chips are hybridized with fluorescently labeled
cDNA pools obtained from a sample of interest (mutant, treatment,
developmental stage) and a control. The cDNA pools from the two samples
are labeled with different fluorescent dyes, generally Cy-3 and Cy-5, that
result in a green and red fluorescence, respectively, upon excitation with a
laser (Schulze and Downward, 2001; Wayne and McIntyre, 2002; Gracey
and Cossins, 2003). After the hybridization, it is possible to identify whether
a given gene was not expressed (no signal), expressed equally in both
samples (yellow fluorescent signal from overlap of green and red
fluorescence), or higher expression in one or the other sample (red or green
signal). Analysis of the promoter sequences of genes coordinately expressed
with the subset of lignin biosynthetic genes revealed that the AC-element
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Chapter 3
(PAL-box) was significantly over-represented. Among these genes were five
genes encoding transcriptional activators of the R2R3 myb, and Dof classes.
Dof - Downstream-of-fibroblast growth factor receptor - transcriptional
activators were first identified in the fruit fly (Drosophila melanogaster).
Given that this set of five transcription factors had expression patterns
similar to the monolignol biosynthetic genes, they may be involved in the
regulation of lignin deposition.
12.2
Monolignol transport and polymerization
Lignin is a complex polymer generated via oxidative coupling of
monolignol radicals (see Section 10). The polymerization of lignin occurs in
the plant cell wall, so the monolignols need to be transported from the
cytosol, where they are synthesized, to the cell wall. There is evidence that
in several species, including conifers and Arabidopsis, the monolignols are
glycosylated prior to storage and transport, which would reduce their
toxicity and make them less reactive. The enzymes responsible for the
glycosylation reaction are UDP-glucosyltransferases (UGT’s; EC 2.4.1.111)
that generate coniferin (coniferyl alcohol 4-O-glucoside) and syringin
(sinapyl-4-O-glucoside) from coniferyl alcohol (3.79) and sinapyl alcohol
(3.81), respectively (Lim et al., 2001). Prior to polymerization these
glucosides need to be converted to the corresponding aglycones by specific
glucosidases, such as coniferin –glucosidase (EC 3.2.1.126).
The Arabidopsis genome contains more than 100 UGT genes. In order
to determine which of these UGTs were capable of forming either
glucosides – involving a linkage between glucose and the hydroxyl group on
C4 of the phenylpropanoid – or glucose esters – involving an ester linkage
between the carboxyl group of hydroxycinnamic acids and the hydroxyl
group of glucose – cDNA’s or genomic clones representing 36 Arabidopsis
genes were expressed in E. coli, and the activity of the resulting recombinant
proteins against 11 different intermediates from the monolignol biosynthetic
pathway was assayed. This analysis identified three genes encoding UGTs
catalyzing the formation of cinnamate glucose esters and two genes –
UGT72E2 and UGT72E3 – encoding UGTs that catalyze the formation of
cinnamate glucosides. The recombinant protein obtained from expression of
the UGT72E gene showed activity towards both coniferyl alcohol and
sinapyl alcohol, whereas the UGT72E3 recombinant protein only showed
activity towards sinapyl alcohol.
Biosynthesis of phenolic compounds
117
Polymerization of lignin occurs through an oxidative coupling
mechanism, whereby monolignol radicals react with radical sites on the
lignin polymer. The two most common monolignols, coniferyl alcohol
(3.79) and sinapyl alcohol (3.81), give rise to guaiacyl (G) and syringyl (S)
residues, respectively, in the ligin polymer. p-Coumaryl alcohol (3.70) gives
rise to p-hydroxyphenyl (H) residues. The latter is not a very common lignin
residue. It is incorporated in compression wood of gymnosperms in response
to gravitropic stress (Higuchi, 1985), and to a small extent (~5%) in the
lignin of graminaceous species.
There has been some controversy regarding the enzyme catalyzing the
formation of the monolignol radicals. Histochemical studies of lignifying
tissues with a chromogenic substrate only resulted in oxidation of the
substrate in the presence of H2O2 (Harkin and Obst, 1973). This observation
combined with the broad distribution of peroxidases (EC 1.11.1.7) in the
plant kingdom and the more limited distribution of laccases (EC 1.10.3.2)
led Higuchi (1985) to propose a dominant role of peroxidases in
lignification. Laccases were subsequently shown to be present and active in
lignifying tissues of various plant species, including sycamore maple (Acer
pseudoplatanus; Sterijades et al., 1992) and loblolly pine (Pinus taeda; Bao
et al., 1993; see also Chapter 2, section 1.8.2). The current view is that both
laccases and peroxidases can generate monolignol radicals, but it is unclear
whether there is a preference for one or the other as a function of the
species, the tissue, the developmental stage, or the environmental conditions.
Both peroxidases and laccases are encoded by large multigene families
(Raes et al., 2003), which has made it difficult to study the specific role of
these enzymes in lignification. Transgenic approaches have been used to
down-regulate or over-express laccase or peroxidase genes, in an attempt to
better understand their function. Down-regulation of three laccase genes in
poplar (Populus trichocarpa) using antisense technology did not have an
impact on lignin content or composition, but down-regulation of one of
these laccase genes did result in alterations in xylem cell wall structure and
an increase in the level of soluble phenolics (Ranocha et al., 2002).
Similarly, a mutation in an Arabidopsis laccase gene resulted in irregularly
shaped xylem cells (Brown et al., 2005). In contrast, down-regulation of an
anionic peroxidase gene from hybrid aspen (Populus sieboldii x P.
gradidentata) via an antisense construct resulted in reduced lignin content
and a reduction in G-residues in the lignin (Li et al., 2003).
Given that there are several monolignols and that these monolignols can
be linked to each other in a number of different ways (see Chapter 1, section
3.12), the question arises how the plant is able to control lignin subunit
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Chapter 3
composition and the distribution of inter-unit linkages. The prevailing view
is that lignin subunit composition is determined predominantly by the flux
of monolignols. In grasses, for example, p-coumaryl alcohol (3.70) is
secreted early on during the lignification process, resulting in the relatively
high abundance of H-residues in cell walls that have just begun to lignify.
Coniferyl alcohol (3.79) and sinapyl alcohol (3.81) are secreted into the cell
wall later on (Terashima et al. 1993). The distribution of interunit linkages
appears to be under chemical control. Based on in vitro formation of
dehydrogenation polymers (DHPs) the distribution of interunit linkages is
affected by the local concentration of monolignol radicals (Syrjanen and
Brunow, 2000), the presence of polysaccharides (Terashima et al., 1995),
proteins (McDougall et al., 1996), and lignin (Guan et al., 1997). When
DHPs were formed in the presence of isolated primary cell walls of maize,
the resulting lignin polymer resembled native maize lignin remarkably well
(Grabber et al., 1996). The model in which lignin polymerization is
considered to be under chemical control is referred to as the ‘random
coupling’ model, or the ‘combinatorial coupling’ model. According to this
model, lignin has little predetermined structure to it. From a biological point
of view this is to the plant’s advantage when it comes to the role lignin plays
in defense against pathogens and insect pests. An invading organism would
have to develop a host of hydrolytic enzymes in order to effectively dissolve
the barrier created by lignified tissue (Denton, 1998).
An opposing view on the polymerization of lignin was proposed by
Lewis and Davin (1998). They reasoned that an abundant and important biopolymer like lignin should be under strict biological control, in a manner
analogous to the other biological polymers, including cellulose, chitin, and
proteins. According to these authors there are two major pieces of evidence
for biological control of lignin polymerization: (1) the DHP formed by the in
vitro polymerization of coniferyl alcohol (3.79) in the presence of
peroxidase and H2O2 displays a very different distribution of interunit
linkage than the lignin formed in planta (Nimz and Ludemann, 1976) and
(2) immunohistochemical analyses using antibodies against specific lignin
substructures show that the different antibodies recognize different parts of
the cell wall, suggesting that lignin subunit composition varies depending on
the exact position in the cell wall (Joseleau and Ruel, 1997). Given that both
lignans and lignin are synthesized from monolignols, Lewis and Davin
(1998) hypothesized that proteins resembling the dirigent protein (see
Chapter 1, Section 3.11, and this chapter, Section 11) could be responsible
for the polymerization of lignin.
Biosynthesis of phenolic compounds
119
This hypothesis was furthered by Gang et al. (1999), who provided
evidence for the existence of epitopes in secondary cell walls of Forsythia
that were recognized by polyclonal antibodies raised against the dirigent
protein. In addition, homology searches of DNA sequence databases
revealed the existence of putative genes resembling the gene encoding the
Forsythia dirigent protein. According to the proposed model, these dirigentlike proteins could stipulate lignin subunit composition, as well as the
formation of specific interunit bonds.
The publication by Gang et al. (1999) initiated a heated debate on
lignification, because the hypothesis that dirigent-like proteins were
involved in lignification was presented as ‘a new paradigm’. The existing
random coupling model agreed, however, surprisingly well with what was
known about lignin polymerization and the establishment of lignin subunit
composition, as discussed above. The continuing effort to replace the
random coupling model with the new dirigent model (Lewis, 1999), which
was touted as the solution to ‘the mystery of specificity of radical precursor
coupling in lignan and lignin biosynthesis’ (Davin and Lewis, 2000),
resulted in a series of rebuttals in which the shortcomings of the dirigent
model were pointed out (Sederoff et al., 1999; Hatfield and Vermerris, 2001;
Ralph et al., 2004). One issue the dirigent model did not adequately address
was optical activity. According to the model proposed by Lewis and
colleagues, the stereospecificity of the dirigent-like proteins would result in
an optically active lignin polymer. Lignin, however, is not optically active
(Freudenberg et al., 1965; Ralph et al., 1999; Akiyama et al., 2000). To
address this issue, Davin and Lewis (2000) proposed that lignin exists
predominantly as a linear polymer, and that the lack of optical activity
results from the synthesis of a lignin strand with the opposite chirality,
originating from self-replicating polymerization templates. This was
schematically represented by Davin and Lewis (2005) based on chemical
analyses aimed at resolving the primary structure of a lignin hexamer with
electron spray ionization-mass spectrometry (Evtuguin and Amada, 2003).
The experiments reported by Evtuguin and Amada (2003) were, however,
not based on the assumption that lignin as a whole was a linear polymer.
Rather, they were interested in defining how different monolignols were
coupled to each other. In addition, mass spectrometry does not allow the
distinction between different stereo-isomers, so that stereo-selective
coupling cannot be inferred from these data.
The articles dealing with the dirigent model are of interest not only from
a scientific but also from a philosophical viewpoint. In the case of the
dirigent hypothesis a completely new model was proposed by extrapolating
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Chapter 3
data obtained from experiments on a different class of compounds (lignans).
This came at a time when the existing random coupling model was able to
explain the majority of the observations. Based on the way the dirigent
hypothesis was presented in review articles (Lewis, 1999; Davin and Lewis,
2000), the newly proposed model was essentially elevated to a theory in a
matter of one year.
Theories are formulated after a process during which a new hypothesis
is tested. The experiments that allow hypothesis testing are generally set up
in such a way that a hypothesis can be disproved. Only after repeatedly
being unable to disprove a hypothesis can one conclude that all the data
support the hypothesis. At that point it becomes possible to predict the
outcome of newly designed experiments. If the outcome is indeed
consistently predicted correctly, it is appropriate to elevate the hypothesis to
a theory.
In the case of the dirigent model, the hypothesis testing phase is far from
complete. For example, if dirigent-like proteins are involved in lignin
polymerization, then the inactivation of the gene encoding these proteins
would be expected to result in a change in lignin distribution or lignin
composition. Plants in which specific genes are down-regulated are
nowadays relatively easy to obtain through insertional mutagenesis or
transgenic approaches. This approach is therefore feasible, and would allow a
direct evaluation of the dirigent model. The need for such experiments has
been expressed several times (Hatfield and Vermerris, 2001; Boerjan et al.,
2003), but no publications in which analyses of such mutants or transgenic
plants are discussed have been published to date. Instead, a number of
mutants and transgenic plants in which lignin biosynthetic genes were downregulated (Anterola and Lewis, 2002; Patten et al., 2004) were reanalyzed,
and, in many cases, were claimed to be flawed. Until more detailed genetic
studies on dirigent-like proteins have been carried out in the context of
lignification, the dirigent model needs to be treated solely as a hypothesis.
12.3
Modification of lignin for agro-industrial
applications
Lignin biosynthesis has received considerable attention during the past
decade, largely because of the economic cost associated with the presence of
lignin in agro-industrial feedstocks. Three industrial processes can benefit
from the modification of lignin content and lignin subunit composition: 1)
the pulp and paper industry, 2) the animal feed industry, and 3) the bioprocessing industry. Consequently, transgenic approaches have been
Biosynthesis of phenolic compounds
121
implemented to modify lignin composition in a variety of commercially
important species.
12.3.1
Pulp and paper industry
A substantial amount of commercially grown wood is destined for the
production of paper. Paper production requires the isolation of the cellulose
fibers from the wood. After debarking, the logs are chipped and the chips are
subsequently pulped. Pulping results in the physical and/or chemical
breakdown of wood so that discrete fibers are liberated. These fibers can
then be dispersed in water and reformed in a web (Biermann, 1996).
Chemical and/or mechanical methods are used to pulp wood. Mechanical
pulping uses grindstones, sometimes in combination with steam, to liberate
fibers. Lignin is not removed during this process. The paper yield resulting
from mechanical pulping is high, but the paper is weak and exposure to light
and air causes the paper to yellow. An example of paper obtained from
mechanical pulping is the paper used for newspapers. In contrast, highquality paper relies on the use of chemicals that can separate cellulose from
lignin. Soda pulping uses sodium hydroxide in the cooking liquid to separate
cellulose fibers. Sulfite pulping is another chemical pulping method; it relies
on mixtures of sulfurous acid and/or its alkali salts to solubilize lignin. Both
methods became less popular with the invention of Kraft (or sulfate)
pulping, which relies on the action of sodium sulfide and sodium hydroxide
at high pH and temperatures between 160 and 180°C. Kraft pulping is useful
for any wood species and the resulting paper is strong. A major disadvantage
of Kraft pulping is the low yield and the substantial environmental pollution.
The black liquor that is left over after the Kraft process contains reduced
sulfides which are odiferous compounds. They are released in the air during
combustion or evaporation of the liquor. Also the bleaching process, which
is used to create white paper, results in pollution, most notably because of
the formation of dioxins.
Experimental methods that aim to prevent the environmental polution
include organosolv pulping (use of organic solvents to remove lignin) and
biological pulping, which uses white rot fungi or lignin degrading enzymes
(Biermann, 1996). The removal of lignin is a major bottleneck in the
production of high quality paper, regardless of the pulping method that is
used. A reduction in lignin content and/or change in lignin chemistry that
would allow a more efficient separation of lignin and cellulose would,
therefore, be beneficial.
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Chapter 3
A number of tree species have been transformed with the attempt to
either down- or up-regulate lignin biosynthetic genes to improve pulping
characteristics. This was considered a relatively uncontroversial application
of genetic modification, given that the wood was not intended for human
consumption but purely for industrial processing, and that the environmental
benefits were apparent. Nonetheless, environmental activists destroyed trials
with transgenic poplars in the UK.
Pilate et al. (2002) reported on a long-term study (four years, two sites)
to evaluate both the industrial processing and the ecological impacts of
transgenic poplar trees (Populus tremula x Populus alba) in which the CAD
or the COMT gene had been down-regulated via the introduction of
antisense constructs. The change in lignin content and composition did not
result in increased manifestation of pests and diseases, or in changes in the
distribution of species living in the trees. The decomposition of the
transgenic trees, however, measured as the release of CO2 from the soil in
which the trees were growing, was more rapid, underscoring the role of
lignin in the prevention of decay. Wood harvested from the transgenic trees
with down-regulated CAD activity was more easily delignified in the Kraft
pulping process, and required 6% less alkali. In contrast, wood obtained
from the trees with down-regulated COMT activity required 15% more
active alkali to obtain a similar degree of delignification as the the wild-type
controls. The poor delignification properties were attributed to a more
heavily cross-linked lignin resulting from the reduction of S-residues and the
concomitant increase in G-residues. The G-residues, derived from coniferyl
alcohol, are able to participate in interunit linkages involving C5, which tend
to be more difficult to break. This is consistent with a report by Huntley et
al. (2003) in which transgenic poplar trees with enhanced F5H (C5H)
activity were evaluated. As a result of the overexpression of the F5H gene,
the lignin of these trees contained more S-residues. The ratio of S:G residues
had increased from 1.90 in the wild-type to 14.17 in the most extreme case.
This change drastically increased pulping efficiency. The pulping severity
required to reach a certain degree of delignification (indicated by the kappa
number) was lower with increasing S:G ratios. The paper obtained from the
transgenic trees required considerably less chemicals for bleaching, and
when the same pulping and bleaching conditions were used for wild-type
and transgenic wood, the paper from the transgenic wood was considerably
brighter. The authors concluded that the use of these transgenic poplars
could increase pulp throughputs by >60% while at the same time reduce the
amount of chemical needed.
Biosynthesis of phenolic compounds
12.3.2
123
Forage and silage quality
The term forage refers to vegetative parts of crop plants that are fed to
animals. The animals either graze the forage while it is still in the field, or
they eat it after the harvest as dried feed. Examples of the latter include hay
and straw obtained from wheat, maize, sorghum, sudan grass, millets and
various forage grasses. Silage refers to the material prepared from chopped
plants, including both vegetative and reproductive tissues, which were
harvested while the plants were still green. The chopped plants are preserved
as a result of organic acids secreted by microbes that were either native or
that originated from a commercially available product. Maize is the most
common source of silage. Both forage and silage provide energy to ruminant
animals in the form of cell wall polysaccharides that are hydrolyzed in the
rumen. Lignin present in the cell wall has been shown to limit the rate of
digestion of forage and silage, largely because it shields the cell wall
polysaccharides from hydrolytic enzymes (Ralph and Helm, 1993).
A class of mutants of maize (Zea mays), sorghum (Sorghum bicolor),
and pearl millet (Pennisetum glaucum) are known as brown midrib mutants.
These mutants have reduced lignin content and altered lignin subunit
composition, and have therefore been suggested as good candidates to
incorporate into breeding programs (Cherney et al., 1990; Cherney et al.,
1991; Barrière et al., 1994).
The brown midrib mutants of maize, bm1, bm2, bm3 and bm4, are
naturally occurring mutants that have brown vascular tissue in their leaves
and stems as a result of changes in lignin content and lignin composition.
The bm1 mutation affects the lignin biosynthetic enzyme cinnamyl alcohol
dehydrogenase (CAD), which results in an accumulation of coniferaldehyde
end groups in the lignin, and a decrease in S/G ratio (Halpin et al., 1998;
Marita et al., 2003). The lignin in the bm2 mutant (Burnham and Brink,
1932) contains fewer G-residues (Chabbert et al., 1994) and shows a
disturbance in the tissue-specific patterns of lignification (Vermerris and
Boon, 2001). The bm3 mutant contains a defective caffeic acid Omethyltransferase (COMT) gene (Vignols et al., 1995), which results in a
reduction in S-residues, and the incorporation of 5-hydroxyguaiacyl residues
that participate in the formation of benzodioxane structures (1.89; Marita et
al., 2003). Based on NMR studies the lignin of stems of the bm4 mutant is
not drastically different from that of the wild-type control, but additional
analyses indicated changes in the lignin subunit composition that were
similar to the changes in the bm2 mutant (Barrière et al., 2004).
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Chapter 3
A collection of 28 brown midrib mutants exists in sorghum, and
contains both chemically induced (Porter et al., 1978) and spontaneous
mutants, some of which have been shown to be allelic (Bittinger et al., 1981;
Saballos et al., 2005). The sorghum brown midrib mutations are abbreviated
as bmr, because bm in sorghum is the abbreviation for the bloomless gene.
Some of the bmr mutants, notably bmr6, bmr12, bmr18, and bmr26 have
been characterized chemically. The lignin of the bmr6 mutant contains
higher proportions of coniferaldehyde (3.76) and sinapaldehyde (3.78) and
has reduced CAD activity (Pillonel et al., 1991). The Bmr6 gene, however,
has not yet been cloned. The mutants bmr12, bmr18 and bmr26 are allelic.
The lignin composition in these mutants resembles that of the maize bm3
mutant (Suzuki et al., 1997), and these mutants were shown to carry point
mutations resulting in premature stop codons in the COMT gene (Bout and
Vermerris, 2003).
There are two brown midrib mutants in pearl millet, one chemically
induced (Cherney et al., 1988) and one spontaneous mutant (Degenhart et
al., 1995). Analyses of the cell walls of these mutants bmr mutant indicated
changes consistent with a reduction in COMT activity (Hartley et al., 1992;
Degenhart et al., 1995; Lam et al., 1996), but the corresponding gene has not
yet been cloned.
Digestibility studies with brown midrib mutants of maize (Barnes et al.,
1971; Barrière et al., 1994; 2004; Fontaine et al., 2003), sorghum (Porter et
al., 1978; Akin et al., 1986) and pearl millet (Cherney et al., 1990; Akin et
al., 1991) have indeed shown that some of the mutants are considerably
more easily digestible. The fact that some of the mutants do not show much
improvement indicates that not all changes in cell wall composition are
automatically of practical use.
12.3.3
Ethanol production from ligno-cellulosic biomass
The global demand for energy has been growing and is expected to
continue to grow during the next decades. While oil supplies are currently
still abundant, it is anticipated that peak oil production will be reached
between 2010 and 2025. After that point, it will become increasingly
expensive to pump and distribute oil (Belyaev et al., 2002). This, together
with the desire of oil-importing countries to become less dependent on
foreign oil, the uncertain political situation in many oil-exporting countries,
and the need to curb the emission of greenhouse gases, has stimulated a
search for alternative and renewable energy sources, including ethanol and
bio-diesel. Ethanol is currently produced from sugar cane in Brazil, and
Biosynthesis of phenolic compounds
125
from corn grain in the US. The corn grain is processed to yield simple sugars
that are converted to ethanol through fermentation. In the US this is carried
out by a well-developed industry that produced 4 billion gallons (15 million
liters) in 2005. Yet the current volume of ethanol represents a mere 2% of
the volume of gasoline consumed in the US every year. In order for ethanol
to contribute significantly as an alternative fuel, its production needs to go
up drastically. This will require the use of alternative sources of fermentable
sugars, such as plant-derived lignocellulosic biomass, which is an abundant
source of the polysaccharides cellulose and hemicellulose (Ladisch et al.,
1978). One of the first lignocellulose substrates to be used in the transition
from a starch-based fuel ethanol industry to a lignocellulose-based ethanol
industry will likely be corn stover, the agricultural residue resulting from
corn grain production. A next step may be the development of specialized
biomass crops grown specifically for the production of fermentable sugars.
Chang and Holtzapple (2000) identified lignin removal the dominant
factor improving enzyme digestibility. Draude et al. (2001) showed that a
removal of 67% of the lignin from softwood pulp resulted in a 174%
increase in the ultimate yield of reducing sugars, an 88% increase in the
ultimate yield of glucose, and an increase in the initial hydrolysis rate (over
the first hour of hydrolysis) by 111%. Charles et al. (2003) confirmed these
results in softwood pulps with similar numbers, and Yang and Wyman
(2004) have shown similar results with corn stover. There appear to be two
primary affects that lignin has on the enzymatic hydrolysis of cellulose
within this matrix. The first effect is to prohibit cellulose fiber swelling,
which reduces the enzyme accessible surface area. The other significant
effect is through the irreversible adsorption of cellulases to lignin, thus
preventing their action on the cellulose (Palonen et al., 2004). This ‘titration’
effect necessitates the use of more enzymes in order to saturate these nonproductive adsorption sites on the surface of the biomass. This leads to
prohibitively high enzyme costs for processing purposes. Pretreatment
strategies aimed at removing lignin from the ligno-cellulosic biomass are
currently under development (Mosier et al., 2005).
13.
HYDROXYCINNAMIC ACID BIOSYNTHESIS
As discussed in Section 7, the general phenylpropanoid pathway
originally included the biosynthesis of the hydroxycinnamic acids caffeic
acid (3.32), ferulic acid (3.33), 5-hydroxyferulic acid (3.34), and sinapic acid
(3.35) from p-coumaric acid (3.30), as well as the corresponding CoA-esters
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Chapter 3
through the action of the enzyme 4CL (see Figure 3-4). In vitro enzyme
activity assays that became possible after the expression of recombinant
proteins in heterologous systems demonstrated, however, that the
hydroxylation and methoxylation reactions do not occur at the level of the
acid, but instead at the more reduced forms such as the CoA esters,
aldehydes and alcohols (Humphreys et al., 1999; Osakabe et al. 1999;
Humphreys and Chapple, 2002).
The biosynthetic pathway towards ferulic acid and sinapic acid was
redrawn after analysis of the reduced epidermal fluorescence1 (ref1) mutant
of Arabidopsis. When exposed to UV-radiation, the leaves of this mutant
display reduced epidermal fluorescence as a result of lower levels of UVabsorbing sinapate esters. Sequence analysis of the cloned REF1 gene
indicated this gene encodes an NADP+-dependent aldehyde dehydrogenase
with in vitro enzyme activity against both coniferaldehyde (3.76) and
sinapaldehyde (3.78). Many different species were shown to contain
aldehyde dehydrogenase activity against coniferaldehyde and sinapaldehyde
(Nair et al., 2004).
So it appears that the hydroxycinnamic acids are - at least in part synthesized through the oxidation of aldehydes, rather than via ring
substitutions of the free acids. There is evidence, however, that this is not
the exclusive route towards the substitution pattern of the phenyl ring, at
least not in all species. The xylem of poplar in which the lignin biosynthetic
enzyme caffeoyl-CoA O-methyltransferase had been down-regulated
through genetic engineering accumulated caffeic acid 3-O-glucoside and
sinapic acid 4-O-glucoside (Meyermans et al., 2000). This suggests that
these glucosides are precursors of the Coenzyme A esters in the lignin
biosynthetic pathway, and therefore that the ring substitution probably
occurred at the level of the free acids.
14.
BIOSYNTHESIS OF SINAPOYL ESTERS
Sinapoyl esters are phenolic compounds found in members of the
Brassicaceae, which includes the model plant Arabidopsis thaliana. The two
major sinapoyl esters are sinapoyl malate (3.92) and sinapoyl choline (3.93),
which accumulate in leaves and seeds, respectively. Sinapoylmalate plays a
role in the protection against UV-radiation (Landry et al., 1995), whereas
sinapoyl choline may be used as a storage form of choline in seeds (Shirley
and Chapple, 2003). The precursor of these two esters is sinapate (3.35).
Biosynthesis of phenolic compounds
127
Sinapate is synthesized via the oxidation of sinapaldehyde (3.79) by an
aldehyde dehydrogenase, as described in Section 13 of this chapter.
Sinapaldehyde, in turn, is derived from the amino acid phenylalanine (3.27)
via the general phenylpropanoid pathway (see Section 7), followed by a
number of the hydroxylation and methylation reactions described in Section
10.
The first step in the biosynthesis of sinapoyl esters from sinapate (3.35)
is glycosylation of the acid by the enzyme UDP-glucose:sinapic acid
glucosyltransferase (SGT) to yield 1-O-sinapoylglucose (3.91). In leaves the
enzyme sinapoylglucose:malate sinapoyltransferase (SMT) converts
sinapoyl glucose (3.91) to sinapoyl malate (3.92). In seeds the enzyme
sinapoylglucose:choline sinapoyltransferase (SCT) performs a similar
reation to yield sinapoylcholine (3.93). This latter compound can be
converted back to sinapate through the action of the enzyme
sinapoylcholinesterase (SCE).
The elucidation of sinapoyl ester metabolism was aided by the
availability of mutants. The sng1 (sinapoyl glucose accumulator 1) mutant
of Arabidopsis had been identified based on a mutant screen for alterations
in the composition of fluorescent compounds in the leaves. The screen was
performed by thin layer chromatography and revealed that the leaves of the
sng1 mutant contained less sinapoylmalate and instead accumulated the
precursor sinapoyl glucose (Lorenzen et al. 1996).
The Arabidopsis SNG1 gene was cloned using a map-based cloning
approach. The gene was shown to encode a serine carboxypeptidase-like
(SCPL) protein (Lehfeldt et al., 2000). This class of enzymes has been
shown to be involved in protein degradation, whereby the peptide bond
between the penultimate and C-terminal amino acid residues of the protein
or peptide substrates is cleaved. Expression of the Arabidopsis SNG1
protein in E. coli demonstrated SMT activity, and hence that this enzyme
catalyzes a transesterification reaction, as opposed to the hydrolytic reaction
typical for serine carboxypeptidases.
The SCT gene was also cloned based on the availability of a mutant. The
Arabidopsis sng2 mutant (sinapoylglucose accumulator 2) was identified in a
mutant screen whereby seed extracts were analyzed with thin layer
chromatography for the accumulation of sinapoylglucose (Shirley et al.,
2001). The Arabidopsis SNG2 gene was cloned via a combination of mapbased cloning and a candidate gene approach. The candidate gene in this case
was an SCPL-gene that had been identified during the sequencing of the
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Chapter 3
Arabidopsis genome, and that mapped to the same chromosome as the SNG2
locus. This candidate gene indeed turned out to be the SNG2 gene, as was
demonstrated via complementation of the mutant. Activity towards
sinapoylglucose was shown by expressing the SNG2 cDNA in E. coli More
detailed kinetic studies of recombinant SCT isolated from yeast are
described by Shirley and Chapple (2003).
O
O
O
O
Glc
UDP
UDP-Glc
a
H3CO
H3CO
OCH3
OCH3
OH
OH
(3.35)
(3.91)
choline
malate
b
choline
d
glucose
H2O
glucose
c
CH3
H 3C
N
O
CH3
O
O
O
O
O
OCH3
H3CO
O
O
OCH3
H3CO
OH
OH
(3.93)
(3.92)
Figure 3-13. Sinapoyl ester metabolism catalyzed by the enzymes (a) UDP-glucose:sinapic
acid glucosyltransferase (SGT), (b) sinapoylglucose:malate sinapoyltransferase (SMT), (c)
sinapoylglucose:choline sinapoyltransferase (SCT), and (d) sinapoylcholinesterase (SCE).
Biosynthesis of phenolic compounds
15.
129
COUMARIN BIOSYNTHESIS
In plants coumarins and hydroxycoumarins are believed to be
synthesized from trans-cinnamic acid (3.29) and trans-p-coumaric acid
(3.30), respectively, but the exact mechanism for its synthesis is still
unknown. One possible biosynthetic route toward coumarin is via ohydroxylation of 3.29 to give coumaric acid (3.94), followed by
glycosylation to result in trans-coumaric acid-2-O-glucoside (3.95) (Figure
3-14).
OH
O
O
OH
OH
O
OH
OH
a
c, d
+Glc
(3.29)
(3.94)
O
OH
O-Glc
b
-Glc
(3.95)
d
OH
O
OO
O
Glc
-Glc
(3.96)
Figure 3-14. Possible biosynthetic route towards coumarin. (a) 2-hydroxylase, (b) glucosyl
transferase, (c) –glucosidase, (d) dimethylallyl transferase and/or UV light. The enzymes
have not yet been identified.
Either the aglycone resulting from the action of a -glucosidase or the
glucoside, or possibly both, undergo cis-trans isomerization under influence
of UV-light or possibly mediated by a dimethylallyl transferase. The last
step of the biosynthetic pathway is an intramolecular esterification reaction,
which can occur spontaneously, to yield coumarin (3.96). The enzymes that
involved in these reactions have not been purified.
16.
STILBENE BIOSYNTHESIS
Stilbene synthase shows similarity to chalcone synthase, which is not
surprising given that stilbenes (3.97) also originate from the condensation of
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Chapter 3
p-coumaroyl-CoA (3.31) with three malonyl-CoA residues (3.48) (Schröder
and Schröder, 1990; Tropf et al., 1994) (Figure 3-15). Since stilbene
synthesis is relatively straightforward, stilbene synthase has been a target for
genetic engineering of disease resistance in plants, as will be discussed in
Chapter 6.
OH
OH
HO
O
S-CoA
O
3x
(3.31)
a
(3.97)
O
OH
CoA-S
O
(3.48)
Figure 3-15. Biosynthesis of stilbene from p-coumaroyl-CoA with three molecules malonylCoA, catalyzed by the enzyme stilbene synthase (a; E.C. 2.3.1.95).
17.
BIOSYNTHESIS OF GALLOTANNINS AND
ELLAGITANNINS
Gallotannins and ellagitannins make up the hydrolysable tannins and are
derived from 1,2,3,4,6-penta-O-galloyl- -D-glucopyranose (3.102). In oak
leaves, and presumably many other plants that synthesize hydrolysable
tannins, this compound is synthesized from –glucogallin (1-O-galloyl- -Dglucopyranose; 3.98). The subsequent esterification of gallic acid residues
(3.47; see Section 8) occurs in a specific sequence: C6 – C2 – C3 – C4
(Figure 3-16). When enzyme preparations obtained from oak leaves were
provided with UDP-glucose, –glucogallin was formed, but also di- and
trigalloylglucoses (Gross, 1983). This suggested that –glucogallin (3.98)
was both an acceptor and a donor of gallic acid residues. The different steps
towards the biosynthesis of pentagalloylglucose are catalyzed by different
enzymes. With the exception of the enzyme catalyzing the formation of –
glucogallin, these enzymes are very large, with molecular weights between
260 and 450 kDa.
Gallotannins contain an additional 10-12 gallic acid moieties per
molecule. This is effectively a continuation of the esterification reactions
that resulted in the formation of pentagalloylglucose (3.99). The major
Biosynthesis of phenolic compounds
131
difference between the formation of gallotannins and pentagalloylglucose in terms of the chemistry - is that the additional gallic acid moieties have to
react with phenolic hydroxyl groups, as opposed to the aliphatic hydroxyl
groups of the glucose molecule. This process results in the formation of
characteristic meta-depside groups (1.91).
OH
O
H OH
OH
UDP-Glc UDP
H O
HO
H
OH
HO
Glc
βGG
Glc
OH
O
HO
βGG
OH
H
H
O
OH
(3.47)
OH
(3.98)
G
G
H O
βGG
H O
Glc
H O
H O
HO
HO
O
HO
H
OH
H
O
HO
H
G
H
O
H
(3.99)
(3.100)
G
G
H
G
G
H O
βGG
H O
H O
Glc
H O
G
O
HO
O
O
H
G
H
(3.101)
O
G
H
O
O
G
H
G
H
O
G
G
H
(3.102)
Figure 3-16. Biosynthesis of 1,2,3,4,6-penta-O-galloyl- -D-glucopyranose (3.102) from
gallic acid (G; 3.47) and UDP-Glucose. The most recently added gallic acid residue is
indicated by a G in bold face. The intermediates are –glucogallin ( GG; 3.98), 1,6-di-Ogalloyl- -D-glucopyranose (3.99), 1,2,6-tri-O-galloyl- -D-glucopyranose (3.100), and 1,2,3,6tetra-O-galloyl- -D-glucopyranose (3.101).
Using cell-free extracts from sumac (Rhus typhina), Hofmann and Gross
(1990) provided evidence that the addition of the gallic acid residues
occurred in a manner similar to the acylation of pentagalloylglucose, with –
glucogallin (3.95) serving as a donor of gallic acid residues. The
biosynthesis of hexa, hepta, and octa galloylated gallotannins appears to be
catalyzed by several gallotannin synthesizing -glucogallin-dependent
132
Chapter 3
galloyltransferases that have a preferred but not unique substrate when it
comes to the degree of substitution (penta-, hexa- or hepta- galloylglucose
molecules) and the substitution pattern, i.e. the location of the meta-depside
residues. As a consequence, a particular gallotannin molecule can have
several biosynthetic origins (Niemetz and Gross, 2005).
Ellagitannins are formed from the oxidative coupling between gallic
acid residues in pentagalloylglucose molecules leading to the formation of
C-C coupled 3,4,5,3′,4′,5′-hexahydroxydiphenoyl (HHDP) residues (1.97;
1.98). Tellimagrandin II (3.103) is a monomeric ellagitannin in which the
4
C1 conformation can be observed. The two galloyl residues are coupled via
a 4,6-linkage.
OH
HO
HO
HO
HO
HO
O
HO
HO
O
OH
OH
O
O
O
HO
OH
O
OH
HO
O
O
O
O
O
HO
HO
O
HO
OH
OH
HO
(3.103)
HO
OH
HO
HO
OH
O
O
HO
OH
O
O
O
O
O
O
O
O
OO
HO
O
O
OH
O
O
O
OH
O
HO
HO
OH
HO
O
OH
OH
OH
HO
O
O
OH
OH
HO
HO
OH
O
OH
(3.102)
O
O
O
HO
O
O
O
O
OH
OH
O
O
OH
HO
OH
HO
O
O
OH
OH
OH
HO
OH
HO
OH
(3.104)
Figure 3-17. Biosynthesis of Tellimagrandin II (3.103) and Cornusiin E (3.104) from
pentagalloylglucose (3.102) by polyphenol oxidases of the laccase class.
Biosynthesis of phenolic compounds
133
Other linkages, namely 1,6-, 3,6- and 2,4-O HHDP linkages, are
possible, but require the less stable 1C4 conformation of the glucose
molecule. Linkages, both C-C and C-O, can also be formed between galloyl
residues of different ellagitannin monomers, thereby giving rise to dimers,
trimers, and tetramers. A wide range of compounds can thus be synthesized.
The biosynthesis of ellagitannins is not well understood. This is due to
the many different compounds that exist in this class, and due to their
complex chemical structure, which requires sophisticated analytical tools for
identification. Niemetz and Gross (2003) isolated two enzymes that catalyze
the formation of Tellimagrandin II (3.103) and Cornusiin E (3.104) from
pentagalloylglucose. Both enzymes, pentagalloylglucose: O2 oxidoreductase
and Tellimagrandin II: O2 oxidoreductase, require oxygen and were shown
to belong to the laccase class polyphenol oxidases.
134
18.
Chapter 3
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ISOLATION AND IDENTIFICATION OF PHENOLIC COMPOUNDS 151
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ISOLATION AND IDENTIFICATION OF
PHENOLIC COMPOUNDS
A practical guide
1.
INTRODUCTION
In the previous chapters we have discussed the different classes of
phenolic compounds, their chemical properties, and their biosynthesis. The
characterization of phenolic compounds relied on the ability to isolate them
from plant tissues. In this chapter we will discuss methods to isolate and
characterize phenolic compounds, and methods to visualize them in planta.
Chapter 5 focuses on techniques for the identification and characterization
of some of these compounds using recently developed mass-spectrometrybased techniques.
2.
ISOLATION OF PHENOLIC COMPOUNDS
Soluble phenolic compounds can be isolated easily from plant tissue by
extraction into methanol or methanol acidified with 0.1% (v/v) HCl. Since
most phenols are present as glycosides one must use caution in their
isolation to avoid hydrolysis. Precautionary techniques should include
isolation in the dark and under cold conditions. This is particularly true
when isolating anthocyanins, as they are easily broken down.
There are several ways to estimate the total amount of phenolic
compounds present in plant tissue, but it is important to keep in mind that
151
152
Chapter 4
none of these methods will detect all phenolic compounds. As a
consequence, it is often necessary to perform several analyses.
2.1
Total phenolic content: the Folin-Ciocalteu reagent
Some of the commonly used methods to determine the content of
phenolics are approximately 100 years old, and were initially developed by
Folin and colleagues at Harvard Medical School to study the metabolism of
proteins in humans. Folin and Denis (1912b) reported on a colorimetric
method to detect the amino acid tyrosine (3.28) in protein hydrolysates. This
method relied the reduction of a mixture of phosphotungstic (WO42-)phosphomolybdic (MoO42-) reagent by the phenolic hydroxyl group of
tyrosine, resulting in the formation of a blue product. The color intensity
could be quantified based on absorbance readings using an early version of
the spectrophotometer. The Folin-Denis reagent is prepared by mixing
sodium tungstate and (phospho)molybdic acid in phosphoric acid, boiling it
for 2 hours, followed by cooling, diluting and filtering it (Folin and Denis,
1912a). This method was subsequently applied to the determination of
phenolics in urine (Folin and Denis, 1915). A modification of this method
was reported by Folin and Ciocalteu (1927). The modification consisted of
the addition of lithium sulfate and bromine to the phosphotungsticphosphomolybdic reagent at the end of the boiling period, followed by
cooling and dilution. The addition of the lithium prevents the formation of a
precipitate that would interfere with the quantification of the color intensity.
The resulting reagent, referred to as the Folin-Ciocalteu reagent, was used to
determine the content of tyrosine and tryptophan in protein hydrolysates, but
can be used to determine the content of phenolics from a wide range of
sources. Below follows a protocol for this method.
1. Dilute an aliquot of the sample 10:1 with water (9 parts water to 1 part
sample). This is not necessary if the phenol content is low.
2. Add 2 ml of freshly prepared 2% (w/v) sodium carbonate (anhydrous) to
0.1 ml of the sample extract (diluted if necessary).
3. Mix vigorously on a Vortex mixer.
4. Let stand for 5 min.
5. While mixing on a Vortex add 0.1 ml of a 1:1 dilution of Folin-Ciocalteu
reagent. This reagent can be purchased from chemical supply companies,
such as Merck. If the reagent has a green color, it is no longer good and
should be replaced.
6. Allow the sample to stand for a minimum of 30 minutes, but not more
than one hour.
Isolation and identification of phenolic compounds
153
7. Read the absorbance in a spectrophotometer at 750 nm.
For blanks one can use ethanol, water or methanol, whichever the tissue
extract was dissolved in last. Since this is a spectrophotometric assay, it is
important to have a standard curve to relate the absorbance value to a
concentration. Common compounds used to generate a standard curve are
chlorogenic acid (1.18) or gallic acid (1.5). The concentration of phenolic
compounds is then reported as chlorogenic acid or gallic acid equivalents,
respectively.
Scalbert et al. (1989) used a slight modification of this method, whereby
a 2.5 mL aliquot of the Folin-Ciocalteu reagent (diluted 10 times in water)
and 2 mL of a 75g/L solution of sodium carbonate are added to 0.5 mL of
the extract (diluted in methanol), followed by a 5 min. incubation in a 50°C
waterbath. A potential complication of this method is the deglycosylation of
phenolic compounds due to the heating.
The colorimetric assay based on the protocol developed by Folin and
Ciocalteu (1927) can be used to determine the concentration of soluble
phenolics, such as anthocyanins in the example above, as well as complex
phenolics such as hydrolysable and condensed tannins. Swain and Hillis
(1959) pointed out that variation in phenolic composition (e.g. tannins
versus flavonoids) can influence the efficiency of the reduction of the FolinCiocalteu reagent, so that comparisons between samples may not always be
appropriate, depending on what the origin of the samples is. Apple et al.
(2001) investigated the appropriateness of the Folin-Ciocalteu reagent in
comparisons of leaf samples obtained from different tree species. They
argued that the use of this reagent offers an efficient method to estimate the
reducing capacity of the sample, but due to variation in phenolic content and
composition between samples, comparing phenolic contents across samples
may not be meaningful. To demonstrate this, Apple et al. (2001) determined
the tannin content of a set of leaves from sixteen tree species, a subset
collected at different times of the year, and mixtures of commercially
available tannins, using the Folin-Ciocalteu reagent as a measure of total
tannin content, the butanol-HCl assay (Bate-Smith, 1977) to determine the
content of condensed tannins (see Section 1.1.2.1), and the potassium iodate
method (Haslam, 1965; Bate-Smith, 1977; Schultz and Baldwin, 1982; see
Section 1.1.3.1) to determine the content of hydrolysable tannins. Since the
tannins were extracted from the leaves, they could perform the reactions
with the same amount of starting material for each of the samples, and
evaluate to what extent the composition of the sample impacted the reducing
capacity. The total phenolic content obtained with the Folin-Ciocalteu
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Chapter 4
reagent was primarily correlated with either the content of hydrolysable
tannins, or the content of condensed tannins, or the combination, depending
on the actual sample, confirming that the phenolic composition affected the
reducing potential of the sample. They concluded that the use of the FolinCiocalteu assay resulted in over- or underestimation of the phenolic content
values obtained with other methods.
2.2
Determining the content of condensed tannins
Scalbert et al. (1989) described several methods to determine the
concentration of hydrolysable and condensed tannins (proanthocyanidins;
see Chapter 1, section 3.13.1) in wood extracts. Tannins are complex and
heterogeneous: In addition to the distinction between the flavonoid-based
condensed tannins and the gallic acid-based hydrolysable tannins, each
group can display a large degree of chemical variability that can affect the
efficacy of the different assays. Interference of chemically related nontannin compounds, such as flavonoids, can in certain cases bias the results.
2.2.1
The butanol-HCl assay
A colorimetric assay involves the oxidative cleavage of
proanthocyanidins with ferrous sulfate. To 0.5 mL of aqueous plant extract
is added a 5-mL portion of an acidic solution of ferrous sulfate (77 mg of
FeSO4.7H2O dissolved in 500 mL of 2:3 HCl/n-butanol). The tubes are
loosely covered and placed in a water bath at 95°C for 15 min. The
absorbance is read at 530 nm. The concentration of proanthocyanidins is
expressed as cyanidin equivalents (used for the standard curve). The
molecular extinction coefficient mol that can be used to convert the
absorbance values to a concentration is equal to 34700 L mol-1cm-1.
2.2.2
The vanillin assay
An alternative colorimetric method relies on the reaction with vanillin
under acidic conditions. A 2-mL aliquot of a freshly prepared solution of
vanillin (1 g/100 mL) in 70% sulfuric acid is added to 1 mL of aqueous plant
extract. The mixture is incubated in a 20°C-waterbath and after exactly 15
min. the absorbance at 500 nm read. The concentration of proanthocyanidins
is expressed as (+)-catechin equivalents (used for the standard curve). This
assay is specific for flavonols. As a consequence, when using this assay to
determine the concentration of condensed tannins, widely distributed
monomeric flavonols, such as catechin (1.39) and epicatechin (1.90), can
interfere (Hagerman and Butler, 1989).
Isolation and identification of phenolic compounds
2.2.3
155
Precipitation of condensed tannins with formaldehyde
A third method relies on the precipitation of proanthocyanidins with
formaldehyde. First, the ‘total phenolic content’ is measured using the FolinCiocalteu reagent as described before. A 0.5 mole equivalent of
phloroglucinol (1.3) is added for every gallic acid equivalent in the extract.
To 2 mL of this plant extract and phloroglucinol is added 1 mL of a 2:5 HCl
/H2O solution and 1 mL of an aqueous solution of formaldehyde (13 mL of
37% formaldehyde diluted to 100 mL in water). After an overnight
incubation at room temperature, the unprecipitated phenols are estimated in
the supernatent by the Folin-Ciocalteu method. The precipitate contains the
proanthocyanidins and the known amount of phloroglucinol, which is
always quantitatively precipitated.
2.3
Determining the content of gallotannins
Gallotannins are hydrolysable tannins and contain a gallic acid residue
esterified to a polyol (see Chapter 1, section 3.13.2).
2.3.1
The potassium iodate assay
Gallotannins, can be detected quantitatively with the potassium iodate
assay. This assay was first described by described Haslam (1965), and is
based on the reaction of potassium iodate (KIO3) with galloyl esters, which
will form a red intermediate and ultimately a yellow compound. The
concentration
of
the
red
intermediate
can
be
measured
spectrophotometrically at 550 nm. The reaction was initially performed by
adding 1.5 mL of a saturated potassium iodate solution to 3.5 mL of tannin,
followed by a 40-min. incubation at 0°C for (Haslam, 1965). Since the red
intermediate turns yellow over time, it is important to be consistent in terms
of the time and temperature of the reaction. Bate-Smith (1977)
recommended performing the reaction at 15°C until a maximum absorbance
was reached (regardless of the time). The formation of the precipitate –
resulting from the absence of free hydroxyl groups on the pentagalloyl
parent residue – can be avoided by changing the solvent of the reaction.
Aqueous acetone (20% (v/v) or methanol above 10% (v/v) generally
resulted in less precipitation than just water. Since most standard curves for
this assay are made with tannic acid, the concentration of hydrolysable
tannins is expressed as tannic acid equivalents (TAE).
156
2.3.2
Chapter 4
The rhodanine assay
Despite the changes Bate-Smith (1977) made to the original protocol,
the time- and temperature sensitivity of the iodate assay, as well as the
cross-reactivity with ellagitannins negatively impact the reproducibility of
this method. Inoue and Hagerman (1988) developed the rhodanine assay.
Rhodanine (2-thio-4-ketothiazolidine; 4.1) reacts with the vicinal hydroxyl
groups of gallic acid to produce a red complex that can be detected
spectrophotometrically at 520 nm. This reaction is specific for gallic acid,
and can thus be used for the detection of gallotannins. This requires acid
hydrolysis of the gallotannins before the reaction with rhodanine. Tannins
are extracted from the plant tissue in 1 mL 70% (w/v) acetone in water per
100 mg dry sample in a sonicator at 4°C. The extract is then filtered through
a sintered glass filter. The filtrate containing the tannins is collected into a
glass ampule. The residue in the filter is then washed with 5 mL 2N sulfuric
acid, which is added to the ampule. The ampule is frozen and vacuumsealed, and then heated at 100°C for 26 h. to hydrolyze the gallotannins.
O
HN
S
S
(4.1)
The contents of the ampule are diluted in water to a final volume of 50 mL.
A 1-mL sample is then taken for the assay. To this sample 1.5 mL 0.667%
(w/v) rhodanine in methanol is added. After exactly 5 min. 1 mL 0.5N KOH is
added. After 2.5 min. water is added to a final volume of 25 mL. The
absorbance is read at 520 nm after a 5-10 min. incubation. A standard curve is
made by reaction of gallic acid in 0.2N sulfuric acid with the rhodanine
solution. Hagerman and Butler (1989) argued that this assay is more suitable
than the potassium iodate assay for the determination of hydrolysable tannins,
although it has to be kept in mind that the rhodanine assay is sensitive to any
gallic acid ester, including those in non-tannin compounds.
A helpful review of the different methods for the analysis of tannins and
the rationale for choosing one over the other was presented by Hagerman
and Butler (1989). They also pointed out that it was critical to choose a
suitable standard to compare the experimental data with. Given the
Isolation and identification of phenolic compounds
157
difficulty of isolating chemically pure tannins, they recommended using
either non-tannin standards (such as gallic acid or cathechin), in which case
the data are expressed in ‘equivalents of the standard’, or using
commercially available tannic acid preparations. In the latter case it is
critical to indicate the source of the standard, since the commercial
preparations vary from each other considerably.
2.4
Determining the content of ellagitannins
Ellagitannins are characterized by the presence of hexahydroxydiphenoyl
esters (HHDP; 1.94) with a polyol such as glucose. Acid hydrolysis of
ellagitannins will result in the release of HHDP units, which will
spontaneously form ellagic acid (1.96), as described in Chapter 1, section
3.13.3. Hence, the quantification of ellagic acid reflects the content of
ellagitannins present in the sample.
2.4.1
Nitrous acid oxidation
Ellagic acid concentration can be determined through an oxidation
reaction with nitrous acid, and two methods have been described by BateSmith (1977) and by Wilson and Hagerman (1990), respectively.
Ellagitannins can be isolated from plant tissue by extraction of freezedried and ground tissue in 2N sulfuric acid (5-10 mg of sample/mL of acid),
followed by freezing in a dry ice/2-propanol bath, vacuum sealing, and
heating at 100°C for 10 h. in glass ampules. After hydrolysis, the ampules
are cooled to room temperature, opened, cooled in an ice bath for 10 min.
and filtered through a membrane filter. The residue on the filter (containing
the ellagic acid) is then washed with several volumes of ice-cold wash
solvent (acetone/H2O/concentrated HC1 (70:30:1 v/v/v) and air-dried. When
the residue is dry, both sample and filter are transferred to a test tube and
dissolved in 10 mL pyridine. Undissolved material is removed by a second
filtration. Samples are filtered through a glass-fiber filter supported on a fine
sintered glass filter to remove insolubles (undissolved membrane filter and
plant material). The final filtrate is assayed for ellagic acid (Wilson and
Hagerman, 1990).
Scalbert et al. (1989) summarized the method of Bate-Smith (1977): In a
tube sealed with a Teflon-lined screw cap, 0.2 mL of the aqueous plant
extract (1 mL if the extract is not very concentrated) is added to 1.8 mL of
1:1 methanol/water (1 mL of 9:1 methanol/water if the sample is not
concentrated) and 0.16 mL of aqueous 6% (v/v) acetic acid. Nitrogen is
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Chapter 4
bubbled for 5-10 min and 0.16 mL of an aqueous solution of 6% (w/v)
sodium nitrite (NaNO2) is added. Nitrogen is bubbled through the solution
for a few more seconds, after which the tube is sealed and incubated in a
25°C water bath for 100 min. A blue product is formed during the reaction,
which is quantified by reading the absorbance with a spectrophotometer at
590 nm. The concentration of HHDP esters is expressed as 4,6hexahydroxydiphenoyl-glucose equivalents ( mol= 2169 L mol-1cm-1).
2.4.2
The NaNO2/HCl assay
Wilson and Hagerman (1990) described an alternative spectrophoto
metric procedure to quantify ellagic acid. This method has three advantages
over the procedure developed by Bate-Smith (1977): 1) it is not sensitive to
gallic acid, which can result in an overestimate of the ellagitannin content,
2) it is more sensitive, and 3) it is more convenient because oxygen does not
interfere with the reaction.
To maximize the formation of a red-colored product in the assay
developed by Wilson and Hagerman (1990), (the sample containing) ellagic
acid is dissolved in pyridine (which needs to be kept in a fume hood) to a
volume of 2.1 mL. A volume of 0.1 mL concentrated HCl is added, and the
mixture is brought to 30°C. After a volume of 0.1 mL 1% (w/v) NaNO2 is
added, the mixture is quickly mixed and the absorbance at 538 nm is read
immediately afterwards (A538,t0). A red product that decays over time forms
while the sample is incubated. After 36 min. at 30°C the red product reaches
its maximum concentration, and the absorbance (A538,t36) is recorded again.
The ellagic acid concentration in the sample is a function of the difference
between A538,t0 and A538,t36. Glass or quartz cuvettes need to be used, because
pyridine will dissolve plastic cuvettes. The assay is also sensitive to residual
detergent in the test tubes, so that new glass test tubes need to be used for
each assay.
This procedure is based on the formation of the electophile NO+, which
can react with an ellagic acid residue (4.2) at two sites, either via a
substitution reaction which results in 4.3, or an addition reaction that results
in the nitrosyl dienone 4.4. These compounds can decay to form the quinine
oxime 4.5, which under alkaline conditions forms the red product 4.6. When
related compounds, such as gallic acid, phloroglucinol, hydroxycinnamic
acids, and phenol are subjected to this assay, a yellow-brown product is
formed, which does not interfere with the spectrophotometric detection of
ellagic acid.
Isolation and identification of phenolic compounds
NO
159
O
HO
O
OH
O
NOH
HO
HO
O
O
O
O
O
OH
NO
OH
OH
NO
O
HO
HO
(4.3)
O
O
NO
OH
O
O
O
ON
(4.2)
NOH
O
H
O
(4.5)
O
OH
OH
HO
O
NO
O
H
O
NO
O
O
O
(4.4)
O
O
O
O
NO
O
(4.6)
Figure 4-1. Formation of a red chromophore (4.6) from the reaction of ellagic acid with the
electrophile NO+, based on Wilson and Hagerman (1990).
2.5
Determining lignin content
Lignin is a complex phenolic cell wall polymer that is chemically crosslinked with hemicellulose and cell wall proteins. Most of the methods to
determine lignin content are based on the removal of all other cell wall
constituents, typically through acid hydrolysis, which will readily remove
hemicellulose under mild conditions, and non-crystalline cellulose under
more severe conditions. Several different methods will be discussed below.
The different methods have also been extensively reviewed and compared
by Hatfield et al. (1994), Brinkmann et al. (2002), Fukushima and Hatfield
(2004), and Hatfield and Fukushima (2005).
160
2.5.1
Chapter 4
Klason lignin
Klason lignin is the fraction of lignin that remains as a solid residue
after the polysaccharides in a cell wall extract have undergone hydrolysis in
sulfuric acid. The cell wall extract can be obtained by several different
methods, which have in common that soluble sugars, proteins, and unbound
phenolics are removed from dried and ground plant tissue. Ground woody
tissue is commonly extracted in a 2:1 (v/v) mixture of benzene (or the less
noxious toluene) and ethanol, followed by extraction in ethanol, and finally
water. Theander and Westerlund (1986) recommended an extraction
protocol for herbaceous species. This involves extraction of 1-3 gram dried
and ground samples in an ultrasonic waterbath, first in 80% (w/v) ethanol (3
x 75 mL; 15 min. each) and then in petroleum ether (boiling point of 6070°C; 2 x 50 mL, 10 min. each). After removal of the solvent, the samples
are dried. The starch is removed from the samples by resuspending 1.5-2 g
of the dried residue in 75 mL 0.1 M sodium acetate buffer pH 5.0,
containing α-amylase (100 L, Termamyl 120L). The suspension is kept in a
glass tube with a screwcap and placed in a boiling water bath, with
occasional shaking. After cooling to 60°C, the content in the tube is
incubated with 500 µL amyloglucosidase suspension and kept capped at
60°C overnight in a shaking water bath. After centrifugation for 15 min. the
supernatant is removed and filtered into an evaporation flask. The insoluble
residue remaining in the tube is washed successively by suspension
(ultrasonication, 5 min.) and centrifugation in water (2 x 50 mL), ethanol (2
x 50 mL), and acetone (50 mL). The remaining insoluble residue (containing
the water-insoluble fiber fraction) is dried with warm air and weighed.
Klason lignin is a measure of the total amount of lignin; it is not
informative with respect to lignin composition. Despite attempts to extract
proteins prior to the lignin determination, Klason lignin tends to be
contaminated with proteins (Monties, 1989). In addition, covalently linked
hydroxycinnamic acids can be included as part of the lignin. In order to
measure Klason lignin in herbaceous plants, the proteins and
hydroxycinnamic acids should ideally be removed by an acidic detergent
before the sulfuric acid treatment is applied. This then leads to acid
detergent lignin (see 1.1.6) and is also referred to as ‘core lignin’, and acidsoluble lignin, or ‘non-core lignin’. The distinction between core and noncore lignin should not be used, because the non-core lignin fraction contains
mostly hydrolyzed hydroxycinnamic acids (Ralph and Helm, 1993; Jung and
Deetz, 1993), which are distinct from the actual lignin polymer.
Isolation and identification of phenolic compounds
161
The Klason lignin procedure was first developed by Klason in the early
1900s and is based on the acid hydrolysis of all cell wall polysaccharides,
with the remaining residue being the lignin. A protocol optimized for small
samples based on the Klason method was described by Hatfield et al.
(1994), which in turn was based on a protocol described by Theander and
Westerlund (1986). The procedure involves the hydrolysis of all
polysaccharides in a 100-mg sample of extracted cell walls, by treating it
with 1.5 mL 12 M sulfuric acid that has been chilled on ice. The mixture is
kept on ice for 30 min. and then shaken at 30°C for 2 h. Deionized water
(9.75 mL) is added to the mixture, after which a secondary hydrolysis is
performed for 1 h. in an autoclave at 121°C. The black liquid that results is
filtered through a Whatman GF/A glass filter placed in a Hirsch funnel. The
solid residue on the filter is washed 3 times with 5 mL of hot deionized
water, and then dried for 48 h. in a 55°C oven. The dried residue contains
the isolated lignin and minerals. The filter and residue are weighed. The
lignin is then removed by a 5-hour incubation in a 500°C furnace. The
samples are weighed again, and the loss in mass resulting from the
incubation in the furnace is attributed to lignin. The Klason lignin assay is
therefore a gravimetric method, and lignin content is reported as mg/g.
2.5.2
Acid detergent lignin
Acid detergent lignin (ADL) is part of a sequential analysis developed to
determine characteristics related to digestibility and feed quality of forage
samples, such as alfalfa and various grasses (Van Soest, 1967). This analysis
includes the gravimetric determination of neutral detergent fiber (NDF), acid
detergent fiber (ADF) and ADL (Van Soest, 1963; Van Soest and Wine,
1967). NDF is a measure of the total cell wall content (cellulose,
hemicellulose and lignin). Subtracting ADF from NDF results in an estimate
of the hemicellulose content, whereas subtracting ADL from ADF results in
an estimate of the cellulose content (Jung, 1997).
NDF content is determined by extracting a 0.5-g sample in boiling
neutral detergent solution (3% (w/v) sodium dodecyl sulfate (SDS; sodium
lauryl sulfate), 18 mM sodium tetraborate decahydrate (Na2B4O7 .10 H2O),
32 mM sodium phosphate (dibasic; Na2HPO4), 50 mM Na2-EDTA, and 1%
(v/v) ethylene glycol (2-ethoxyethanol) for 1 h. The suspension is filtered,
and the cell wall residue on the filter is washed in hot water and acetone.
After drying overnight at 100°C the sample is weighed.
ADF is determined on the dried NDF sample, by boiling for 1 h. in 200
mL acid detergent solution (0.5 M sulfuric acid, 2% (w/v) CTAB
162
Chapter 4
(hexadecyltrimethylammonium bromide)), followed by filtration, washing in
hot water, and rinsing in acetone. The resulting ADF residue is weighed
after overnight incubation at 100°C. The ADF residue is then hydrolyzed in
12 M sulfuric acid for 3 h. The insoluble residue that remains is collected by
filtration, washed with hot water, rinsed with acetone, and dried overnight at
100°C. ADL is represented by the difference in weight between this dried,
residue containing lignin and minerals, and the residue remaining after
heating for 5 h. at 450°C, containing only minerals.
Hatfield et al. (1994) compared the Klason and ADL methods on
samples derived from a variety of species representing different maturities
(and hence degrees of lignification). While both methods showed in increase
in lignin content with increasing maturity, the Klason lignin content was
consistently higher than the ADL content for all samples. This difference
could be the result of protein contamination of the Klason lignin. As part of
the ADL determination, proteins are removed by the neutral detergent
solution. In order to investigate this in more detail, Hatfield et al. (1994)
determined N-content of the different samples and reported a negative
correlation between N-content and lignin content, suggesting that proteins
were not responsible for the higher Klason lignin values. This was further
substantiated by performing the procedure on isolated cell wall
polysaccharides to which nitrogen (either bovine serum albumine, lysine or
ammonia sulfate) was added. The addition of nitrogen did not affect the
amount of residue that was recovered. The N-content of both Klason lignin
and ADL was attributed to cell wall proteins cross-linked with the other cell
wall constituents. The difference between ADL and Klason lignin is
therefore most likely the result of some of the lignin being solubilized in the
acid detergent solution (Hatfield and Fukushima, 2005). Based on pyrolysisgas chromatography-mass spectrometry (see 1.3.4), lignin subunit
composition did not significantly differ between the two types of residues.
The ADL contained some contaminants from the extraction procedure.
Hatfield et al. (1994) concluded that while both methods are of value for the
characterization of forage samples, the Klason lignin determination resulted
in a better representation of the actual lignin content.
2.5.3
Thioglycolic acid lignin
The reaction of thioglycolic acid (4.7) with the benzyl alcohol groups in
lignin (4.8) under acidic conditions and at elevated temperatures results in
the formation of thioethers that render the derivatized lignin (4.9) soluble in
an alkaline solution (Figure 4.2).
Isolation and identification of phenolic compounds
HO
HO
OH
R3
∆ , HCl
O
O
HS
OH
OH
R3
R3
O
163
R3
O
O
O
OH
(4.7)
O
S
R2
R2
O
HO
HO
R1
O
HO
R1
O
HO
O
S
HO
R1
R1
O
O
R1
O
(4.8)
R1
O
(4.9)
Figure 4-2. Reaction of thioglycolic acid with lignin. Adapted from Hatfield and Fukushima
(2005).
The method was first described by Browning (1967) for 40-gram
samples and then adapted for smaller samples (15 mg) by Bruce and West
(1989). In this modified protocol, which is the most practical, the sample is
incubated for 4 h. at 95°C in 1 mL 2 M HCl and 0.2 mL thioglycolic acid.
The insoluble residue containing the lignin is recovered by centrifugation,
washed three times in water, and the pellet is dissolved in 0.5 M NaOH.
Non-lignin materials will not dissolve and are removed by centrifugation.
The supernatant containing the thioglycolate lignin is then recovered by
precipitation after addition of HCl and a 4-h. incubation at 4°C. The
thioglycolate lignin is again recovered by centrifugation, dried, and
dissolved in 0.5 M NaOH. An aliquot is diluted 40 times in 0.5 M NaOH
and the absorbance at 280 nm is read. An in-vitro generated
dehydrogenation lignin polymer (DHP) is used as a standard. The DHP is
made by polymerizing coniferyl alcohol in the presence of hydrogen
peroxide and horseradish peroxidase.
164
Chapter 4
It is unclear how accurate this method is for the determination of lignin
content in grasses, since a fraction of the lignin from grasses appears to be
readily soluble in acidic solutions, such as used in this procedure. This
would result in underestimation of the actual lignin content (Hatfield and
Fukushima, 2005).
2.6
Acetylbromide lignin
The acetyl bromide procedure was developed by Johnson et al. (1961) to
determine lignin content in small samples from woody species. This method
uses acetyl bromide (4.10) to acetylate unbound hydroxyl groups in the
lignin (4.11), while the hydroxyl group on the α-carbon is substituted with a
bromine group. The substituted lignin derivative (4.12) is soluble under
acidic conditions, and its concentration can be measured with a
spectrophotometer at 280 nm.
HO
OH
OH
O
R3
O
R3
O
O
Br
CH3
O
O
H3 C
R2
CH3
R2
HO
O
R1
HO
OH
R3
O
O
O
(4.10)
HO
O
R3
∆ , CH3COOH
OH
O
OH
Br
Br
O
O
HO
HO
R1
O
Br
Br
HO
R1
O
R1
O
O
R1
O
(4.11)
O
R1
O
(4.12)
Figure 4-3. Reaction of acetyl bromide with lignin. Adapted from Hatfield and Fukushima
(2005).
Acetyl bromide lignin is obtained from isolated cell walls. These can be
obtained by extraction of ground woody samples in ethanol and chloroform.
Isolation and identification of phenolic compounds
165
The isolated cell walls or the dioxane lignin are dissolved by incubation in
2.5 mL of 25% (w/v) acetyl bromide in glacial acetic acid at 70°C for 30
min. The lignin is extracted by adding the dissolved cell walls to a flask
containing 10 mL 2 M sodium hydroxide and 12 mL glacial acetic acid. A
1.75-mL volume of 0.5 M hydroxylamine is then added, and the total
volume is brought up to 50 mL with acetic acid. The lignin content is
determined spectrophotometrically by measuring the absorbance at 280 nm.
Modifications have been made to the original protocol in order to apply
this method to herbaceous species. Iiyama and Wallis (1990) added 100 µL
perchloric acid (HClO4) to improve the dissolution of wall material. This
decreased the overall time required for the procedure and made the use of
hydroxylamine unnecessary.
Hatfield et al. (1999) reexamined the different protocols used for the
determination of lignin content in herbaceous species, using alfalfa stems
and corn rind as representative samples. They performed the initial
incubation at 50°C for up to 4 h., at 60°C for up to 2 h., or at 70°C for up to
1 h., and with or without 100 µL perchloric acid. They concluded that the
addition of perchloric acid indeed raises the overall absorbance values, but
that this increase is the result of oligomeric xylan-degradation products that
also absorb light in the 250-300-nm range. In order to avoid excessive xylan
degradation, they recommended performing the extraction in acetyl bromide
at 50°C for a period of 2-4 h, in the absence of perchloric acid.
As for any spectrophotometric method, a standard curve needs to be
made. Since lignin is very heterogeneous in nature (variation in subunit
composition and interunit linkages within and between species), finding a
good standard is not trivial. Fukushima and Hatfield (2001) recommended
using lignin extracted with dioxane-HCl, rather than isolated cell walls,
because it contains little protein and polysaccharide residues. Isolating
dioxane lignin requires adding 100 mL of acidified dioxane (90 mL dioxane
and 10 mL 2 N HCl) to 5 g dry cell wall material. The flask is then
connected to a reflux condenser, nitrogen gas is blown onto the liquid
surface, and the solution is refluxed for 30 min. under nitrogen gas. The
solution is then cooled and filtered through a Whatman GF/C glass fiber
filter, and collected in a flask. The residue on the filter is washed in 20 mL
96% dioxane, and then added to the filtrate. In order to neutralize the
solution, 4 g sodium bicarbonate (NaHCO3) is added to the flask, followed
by a 3-min. incubation on a rotary shaker until the pH was neutral. The
solution is filtered through a 0.45-µm nylon membrane, and subsequently
concentrated to 10-15 mL under reduced pressure on a rotary evaporator
166
Chapter 4
using a waterbath set at 40°C. The solution is then added drop-wise to a
250-mL centrifuge bottle containing 200 mL of rapidly stirring distilled
water. Any insoluble residue remaining in the flask is washed with 2.0 mL
96% dioxane solution and added drop-wise to the water. The drop-wise
addition to the water should result in the precipitation of the lignin, which
can be stimulated by the addition of 2 g anhydrous Na2SO4. The lignin
precipitate can be collected by centrifugation (9000g for 20 min.). The pellet
is then dissolved in 100% dioxane, filtered through a 0.45-µm nylon
membrane, and added drop-wise to 200 mL stirring anhydrous ether in a
centrifuge bottle. The lignin precipitates and is collected by centrifuging at
9000g for 15 min. at 0°C. In order to remove all hydrophobic non-lignin
contaminants, the lignin pellet is again dissolved in dioxane, filtered, and
added to ether. The ether is then removed and 60 mL petroleum ether is
added to wash the lignin. After removal of the petroleum ether, the lignin
residue is freeze dried.
Lignin content determined with the acetyl bromide method using dioxin
lignin as a standard, showed the best correlation with in vitro dry matter
digestibility for a diverse set of forage samples (Fukushima and Hatfield,
2004). Even though negative correlations between lignin content and
digestibility were identified for the other methods, including Klason lignin
and acid detergent lignin, the correlation coefficients were not as high.
Fukushima and Hatfield (2004) pointed out, however, that it is important to
calculate the lignin content of a given sample based on the standard derived
from that same type of sample.
3.
IDENTIFICATION AND CHARACTERIZATION
OF PHENOLIC COMPOUNDS
In the previous section methods to isolate (certain classes of) phenolic
compounds were described. In general, however, these methods do not
provide information on specific chemical composition. In order to
characterize mixtures of phenolic compounds, a variety of separation and
identification methods exist. They will be described below.
3.1
Thin layer chromatography
A common, simple, inexpensive and relatively fast method for the
separation of phenolic compounds from a mixture is thin layer
chromatography (TLC). A small amount of the extract (40-100 µl) is applied
approximately 2 cm from the bottom of a thin layer chromatography
Isolation and identification of phenolic compounds
167
plate, which is a matrix (typically cellulose or silica gel) attached to an inert
carrier material, such as glass or plastic. The solvent is allowed to dry, either
at room temperature, under a gentle stream of nitrogen, or with the use of
warm air (a hair dryer can be convenient for this purpose). Application of
multiple small amounts with drying in between applications will result in
tighter spots and better resolution later on. A sharp pencil can be used to
scratch off the matrix in order to clearly delineate individual lanes running
along the length of the TLC plate. This will avoid samples from mixing later
on.
The TLC plate is then placed in a glass container with a solvent filled to
approximately 1 cm from the bottom. The solvent will move to the top of the
TLC plate as a result of capillary action. Since each compound in the
mixture will have a unique way of interacting with the matrix and the
solvent, some compounds will move faster towards the top of the TLC plate
than others. The Rf-value is the ratio of the distance of the compound has
migrated divided by the distance the solvent has migrated, and has by
definition a maximum value of 1. The Rf-value tends to be constant for a
given combination of compound, solvent, and matrix so that comparisons
can be made between separations performed at different times. If a given
compound is colored, it is easy to determine the Rf-value. For non-colored
compounds staining methods are available (see section 1.3).
The identification of phenolic compounds separated by TLC is
somewhat challenging. The most common strategy is to include a set of
reference compounds on the TLC plate. These compounds are applied
individually, and if the mixture contains any of the reference compounds,
they can be identified based on the Rf-value. Note that this approach always
leaves some room for uncertainty, because two different compounds can
have the same Rf-value. Further characterization is necessary to establish
compound identity with more confidence. This can be achieved by scraping
off the area on the TLC plate where the compound of interest has migrated
to, followed by solvent extraction of the matrix, and more detailed chemical
analyses, such as, for example gas chromatography-mass spectrometry or
mass spectrometry (see Section 1.5 and Chapter 5).
Below is a step-by-step protocol for TLC. In this example the goal is to
separate anthocyanins isolated from flower petals.
1. Pick the petals and place them in 1 ml methanol acidified with 0.1 or 1%
(v/v) HCl.
168
Chapter 4
2. Let the petals remain in the methanol overnight in the refrigerator.
Alternatively, the tissue can be gently crushed with a glass rod or pipette.
3. Remove the supernatant with a Pasteur pipette and put it into a clean
glass vial or a microfuge tube. Keep the material in the dark and in the
cold as the compounds may break down easily.
4. Cellulose TLC plates are recommended for the separation of
anthocyanidins, but silica gel will also work. If you use silica gel plates,
it is helpful to wash the plates first with a mixture of chloroform and
methanol (1:1 v/v). Simply run the solvent up the plates and let them air
dry. This helps to remove some of the debris that is often associated with
the plates. The plates will work even if you do not take the time to do
this, but using pre-washed plates often provides a cleaner separation of
compounds.
5. Spot a thin, fine band of pigment on the plate. To see as many spots as
possible you need to put quite a lot of material onto the plate. It is best to
apply the pigments in a band, while removing the solvent with a gentle
flow of nitrogen.
6. There are many possible carriers, most of them mixtures of organic
solvents. Table 4.1 lists some options that can be used for the separation
of anthocyanins from petals.
Table 4-1. Solvent mixtures for thin layer chromatograpy of phenolic compounds
Solvent mixture
Ratio
Layer
n-butanol-acetic acid-water
4:1:5
upper layer
acetic acid-HCl-water (Forestal solvent)
30:3:10
miscible
ethyl acetate-formic acid-water
85:6:10
upper layer
ethyl acetate-water-formic acid-HCl
85:8:6:1
upper layer
n-butanol-2N hydrochloric acid
1:1
upper layer
n-butanol-acetic acid water
4:1:1
upper layer
The oxidation reaction of phenolic compounds can be used for the
purpose of detection. While the phenolic compound is oxidized, a reagent is
reduced, and the reduction can be monitored by a change in color. Two
common reagents are ammoniacal silver nitrate and the Folin-Denis reagent.
Reactions of phenolic compounds with ammoniacal silver nitrate result in
the formation of metallic silver. A simple procedure is to mix equal volumes
Isolation and identification of phenolic compounds
169
of 0.1 N NH4OH and 0.1 N AgNO3 and to apply this as a spray to a thin
layer chromatogram at room temperature. The oxidized phenols appear as
brown spots because of the silver. The reaction is, however, not specific to
phenolic compounds. The Folin-Denis reagent, discussed in Section 1.1.1,
and produces a blue color upon reaction with phenolic compounds.
An alternative way to visualize certain phenolic compounds is with the
use of a hand-held UV lamp in the dark. The presence of the phenolic
compounds can be observed by the fluorescence.
3.2
Liquid chromatography: HPLC and LC-MS
The principle of liquid chromatography was described in Chapter 3,
Section 2. Liquid chromatography can be used for the separation of proteins,
as well as for the separation of individual phenolic compounds from
complex mixtures, based on the compounds’ variation in affinity for a resin
packed in a column. Changing the pH and/or ionic strength of the solution
will allow all compounds of interest to elute, ideally in a sequential manner.
The identification of the compounds is based on a combination of the
retention time and either a UV/vis-spectrum or a mass spectrum. The
absorbance spectrum in the UV and visible range of the light spectrum for
each of the eluted compounds can be obtained by so-called diode array
detectors, which will record the absorbance of a compound across a wide
range of the spectrum. The mass spectrum is obtained with a mass
spectrometer that is connected to the liquid chromatography equipment.
Mass spectrometry is described in detail in Chapter 5. Large searchable
databases exist to aid with the identification of compounds based on mass
spectral data. The ultimate way to confirm a compound’s identity is to show
co-elution of the compound identified in the sample and the candidate
reference compound.
Scalbert et al. (1989) described the chromatographic separation of
cyanidin and delphinidin from methanolic extracts of oak heartwood using a
C-18 Novapak column. The elution solvent was a mix of two solvents A and
B that changed in composition from 0-100% B in a linear fashion over a
period of 20 min. Solvent A was a mixture of 94:5:1 H2O/methanol/H3PO4,
and solvent B was 99:1 methanol/H3PO4, at a flow rate of 1.7mL/min. In this
case a dual-channel spectrophotometer was used with two wavelengths
selected for detection: 280 and 530 nm. Under these conditions, and based
on reference compounds, delphinidin eluted after 13.9 min and cyanidin
after 14.8 min.
170
Chapter 4
Tamagnone et al. (1998) described the impact of over-expression of
Myb transcription factors in tobacco on the phenolic composition of leaf
extracts, and used HPLC coupled to a diode array detector to evaluate
change in chemical composition. Leaf material frozen in liquid nitrogen and
ground to a fine powder was extracted in cold methanol (chilled on dry ice).
Cell debris (mostly cell walls) was removed by centrifugation (5 min. at
50g). The supernatant was further clarified by additional centrifugation
(2,000g for 30 min.) and then analyzed by HPLC using a C-18 column with
a flow rate of 1 mL/min. The solvents consisted of 10% (v/v) methanol in
water with 1 mM trifluoric acid (TFA) (solvent A), and 80% (v/v) methanol
in water with 1 mM TFA (solvent B). The solvent gradient started out with
90% solvent A and 10% solvent B, and ended 60 min. later with 100%
solvent B. The diode array detector provided a full absorbance spectrum on
each of the eluted compounds.
Morreel et al. (2004) extracted wood from transgenic poplars in which
the monolignol biosynthetic gene caffeic acid O-methyltranferase was
down-regulated. They used methanol to extract frozen (liquid N2) and
ground xylem tissue from debarked stems. After removing cell debris by
centrifugation, the supernatant was freeze-dried and extracted with 1:1
cyclohexane:water acidified with 0.1% (v/v) TFA. The samples were then
separated on a C-18 column with 1% aqueous triethylammonium acetate
(TEAA) as solvent A, and a 25:75 mixture of methanol and acetonitrile
containing 1% TEAA as solvent B, at a flow rate of 0.3 ml/min, going from
100% solvent A to 100% solvent B in 40 min. The eluted compounds were
detected with a diode array spectrophotometer which obtained the
absorbance spectrum of each compound in the range between 200 and 450
nm. In addition, a mass spectrum was obtained for each of the compounds
after the eluate was vaporized.
These three examples illustrate technology developments over time
(dual-channel detector, diode array detector, mass spectrometer). Note that
while the overall methodology is very similar (methanolic extracts,
methanol-based, acidified solvents used for HPLC, detection of eluted
compounds), the exact conditions for successful separation need to be
defined for each system.
3.3
Gas chromatography
The principle of gas chromatography (GC) is similar to that of liquid
chromatography or TLC, in that compounds in mixtures are separated from
each other based on their affinity for a resin. GC is performed on volatile
Isolation and identification of phenolic compounds
171
samples, using a long (20-30 meter) and very thin (<0.5 mm internal
diameter) column coated on the inside with a silica-based solid phase
applied as a thin (~0.2 µm) film. The column is placed in an oven in which
the temperature can be changed quickly, and with great accuracy. The
samples are generally dissolved in a volatile organic solvent that has little
affinity for the solid phase. A small volume (1-5 µL) is loaded onto the
column through a septum using a syringe with a thin needle. The sample is
then moved through the column with an inert carrier gas such as helium. The
interaction of the compound with the solid phase is temperature-dependent,
and elution of the compounds of interest is achieved by increasing the
temperature. Detection of the compounds at the end of the column is
nowadays mostly based on mass spectrometry (see Chapter 5), although
flame ionization detectors (FID) are also used for routine analyses of
samples in which there are only a limited number of compounds that can be
easily identified based on retention time. In order to increase the volatility of
phenolic compounds, chemical modifications such as methylation,
acetylation or silylation can be employed.
An example of the use of GC for the identification of phenolic
compounds can be found in Dias and Grotewold (2003). In order to identify
the role of the ZmMyb-IF35 transcription factor, the phenolic composition
of transgenic maize cell lines in which the gene encoding this transcription
factor was over-expressed was examined. They extracted phenolic
compounds by homogenizing the tissue in methanol, followed by 15 min.
centrifugation at 13,000g to remove insoluble debris. The methanol was then
evaporated and the extract was hydrolyzed by boiling in 2 M HCl for 20
min. The acid was evaporated and the pellet was redissolved in methanol,
adjusted for weight, so that the final concentration was 0.1 mg/µl. One µl of
this extract was injected in a GC, with a mass spectrometer as a detector.
The initial temperature of the GC column was 40°C for 1 min., and the
temperature was increased to 310°C at a rate of 11°C/min. This resulted in a
gas chromatogram with more than 40 peaks representing the compounds in
the methanol extract. Some of these peaks were uniquely associated with the
presence of the transgene, thus providing clues regarding the function of this
transcription factor in phenolic metabolism.
3.4
Methods for the identification of lignin subunit
composition
Chemical analyses to determine lignin content and composition are
difficult because of the complex nature of lignin. In addition, they often
result in loss of information regarding the original structure. A large number
172
Chapter 4
of analytical techniques have been developed to investigate the content and
composition of lignin. Reviews and protocols of various techniques can be
found in Monties (1989), Lewis and Yamamoto (1990), Chen (1991), Lin
and Dence (1992), and Sjöström and Alen (1999). This section presents a
brief description of the different methods available.
3.4.1
The nitrobenzene oxidation
Nitrobenzene oxidation involves the treatment of cell wall material with
sodium hydroxide containing nitrobenzene. The method was developed by
Freudenberg (1939). Nitrobenzene oxidation degrades the phenylpropane
structure from a C6-C3 unit to a C6-C1 unit. After incubation the soluble
fraction is acidified and extracted with ether. The degradation products – phydroxybenzaldehyde (4.13), vanillin (4.14), and syringaldehyde (4.15), and
the corresponding acids p-hydroxybenzoic acid (4.16), vanillic acid (4.17),
and syringic acid (4.18) – can be separated on an HPLC column to
determine the lignin composition. Alternatively, gas chromatography (GC)
or gas chromatography-mass spectrometry (GC-MS) can be used.
The lignin composition can be expressed as the S/V ratio, whereby the
‘V’ (vanillin (4.14), vanillic acid (4.17)) reflects guaiacyl residues and the
‘S’ (syringaldehyde (4.15), syringic acid (4.18)) reflects syringyl residues.
(Monties, 1989). Nitrobenzene oxidation does not affect the aromatic C-C
bonds, but only the bonds from the so-called uncondensed units, so that the
estimates of lignin composition can be somewhat biased (Lewis and
Yamamoto, 1990). In grasses these methods cannot distinguish between
aromatic compounds derived from lignin and from p-coumaric and ferulic
acid esterified to the cell wall (Lapierre, 1993), so that this method is seldom
used for the analysis of lignin from grasses.
An example of the use of the nitrobenzene oxidation to elucidate
differences in lignin subunit composition between wild-type and mutant
Arabidopsis plants can be found in Chapple et al. (1992). They dried and
ground stem tissue of Arabidopsis and extracted 50 mg with methanol (three
times 1 mL) and deionized water (twice, 1 mL) at 60°C. Esterified phenolics
Isolation and identification of phenolic compounds
O
O
O
OCH3 H3CO
OCH3
OH
OH
OH
(4.13)
(4.14)
(4.15)
O
OH
O
173
O
OH
OH
OCH3 H3CO
OCH3
OH
OH
OH
(4.16)
(4.17)
(4.18)
were saponified in 1 M NaOH (37°C, 60 min.), after which the tissue was
rinsed twice and resuspended in water. Sodium hydroxide solution was
added to an aliquot representing 20 mg of dry tissue so that the final
concentration was 2 M NaOH in a volume of 980 µL. Nitrobenzene (20 µL)
was added to this suspension, followed by a 2-hour incubation at 160°C.
After cooling the sample, a 250-µL aliquot was added to 750 µL 1 M acetic
acid to neutralize the sample. A 100-µL aliquot was then reduced (addition
of 1 mL 2% (w/v) sodium borohydride in DMSO; 90 min. at 40°C),
acetylated to increase the volatility (4 mL of acetic anhydride plus 250 µL of
1-methylimidazole as catalyst; 10 min. at room temperature), and extracted
in methylene chloride (CH2Cl2). The break-down products of the lignin were
subsequently analyzed on a GC with an FID, using p-hydroxybenzaldehyde
(4.13), vanillin (4.14), 5-hydroxyvanillin, and syringaldehyde (4.15) as
reference compounds.
3.4.2
Thioacidolysis
Thioacidolyis is an acidolytic degradation method used to analyze lignin
subunit composition. The method relies on the use of boron trifluoride
etherate ((C2H5)2-O-BF3) and ethanethiol (C2H5-SH) to cleave β-O-4 bonds
(1.82) in the lignin (4.19). The reaction mechanism is shown in Figure 4.4.
174
Chapter 4
First the α–carbon is substituted by BF3, resulting in compound 4.20. The
activated α–carbon makes this compound reactive with ethanethiol, resulting
in the formation of 4.21. The thioethyl group now attacks the activated –
carbon of 4.21, resulting in the intermediate 4.22. Attack of ethanethiol
results in the formation of 4.23, and through a similar mechanism the –
carbon is substituted, resulting in the formation of 1,2,3-trithioethane
phenylpropanoid monomer 4.25 (Lapierre et al., 1986; Chen, 1991; Rolando
et al., 1992; Lapierre, 1993).
The thioacidolysis reagent is prepared fresh for each analysis by mixing
2.5 mL BF3-etherate and 10 mL of ethanethiol in a flask. The volume is
adjusted to 100 mL with dioxane. The cell wall extract (20 mg) is added to
10 mL of the thioacidolyis reagent, along with an internal control in order to
be able to quantify the products later on. Docosane dissolved in methylene
chloride is a suitable internal standard. The reaction is performed in glass
tubes that can be closed with Teflon-lined screw caps. This is important,
because the reagent is very odiferous. The actual thioacidolysis is performed
for 4 h. at 100°C in an oil bath. Deionized water is added to the cooled
reaction to get a volume of 30 mL. Sodium bicarbonate (0.4 M NaHCO3) is
added to increase the pH to a value between 3 and 4. The reaction mixture is
then extracted in 3 x 30 mL methylene chloride. Water is removed from the
combined organic extracts by addition of Na2SO4 and the methylene
chloride is removed via evaporation at 40°C. The resulting residue is
redisolved in 0.5 mL methylene chloride, silylated, and injected in a GC-MS
for quantitation. The internal standard is used to correct for loss of
compounds during the extraction.
Thioacidolysis allows the distinction between products derived from
lignin and products derived from p-coumaric and ferulic acids, and the
distinction between products derived from cinnamaldehydes and cinnamyl
alcohols. Recent improvements have made it possible to estimate the
fraction of free phenolic groups in uncondensed lignin (see Section 1.3.1),
and to depolymerize the dimers, so that they can be included in the analysis
of the lignin composition.
Thioacidolysis allows the distinction between products derived from
lignin and products derived from p-coumaric and ferulic acids, and the
distinction between products derived from cinnamaldehydes and cinnamyl
alcohols. Recent improvements have made it possible to estimate the
fraction of free phenolic groups in uncondensed lignin (see Section 1.3.1),
and to depolymerize the dimers, so that they can be included in the analysis
of the lignin composition.
Isolation and identification of phenolic compounds
γ
HO
R1
O
α
HO
R5
R5
R1
β
175
O
O
F3 B
O
R3
R3 (C H ) O-BF
2 5 2
3
C2H5-SH
R1-OH
R5
R3
R5
R3
OR4
OR4
(4.19)
(4.20)
HO
HO
R5
C 2 H 5S
R5
C2 H5S
O
O
R3
R5
R3
R3
BF3
(C2H5)2O-BF3
C2H5-SH
R5
R3
OR4
OR4
(4.21)
(4.22)
OH
SC2H5
OH
C2H 5S
H
S-
C2H 5S
C
2
C2H 5S
SC2H5
SC2H5
H
5
(C2H5)2O-BF3
C2H5-SH
R5
R3
OR4
(4.23)
R5
R3
OR4
(4.24)
H2 O
R5
R3
OR4
(4.25)
Figure 4-4. Reaction mechanism for the formation of 1,2,3- trithioethane phenylpropanoid
monomers (4.25) from lignin (4.19). R1 is either an aryl group or a hydrogen atom. In Hresidues both R3 and R5 are hydrogen atoms, in G-residues R3 is a methoxyl group and R5 is
a hydrogen atom, and in S-residues both R3 and R5 are methoxyl groups. R4 is either a
hydrogen atom or an alkyl group. The wavy bonds indicate that both the S- and R-stereoisomers are present.
176
Chapter 4
Examples of the use of thioacidolysis can be found in Hoffmann et al.
(2004), who describe the effects on lignin subunit composition resulting
from silencing the gene encoding hydroxycinnamoyl-CoA shikimate/quinate
hydroxyltransferase (HCT) in tobacco, and in O’Connell et al. (2002) who
investigated the effects of down-regulating cinnamoyl-CoA reductase in
tobacco on lignin subunit composition.
3.4.3
Derivatiation Followed by Reductive Cleavage
A recently developed method based on acetylbromide cleavage is
‘Derivatization Followed by Reductive Cleavage’ (DFRC; Lu and Ralph,
1997). This method uses acetylbromide to acetylate alcohols and phenols in
lignin (4.26). In addition, the α-carbon is brominated (4.27). Cleavage of the
-O-4 linkage is catalyzed by zinc. The monomeric residues (4.28) that are
formed are acetylated to yield diacetylated monolignols (4.29) and separated
using gas chromatography. The DFRC reaction is presented in Figure 4-5.
DFRC is achieved by heating the sample in 2.5 mL acetyl bromide with
HCl, followed by removal of the reagent by evaporation. The residue that
remains is dissolved in 2.5 mL of dioxane/acetic acid/ H2O (5:4:1). To this
solution is added 50 mg zinc dust, to catalyze the cleavage of β-bromoethers. After stirring for 30 min. the zinc is removed by filtration through
glass wool. Dichloromethane (10 mL) is then added to the filtrate along with
10 mL saturated NH4Cl solution, and the internal standard tetracosane is
added. After mixing, phase separation, and removal of the organic phase, the
aqueous phase is extracted twice more in dichloromethane. The combined
dichloromethane extracts are then combined and dried. The resulting residue
is dissolved in 1.5 mL dichloromethane, acetylated with 0.2 mL acetic
anhydride and 0.2 mL pyridine. Ethyl acetate (2.5 mL) is added after 90
min. and the mixture is evaporated. Ethanol is added during the evaporation
to get rid of the pyridine. Pyridine-free samples are dissolved in 200 µL
dichloromethane and a 1-2 µL is injected in a gas chromatograph for
analysis using the reference compounds p-acetoxycinnamyl acetate (4.29a),
coniferyl diacetate (4.29b), and sinapyl diacetate (4.29c) for peak
identification.
Advantages of the DFRC method over thioacidolysis include the better
yield, simpler and milder reaction conditions, and the fact that esters are not
cleaved. Especially in the analysis of lignin in grasses, with large amounts of
esterified p-coumaric acid (1.13), this is an important consideration.
Isolation and identification of phenolic compounds
γ
HO
R 1O
α
AcO
R5
R5
β
Br
O
O
R3
R3
Zn
AcBr
R5
177
R5
R3
R3
O
O
(4.26)
(4.27)
OAc
OAc
a. R3 = R5 = H
b. R3 = OCH3 R5 = H
c. R3 = R5 = OCH3
Ac2O/Py
R5
R3
OH
(4.28)
R5
R3
OAc
(4.29)
Figure 4-5. Reaction mechanism for the derivatization followed by reductive cleavage based
on Lu and Ralph (1997). Reaction of the lignin with acetylbromide (AcBr) results in the
acetylation of the –carbon, while the α-carbon is brominated. Zinc (Zn) catalyzes the
cleavage of the ether bond between the -carbon of one residue and the O-4 position of the
adjacent residue. The resulting monomer is acetylated with acetic anhydride (Ac2O) and
pyridine (Py). R1 can be a proton or an aryl group. In H-residues R3 and R5 are protons, in Gresidues R3 is a methoxyl group and R5 is a proton, whereas in S-residues both R3 and R5 are
methoxyl groups. The wavy bonds indicate that both the S- and R- (4.26, 4.27) or E- and Zstereo-isomers (4.28, 4.29) are present.
An example of the use of the DFRC method can be found in Zhang et al.
(2006), who analyzed the lignin subunit composition in the gold hull and
internode2 mutant of rice. This mutant has a defective OsCAD2 gene and
accumulates coniferaldehyde residues in its lignin.
178
3.4.4
Chapter 4
Analytical pyrolysis
Pyrolysis (Py) is the rapid thermal degradation of a compound in the
absence of oxygen. Pyrolysis of plant cell wall material results in the release
of a pyrolysate of low molecular weight compounds that are break-down
products of both polysaccharides and lignin. Lignin breaks down to
substituted monomeric phenols; the propanoid chain is often reduced to one
or two carbons or is lost altogether (Meier and Faix, 1992). The pyrolysate is
generated in an oven or through the use of a heated filament to which the
sample is applied. The resulting pyrolysate is led directly into a mass
spectrometer (Py-MS) or a gas chromatography coupled to a mass
spectrometer (Py-GC-MS) (Boon, 1989; Meier and Faix, 1992; Lapierre,
1993).
Analytical pyrolysis requires only small samples (10-1,000 mg) and is
fast compared to most other analytical methods (Mulder et al., 1991;
Lapierre, 1993). This makes it an attractive method for lignin analysis.
Another major advantage is that it is not necessary to isolate the lignin prior
to pyrolysis, because there is little similarity between carbohydrate and
lignin pyrolytic fragments and their mass spectra are distinctly different
(Mulder et al., 1991; Meier and Faix, 1992; Boon, 1992). Pyrolytic
disintegration of lignin is mostly due to the cleavage of ether-bonds. This
may bias the quantitation of S and G units since S-units are more often
involved ether-bonds than G units (Lapierre, 1993). In general, the
reproducibility of results obtained with pyrolysis is good when the same
experimental unit is used. Results from different experimental settings tend
to be less similar.
Examples of the use of Py-MS or Py-GC-MS for the characterization of
mutants in which lignin biosynthesis was disrupted can be found in
Vermerris and Boon (2001) and Bout and Vermerris (2003), whereas
Fontaine et al. (2003) used Py-GC-MS to investigate the chemical basis of
forage quality.
3.4.5
Nuclear magnetic resonance
Nuclear magnetic resonance (NMR) is considered the most informative
method to analyze lignin structure, because it does not rely on chemical
degradation of the lignin prior to the analysis. As a consequence, NMR
analysis of lignin provides information on the different types of interunit
linkages, including the C-C bonds that are typically not broken by the
chemical degradation methods or pyrolysis.
Isolation and identification of phenolic compounds
179
NMR is a spectroscopic technique that is based on the ability to
influence the spin of certain nuclei. Spin is an electromagnetic property of
electrons, protons and neutrons that can be described in quantum chemical
terms. Its value is +½ or -½. A nucleus with a non-zero net spin can be
considered as a small magnet. If a population of such nuclei are placed in a
magnetic field, the nuclei will align with the magnetic field, and an
equilibrium will be established whereby the majority of the nuclei will be in
the energetically lower state (alignment of the North-pole of the nuclei with
the South-pole of the magnet), hence creating a net magnetic field in the
direction of the external magnetic field.
Transition from the low- to the high-energy state can occur by the
absorption of a photon with energy in the radio frequency range. The exact
amount of energy required depends on the nucleus itself, but also on its
immediate environment, since the nucleus experiences a shielding effect
from neighboring nuclei that also act like small magnets. The principle of
NMR is based on the detection of nuclei with a net non-zero spin, by placing
a sample in a strong magnetic field and subjecting it to energy in the radio
frequency range that will reverse the net magnetization of the nuclei. The
relaxation time (T1) is the time required to let the nuclei return to their
equilibrium state after the radio frequency energy has been absorbed, and
provides information on the chemical environment of the nucleus of interest.
The NMR spectrum that is obtained after several mathematical
manipulations of the output signal reflects the different energy levels
associated with nuclei in their specific environments. The variable on the xaxis of the spectrum is the chemical shift, indicated by the symbol , and
measured in part per million (ppm). The chemical shift of a nucleus is
defined as the difference between the resonance frequency of the nucleus
and a standard, relative to the standard. Common standards are
tetramethylsilane (TMS; Si(CH3)4) and deuterated d6-acetone ((CD3)2CO).
The use of chemical shift allows standardization of spectra across different
experimental platforms (manufacturers, models, temperatures, etc.).
NMR can detect nuclei with a net non-zero spin, as long as the
abundance of that nucleus is high enough. For example, the 13C nucleus has
a net spin of ½, and is the only carbon nucleus with a non-zero spin. Its
natural abundance is, however, only 1.1%, which limits the sensitivity. As a
consequence, large samples need to be analyzed in order to get detailed
compositional data, or the sample needs to be analyzed for prolonged
periods, or, if the sample is derived from plants, the plants can be grown in
an environment enriched with 13CO2. For biological materials in general, 1H
NMR, 13C NMR or 31P NMR are most commonly used. For the analysis of
180
Chapter 4
lignin 13C NMR, or a combination of 1H and
being referred to as 2D-NMR.
13
C NMR is used, the latter
The low abundance of 13C in biological samples has resulted in the
development of new techniques that have enhanced the sensitivity of NMR.
This includes high frequency NMR spectrometers in combination with
recent advances such as Cross Polarization/Magic Angle Spinning
(CP/MAS). The cross-polarization technique transfers magnetic impulses
from abundant nuclei such as protons onto rare nuclei (such as 13C), which
enhances the signal from the rare nucleus. Magic angle spinning, whereby
the sample is at a 54.7° angle with respect to the magnetic field, sharpens the
bandwidths. Structural information can also be obtained through
heteronuclear single quantum coherence (HSQC), HSQC-total correlation
spectroscopy (HSQC-TOCSY), and heteronuclear multiple bond correlation
(HMBC) experiments. A detailed description of these techniques goes
beyond the scope of this book, but several excellent references exist. For a
more detailed general description, see Friebolin (2004) or Keeler (2005),
whereas Ralph et al. (1999) provide a description of NMR specifically for
the characterization of lignin.
13
C-NMR is the most informative technique for the elucidation of
chemical structures, but generally requires large sample sizes as a result of
the low abundance of 13C. As a consequence, micro-sampling to obtain
tissue specific information is practically impossible. In addition, the amount
of time required for the acquisition of NMR spectra makes this technique
unsuitable for the analysis of large samples.
NMR analysis of cell walls traditionally relied on the isolation of lignin
from the cell wall, with the associated risk that the NMR spectrum was not
completely representative of native lignin. A method to dissolve cell walls in
their entirety was recently developed (Lu and Ralph, 2003), and this has
made analysis of cell walls using NMR appreciably easier. Ground cell wall
samples are extracted with organic solvents to remove non-cell wall
materials, followed by ball milling. Milled wood lignin (MWL) is obtained
by extracting the ball-milled residue in a mixture of dioxane and water
(96:4), followed by freeze-drying, and washing with water. Approximatley
600 mg of the MWL is suspended in 10 mL dimethylsulfoxide (DMSO),
and 5 mL of N-methylimidazole is added. A clear solution is formed in up to
3 h. Acetylation of the lignin is achieved by adding 3 mL acetic anhydride
followed by 1.5 h. of stirring. The brown solution is added to 2 L of water
and allowed to sit overnight, after which the precipitate is recovered by
filtration through a 0.2 µm filter. After washing the residue with 250 mL of
Isolation and identification of phenolic compounds
181
water, it is dried, dissolved in deuterochloroform (CDCl3) to a concentration
of 150 mg/mL, and subjected to NMR spectroscopy.
Advanced NMR techniques have resulted in the elucidation of novel
lignin structures, involving, for example, the benzodioxan structure
involving -O-4 and α-O-5 linkages between two monolignol residues (1.89;
Ralph et al., 2001; Marita et al., 2003). In addition, NMR has been used to
determine the impact of mutations and introduction of transgenic constructs
on lignin subunit composition (Ralph et al., 1997; Marita et al., 1999; Marita
et al., 2003).
3.4.6
Fourier-transform infrared spectroscopy and near infrared
reflectance spectroscopy
Fourier-transform infrared spectroscopy (FT-IR) and near infrared
reflectance (NIR) spectroscopy are methods that rely on the detection of
vibration of molecular bonds. These vibrations can either involve the
distance between the atoms (stretch) or the angle between the atoms (bend).
Changes between two vibration energy states that also impact the dipole
moment can be induced by absorption of infrared light, and this absorption
can be monitored and represented in a spectrum. Various functional groups
have characteristic absorptions and their presence can be used for diagnostic
purposes. FT-IR spectra of ligno-cellulosic materials are typically complex,
because of overlap in signal between various functional groups.
FTIR spectra can be acquired in transmission mode with an FTIR
microscope after drying a cell wall suspension on a BaF2 microscope slide
(Sené et al., 1994), or by pressing a thin potassium bromide pellet containing
finely ground plant tissue and measuring the absorbance of infrared light
going through the pellet with the use of an FTIR spectrometer (Vermerris et
al., 2002). Alternatively, a suspension of isolated cell walls can be placed on
a gold-plated reflective surface under a microscope equipped with an FTIR
spectrometer (Yong et al., 2005).
NIR spectra are acquired using dried tissue, either whole or ground. The
reflectance is measured relative to a so-called white reference, which is a
highly reflective surface, such as Gore-Tex® or white ceramic. Near-infrared
reflectance spectra, or absorbance spectra, defined as log(1/R), with R being
the reflectance, look rather flat. For analysis purposes the second derivative
of the spectra is often used, because it enhances certain spectral features.
182
Chapter 4
NIR spectroscopy is popular for the analysis of large numbers of
samples because of the speed at which the spectra can be obtained. The lack
of distinct spectral features, however, necessitates the need for calibration
using a set of standards that have been analyzed via wet-chemical methods.
Brinkmann et al. (2002) described the use of NIR spectroscopy to predict the
lignin content leaves and stems of beech (Fagus sylvatica). They used lignin
content determined with either acid detergent lignin method (see Section
1.1.5.2) or the thioglycolic acid method (see Section 1.1.5.3), and in both
cases were able to define a calibration model that allowed accurate
prediction of the lignin content using NIR spectroscopy. Interestingly,
different variables (absorbances at given wavelengths) were selected for the
model, depending on the way lignin content had been established, indicating
that the two lignin determinations rely on the detection of different structural
features. Indeed, the acid detergent lignin was shown to contain considerable
amounts of protein.
When the spectra of several (groups of) samples are compared, it is
often helpful to analyze the difference spectrum, which is obtained by
digitally subtracting one spectrum from the other. The analysis of signal that
thus remains can help to establish what is different between the two samples
(Faix, 1992; Sené et al., 1994). Recent developments include the application
of multivariate statistics to the analysis of FTIR and NIR spectra (Johnson
and Wichern, 1992; Kemsley, 1998; Chen et al., 1998; Mouille et al., 2003).
This makes sense given the high-dimensionality of the spectral data. There
are typically over 1000 measurements per sample, with a high degree of
correlation between certain data points. The spectral data are typically first
compressed by principal components analysis (PCA) or partial least squares
(PLS). These manipulations result in the generation of a small (<10) set of
new variables in which the original variables (absorbance values across the
entire spectrum) are combined, taking into account either the variancecovariance structure of the data, or correlations between variables. These
new variables can then be used in further analyses, such as discriminant
analysis, in which a sample of unknown identity is classified into predefined groups based on its spectral features and a classification model
derived from a large group of samples with known identity. This approach is
now applied to the identification of maize and Arabidopsis mutants with
altered cell wall composition (Yong et al., 2005). A useful reference book on
the application of NIR spectroscopy for the analysis of biological samples is
written by Siesler et al. (2002).
Isolation and identification of phenolic compounds
183
4.
VISUALIZATION OF PHENOLIC COMPOUNDS
IN PLANTA USING HISTOCHEMICAL STAINS
4.1
An overview of histochemical staining protocols
A number of histochemical stains are available to visualize phenolic
compounds in the plant, either in thin sections, or applied to whole tissues.
De Neergaard (1997) published a series of detailed protocols that provides
an excellent source of information. Below follows a summary of the
available reagents and the specific compounds they detect.
Aniline sulfate is dissolved in 0.1N sulphuric acid in aqueous solution
or 60% (v/v) ethanol to a final concentration of 1-6 % (w/v). This stain
reacts with lignin, which will turn yellow.
Chlorine sulfite can be used to detect syringyl lignin. The specimen is
placed above a tissue soaked in a sodium hypochlorite (‘bleach’) solution for
30 minutes. A 3% (w/v) solution of sodium sulfite (Na2SO3) is applied to the
specimen for 5-10 minutes. Lignin will stain orange to red.
4′,6 diamino 2-phenylindole (DAPI) is a fluorescent stain that reacts
with DNA and phenolic compounds. It can be used on live tissue at a
concentration of 0.002% (w/v) or in fixed tissue at a concentration of 0.010.05% (w/v). Several buffers, such as TRIS or potassium phosphate can be
used, depending on the exact application; the pH is generally around 7.5.
The staining solution needs to be stored at 4°C. The specimens are incubated
in the dark for a minimum of 30 minutes, up to an overnight incubation
(4°C) and then exposed to UV light with an excitation wavelength of 365
nm.
Ethidium bromide is a fluorescent dye that can be used for the
visualization of DNA as well as lignin and other phenolic substances. An
aqueous solution of 0.1 % (w/v) is prepared and applied to tissue sections
for 5-10 minutes. The specimen is exposed to UV light, which results in an
orange fluorescence as a result of the ethidium bromide.
Ferric chloride (FeCl3) is prepared as a 2% (w/v) aqueous solution,
although for embedded tissues a 10% solution is recommended. The
specimen is incubated in the solution for 5-10 min. Phenolic compounds,
including tannins will stain yellow or orange with this reagent.
184
Chapter 4
The Mäule reagent is used to distinguish between guaiacyl and syringyl
residues in the lignin. A freshly prepared aqueous solution of 1% (w/v)
potassium permanganate (KMnO4) is applied to the specimen for at least 30
min. After washing in distilled water for 2 min. and removing all liquid, a
15-20% aqueous solution of HCl is applied. The HCl is removed, and a 10%
solution of NH3 is applied. Syringyl residues result in the development of a
red color. Absence of syringyl residues results in the development of a
yellow color. Chapple et al. (1992) used this reagent to demonstrate the lack
of syringyl residues in the lignin of the Arabidopsis fah-1 mutant, in which
the activity of the monolignol biosynthetic enzyme ferulate 5-hydroxylase
(coniferaldehyde/ coniferyl alcohol 5-hydroxylase) is reduced.
Overexpression of a wild-type copy of the defective F5H gene in the fah1
mutant resulted in a predominance of syringyl residues, as evident from the
red color of the lignified tissues in the stem (Meyer et al., 1998).
Methylene Blue is used to detect pectin and phenolic compounds. It is
applied to the specimen as a 0.01-0.15% (w/v) aqueous solution, incubated
for 30 min. at room temperature, or for 5 min. at 60°C, and then removed by
washing in distilled water. Methylene Blue is a blue dye, but it produces a
red coloration as a result of the reaction with the phenolic or pectic
substances. This phenomenon is referred to as metachromasia, and the cell
or tissue components that exhibit it are called metachromatic.
Toluidine Blue O is dissolved in a buffer with a pH between 4 and 8, or
in 70% ethanol or in a 0.05% (w/v) borax solution and kept at 4°C. The
specimen is stained for 1-10 min. washed for 1-2 min. in distilled water, or
in the buffer used to dissolve the Toluidine Blue O, or in ethanol until all
excess stain is removed. When the specimen is viewed under the
microscope, different cell components produce colors different colors
(metachromasia): DNA is bluish-green, RNA is violet, the middle lamella is
red, non-lignified cell walls are red-violet or blue-violet, and polymerized
phenolics such as lignin are green or blusih-green. This stain is therefore of
general use.
Phloroglucinol-HCl is a 2% (w/v) solution of phloroglucinol (4.30)
dissolved in a 2:1 mixture of ethanol and concentrated HCl. This reagent
reacts with cinnamaldehyde end groups (4.31) in the lignin, resulting in the
cationic chromophore (4.32), and which appears as a burgundy-red
compound (Adler et al., 1948; Geiger and Fuggerer, 1979; Pomar et al.,
2002; see Figure 4-6). This procedure is known in the literature as the
Wiesner reaction and is also used as a general stain for lignin. This may not
be appropriate if samples differing in cinnamaldehyde content are being
Isolation and identification of phenolic compounds
185
compared. This is probably the most common stain used to detect lignin,
because the reagent is easy to make and the reaction can be readily
monitored. Pictures of the use of the Wiesner reaction can be found in
Halpin et al. (1998), Vermerris et al. (2002), and Pomar et al. (2002).
Vanillin-HCl is prepared by making a 0.05% (w/v) solution in 50%
ethanol. This is then mixed in a 2:5 ratio with concentrated HCl. The mix is
applied to the specimen for 1 min. Alternatively, the sample is immersed in
OH
HO
OH
HO
HO
OH
OH
(4.30)
O
OH
OH
HCl
OCH3
OCH3
OCH3
O
O
O
Cl
(4.31)
Cl
(4.32)
Figure 4-6. Reaction of phloroglucinol with coniferaldehyde end-groups in the lignin under
acidic conditions results in the formation of a red chromophore.
a saturated solution of vanillin in 95% ethanol for 15-30 min. The section is
then placed on a microscope slide, one drop of 9N HCl is added and the
section is immediately viewed under the microscope. This stain will produce
a red color in the presence of tannins. When pictures need to be taken, it is
important to realized that this stain is not permanent.
4.2
Visualizing plant-pathogen interactions involving
phenolics with histochemical stains
As most phenols are found as esters or glycosides, knowledge of their
location in a cell or tissue is essential. It is typical that such phenols are
sequestered or stored in the cell vacuole. This is important since all phenols
are weak acids (see Chapter 2) and as such they are relatively toxic even to
186
Chapter 4
the cells in which they are synthesized. The toxicity of the compounds is
significantly reduced when they are present as either esters or glycosides.
The enzymes that cleave them are sometimes specific esterases and
glycosidases but nonspecific enzymes may also cleave them. The enzymes
are also located within the cell vacuole and in some cases have been shown
to be present within the cell wall. Under environmental stresses, such as
heat, cold and high light intensity, appropriate enzymes can be produced
resulting in compound breakdown. The most typical situation, however, is
that of disease where an invading pathogen such as a fungus perturbs cells or
tissues causing them to respond by the synthesis of enzymes. It is also not
uncommon for a single cell of the host to respond to the presence of an
invading pathogen, a condition often referred to as a hypersensitive
response. Although the hypersensitive response is a restricted phenomenon
with the involvement of a very limited number of host cells, there is
substantial evidence that host cells that surround the site of attempted
infection are themselves stimulated or activated to a condition that allows
for a much more rapid response if they themselves are perturbed by a
pathogen that is attempting to infect the cell or tissue. The best-documented
examples of this phenomenon are found in the barley powdery mildew
disease interaction. The powdery mildew pathogen is Erysiphe graminis
(Blumeria graminis), which is an obligate pathogen. It was shown that
when a conidium of a compatible pathogen attempts to penetrate into a
susceptible host, the penetration attempt is successful. However, if a
conidium of an incompatible pathogen attempts to penetrate, the host cell
responds in an incompatible manner and expresses resistance (Shiraishi et
al., 1995).
Papillae are structures made by the plant in an attempt to contain and
eliminate a pathogenic fungus. Papilla formation in maize leaves inoculated
with Colletotrichum graminicola was reported by Politis and Wheeler
(1973). These authors reported that even ‘massive’ papillae were unable to
prevent penetration by the fungus. Their data were, however, not based on a
time-sequence study of the events leading up to and including penetration
and they did not present data on the number of successful penetrations.
Papilla formation was also reported in isolated maize root cap cells after
inoculation with C. graminicola (Sherwood, 1985). Differences in the
frequency of papilla formation and penetration by the fungus were found
among different maize lines.
Inoculation of maize roots with the non-pathogen Phytophthora
cinnamomi also resulted in production of papillae (Hinch and Clarke, 1980).
Callose was reported as the major component of the papillae, and
carbohydrates but not proteins were identified. Lignin did not appear to be
Isolation and identification of phenolic compounds
187
Figure 4-7 (left). Characteristics of papilla formation by the epidermis of maize mesocotyls in
response to attempted penetration by Colletotrichum graminicola and Helminthosporium
maydis. a) Papilla formed by inbred B73Ht in response to C. graminicola, b) granulation
(arrow) in B73Ht in response to H. maydis, c) granulation (arrow) and papilla formation in a
single epidermal cell of the inbred B73Htrhm in response to H. maydis. All specimens stained
with Toluidine Blue-O. Bars represent 10 µM. Reprinted from Physiol. Mol. Plant Pathol.
Vol. 31, Cadena-Gomez, G., and Nicholson, R.L., Papilla formation and associated
peroxidase activity: A non-specific response to attempted fungal penetration of maize, pages
51-67, 1987, with permission from Elsevier.
Figure 4-8 (right). Histochemical reactions of maize (B73Htrhm) papillae. a) Specimen stained
with Toluidine Blue-O. Blue-green color of the papilla and associated host cell wall indicate
the presence of lignin. b) Specimen stained with phloroglucinol-HCl. Red to red-brown color
indicates the presence of cinnamaldehyde end groups in lignin. c) Specimen stained for the
presence of peroxidase by the syringaldazine procedure. Red color indicates the presence of
peroxidase. Specimens were taken between 12 and 24 h after inoculation. Reprinted from
Physiol. Mol. Plant Pathol. Vol. 31, Cadena-Gomez, G., and Nicholson, R.L., Papilla
formation and associated peroxidase activity: A non-specific response to attempted fungal
penetration of maize, pages 51-67, 1987, with permission from Elsevier.
188
Chapter 4
present. The authors reported that papillae formed after penetration of roots
may represent a wound response that allows repair of damaged cells or
provides a barrier to toxic products produced by the fungus.
In maize a variety of stain procedures were used with light microscopy
to demonstrate that papillae formed in response to attempted infection by
Colletotrichum graminicola and Helminthosporium maydis are composed of
lignin (Cadena-Gomez and Nicholson, 1987; Figures 4-7 and 4-8).
Different histochemical tests have been used for peroxidase
identification. Benzidine (4.33) has been used as a staining reagent, as well
as guaiacol (4.34) and pyrogallol (4.35). However, until the 1970s, no
reliable methods were known that allowed a sharp discrimination between
oxidase and peroxidase activities (Maehly and Chance, 1954). Harkin and
Obst (1973) reported the syringaldazine (4.36) histochemical test for
peroxidase. This test permitted the proof of exclusive peroxidase
participation in the lignification process.
H2N
NH2
(4.33)
HO
OCH3
OH
OH
OH
(4.34)
(4.35)
OCH3
H3CO
OH
N
N
OCH3
HO
H3CO
(4.36)
Cytochemical identification of peroxidase was conducted in infected
wound margins in the cell walls and degenerating cytoplasm of wheat leaves
with 3,3′-diaminobenzidine (Thorpe and Hall, 1984). The same substrate
Isolation and identification of phenolic compounds
189
was used to demonstrate peroxidase in the epidermis of wheat roots (Smith
and O’Brian, 1979), and in roots and hypocotyls of cotton seedlings
(Mueller and Beckman, 1978), and in bean leaves infected by the rust
fungus Uromyces apendiculatus (Deising et al., 1992).
Peroxidase activities were also shown to occur during the bean rust
infection process (Mendgen, 1975). In reed canary grass (Phalaris
arundinacea L.), peroxidase was identified with the pyrogallol test (Vance
and Sherwood, 1976).
190
5.
Chapter 4
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Recommendations for further reading
De Neergaard, E., 1997, Methods in Botanical Histopathology, Danish
Government Institute of Seed Pathology for Developing Countries,
Copenhagen, pp. 216.
Friebolin, H., 2004, Basic One- and Two-Dimensional NMR Spectroscopy,
John Wiley & Sons, pp. 430.
Johnson, R. A., and Wichern, D. W., 1992, Applied Multivariate Statistical
Analysis, Prentice Hall, Englewood Cliffs, NJ, pp. 767.
Keeler, J., 2005, Understanding NMR Spectroscopy, John Wiley & Sons,
pp. 476.
Kemsley, E.K., 1998, Discriminant Analysis and Class Modelling of
Spectroscopic Data, John Wiley & Sons Ltd, Chichester, UK, pp. 179.
Siesler, H. W., Ozaki, Y., Kawata, S., and Heise, H. M., 2002, Near Infrared
Spectroscopy. Principles, Instruments, Applications, Wiley-VCH,
Weinheim, Germany, pp. 348.
Sjöström, E., and Alen, R., 1999, Analytical Methods in Wood Chemistry,
Pulping and Papermaking, Springer-Verlag, Berlin, pp. 316.
ANALYSIS OF PHENOLIC COMPOUNDS WITH MASS
SPECTROMETRY
197
Chapter 5
ANALYSIS OF PHENOLIC COMPOUNDS WITH
MASS SPECTROMETRY
Karl V. Wood
Department of Chemistry, Purdue University
1.
THE PRINCIPLES OF MASS SPECTROMETRY
Mass spectrometry is a very valuable analytical tool based on the simple
premise of determining the molecular weight of the compound of interest. In
fact, mass spectrometry involves the measurement of the mass (m) of a
compound as a function of charge (z), m/z. In most mass spectrometer
experiments the charge on an ion is one, such that the molecular weight of
the ion is equal in value to m/z. Mass spectrometry can be divided into three
steps: ionization, mass analysis and detection. Sample introduction is really
a distinct component for obtaining quality mass spectra and will be
discussed separately.
1.1
Ionization
Once the sample has been introduced into the mass spectrometer, it must
be ionized. There are a number of different ionization techniques which span
a wide-range of different compound classes. The original ionization
technique was termed electron impact (EI). EI involves the interaction of the
compound of interest (M) with 70 eV electrons that have been emitted from
a filament wire (1 eV = 1.6*10-19 Joule). This results in a relatively energetic
reaction in which the compound of interest (M) loses an electron, thereby
forming a radical cation.
197
198
Chapter 5
M + e- → M+. + 2e-
(1)
Using appropriate electric fields the radical cation M+. can now be sent
to the mass analyzer for subsequent mass analysis. This radical cation can
also undergo fragmentation.
M+. → F+ + e-
(2)
The fragment ions (F+) formed can be used to provide additional
structural information about the original compound.
Another conventional ionization technique termed chemical ionization
(CI), utilizes a reagent gas (such as isobutane, methane, ammonia) to form
reagent ions (RH+) which can undergo ion-molecule reactions with the
compound of interest to form protonated molecules.
M + RH+ → (M+H)+ + R
(3)
The protonated molecules are much less energetic than the related
radical cations formed during electron impact and are less prone to
fragmentation. Both EI and CI require volatile samples. This puts a
tremendous limitation on the types of molecules that can be ionized, in
particular, compounds found in biological systems.
1.2
Mass Analysis
After the compound of interest has been ionized the resulting ion must
be mass analyzed. There are a number of commercially available mass
analyzers, including but not limited to quadrupole, time-of-flight, ion trap
and double-focusing sector analyzers. A quadrupole mass analyzer utilizes
four concentric quadrupole rods with the opposite rods having opposite
polarity. With appropriate direct current (DC) and radio frequency (rf)
voltages, ions can be made to pass through the rods as a function of their
mass-to-charge ratio.
An ion trap mass analyzer has a variety of differing physical
arrangements of its electrodes, but the primary objective remains the same to
allow the ions to enter and then to trap them in space between the electrodes.
Unlike the fly-through mass analysis scheme of a quadrupole, the ion trap
mass analyzer stores the ions. They are then ejected to the detector as a
function of the mass-to-charge ratio, typically by scanning the rf voltage.
Analysis of phenolic compounds with mass spectrometry
199
The time-of-flight mass analyzer utilizes the physical characteristics of
the ions themselves. Smaller ions travel faster than larger ions. The ions
formed are accelerated such that ions with the same m/z have similar kinetic
energies. This enables the arrival times of the ions at the detector to be
related to their mass-to-charge ratio. Two improvements for reducing kinetic
energy spread – and ultimately resolution – have been introduced in
conventional linear time-of-flight mass analyzers: delayed extraction and the
reflectron. In delayed extraction there is a measured delay in the time
between initial ion formation and acceleration down the flight tube, resulting
in reduced kinetic energy spread of ions with the same m/z. A reflectron is
an electrostatic ion focusing element. Ions of the same m/z but with different
kinetic energies require more or less time in order to be reflected, with, as
the net result, simultaneous arrival at the detector.
The double-focusing sector mass spectrometer is used primarily to
obtain high-resolution mass measurements, that is, determining the mass to
better than 5 ppm accuracy. This mass analyzer is comprised of both a
magnetic sector and an electric sector, utilized in combination. The magnetic
sector separates the ions based on momentum and the electric sector
separates the ions based on kinetic energy. The result is ions that have very
little spread in energy, thus enabling their mass-to-charge ratios to be
measured very accurately.
1.3
Detectors
Once the ions of the compound of interest have been mass analyzed, the
ions are directed to the detector of the mass spectrometer. Most detectors are
based on the premise that the incoming ion will impact the surface of the
detector, thereby forming many secondary electrons. Depending on the
geometry of the detector these secondary electrons then impact another part
of the detector surface. This process continues resulting in a 105 and higher
amplification of the original ion beam. Parameters such as mass and velocity
of the impacting ion as well as chemical characteristics of the impacted
surface affect the ultimate current amplification in any detector.
The information obtained from the detector is used to generate the mass
spectrum. The mass spectrum is a plot of the intensity of the individual massanalyzed ions plotted as a function of m/z. Usually the most intense ion,
termed the base peak, is given a relative abundance of 100% and the rest of
the ions in the mass spectrum are normalized to this intensity. Figure 5.1
shows the EI mass spectrum of 2-methoxy-4-vinyl phenol (molecular weight
150 Daltons). The base peak is the molecular ion at m/z 150, a radical
200
Chapter 5
intensity
cation. The three most intense fragment ions are m/z 135 (loss of CH3.), m/z
107 (loss of CH3. and CO), m/z 77 (C6H5+ ; loss of .OCH3., C2H2, and .OH),
and m/z 51 (C4H3+ ; loss of C2H2 from m/z 77).
H3CO
OH
Figure 5-1. Mass spectrum of 2-methoxy 4-vinylphenol
1.4
Sample introduction
Samples submitted for analysis can be introduced into the mass
spectrometer in many forms. Probably the most common means are (1)
direct introduction, (2) gas chromatography (GC), and (3) liquid
chromatography (LC).
In direct introduction the sample can be introduced via a sample probe
or plate through a vacuum lock, and can subsequently be ionized via EI, CI
or matrix-assisted laser desorption ionization (MALDI; see Section 2.4).
Alternatively, the sample can be introduced as a liquid stream into an ion
source at atmospheric pressure, after which it is subjected to electrospray
ionization (ESI; see Section 2.3). Direct injection does not offer any form of
sample separation.
In contrast, both gas and liquid chromatography enable the samples of
interest to be separated into individual components prior to introduction into
the mass spectrometer ion source. Gas chromatography involves sample
introduction with the requisite that the sample components must be
volatilized prior to separation, and results in a gas sample being introduced
to the mass spectrometer (i.e. EI, CI). Figure 5-2 shows the chromatogram
obtained after a mixture of three simple phenolic compounds - phenol, o-
Analysis of phenolic compounds with mass spectrometry
201
cresol and 2,5-dimethylphenol – was injected in the gas chromatograph.
This analysis utilized a 15-m long DB5 capillary column with an initial
column temperature of 50°C heated to 320°C at 15°C per minute. The
retention time is the time that elapses between injection of the mixture and
the elution (detection) of an individual compound and depends on the
volatility of the compound combined with the affinity of the compound for
the stationary phase inside the capillary column. Liquid chromatography
involves a liquid sample being separated into individual components and
these components being introduced into a mass spectrometer ion source at
atmospheric pressure (i.e. ESI).
It is important to remember the few restrictions imposed by electrospray
when considering an LC-MS analysis. Common solvents like methanol, water,
acetonitrile and volatile salts (below 25 mM) like ammonium acetate and
ammonium bicarbonate are acceptable in the mobile phase, whereas phosphate
salts/buffers, mineral acids or other nonvolatile components cannot be used.
Unfortunately, this conflicts with many of the routine mobile phases used for
the analysis of phenolic compounds and anthocyanins, necessitating changes in
methods when going from LC to LC-MS analyses.
intensity
p-cresol
phenol
2,5-dimethylphenol
retention time
Figure 5-2. Gas chromatogram obtained after injecting a mixture of three phenolic
compounds into a gas chromatograph equipped with a 15-m long DB5 column. The
temperature was increased from 50 to 320°C at 15°C per minute.
202
Chapter 5
Many other analytical techniques can be coupled to mass spectrometers.
These so-called hyphenated techniques, like GC-MS and LC-MS, include but
are not limited to ICP-MS (inductively coupled argon plasma), SCF-MS
(supercritical fluid), NMR-MS (nuclear magnetic resonance) and IR-MS
(infrared).
2.
NEW DEVELOPMENTS IN MASS
SPECTROMETRY
Direct mass spectrometric analysis of thermally labile and nonvolatile
molecules, like many of the anthocyanins and other phenolic compounds,
has not always been possible. In the days of EI and CI it was necessary to
derivatize these molecules before obtaining mass spectrometric data.
However, with the advent of soft ionization techniques - initially fast atom
bombardment (FAB; Fenselau, 1983) and plasma desorption (PD; Sundqvist
and Macfarlane, 1985) - followed by the development of electrospray
ionization (ESI; Cole, 2000) and matrix-assisted laser desorption ionization
(MALDI; Zenobi and Knochenmuss, 1998), the mass spectrometry of these
compound classes became possible, even routine. These techniques provide
molecular weight information through formation of (M+H)+ or (M+Na)+
ions, or in the case of pre-charged ions like anthocyanins, the cations
themselves. Mass spectrometry not only provides information on the
molecular weight of a compound of interest, but with the advent of tandem
mass spectrometric techniques structurally significant information can also
be generated. In tandem mass spectrometry (ms/ms) the ion of interest is
selected and given excess energy. This can be done in a variety of ways,
including collisions with neutral atoms (Busch et al., 1988). The excited
molecule can then undergo fragmentation, with the resulting ions providing
structural information about the original precursor ion.
2.1
Fast atom bombardment
FAB mass spectrometric analyses require a high-energy atom beam,
usually 6-10 keV. The atom beam, typically xenon, is directed at the sample
which is dissolved in a matrix. Typical matrices include glycerol,
thioglycerol, m-nitrobenzyl alcohol and a mixture of dithiothreitol and
dithioerythritol. The continual bombardment of the sample/matrix mixture
results in desorption of both species. Ions are formed, either as pre-formed
ions from the matrix or in the gas phase immediately above the sample
surface.
Analysis of phenolic compounds with mass spectrometry
2.2
203
Plasma desorption ionization
Plasma desorption mass spectrometry utilizes a 252Cf (californium) ionizing
source which produces MeV fission fragments. The interaction of the fission
fragments with the sample produces ions which are mass analyzed. The samples
are applied to a nitrocellulose-coated mylar target, either by droplet or
electrospraying, allowed to adsorb, and then washed with a 0.1% trifluoroacetic
acid solution. Typically the samples are allowed to air dry prior to being
introduced into the mass spectrometer. The sample ions that are formed, are
accelerated into a time-of-flight mass spectrometer for mass analysis.
287
B
287
relative abundance
relative abundance
A
271
303
271
303
200
240
280
320
360
200
m/z
240
280
320
360
240
280
320
360
m/z
D
C
relative abundance
relative abundance
271
287
200
240
280
320
360
m/z
200
m/z
Figure 5-3. Plasma desorption mass spectra of anthocyanidins extracted from A.
chrysanthemum, B. begonia, C. carnation, and D. phlox. The data in this figure was published
in the article ‘Plasma desorption mass spectrometry of anthocyanidins’, Rap. Comm. Mass
Spectrom. 7:400-403, by Wood, K. V., Bonham, C. C., Ng, J., Hipskind, J. and Nicholson, R.
L. 1993. Copyright John Wiley and Sons. Reproduced with permission.
204
Chapter 5
Accelerating potentials between 15-20 keV are typically used, with data
being collected from anywhere between fifteen minutes to an hour depending
on the sample. The useful mass range for PDMS is up to m/z 5000. Typically
the observed ion is the protonated molecule. PDMS, however, has proven to
be particularly well-suited for the analysis of pre-charged species like
anthocyanidins. This can be seen in Figure 5-3, which shows the plasma
desorption mass spectrum of anthocyanidins extracted from chrysanthemum,
begonia, carnation and phlox (Wood et al., 1993). Note the presence of
cyanidin (m/z 287; 1.40), as a dominant ion in the first three mass spectra.
Pelargonidin (m/z 271; 1.39) and delphinidin (m/z 303; 1.42) are also
observed. The phlox sample analyzed was white phlox which explains the
absence of any anthocyanidins.
2.3
Electrospray ionization
FAB and PD have been replaced by electrospray ionization (ESI) and matrixassisted laser desorption ionization (MALDI) in the analytical mass
spectrometry laboratory, because both of these newer techniques have a
wider mass range of analysis and have lower detection limits. ESI and
MALDI have become invaluable ionization techniques for nonvolatile
components. This is particularly true for a wide range of biological
molecules including proteins, peptides, nucleic acids, etc. Samples can be
analyzed by ESI using either direct injection or introduction through liquid
chromatography.
Typically ESI forms protonated molecules with little or no fragmentation.
High molecular weight molecules can also be ionized using ESI. In this case
the molecule becomes multiply-charged and the molecule of interest is
observed at its respective m/z. For example, a protein with a molecular weight
of 20,000 Da that has 20 protons attached to it, would be detected at m/z
20,020/20, which is equal to 1,001. Figure 5-4 shows the ESI mass spectrum of
horse heart myoglobin, with a molecular weight 16,951 Daltons. The M+16H+
and M+15H+ ions result in the peaks at m/z 1060.5 ((16,951 + 16)/16) and
1131.1((16,951 + 15)/15), respectively.
In the case of a protein of unknown molecular weight, the molecular
weight can be determined by using equations (4) and (5) and knowing the m/z
values of two adjacent multiply-charged ions.
n = (X1 – 1)/(X2 – X1)
(4)
Analysis of phenolic compounds with mass spectrometry
205
X1 is the mass-to-charge ratio of the ion with the lower m/z, X2 is the massto-charge ratio of the ion with the higher m/z, and n is the charge on the ion
with the higher m/z. Knowing the charge on a given m/z ion, the molecular
weight of the protein can be calculated by using the following equation.
MW = nX2 – n
(5)
relative abundance
In the example of the horse heart myoglobin, we select X1 = 1060.5 and X2
= 1131.1, n = 15, and MW = 15*1131.1 – 15 = 16,951.5. This is consistent
with the known molecular weight of the protein.
500
1000
1500
m/z
2000
Figure 5-4. Electrospray ionization mass spectrum of horse heart myoglobin.
206
Chapter 5
The choice of solvents and related components, which must be volatile, is
very important for obtaining high-quality electrospray spectra. Even though
sample introduction is through a liquid medium, the solvent eventually needs
to be volatilized. If a nonvolatile solvent or buffer is used, the result is frequent
plugging of the capillary tubing and/or some of the beam-defining components.
In ESI the solvated sample is passed through a needle held at a high potential
(3-10 kV). As the molecule exits from the needle, the resulting spray
undergoes electrostatic nebulization, which places (a) charge(s) on the droplet.
The charged droplet passes through a variety of focusing elements, which
are differentially pumped. One result is desolvation of the droplet.
Depending on the size and chemical makeup of the analyte, the resulting
stable ion can have a single charge, or depending on the repulsive forces,
may be multiply charged. Mass analysis of this ion can be carried out with
any type of mass analyzer, including magnetic sector, quadrupole, ion trap
or time-of-flight. Sample concentrations that can routinely be analyzed are
sub-picomole/microliter. The detection limit for many components, however,
is considerably lower. Table 5-1 gives the results of the ms/ms analysis of a set
of betaine analogs. Notice the structurally diagnostic fragment ions available
for each of these individual betaines (Wood et al., 2002).
2.4
Matrix-assisted laser desorption ionization
MALDI typically utilizes a nitrogen laser at 337 nm as the ionization
source. The sample is mixed with a matrix, and allowed to dry prior to
insertion into the mass spectrometer. Crystallization of the sample within the
matrix is an important component of successful MALDI analysis. A variety
of matrices, present in great excess relative to the sample amount, are used
to span the range of compound classes amenable to MALDI mass analysis.
Formation of sample ions, upon laser irradiation, involves a proton transfer
reaction involving the matrix (which absorbs the UV photon) and the
analyte. The ions are then accelerated into a time-of-flight mass analyzer for
mass analysis. Typical matrices include α-cyano-4-hydroxycinnamic acid,
sinapinic acid and 2,5-dihydroxybenzoic acid. MALDI can be done
routinely to m/z 100,000 and there are many examples of analyses going
well above this mass range. Figure 5-5A shows a MALDI mass spectrum of
grape anthocyanins over the mass range m/z 450 to 550 (Sugui et al., 1999).
Two series of ions are evident, representing monoglucosides (1-5) and
acetylglucosides (6-10) of the following aglycones: cyanidin (1, 6; 1.40),
peonidin (2, 7; 1.41), delphinidin (3, 8; 1.42), petunidin (4, 9; 1.43) and
malvidin (5, 10; 1.44). These results were obtained with a first-generation
(continuous mode) MALDI mass spectrometer that did not have delayed
Analysis of phenolic compounds with mass spectrometry
207
extraction capabilities. Figure 5-5B shows a similar mass range for the
analysis of anthocyanins from a different grape variety, with improved mass
resolution using a MALDI mass spectrometer having delayed extraction
capabilities. Note the presence of the monoglucosides of cyanidin (m/z 449),
peonidin (m/z 463), petunidin (m/z 479) and malvidin (m/z 493). Both
MALDI mass spectra were obtained in the reflector mode.
relative intensity
A
relative intensity
B
450
475
500
m/z
Figure 5-5. A. MALDI mass spectrum of the anthocyanin pigments in the grape variety
Marechal Foch. This figure is from the article ‘Matrix-assisted laser desorption ionization
mass spectrometry analysis of grape anthocyanins’, Am. J. Enol. Vitic. 50:199-203 by Sugui,
J. A., Wood, K. V., Yang, Z., Bonham, C. C. and Nicholson, R. L. 1999. Reprinted by
permission of the American Society for Enology and Viticulture. B. MALDI mass spectrum
of grape anthocyanins acquired on a MALDI mass spectrometer with delayed extraction
capabilities. Peak identities are discussed in the text.
208
Chapter 5
MALDI is considered the most sensitive of these techniques, with
detection limits in the femtomoles/microliter range being relatively behind
these other two, and this probably explains the general lack of continued
interest in them in recent years. Of course statements concerning the relative
sensitivities of the four ionization techniques have to take into account the
chemical differences between compound classes, which plays a major part in
the ionization of a given compound by a given ionization technique.
3.
QUANTITATION
In addition to providing molecular weight confirmation and structural
information, mass spectrometry can also provide quantitative results
(Hoffmann and Stoobant, 2001; Watson, 1997). This is most often done
using either a calibration curve (external calibration) or an internal standard,
like an isotopically labeled analog or a compound with a closely related
structure (internal calibration). Standard addition, the addition of known
amounts of the compound of interest to the unknown sample is another
frequently used quantitative method. Mass spectrometry is a very universal and
sensitive analytical technique. As the methodology has matured over the years
it has been used to solve a myriad of far-ranging analytical problems including
those involving complex plant phenolics such as anthocyanins.
The data presented in Table 5-1 were reprinted from Phytochemistry 59:
Wood K. V., Bonham, C. C., Miles, D., Rothwell, A. P., Peel, G., Wood, B.
C., and Rhodes, D., Characterization of betaines using electrospray MS/MS,
p. 759-765, copyright 2002, with permission from Elsevier.
Analysis of phenolic compounds with mass spectrometry
209
Table 5-1. Electrospray MS/MS spectra of a series of betaine analogs. The
product ions in bold face are the base peaks of the MS/MS spectra.
Compound
glycine-betaine
Structure
OH
H3 C
N
+
H3 C
d9-glycine-betaine
Parention [M]
Productions
(m/z)
(m/z)
118
59 [M-CH2CO2H]
58 [M-CH3CO2H]
CH3
O
OH
D 3C
N
127
68 [M-CH2CO2H]
66 [M-CH2DCO2H]
132
60 [M-H2CHCO2H]
73 [M-N(CH3)3]
141
69 [M-H2CHCO2H]
73 [M-N(CD3)3]
138
94 [M-CO2]
110 [M-CO]
141
97 [M-CO2]
113 [M-CO]
144
84 [M-C3H6-H2O]
102 [M-C3H6]
98 [M–HCO2H]
58 [CH2N(CH3)2]+
150
90 [M-C3H6-H2O]
108 [M-C3H6]
104 [M–HCO2H]
64 [CH2N(CD3)2]+
+
D3 C
CD3
O
β-alanine-betaine
HO
H 3C
O
N+
H3C
CH3
HO
d9-β-alanine-betaine
D 3C
O
N+
D 3C
CD3
trigonelline
OH
N
CH3
O
d3-trigonelline
OH
N
CD3
O
proline-betaine
OH
N
CH3
H3 C
O
d6-proline-betaine
OH
N
D3 C
CD3
O
210
4.
Chapter 5
REFERENCES
Busch, K. L., Glish, G. L. and McLuckey, S. A., 1988, Mass
Spectrometry/Mass Spectrometry: Techniques and Applications of
Tandem Mass Spectrometry, VCH Publishers, New York.
Cole, R. B., 2000, Some tenets pertaining to electrospray ionization mass
spectrometry, J. Mass Spectrom. 35: 763-772.
Fenselau, C., 1983, Fast atom bombardment (review), in Ion Formation
from Organic Solids: Proceedings of the Second International
Conference, Springer Series in Chem. Phys Vol.25., A. Benninghoven,
ed., Springer-Verlag New York, pp 90-100.
Sugui, J. A., Wood, K. V., Yang, Z., Bonham, C. C. and Nicholson, R. L.,
1999, Matrix-assisted laser desorption ionization mass spectrometry
analysis of grape anthocyanins, Am. J. Enol. Vitic. 50: 199-203.
Sundqvist, B. and Macfarlane, R. D., 1985, 252Cf-plasma desorption mass
spectrometry, Mass Spectrom. Rev. 4: 421-460.
Wood, K. V., Bonham, C. C., Miles, D., Rothwell, A. P., Peel, G., Wood, B.
C. and Rhodes, D., 2002, Characterization of betaines using electrospray
MS/MS, Phytochem. 59: 759-765.
Wood, K. V., Bonham, C. C., Ng, J., Hipskind, J. and Nicholson, R. L.,
1993, Plasma desorption mass spectrometry of anthocyanidins, Rap.
Comm. Mass Spectrom. 7: 400-403.
Zenobi, R. and Knochenmuss, R., 1998, Ion formation in MALDI mass
spectrometry, Mass Spectrom. Rev. 17: 337-366.
For further reading we suggest several excellent books on the
specifics of mass spectrometry:
Herbert, C. G. and Johnstone, R. A. W., 2003, Mass Spectrometry Basics,
CRC Press, Boca Raton, FL.
Hoffmann, E. de and Stroobant, V., 2001, Mass Spectrometry: Principles
and Applications, John Wiley, New York.
Watson, J. T., 1997, Introduction to Mass Spectrometry, 3rd Edition,
Lippincott-Raven, Philadelphia.
THE ROLE OF PHENOLS IN PLANT DEFENSE
211
Chapter 6
THE ROLE OF PHENOLS IN PLANT DEFENSE
1.
PREFORMED ANTIMICROBIAL AND
INSECTICIDAL METABOLITES
When considering substances produced by plants that act as agents that
protect the plant from pathogens and insect pests, we must first consider
whether the compounds are present prior to the time of infection or whether
they are synthesized in response to infection. When compounds are present
prior to attempted infection they are known as preformed antimicrobial
metabolites. Such preformed compounds are part of a passive resistance
mechanism. In general, such preformed metabolites are toxic to a broad
spectrum of fungi and bacteria, but the compounds have a relatively low
level of toxicity. Thus, preformed compounds are present in all plant species
and help plants to ward off pathogens that are not considered as highly
aggressive organisms. They are also referred to as phytoanticipins (Van
Etten et al., 1994).
When one considers resistance expression in plants, it is necessary to
consider whether resistance expression is part of a passive or active response
system. There are several situations that could arise:
1. Compound “A” is present in the plant and is toxic to the potential
pathogen. The compound is present in cells or tissues that the pathogen
must come into contact with, at some time during the attempted
infection. The compound is not further metabolized, but may or may not
be changed by the pathogen, and the compound as such then accounts
211
212
Chapter 6
for toxicity to the pathogen. This example represents a form of passive
resistance.
2. A preformed substance is degraded or metabolized to a different
compound by the host in direct response to the pathogen and it is this
compound that accounts in part for toxicity to the pathogen. Because the
host changes the compound, this would be considered a mechanism of
active resistance.
There are several criteria that must be satisfied before it can be decided
whether a particular compound plays a significant role in the resistance to a
pathogen. These are as follows:
1. The compound must be present in those parts of the plant where the
pathogen will come in contact with it. For example, apple leaves contain
phloridzin (6.1) and its aglycone phloretin (6.2), but these compounds
are not present in the fruit.
2. The compound must be present at concentrations high enough to affect
the pathogen. For example, maize contains the preformed cyclic
hydroxamic acid DIMBOA (2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3one; 6.3). The concentration of DIMBOA, however, decreases over time
and is initially not high enough for it to serve as a fungi-toxic agent.
3. Directly related to the criterion above is the requirement that the
compound be “available” in the host in a form or place where it can
express its toxicity. This may ultimately be a question of whether the
compound is changed when it is extracted. Is the compound
compartmentalized in a cell and possibly inaccessible to the pathogen?
Is it in only one tissue?
4. Another criterion that must be met deals with the time of appearance of
the compound. Thus, even if preformed, the compound must be at a
sufficient in vivo concentration by the time resistance is being expressed.
OH
OH
OH
HO
OH
O
O
OH
HO
OH
OH
O
Glc
(6.1)
(6.2)
The role of phenols in plant defense
213
OH
H3CO
N
O
O
OH
(6.3)
Phenolic substances are often thought of when referring to preformed
rsistance compounds. The following classes of metabolites, however, should
also be included: alkaloids, carbohydrates that can bind bacteria, proteins
that act as lectins, amino acids, terpenoids, and polyacetylenes. Some of
these compounds will be included in the discussion below.
1.1
Chlorogenic acid
Chlorogenic acid (6.4) is an example of a preformed compound that has
a relatively low level of toxicity to most microorganisms. It is effective
against microorganisms considered as weak pathogens of potato. In potato
tubers, chlorogenic acid is present in the periderm (Kojima et al., 1985) and
is toxic to the organism that causes potato scab, Streptomyces scabies. In
general, there is more of the compound in tubers of cultivars that are
resistant to the pathogen than in tubers of cultivars that are susceptible.
Furthermore, more of the compound is present during the time of tuber
expansion. Chlorogenic acid also affects the growth of the vascular pathogen
Verticillium albo-atrum and is present in the vascular tissue of the potato
(Dao and Friedman, 1994). The mechanism by which ortho-dihydroxy
phenolic compounds such as chlorogenic acid provide defense against insect
pests was studied by Felton et al. (1989). They investigated the fate of
chlorogenic acid in tomato (Lycopersicon esculentum) on the feeding
behavior of the tomato fruit worm (Heliothis zea) and beet army worm
(Spodoptera exigua). Upon feeding by the insects, polyphenol oxidases that
are compartmentally separated from chlorogenic acid in the plant, come in
contact with their substrate and convert chlorogenic acid to the toxic
chlorogenoquinone (6.5; Figure 6-1). This quinone is a highly reactive
electrophile and will react with nucleophilic –SH and –NH2 moieties in
proteins (as indicated by structure 6.6; Matheis and Whitaker, 1984). This
results in the cross-linking of proteins with chlorogenic acid,, which reduces
the availability of free amino acids and proteins to the insect.
214
Chapter 6
O
OH
O
HO
O
O
O
O
R2
OH
HO
R3
HN
R1
O
PPO
HN
HO
O
OH
O
(6.4)
(6.5)
(6.6)
O
O
O
O
O
HN
R1
R2
O
O
R1
(6.6)
HN
R3
R2
O
O
N
N
NH
O
R1
R3
N
R1
O
O
OH
OH
(6.7)
(6.8)
Figure 6-1. Oxidation of chlorogenic acid (6.4) by polyphenoloxidase (PPO), resulting in
chlorogenoquinone (6.5), which can react with nucleophilic groups in proteins (6.6) to give
the cross-linked compound 6.7, which can react with another protein molecule to yield 6.8.
The quinate residue in structures 6.5, 6.7 and 6.8 is represented by R, whereas R1 and R2
indicate different amino acid residues.
1.2
Phloridzin and phloretin
In apple, the glycoside phloridzin (1.27) and its aglycone phloretin are
thought to inhibit the apple scab fungus Venturia inaequalis. Phloridzin is
also an ortho-dihydroxyphenolic compound, and, like chlorogenic acid, can
also be easily converted to a reactive quinone upon attack by a pathogen.
Raa (1968) demonstrated that oxidation products of phloridzin inhibit fungal
The role of phenols in plant defense
215
pectinases. Fungal pectinases hydrolyze pectin, a cell wall compound that is
abundant in the middle lamella and plays a role in cell adhesion. Thus, by
inhbiting pectinases, the ability of the fungus to hydrolyze and invade the
plant cell wall would be compromised. Although phloridzin and phloretin
are toxic at high concentrations, based on the poor correlation between
resistance to scab and the concentration of phenolic compounds such as
phloridzin, they are probably not the factors that account for the actual
resistance of apple cultivars to this fungus (Nicholson and Rahe, 2004). This
was further substantiated by Leser and Treutter (2005), who investigated the
effect of nitrogen supply on the scab susceptibility of the susceptible apple
cultivar ‘Golden Delicious’ and the resistant cultivar ‘Rewena’. Increased
nitrogen supply was hypothesized to stimulate growth and decrease the
levels of phenolic compounds. This was shown to indeed be the case.
Consistent with this hypothesis, the susceptible cultivar became more
susceptible under high nitrogen supplements. The resistant cultivar,
however, did not become susceptible, even though the levels of phenolic
compounds, including phloridzin, decreased.
1.3
Cyanogenic glycosides
Many plants contain cyanogenic glycosides. Toxicity of the cyanogenic
glycoside results when the compound is enzymatically cleaved to release
hydrogen cyanide (HCN) that is toxic to the pathogen. Sorghum contains the
cyanogenic glycoside dhurrin (6.9). This compound is of interest to both
pathologists and entomologists as an example of a preformed resistance
compound and acts as an insect feeding deterrent and as a fungicidal agent
(Starr et al., 1984; Adewusi, 1990).
N
C
O-Glc
OH
(6.9)
216
1.4
Chapter 6
Tuliposides
Tulips contain preformed compounds known as tuliposides, (tuliposide
A, 6.10, and tuliposide B, 6.11). Hydrolysis of the tuliposides results in
formation of aglycones (6.12) and (6.13) which will form butyrolactones
(6.14) and (6.15). These lactone forms are inhibitory to fungi.
O
O
HO
Glc
O
OH
(6.11)
OH
HO
(6.10)
O
HO
O
Glc
H2O
(6.12)
Glc
O
(6.14)
HO
O
Glc
OH
HO
O
H2O
O
O
OH
(6.13)
(6.15)
Figure 6-2. Formation of antifungal butyrolactones from tuliposides via an internal
esterification reaction.
Some fungi are able to metabolize the lactone form to a butyric acid
form that is not inhibitory to a variety of organisms, including the tulip
pathogen Fusarium oxysporum. Two important diseases of tulip are bulb rot
and grey mold. Botrytis tulipae infects all parts of the tulip, including the
pistils, macerating the tissue. In contrast, Botrytis cinerea does not develop
on tulips in the field, but eventually infects various parts of the plant when
kept in a high humidity chamber. B. cinerea never infects the flower pistils
because they contain exceptionally high concentrations of tuliposides.
Tuliposides are stored in cell vacuoles. Importantly, growth of the pathogen
is at first intercellular. Under these conditions the pathogen does not
encounter the tuliposides. It is only when the tuliposides are released from
the vacuoles that the effects of their toxicity can be expressed. B. cinerea
converts tuliposides into inhibitory lactones. B. tulipae converts tuliposides
into hydroxycarboxylic acids which are non-toxic (Schönbeck and
Schroeder, 1972).
The role of phenols in plant defense
1.5
217
Protocatechuic acid
Probably the most commonly referred to compound which accounts for
a form of passive resistance of a chemical nature is protocatechuic acid
(6.16) which is found in yellow and red skinned onions and prevents the
germination of spores of the onion smudge fungus, Colletotrichum
circinans. Thus, protocatechuic acid serves as a barrier to infection prior to
penetration. Once penetration occurs the compound is ineffective.
OH
HO
OH
O
(6.16)
1.6
Lignin
The cell wall polymer lignin (see Chapter 1, Section 3.12, and Chapter
3, Section 12) has been implicated in defense against pests and pathogens, as
a preformed, passive defense compound. It is a suitable defense compound
in that respect, because it hardens the cell wall and thus creates a physical
barrier against invasion. In addition, the chemical structure of lignin is very
complex because of the different lignin subunits, and the many different
types of chemical bonds that exist between subunits. Hydrolysis would,
therefore, require a battery of enzymes, which is something most pests and
pathogens do not have access to (Vance et al., 1980; Denton, 1998).
Exceptions are white rot fungi, which can oxidize lignin in order to degrade
it (Chen and Chang, 1985). This is how fallen tree logs eventually
disintegrate. There is evidence that lignin can also be synthesized de novo.
This lignin is synthesized locally, and specifically in response to pathogenic
attack. This mechanism resembles papilla formation (Cadenagomez and
Nicholson, 1987) and will be discussed in the next section.
1.7
C-glycosyl flavones
Maysin (2´´-O-α-l-rhamnosyl-6-C-(6-deoxyxylo-hexos-4-ulosyl)-luteolin;
6.17a), apimaysin (6.17b) and methoxymaysin (6.17c) are C-glycosyl
flavones that confer resistance against the corn earworm (Helicoverpa
zea (Boddie)), a major silk- and kernel-feeding insect pest in
218
Chapter 6
the United States. These three compounds differ from each other in the
substitution pattern of the B-ring: apimaysin has one hydroxyl group on the
4´ position of the B-ring, maysin has a 3´,4´-dihydroxy substitution pattern,
and methoxymaysin has a 4´-hydroxy, 3´-methoxy substitution pattern.
These compounds accumulate in the silks of maize (i.e. the styles attached to
the ovules) and are thought to act in a manner similar to chlorogenic acid
(see Figure 6-1) when insects damage the silks.
R
OH
HO
H
H3C
O
H
H
O
O
HO
OH
O
H
a. R = OH
b. R = H
c. R = OCH3
O
H
H
O
H
H
CH3
OH
HO
OH
H
(6.17)
Maysin is generally the most abundant of these three compounds, and is
typically present at concentrations of 0.3% fresh silk weight, which is very
high for a single compound. As a consequence, the C-glycosyl flavones can
be considered preformed defense compounds.
The concentration of C-glycosyl flavones varies considerably among
different maize lines. Since the concentration of the C-glycosyl flavones
does not have a discrete value, but rather varies along a continuum, it can be
considered a quantitative trait (see Chapter 3, Section 3.4). In order to
identify loci controlling maysin concentration in silks, Byrne et al. (1996)
investigated the role of a number of structural and regulatory genes known
to play a role in flavonoid biosynthesis. They generated an F2 population
derived from the maize inbred lines GT119 and GT114, which had low
(0.031%) and high (0.56%) maysin levels, respectively. They determined the
genotype at a number of loci at or near flavonoid biosynthetic genes, and
concluded that the P1 (‘P-one’; see Chapter 3, Section 9.2) and a locus
referred to as recessive enhancer of maysin (rem1) near the Brown pericarp1
(Bp1) locus accounted for 58 and 11% of the variance, respectively. In
addition, a QTL near the centromere of chromosome 1 was uncovered, but
there were no obvious candidate genes at this locus.
The role of phenols in plant defense
219
The effects of the P1 gene and the tightly linked homolog P2 on maysin
biosynthesis were further investigated by Zhang et al. (2003). These
researchers used introduced the P1 and P2 cDNA’s under control of the
ubiquitin promoter in cultured Black Mexican Sweet maize cells using
microprojectile bombardment. This is a method in which small gold or
tungsten particles coated with an expression construct are introduced into
target cells using a burst of pressure. Based on gene expression studies, P1
and P2 activate the same genes, including phenylalanine ammonia lyase
(Chapter 3, Section 7), chalcone synthase, chalcone isomerase, and
dihydroflavonol 4-reductase (Chapter 3, Section 9), but not genes involved
in the biosynthesis of flavonols and anthocyanins. Increased levels of
flavones were detected in extracts obtained from the transformed cells.
Further evidence for a role of both genes in maysin biosynthesis came from
the observation that maize plants in which both P1 and P2 were deleted did
not produce maysin, whereas plants in which P1 was deleted, but P2 was
still present, still produced maysin, albeit at reduced levels.
Maysin is two times more effective in its ability to inhibit growth of the
corn earworm, which is attributed to the fact that two neighboring hydroxyl
groups (such as on the on the 3´ and the 4´ positions in maysin) will result in
the efficient formation of a toxic quinone, whereas the quinone formation
from apimaysin and methoxymaysin is less efficient (Elliger et al., 1980;
Snook et al., 1994). The genetic basis of the substitution reactions of the Bring have been the subject of several studies. Using an F2 population derived
from the maize inbred lines GT114 (moderately high levels of maysin,
negligible levels of apimaysin) and NC7A (moderately high levels of
apimaysin, maysin, and chlorogenic acid (6.4), Lee et al. (1998) determined
that the rem1 locus identified by Byrne et al. (1996) explained 55% of the
variance for maysin, whereas a QTL that mapped near the Pr1 gene, which
is thought to encode flavonoid 3´ hydroxylase (F3´H), explained 65% of the
variance for apimaysin. Furthermore, the levels of maysin and apimaysin
were independent of each other, suggesting these two compounds are
synthesized via different pathways. Surprisingly, a functional Pr1 gene was
not required for maysin production. Lee et al. (1998) speculated that the
actual gene responsible for maysin biosynthesis may be near Pr1, but does
not have to be Pr1, that a second F3´H gene is responsible for maysin
production, or that the hydroxylation at C3´ occurs at the level of the
hydroxycinnamoyl CoA ester rather than at the level of the flavone.
The genetic control of the substitution of the C-glycosyl flavones was
investigated in further detail by Cortés-Cruz et al. (2003). Two F2
populations were generated from maize inbred lines that differed from each
220
Chapter 6
other in the relative concentrations of maysin, apimaysin, and
methoxymaysin. In both F2 populations the main QTL associated with
levels of chlorogenic acid, maysin and methoxymaysin was located on the
short arm of chromosome 4, whereas the main QTL associated with levels of
apimaysin was located on the long arm of chromosome 5. Presence of a
specific allele in the QTL on chromosome 4 resulted in higher levels of
methoxymaysin and lower levels of maysin and chlorogenic acid. The fact
that a single QTL affects the concentrations of three compounds
(methoxymaysin, maysin and chlorogenic acid) suggests that there may be a
regulatory gene underlying the QTL, or that there is a branched rather than a
linear biosynthetic pathway leading to these different compounds. The QTL
for apimaysin on chromosome 5 coincided with the Pr1 locus, consistent
with the data reported by Lee et al. (1998).
The biosynthetic pathway leading to maysin starts with flavanone
(6.18), which is hydroxylated by flavone 3´ hydroxylase to yield di-hydroxyl
flavanone (6.19) and is the reduced by flavone synthase to the flavone
luteolin (6.20). The next steps were recently investigated in more detail by
McMullen et al. (2004) using two salmon silk mutants, sm1 (Anderson,
1921) and a newly discovered mutant sm2. These mutants have salmon
colored silks instead of green silks as a result of pigment accumulation
throughout the shaft of the silks, as opposed to only in the silk hairs, but do
require a functional P1 gene in order for the mutant phenotype to be
apparent (see also Chapter 3, Section 9.2).
Detailed chemical analyses of flavone composition in the silks in wildtype, sm1, sm2 and sm1-sm2 plants revealed that isoorientin (6.21) is the
only flavone accumulating in sm1-sm2 double mutants, indicating the
synthesis of this compound precedes the action of the gene products of the
functional Sm1 and Sm2 genes. Isoorientin (6.21) is present at high levels in
sm2 but not sm1 mutants, so that a functional Sm2 gene is required for the
formation of rhamnosyl-isoorientin (6.22) from isoorientin. The Sm2 gene
may encode a rhamnosyl transferase, or control the expression of a
rhamnosyl tranferase gene. Rhamnosyl-isoorientin (6.22) accumulates in
sm1 mutants, suggesting that Sm1 encodes a protein that catalyzes the
formation of maysin (6.17a), or otherwise controls the expression of gene(s)
encoding the necessary enzymes.
Taking all of the abovementioned data into consideration, the most
likely biosynthetic pathway leading to maysin is as shown in Figure 6-2,
although further research is needed to fully elucidate the pathway and the
regulatory genes.
The role of phenols in plant defense
221
OH
OH
HO
OH
O
O
HO
(6.18)
(6.20)
b
OH
OH
a
O
OH
OH
HO
O
c
O
OH
OH
(6.19)
O
HO
OH
O
H
H
(6.21)
H
HOH2C
O
H
OH
HO
HO
OH
OH
O
d
H
O
HO
H
OH
H
(6.22)
H
HOH2C
O
H
O
HO
HO
OH
H H
O
H
O
H
H
CH3
OH
OH
HO
OH
H
OH
e
HO
O
H
H3C
H
(6.17a)
H
O
O
HO
OH
O
O
H
H H
O
H
HO
H
CH3
OH
OH
H
Figure 6-2. Biosynthesis of maysin proposed by McMullen et al. (2004) based on the analysis
of flavones in the silks of maize salmon silk mutants. a. flavone 3´ hydroxylase (encoded by
the maize Pr1 gene), b. flavone synthase, c. C-glucosyltransferase, d. putative rhamnosyl
transferase (encoded by the Salmon silk2 gene), e. the step(s) controlled by the Salmon silk1
gene.
222
2.
Chapter 6
COMPOUNDS FORMED IN RESPONSE TO
PATHOGEN ATTACK
Compounds formed in response to stress may occur in at least two ways.
In one response, the plant may form compounds throughout the tissue at a
considerable distance from the infection site (Hammerschmidt, 1999). In
another response, the plant may form compounds specifically at the
infection site. This may include only a few cells and in rare cases, as few as
one or two cells. (Snyder and Nicholson, 1990; Nicholson and Wood, 2001).
In general, such compounds are referred to as either stress metabolites or
more often as phytoalexins. By definition phytoalexins are formed in
response to infection (Aguero et al., 2002; Lo et al., 2002; Hammerschmidt
and Nicholson, 2001; Lo and Nicholson, 1998). Phytoalexins often exhibit
toxicity to specific pathogens. In this case there is a genetic relationship
between the expression of phytoalexin synthesis and the organism that
induces that synthesis (Essenberg et al., 1985).
2.1
3-Deoxyanthocyanidins
The 3-deoxyanthocyanidins are a class of phytoalexins found in
sorghum. These compounds are so fungi-toxic that they are effective at
femtogram levels (Snyder and Nicholson, 1990; Nicholson and Wood,
2001). The synthesis of these compounds is initiated on the endoplasmic
reticulum. Compounds are then trafficked in subcellular inclusions. The
inclusions appear similar to vesicles, but there is no evidence that
membranes surround the inclusions (Snyder and Nicholson, 1990). Nielsen
et al. (2004) recently summarized this defense response. The cytological
response commences when clear, colorless inclusions (less than 0.1 µm in
diameter) accumulate in leaf cells under fungal attack. The inclusions
eventually are seen as red bodies at the infection site. When the 3deoxyanthocyanidins enter the apoplast, the host cell collapses. The
phytoalexins then accumulate in the pathogen and cause its death. Excess
phytoalexins are trapped in host cell walls at infection sites (Lo et al., 1998;
1999).
In the publication by Nielsen et al. (2004) images of the pigmented
inclusions that contain the phytoalexins were prepared by confocal
microscopy. This provided a three-dimensional perspective of inclusion
body formation and visualization of the phytoalexins. A representation of
deoxyanthocyanidin accumulation is shown in Figure 6-3 where inclusions
begin to form by 5 to 8 hours after a fungal appressorium was formed by a
hypha.
The role of phenols in plant defense
223
Figure 6-3. Cell-specific accumulation of 3-deoxyanthocyanidins in Sorghum bicolor in
response to attempted fungal attack. (A) Illustration of changes in inclusion morphology in
cells under fungal attack in response to formation of infectious structures 0–48 h after
inoculation. (B) Site-specific accumulation of 3-deoxyanthocyanidins at site of incipient
penetration, before host cell collapse 24 h after inoculation. (C) Illustration of site-specific
trafficking (arrows) of inclusions in relation to position of fungal infectious structures.
Reprinted from Phys. Mol. Plant Pathol., 65, Nielsen, K. A., Gottfredsen, C. H., BuchPedersen, M. J., Ammitzbøll, H., Mattsson, O., Duus, J. Ø., and Nicholson, R. L., Antimicrobial flavonoid 3-deoxyanthocyanidins in Sorghum bicolor self-organize into spherical
structures, 187-196, Copyright 2004, with permission from Elsevier.
Note that the inclusions at this early time are not pigmented; rather they
are colorless bodies that move through the cytoplasm toward the site of
appressorial attack. Over time, the inclusions take on a yellow color and
eventually become deep red in pigmentation. Inclusion size changes from
less than 1 µm to 20 µm or even larger. Inclusions move to the penetration
site and cluster in the area where the penetration peg has made physiological
contact with the host cell. When the appressorium begins the process of
penetration the inclusions burst, releasing their contents into the cytoplasm.
The deoxyanthocyanidins kill the host cell itself and are taken up by the
224
Chapter 6
pathogen. This is possible because these deoxyanthocyanidins are soluble in
both water and organic solvents. In this manner the pathogen is also killed
and prevented from causing extensive damage and cell death of the host.
2.2
Pisatin
Pisatin (6.23) is an isoflavonoid phytoalexin that is synthesized by pea
(Pisum sativum L.) as a response to infection (Preisig et al., 1989).
Subsequently, it was shown that pathogens capable of demethylating pisatin
were tolerant of this phytoalexin. The enzyme responsible for demethylation
is a specific cytochrome P450 mono-oxygenase released by the fungus
Nectria haematococca (Delserone et al., 1999).
H3CO
O
H
O
H
O
O
(6.23)
2.3
Stilbenes
Aside from being UV-protectants, in a number of species certain
stilbenes act as phytoalexins. Resveratrol (6.24; trans-3,5,4′–
trihydroxystilbene), its cis-isomer, as well as their glucosides and
dehydrodimer trans- -viniferin (6.25) are present in grape leaves and berries
and play a role in the defense against gray rot caused by the fungal pathogen
Botrytis cinerea.
Viniferin is synthesized by a grape peroxidase (Morales et al., 1997).
The fungus in return is able to inactivate resveratrol through the action of a
laccase-like stilbene oxidase (Breuil et al., 1998). This results in the
formation of resveratrol trans-dehydrodimer (6.26), as well as the
corresponding cis-dehydrodimer (6.27), both of which structurally resemble
viniferin.
The role of phenols in plant defense
HO
225
HO
OH
O
HO
OH
OH
OH
OH
(6.24)
(6.25)
HO
HO
O
O
OH
HO
HO
HO
OH
OH
OH
(6.26)
2.4
HO
(6.27)
Salicylic acid
When a specific part of a plant is attacked by a fungal pathogen, distant
parts of the plant may display an enhanced state of resistance involving the
accumulation of pathogenesis-related (PR) proteins, a class of plant proteins
that are normally not present but that are induced upon pathogen attack
(reviewed by Van Loon and Van Strien, 1999). In addition, salicylic acid
(SA; 6.28) and hydrogen peroxide accumulate at the wound site and in other
parts of the plant. This response is referred to as systemic acquired
resistance (SAR) and is thought to be mediated by one or more signaling
molecules in the phloem.
SA was hypothesized to be one of those signaling molecules, because 1)
SA accumulation was shown to be correlated with SAR and resistance
(Uknes et al., 1993), 2) exogenous SA applied to an uninfected plant induced
SAR and resistance in a manner similar to that of an infected plant (Ward
226
Chapter 6
et al., 1991), and 3) transgenic plants expression the nahG gene from
Pseudomonas putida, which encodes a salicilate hydroxylase, were unable to
display SAR (Gaffney et al., 1993).
OH
O
OH
(6.28)
SA was hypothesized to be one of those signaling molecules, because 1)
SA accumulation was shown to be correlated with SAR and resistance
(Uknes et al., 1993), 2) exogenous SA applied to an uninfected plant
induced SAR and resistance in a manner similar to that of an infected plant
(Ward et al., 1991), and 3) transgenic plants expression the nahG gene from
Pseudomonas putida, which encodes a salicilate hydroxylase, were unable to
display SAR (Gaffney et al., 1993). While this latter study demonstrated the
role of SA in initiating SAR, it did not address whether SA was the
signaling molecule that transmitted the SAR signal through the phloem to
other parts of the plant. Vernooij et al. (1994) performed a series of elegant
experiments to investigate the role of SA in signaling. They grafted a scion
from a transgenic tobacco plant expressing the NahG gene onto the root
stock of an untransformed tobacco plant. In addition, an untransformed
scion was grafted onto a transgenic rootstock. Ungrafted plants and plants
where the scion was grafted back on the rootstock from which it came were
used as controls. The root stocks were inoculated with the viral pathogen
tobacco mosaic virus (TMV) to induce SAR. The degree of SAR was
evaluated by challenging the scions of the inoculated plants 7 days later with
TMV or the fungal pathogen Cercospora nicotianae. In the untransformed
graft control, the lesions induced by TMV were 41% smaller compared to a
mock-inoculated control. This indicated that the SAR signal was not
hampered by the graft. When the scions of the transgenic grafted plants were
inoculated, the lesion size was the same as in the corresponding mockinoculated control. This indicated that the expression of the NahG gene
prevented SAR, as had been shown by Gaffney et al. (1993). The TMVinoculated transgenic scions on untransformed root stocks behaved similarly
as the mock-inoculated controls, indicating that SA was required to induce
SAR in the scions. When untransformed scions on transgenic root stocks
were inoculated with TMV, however, they displayed SAR. This reveals that
the NahG-expressing tissues were able to transmit the signal required for
The role of phenols in plant defense
227
SAR. Similar results were obtained when the SAR response was induced by
C. nicotianae, suggesting that SAR is a response to a broad range of
pathogens. These experiments thus demonstrated that SA is required for the
induction of SAR, but that it is not the actual signaling molecule.
After an additional 10 years of research it is still not entirely clear what
the signaling molecule is. Van Bel and Gaupels (2004) recently reviewed the
possible signaling molecules that could induce SAR. The list includes
jasmonic acid, lipid-derived molecules, reactive oxygen species (see Chapter
2, Section 1.9), oligosaccharides, mRNA molecules, calcium, and various
peptides.
2.5
Lignin
There is evidence that lignin can be synthesized de novo. This lignin is
synthesized locally, and specifically in response to pathogen attack. There is
new evidence that this lignin requires different biosynthetic enzymes, which
results in a different subunit composition than the lignin of the vascular
tissue.
Wheat cultivars resistant to Puccinia recondita f. sp. tritici, a fungal
pathogen causing leaf rust, were shown to accumulate more lignin than
susceptible cultivars, based on histochemical stains and a quantitative assay
to detect total phenolics (Southerton and Deverall, 1990). Similar results
were reported for the response of wheat to infection with Fusarium
graminearum, which causes Fusarium head blight. In this case immunogold
labeling against lignin was used to evaluate the accumulation of lignin in
inoculated and non-inoculated spikes of a resistant and susceptible cultivar.
Labeling densities were significantly higher in inoculated spikes of the
resistant cultivar, compared to either non-inoculated spikes and inoculated
spikes of the susceptible cultivar (Kang and Buchenauer, 2000).
Several studies have focused on the role lignin biosynthetic enzymes
play in response to pathogenic attack. Moerschbacher et al. (1990) used
specific inhibitors of the enzymes phenylalanine ammonia lyase (PAL) and
cinnamyl alcohol dehydrogenase (CAD). These inhibitors were applied to
wheat cultivars highly resistant to stem rust (Puccinia graminis Pers f.sp.
tritici Erics. & E. Henn.). Regardless of the inhibitor that was used, de novo
lignification was decreased and fungal development increased. Thus, a strict
correlation between resistance and lignification was demonstrated.
228
Chapter 6
Lignification in response to infection has been found to be associated by
an increase activity of several enzymes of the lignin biosynthetic pathway.
When a wheat lines carrying the stem rust resistance gene Sr5 was compared
with a near-isogenicline without this resistance gene, different patterns of
enzymatic activities were observed (Moerschbacher et al., 1988). Both lines
had an early activation of the enzymes PAL, 4-coumarate:CoA ligase (4CL),
and CAD, but only the resistant genotype exhibited a second significant
increase at the time of the hypersensitive response. Similarly, Mitchell et al.
(1994) measured p-coumaryl, coniferyl and sinapyl alcohol dehydrogenase
activity in lignifying leaves of wheat. Lignification induced by wounding or
elicitors was found to be specifically associated with an increase in sinapyl
alcohol dehydrogenase activity, which is expected to result in a lignin rich in
syringyl units. In a subsequent study Mitchell et al. (1999) investigated the
role of CAD in the defense response of wheat, mimicked by wounding or
the application of the elicitors chitosan and chitin. Three major forms of
CAD were identified, but only one, CAD-C, was found to be induced during
lignification at the wound margin. This particular form had a substrate
preference for sinapyl alcohol. Thus, in agreement with the previous study,
de novo lignin could contain a high level of syringyl units.
Deborah et al. (2001) studied PAL and peroxidase (PO) activity as well
as accumulation of lignin in response to inoculation of rice leaf sheaths with
a pathogen and a non-pathogen. Infection with the non-pathogen Pestalotia
palmarum resulted in a stronger increase in PO and PAL activity and a
higher accumulation of lignin than after inoculation with the pathogen
Rhizoctonia solani.
Altogether, these results indicate that if lignin plays a role in the
resistance against a pathogen, a coordinated activation of lignin biosynthetic
enzymes is required.
Two recent studies, one in maize and one in Arabidopsis, provided
evidence for the existence of multiple copies of the same gene and a
divergence in their function. Pichon et al. (1998) cloned two different maize
cDNA’s encoding cinnamoyl-CoA reductase (CCR) and found that the
corresponding genes are differentially expressed in different parts of the
plant. A similar situation was observed in Arabidopsis (Lauvergeat et al.,
2001). The expression of the two Arabidopisis genes was studied during
plant development, and in response to infection with the pathogenic bacteria
Xanthomonas campestris pv. campestris. The AtCCR1 gene was mainly
expressed in lignifying tissues during development. In contrast, the AtCCR2
gene had a low expression level during development, but was induced when
The role of phenols in plant defense
229
the plant was challenged with the bacteria. With the availability of whole
genome sequences, it is apparent that many of the lignin biosynthetic genes
are respresented by multiple copies, some of which are likely to be involved
in defense responses (Raes et al., 2003).
230
3.
Chapter 6
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PHENOLIC COMPOUNDS AND THEIR EFFECTS ON HUMAN
HEALTH
235
Chapter 7
PHENOLIC COMPOUNDS AND THEIR
EFFECTS ON HUMAN HEALTH
1.
INTRODUCTION
There is a huge body of evidence that phenolic compounds have effects
on human health, and that is the topic of this chapter. Perhaps the oldest
medical application of phenolic compounds is the use of phenol (1.1) as an
antiseptic. Because of its negative side effects on living tissues, including
blister formation, especially at higher concentrations, it is no longer used in
this capacity. The modern antiseptic agents effective against the bacterium
Staphylococcus aureus are, however, still compared to a 5% (w/v) solution
of phenol. Phenol is still used as an oral anesthetic in throat lozenges, at a
typical concentration of 1.4%.
Another very common use of phenolic compounds is in sunscreens. The
presence of the aromatic ring results in the effective absorbance of the UV-B
radiation (between 280 and 315 nm) from the sun and thus prevents
sunburns. The most common active ingredient in many sunscreens is paminobenzoic acid (PABA; 7.1), which is actually not a phenolic compound.
This compound has been widely used since the 1970’s but is less popular
nowadays due to the formation of skin rashes and acne. As a result many
sunscreens are now PABA-free. Alternative active ingredients include
salicylates such as octylsalicylate (7.2), cinnamates such as octyl
methylcinnamate (7.3), benzophenone (7.4) and the related compound
oxybenzone (7.5), and anthranilates, such as menthylanthranilate (7.6). Octyl
methylcinnamate (7.3) is insoluble in water and is therefore commonly
235
236
Chapter 7
used in water-proof sunscreens. The most recently developed sunscreens use
titanium dioxide and/or zinc oxide which reflect the light, rather than absorb
it, and are considered more effective. They work best in relatively thick
layers, which is less desirable from a cosmetic perspective. The more
traditional sunscreens with phenolic compounds that absorb UV radiation
are therefore still very common.
HO
O
O
OH
O
O
(7.2)
O
NH2
(7.1)
H3CO
O
(7.3)
(7.4)
O
O
NH2
O
(7.6)
HO
OCH3
(7.5)
A concern of the widespread use of phenolic compounds is the
estrogenic activity these compounds may display, which impacts the
hormone balance and may result in breast cancer in women. In order to
investigate this, Miller et al. (2001) used recombinant yeast in an estrogen
assay to assess the activity of 73 phenolic additives in sunscreens,
preservatives, perfumes, disinfectants, antioxidants and flavorings. Thirtytwo compounds were shown to have activity in this assay. Twenty-two
exhibited potencies relative to 17b-estradiol that ranged from 1/3,000 to
1/3,000,000. Forty-one compounds were inactive. The major criteria for
estrogenic activity were the presence of an unimpeded phenolic OH group in
a para-position and a molecular weight of 140-250 amu.
Phenolic compounds and their effects on human health
237
Kawamura et al. (2003) performed a similar study, also using a yeastbased assay to detect estrogenic activity, with UV-absorbing compounds in
food plastics, as well as with benzophenone (7.4) derivatives used in
sunscreens. They reported estrogenic activity higher than the known
endocrine disrupting compound bisphenol A (7.7) for several benzophenone
derivatives, including 2,4-dihydroxyphenone and 4-hydroxybenzophenone.
Based on the specific estrogenic activity, they concluded that a hydroxyl
group on the phenol ring of benzophenone has the biggest impact when it is
present at the para-position, followed by the meta- and ortho-positions,
which is consistent with the data reported by Miller et al. (2001).
OH
OH
(7.7)
Aside from medical applications, polyphenols, including the flavonoids
and tannins, are an integral part of human and animal diets, because they
represent one of the most numerous and ubiquitous groups of plant
metabolites (Bravo, 1998). Although traditionally regarded as anti-nutrients,
because of their bad taste, unappealing color, or cause of browning of
tissues, polyphenols and other food phenolics are the subject of increasing
interest because of their possible beneficial effects on health.
This chapter will highlight the positive effects of phenolic compounds
on human health. This is by no means meant to be an exhaustive
presentation of all the literature available on this topic. An entire book could
be dedicated to it, and such books indeed exist. Several references for
further reading are provided at the end of this chapter.
2.
ANTIOXIDANT PROPERTIES
As part of normal metabolism, radicals are generated. Radicals, as
discussed in Chapter 2, are compounds with free (i.e. unpaired) electrons.
Radicals are very reactive, and, when left unchecked, can cause oxidative
238
Chapter 7
damage to the molecules in a cell, and hence have negative impacts on the
cellular metabolism. An excess of radicals can cause oxidative stress.
Compounds that can scavenge radicals are also referred to as antioxidants. The best known anti-oxidants are vitamin C and vitamin E.
Vitamin C is L-ascorbate (7.8), a good reducing agent that prevents
oxidation of other molecules. The oxidized form of L-ascorbate is Ldehydroascorbic acid (7.9). Vitamin E is a mixture of α-, -, -, and tocopherol (7.10a-d). Of these four compounds, α-tocopherol is the most
effective. Vitamin E is lipid-soluble and has the ability to disrupt the chain
reaction during lipid peroxidation (see Chapter 2, Section 1.9).
HO
HO
O
O
O
O
OH
OH
OH
O
(7.8)
O
O
(7.9)
R1
HO
O
R2
R3
a.
b.
c.
d.
R1 = CH3
R1 = CH3
R1 = H
R1 = H
R2 = CH3
R2 = H
R2 = CH3
R2 = H
R3 = CH3
R3 = CH3
R3 = CH3
R3 = CH3
(7.10)
A lack of vitamin C in the diet results in the disease scurvy. Scurvy’s
symptoms include purple lesions on the skin, rotten gums, and, as a
consequence, loss of teeth. This disease was common among 16th and 17th
century sailors who relied on preserved foods and an overall unbalanced
diets on their long journeys. The intake of fresh fruits and vegetables, which
are rich in vitamin C, can effectively prevent scurvy. The biochemical basis
of scurvy is the reduced activity of the enzyme prolyl hydroxylase (E.C.
1.14.11.2), probably because the iron atom that is part of the enzyme cannot
be maintained in its active, ferrous state due to the lack of vitamin C. This
reduced enzyme activity then results in insufficient hydroxylation of
collagen, a structural protein that gives elasticity to the skin and blood
Phenolic compounds and their effects on human health
239
vessels. The lack of elasticity causes the lesions in the skin and the rupture
of blood vessels (Stryer, 1988).
In the current era scurvy is a rare disease, but many other diseases can
arise from low levels of these vitamins. For example, low plasma levels of
α-tocopherol and L-ascorbate correlate with an increased incidence of
myocardial infarction and of some forms of cancer (Gey et al., 1987). In
fact, many diseases are thought to be associated with higher levels of
radicals in the cell (Halliwell, 1991). An example includes rheumatoid
arthritis (RA), whereby joint tissues have an excess of activated neutrophiles
that secrete radicals, such as O2-. Under normal circumstances the radicals
are used to kill pathogenic microorganisms, but in the case of RA, the excess
of activated neutrophiles contribute to the inflammation and swelling, and
hence aggravate the disease symptoms. Other diseases in which reactive
oxygen species are implicated include atherosclerosis, adult respiratory
distress syndrome (ARDS), myocardial infarction and some forms of cancer.
Thus, the ability to scavenge radicals can prevent the onset of a disease,
slow down the progress of the disease, or alleviate its symptoms.
Aside from vitamins C and E many other compounds present in fruits
and vegetables have been shown to have anti-oxidant properties. Among
these compounds there are several classes of phenolic compounds. Aside
from preventing scurvy these compounds have a positive influence on
cardiovascular health.
An interesting case is the prevention of cardio-vascular diseases as a
result of the consumption of wine. Like most fruits grapes are rich in
polyphenols, and the process of wine making results in the concentration of
polyphenols. Wine polyphenols are considered to have beneficial effects on
coronary heart disease and atherosclerosis. The presence of polyphenols in
wine are thought to be the reason for the ‘French paradox’: France was
shown to have a coronary mortality rate close to that of China and Japan in
spite of the high amount of saturated fat and cholesterol levels in the French
diet. The consumption of red wine in France, however, is considerably
higher than in either China or Japan (Staggs, 1996).
Wollin and Jones (2001) investigated the effects and mechanisms of
action of consumption of red wine compared to other alcoholic beverages on
the risk of cardiovascular disease. Of particular interest was the form and
quantity of alcohol consumed. The relationship between alcohol
consumption and mortality is supported by epidemiologic studies suggesting
that different forms of alcohol alter the relative risk for mortality. Evidence
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Chapter 7
from various epidemiologic and experimental studies suggests a protective
effect against the development of cardiovascular disease by moderate
consumption of red wine. They point out that components of red wine that
are thought to be responsible for the protective effects include various
phenolic compounds as well as alcohol content.
The effects of wine and its polyphenol constituents on early indicators
of coronary heart disease such as elevated levels of plasma lipids, platelets
and serum antioxidant activity were discussed in a review by Cooper et al.
(2004). This review also addressed whether the polyphenols or alcohol are
responsible for the beneficial effects of wine on cardio-vascular health. The
authors conclude that red wine polyphenols have little effect on plasma lipid
concentrations, but that wine consumption reduces the susceptibility of lowdensity lipoprotein (LDL) cholesterol to oxidation and increase serum
antioxidant capacity. These effects, however, do depend on the amount of
wine that is consumed and the period of supplementation. It was suggested
that specific polyphenols appear to have endothelium-dependent vasorelaxing abilities. Red wine phenolics also have an inhibitory effect on
platelet aggregation. Evidence suggests that alcohol has a positive
synergistic effect with wine polyphenols on some atherosclerosis risk
factors. Thus, evidence that wine drinking is beneficial for cardiac health
appears positive.
Flavonoids may benefit health in cardiovascular disease by adjusting
adhesion of monocytes (large mononuclear leukocytes in the blood) in the
inflammatory process of atherosclerosis. Most in vitro studies have used
types of flavonoids present in food rather than those that appear in plasma
after food ingestion. Koga and Meydani (2001) tested the effects of plasma
metabolites of the flavonoids (+)-catechin (1.39) and quercetin (1.43) on the
alteration of monocyte adhesion to human aortic endothelial cells and on the
production of reactive oxygen species. Plasma extracts of flavonoid
metabolites were prepared after administration of pure compounds to rats.
The plasma preparations contained sulfate or glucuronide conjugates or
both, as well as methylated forms. Adhesion of U937 monocyte cells to
human aortic endothelial cells was measured, and the production of reactive
oxygen in the endothelial cells was monitored, when the cells were
pretreated with either pure compounds or plasma extracts from control or
treated rats. Adhesion assays were performed with endothelial cells
stimulated with interleukin or cells activated with phorbol myristylacetate.
Reactive oxygen species were measured after challenging the human aortic
endothelial cells with interleukin-1b (IL-1b) or hydrogen peroxide.
Phenolic compounds and their effects on human health
241
Pretreatment of endothelial cells with (+)-catechin (1.39) metabolites
inhibited U937 cell adhesion to interleukin IL-1b-stimulated endothelial
cells, whereas pretreatment with intact (+)-catechin had no effect.
Generation of reactive oxygen species in hydrogen peroxide-stimulated cells
was inhibited by (+)-catechin, its metabolites, and control plasma extract,
whereas reactive oxygen species generation in IL-1b-stimulated cells was
inhibited by (+)-catechin metabolites only. In contrast, quercetin inhibited
U937 cell adhesion to interleukin IL-1b-stimulated cells, whereas its
metabolites were not effective. The authors concluded that metabolic
conversion of flavonoids such as (+)-catechin and quercetin modifies the
biological activity of the flavonoids. It was suggested that metabolites of
flavonoids, rather than their intact forms, contribute to the effects of
flavonoids on reducing the risk of cardiovascular disease.
The seeds of fenugreek (Trigonella spp.) are rich in phenolic
compounds. Kaviarasan et al. (2004) evaluated fenugreek seeds for their
potential to protect erythrocytes from oxidation induced by hydrogen
peroxide (H2O2). Human erythrocytes from diabetic and non-diabetic
subjects were incubated with increasing amounts of fenugreek seed extract
and challenged with H2O2. They were then analyzed for hemolysis (release
of hemoglobin) and lipid peroxidation. Erythrocytes from diabetic subjects
were more susceptible to hemolysis and lipid peroxidation than those from
non-diabetic subjects. It was significant that incubation of the cells with the
polyphenol-rich seed extract significantly reduced the oxidative
modifications in both cell groups. The inhibition of lipid peroxidation was
concentration-dependent. The extract contained 0.75 mM gallic acidequivalents (1.41) of phenolic compounds. The findings demonstrated the
potent antioxidant properties of the phenol-rich fenugreek seeds.
Palmerini et al. (2005) reported that phenols in the Mediterranean diet
are free radical scavengers and have antioxidant properties. Yet the
mechanisms of their effects are not fully understood. Palmerini and coworkers hypothesized an effect on the concentration of Ca2+, which plays an
important role in intracellular signaling and regulates various processes. To
test this hypothesis they incubated human lymphomonocytes with two
phenolic compounds isolated from olive oil: 3,4-dihydroxyphenyl-ethanol
(7.11) and p-hydroxyphenyl-ethanol (7.12). They showed that both of these
compounds increased the intracellular concentration of Ca2+ in a dosedependent manner, both in the presence and in the absence of calcium in the
extracellular medium. This effect was antagonized by the drug nifedipine
(7.13), a calcium channel blocker administered as a muscle relaxing agent to
patients suffering from chest pain.
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Chapter 7
OH
OH
H
N
O
O
H3CO
H
OCH3
NO2
OH
OH
OH
(7.11)
(7.12)
(7.13)
The olives themselves contain many phenolic compounds with
antioxidant properties. Bouaziz et al. (2005) investigated the olive cultivar
“Chemlali” from Tunisia. Oleuropein (7.14), a bitter glycoside esterified
with a phenolic acid, was the major compound present. Phenolic monomers
and twelve flavonoids were also identified. The antioxidant activity of the
extract was evaluated. Acid hydrolysis of the extract enhanced its
antioxidant activity. p-Hydroxyphenyl-ethanol (7.12) and quercetin (1.43)
showed antioxidant activities similar to that of 2,6-di-tert-butyl-4-methyl
phenol (7.15), a reference compound with known antioxidant properties. It
was suggested that a hydroxyl group at the ortho-position on the flavonoid B
ring could contribute to the antioxidant activity of the flavonoids.
HO
O
O
O
HO
C
OCH3
H
OH
O
(7.15)
H O
H O
HO
OH
HO
H
H
(7.14)
OH
H
Phenolic compounds and their effects on human health
243
In addition to the contribution of olives and olive oil to antioxidant
activity in the Mediterranean diet, spices and dressings have also been
shown to have health-promoting activity. In a study by Ninfali et al. (2005),
twenty-seven vegetables, fifteen aromatic herbs and some spices consumed
in Central Italy were studied to determine total phenolic flavonoid content as
well as their antioxidant capacity measured by the oxygen radical
absorbance capacity method. A comparison of antioxidant capacity was
made between different salads, as well as between salads to which aromatic
herbs had been added. Lemon balm and marjoram at a concentration of
1.5% (w/w) increased the antioxidant capacity of a salad by 150% and
200%, respectively. A 200-gram portion of a salad enriched with marjoram
corresponded to an intake of 200 mg phenolics and 4000 oxygen radical
absorbance capacity units. Olive oils, wine and apple vinegars were salad
dressings that provided the highest increase in antioxidant capacity. Of the
spices tested, cumin and ginger made the most significant contribution to the
antioxidant capacity.
Both intact compounds and their metabolites - formed either in human
tissues or meabolized in the colon by microflora - might explain the effects
on health of dietary polyphenols. In order to assess the importance and
biological activities of microbial metabolites in vivo, Gonthier et al. (2003)
measured the microbial metabolites formed in rats fed a diet supplemented
with three levels of catechin (1.43) or red wine powder containing
proanthocyanidins, phenolic acids, flavanols, anthocyanins and flavonols.
This was compared to rats fed an unsupplemented diet. Aromatic acid
metabolites in urine were assayed by an HPLC-electrospray ionization-mass
spectrometry method. The primary metabolites formed from the catechin
diet were 3-hydroxyphenylpropionic acid, 3-hydroxybenzoic acid and 3hydroxyhippuric acid. Their total urinary excretion accounted for 4.7%
(w/w) of the catechin ingested, and that of intact catechins for 45.3% (w/w).
When the diet was supplemented with red wine powder, the same
metabolites observed with the catechin diet were identified in urine, along
with p-hydroxybenzoic (1.4), 3-hydroxyphenylacetic (see 1.11 for a
comparable structure), p-coumaric (1.13), vanillic (1.8), and hippuric (Nbenzoylglycine) acids. These aromatic acids accounted for 9.2% (w/w) of
the total wine polyphenols ingested, whereas intact catechins accounted for
only 1.2% (w/w). It was suggested that the higher excretion of aromatic
acids by rats fed wine polyphenols was due to their poor absorption in the
proximal part of the gut. Some of the microbial metabolites still contained a
reducing phenolic group and should also prevent oxidative stress in inner
tissues. The authors suggested that attention be given in the future to these
244
Chapter 7
microbial metabolites and their biological properties to help explain the
effects of polyphenols that are not easily absorbed through the gut.
Whole grains provide another source of phenolic antioxidants, but whole
grains contain many other compounds that have a positive effect on human
health. They have high concentrations of dietary fiber, starch, and
oligosaccharides, and contain phytate, phyto-oestrogens such as lignans,
plant stanols and sterols, vitamins and minerals. Epidemiological studies
have shown that whole-grain intake is protective against cancer,
cardiovascular disease, diabetes, and obesity (Slavin et al., 2004). Despite
recommendations to consume three servings of whole grains daily, usual
intake in Western countries is only about one serving. Feeding studies show
that consumption of whole grains improves biomarkers such as weight loss,
blood-lipid levels, and the concentration of antioxidants. Although it is
difficult to separate the protective properties of whole grains from dietary
fiber and other components, the disease protection seen from whole grains in
prospective epidemiological studies far exceeds the protection from isolated
nutrients and phytochemicals in whole grains
An interesting question is whether the health-promoting properties of
phenolic compounds is consistent, or whether there are effects of the culture
practice during crop production, the location of the field where the crop is
grown, and the specific cultivar that was selected. Emmons and Peterson
(2001) investigated whether cultivar and location had an effect on phenolic
contents and antioxidant activities of alcohol-soluble extracts from groats
(i.e. the edible part of the grain) of oat (Avena sativa L.). Antioxidant
activities (AOA) and concentrations of eight phenolic compounds having
AOA were measured in three cultivars grown at seven locations in
Wisconsin during 1998. The phenolic compounds included p-coumaric acid
(1.13), ferulic acid (1.15) and avenanthramides (7.16a-d). Avenanthramides
are phytoalexins found in oats. There are several different compounds –
avenanthramide A, B, D and G – that differ in the substitution pattern of the
two aromatic rings, as is shown below. Avenanthramide L (7.17) contains an
additional carbon in the chain linking the two aromatic rings (Okazaki et al.,
2004).
Phenolic compounds and their effects on human health
O
HO
R2
HN
R1
245
R3
HO
O
R2 = H
R3 = OH
b. Avenanthramide B: R1 = OMe R2 = H
a. Avenanthramide A: R1 = H
R3 = OH
c. Avenanthramide D: R1 = H
R2 = H
R3 = H
d. Avenanthramide G: R1 = H
R2 = OH
(7.16)
O
HN
OH
HO
HO
O
(7.17)
There were significant differences among cultivars for AOA
concentrations of all of the phenolic compounds measured, except pcoumaric and ferulic acids, and for total free phenolic contents (FPC).
Location significantly affected the concentrations of five of the phenolics
and total FPC, but did not affect AOA. There were significant cultivar x
location interactions for the concentrations of avenanthramides and for total
FPC. The presence of this interaction means that the cultivar with the
highest FPC level in one location, does not produce the highest FPC levels
in another location. The unexpectedly high concentrations of
avenanthramides from the Sturgeon Bay location were confirmed by
analysis of groats from 1999 and 2000. Based on these observations it
should be possible to improve the AOA and phenolic concentrations of oat
as quantitative traits in a cultivar development program (see Chapter 3,
Section 3.4), but significant location effects may slow down progress.
Similarly, Tarozzi et al. (2004) assessed the impact of cultivation
practices, commercial processing, and storage of fruits and vegetables on
phenolic antioxidants. They investigated the influence of commercial coldstorage periods on antioxidant properties of apples grown by organic or
246
Chapter 7
integrated systems. Regardless of the production method, total phenolics and
total antioxidant activity decreased in apples with the peel intact only in the
first three months of storage. It was suggested that cold storage rapidly
depletes antioxidant properties in apple skin but not in the pulp. Antioxidant
activity was assessed in vitro in terms of intracellular antioxidant,
cytoprotective, and anti-proliferative activities in human colon carcinoma
(Caco-2) cells. Time-related decreases in antioxidant activity after six
months cold storage were found regardless of the cultivation method. These
data suggest that cold storage should be taken into account when evaluating
the cancer-preventive benefits of fruits and vegetables. Furthermore, the
authors concluded that organic production methods of apples do not provide
health benefits. This latter conclusion is in contrast with a study by Halweil
(2003), who concluded that organic produce was richer in health-promoting
phenolic compounds.
3.
DISEASE PREVENTION
From the previous section it is apparent that the antioxidant activity of
polyphenols offers many health benefits. There are also cases where the
impact of phenolic compounds in the diet may not be related to their
antioxidant activity. For example, phenols might exert effects within the
gastrointestinal tract. Such effects could include binding of iron, scavenging
reactive nitrogen, chlorine, and oxygen species, and inhibition of
cyclooxygenases and lipoxygenases (Halliwell et al., 2005). This section
therefore focuses on the role of phenols in disease prevention, where the
precise mechanism is either unknown, or related to activity other than
antioxidant activity.
The stilbene trans-resveratrol (trans-3,5,4′-trihydroxystilbene; 1.60) is
present in grapes and wines. Resveratrol has been shown to have cancer
chemopreventive activity in the three major stages of carcinogenesis:
promotion, initiation, and progression (Jang et al., 1997). In in vitro assays
performed on tumor cell lines, resveratrol was shown to effectively inhibit
cyclo-oxygenase (COX), an enzyme that catalyzes the formation of proinflammatory compounds implicated in tumor cell growth and the
suppression of immune surveillance mechanisms. Furthermore, rats in which
inflammation had been induced through injection of carrageenan showed
less severe symptoms after they were treated with resveratrol. The antipromotion activity of resveratrol was demonstrated by showing a dosedependent inhibitory effect on the formation of free radicals in cultured HL60 promyelocytic leukemia cells treated with the inflammation-inducing
compound 12-O-tetradecanoylphorbol-13-acetate (TPA). In addition,
Phenolic compounds and their effects on human health
247
resveratrol inhibited in a dose-dependent manner the mutagenic effects of
7,12-dimethylbenz(a)anthracene (DMBA) in assays performed on
Salmonella typhimurium strain TM677. Finally, the anti-progression activity
of resveratrol was demonstrated by showing that cultured HL-60 cells
treated with resveratrol were no longer able to proliferate indefinitely, but
rather developed symptoms of terminal differentiation to a non-proliferative
phenotype.
The anti-carcinogenic effects of resveratrol were also tested in vivo, by
examination of mammary glands of mice treated with DMBA and TPA. A
dose-dependent reduction in the formation of tumors was observed when the
mice were treated with resveratrol. Resveratrol is present in high
concentrations in the skins of grapes (50-100 µg/g) and, consequently, in red
wine (1.5-3 mg/l). Based on these experiments, the consumption of grapes
and grape products appears to have beneficial effects.
Resveratrol has also been reported to offer protection against
cardiovascular disease, such as coronary heart disease. The effects of
resveratrol on factors implicated in the development of coronary heart
disease – human platelet aggregation and the synthesis of eicosanoids
(lipids) from arachidonate by platelets – were investigated and compared
with the actions of other wine phenolics - catechin (1.39), epicatechin
(7.18a), and quercetin (1.43) - and the antioxidants α-tocopherol (7.10a),
hydroquinone and butylated hydroxytoluene. Resveratrol and quercetin
demonstrated a dose-dependent inhibition of platelet aggregation, whereas
the other compounds tested were inactive. Resveratrol also inhibited the
synthesis of the eicosanoids in a dose-dependent manner, whereas the other
phenolics were less effective of not effective at all. Removal of the alcohol
from the wine did not diminish the effect on platelet aggregation (PaceAsciak et al., 1995; Goldberg et al., 1995).
While consumption of products rich in resveratrol appears to be
beneficial for the reasons discussed above, little is known about resveratrol
bioavailability in humans and animals. Emilia et al. (1999) developed an
analytical method to measure stilbene present in blood. Resveratrol
administered orally to rats was detected in plasma. Excellent HPLC-based
separation of trans-resveratrol from other compounds in the blood was
achieved, allowing a rapid analysis of the sample for absorption,
distribution, and metabolism studies.
A high dietary intake of fruits and vegetables has consistently been
associated with a reduced risk of various human cancers, including those of
the lung, breast, prostate, and colon. It is unknown which bioactive
248
Chapter 7
compound or compounds in plant foods provide these protective effects, but
flavonoids have been of special interest. There are numerous animal model
studies that suggested that flavonoids influence important cellular
mechanisms related to carcinogenesis, including cell cycle control and
apoptosis. There are limited data from studies with humans. In a review
artice by Neuhouser (2004) four studies are reviewed, in which associations
of flavonoid intake with cancer risk were examined. There is substantial
evidence that flavonoids, especially quercetin (1.43), reduce the risk of lung
cancer.
Green and black tea, obtained from dried leaves of the Chinese tea plant
(Camellia sinensis), are sources of different polyphenolic compounds. The
main polyphenols of green tea are (-)-epicatechin (7.18a), (-)-epicatechin 3gallate (7.18b), (-)-epigallocatechin (7.19a), and (-)-epigallocatechin 3gallate (7.19b), whereas in black tea theaflavin (7.20) and thearubigin (7.21)
are the most abundant. Green tea is prepared by steaming or pan-frying
freshly picked tea leaves. This treatment inactivates oxidases, so that the
catechins in the leaves remain stable and thus contribute to the characteristic
color and smell of green tea. For the production of black tea, the leaves are
left to dry down to approximately 55% of the fresh weight, rolled and then
crushed. Theaflavin and thearubigen arise from oxidation of catechins
during this process. Oolong tea is produced in a similar manner as black tea,
except that the oxidation process is stopped by firing the dried and rolled
leaves (Mukhtar and Ahmad, 2000).
Haqqi et al. (1999) investigated the impact of polyphenols present in
green tea on rheumatoid arthritis (RA). The study used mice as test subjects
and showed that although 92% of ordinary mice developed RA when
injected with a compound that induced RA, less than half of the mice that
consumed the green tea polyphenols developed RA after a similar injection.
The health benefits of both green and black tea were summarized by
Mukhtar and Ahmad (2000). Consumption of tea and its polyphenolic
constituents offers protection against skin cancer induced by either chemical
carcinogens or ultraviolet radiation in mice. Tea consumption also provides
protection against cancers induced by chemical carcinogens that involve the
lung, forestomach, esophagus, duodenum, pancreas, liver, breast, colon, and
skin in mice, rats, and hamsters. As was shown for resveratrol from grapes,
polyphenolics from green tea do not only have anti-carcinogenic properties,
but also protect against coronary heart disease and atherosclerosis, as well as
inflammation. The latter effect was demonstrated by showing anti-
Phenolic compounds and their effects on human health
OH
OH
OH
HO
O
249
OH
HO
R1
O
R1
OH
O
OH
OH
OH
O
a. R1 = H
b. R1 = OH
a. R1 = H
b. R1 = OH
OH
OH
(7.18)
(7.19)
OH
OH
OH
OH
OH
COOH
HO
OH HO
O
COOH
O
O
HO
O
O
HO
O
OH
OH
OH
OH
OH
OH
(7.20)
(7.21)
inflammatory effects of green tea in response to UV-B radiation and the
inflammation-inducing compound TPA.
The mechanisms underlying the protective role of tea polyphenols have
been investigated in a number of studies. Green tea polyphenols were shown
to transcriptionally activate a signaling cascade that resulted in the activation
of detoxifying enzymes involved in the elimination of chemical carcinogens
(Yu et al., 1997). Apoptosis (programmed cell death) is a way to clean up cells
that are no longer needed or cells that have aged to the point where they start
to malfunction. Certain types of cancer have been shown to arise from a
disturbance in the process of apoptosis, whereby damaged cells do not get
eliminated, or whereby carcinogens result in the elimination of functional cells
that are critical. A report by Ahmad et al. (1997) showed that green tea
250
Chapter 7
polyphenolics induced apoptosis and cell cycle arrest in human epidermoid
carcinoma cells. This apoptotic response was specific to cancer cells,
because the induction of apoptosis was also observed in several other types
of cancer cell lines, but not, for example, in normal human epidermal cell
lines. Dong et al. (1997) used a mouse epidermal cell line to examine the
anti-tumor promotion effects of polyphenolics from green and black teas.
They showed that these compounds inhibited the formation of tumor cells
resulting from the application of epidermal growth factor or TPA, and this
occurred in a dose-dependent manner. They also showed that polyphenolics
from tea inhibited the transcriptional activation of genes regulated by the
transcription factor AP-1. This transcription factor has been shown to be
important for the formation of tumor cells.
Ellagitannins are dietary polyphenols containing ellagic acid (1.96)
subunits that are thought to act as cancer chemo-preventive agents. Thus,
they may have properties that contribute to health benefits in humans. Little
is known, however, of their metabolic fate. Cerdá et al. (2005) investigated
the metabolism of different dietary ellagitannin derivatives in humans.
Healthy volunteers in four groups consumed, in a single dose, a different
ellagitannin-containing food. The reported consumption was strawberries
(250 g), red raspberries (225 g), walnuts (35 g), and oak-aged red wine (300
ml). Urine fractions were collected at 8, 16, 32, 40, and 56 hours after
consumption. Neither ellagitannins nor ellagic acid were detected in the
urine samples by liquid chromatography-MS/MS analysis. However, a
microbial metabolite 3,8-dihydroxy-6H-dibenzo-[b,d]-pyran-6-one (urolithin
B; 7.22) conjugated with glucuronic acid was detected in all study
participants independent of the consumed food.
OH
O
O
OH
(7.22)
These authors reported that considerable differences among and between
individuals were found, which identified individuals who excreted high and
Phenolic compounds and their effects on human health
251
low metabolite levels. The variation in excretion of these metabolite levels
was likely reflective of the micro-flora in the colon during ellagitannin
metabolism. These results indicate that urolithin B is a marker of exposure
to dietary ellagitannins and may be useful in intervention studies with
ellagitannin-containing products.
Proanthocyanidins and tannin-like compounds are complex flavonoid
polymers naturally present in cereals, legume seeds and are frequently found
in some fruits and fruit juices. They share some common structural features
with phenolic polymers found in black tea and red wine. The polymeric
nature of proanthocyanidins makes their analysis in food difficult. Thus,
little is known about their consumption, although they probably contribute a
large part of the daily polyphenol consumption. They also share common
physico-chemical properties: they form stable complexes with metal ions
and proteins and are good reducing agents. Many of their biological
attributes of nutritional interest derive from these properties. As metal ion
chelators, they influence the availability of several minerals. The nutritional
significance of the property of forming non-specific complexes with proteins
is not clear. As reducing agents, they may participate in prevention of
cancers of the digestive tract and inner organs. They may also protect lowdensity lipids (LDLs) against oxidation and inhibit platelet aggregation. This
property would act as a preventative of cardiovascular diseases (SantosBuelga and Scalbert, 2000).
Since epidemiological studies have suggested a correlation between high
flavonoid consumption and decreased risk of cancer, cardiovascular disease,
and other age-related diseases, enhancing flavonoid biosynthesis in crops
may result in foods with benefits to human health. Verhoeyen et al. (2002)
attempted to generate transgenic tomatoes with increased levels of
flavonoids. They achieved a 78-fold increase in fruit flavonols achieved
through ectopic expression of the gene encoding chalcone isomerase.
Furthermore, they observed that chalcone synthase and flavonol synthase
transgenes were found to act synergistically to up-regulate flavonol
biosynthesis in tomato tissues.
4.
ACTIVITY AGAINST TOXINS
Plant phenolic compounds have also been suggested to provide a means
for preventing the adverse affects that fungal toxins (mycotoxins) have on
human health as well as serving in their detoxification (Beekrum et al.,
2003). These authors investigated the impact of the plant phenolic
252
Chapter 7
compounds chlorophorin (4-homogeranyl-2,3',4',5'-tetrahydroxystilbene;
7.23), iroko, maakianin, benzoic acid, caffeic acid (1.14), ferulic acid (1.15),
and vanillic acid (1.8) on the growth of Fusarium verticillioides and its
production of the mycotoxin fumonisin B-1. The stilbene chlorophorin was
the most effective compound in inhibiting fungal growth and in reducing
toxin production. As a group these phenols reduced fumonisin levels by
more than 90%. The authors suggested that the widespread occurrence of
fumonisins and the lack of adequate prevention measures require
‘biologically safe’ alternatives to prevent the transfer of fungi and their
hazardous toxins into foods.
OH
OH
OH
OH
(7.23)
Phenolic compounds and their effects on human health
5.
253
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Chapter title
257
Appendix
Alphabetical list of the compounds represented by chemical
structures
The compounds are listed in alphabetical order, whereby numbers, prefixes
such as para or O- have been ignored. For example, ‘p-coumaric acid’ is
listed under ‘C’, and ‘1,2-dihydroxybenzene’ is listed under ‘D’. When
applicable, the compound may be listed multiple times. In the example
above, ‘1,2-dihydroxybenzene’ is also listed under ‘B’, as ‘benzene, 1,2dihydroxy’.
A
acetyl bromide
Acutissimin A
4-aminobutanal,
Anthocyanidin
Anthocyanin
Apigeninidin
Apimaysin
Arbutin
Arogenate
L-ascorbate
Aurones
avenanthramide A, B, D, G
avenanthramide L
4.10
1.95
2.57
3.65
3.66
1.51
6.17b
2.46
3.26
7.8
1.28
7.16
7.17
257
258
B
Benzene
benzene, 1,3,5-trihydroxy
benzene, 1,4-dihydroxy
Benzidine
benzoic acid
benzophenone
benzoquinone, 2,6-dimethoxy
bergenin
betanidin
bisphenol A
butanal, 4-amino
butein
butyrolactones
C
caffeic acid
caffeoyl-CoA
caffeoyl-CoA, D-quinate ester
caffeoyl-CoA, shikimate ester
catechin
catechol
catechin, gallic acid ester
chalcones
chlorogenic acid
chlorogenoquinone
chlorophorin
chorismate
cinnamic acid
coniferaldehyde
coniferyl alcohol
coniferyl aldehyde
Cornusiin E
coumaric acid
Appendix
2.1; 2.2
4.30
2.42
4.33
3.40
1.58; 7.4
1.62
1.23
1.66
7.7
2.57
1.26
6.12; 6.13
1.14; 3.32
3.36
3.73
3.74
1.39; 2.9
2.11; 2.20
1.41
1.25
1.18; 2.24; 6.4
6.5
7.23
3.24
1.12; 3.29
1.75; 3.76;
4.31
1.69; 2.48;
3.79
1.75; 3.76;
4.31
3.103
2.26; 3.42;
3.94
Appendix
259
trans-coumaric acid-2-O-glucoside
p-coumaric acid
coumarin
p-coumaroyl-CoA
p-coumaroyl-CoA, D-quinate ester
p-coumaroyl CoA, shikimate ester
p-coumaryl alcohol
p-coumaryl aldehyde
cyanidin
3.95
1.13; 3.30
2.25; 3.96
3.31
3.71
3.72
1.68; 3.70
3.69
1.46
D
dalbergin
L-dehydroascorbate
2,6-dimethoxybenzoquinone
2-O-digalloyl-1,3,4,6-tetra-O-galloyl- -D-glucopyranose
1,4-dihydroxybenzene
3,4-dihydroxyphenylethanol
3-dehydroquinate
3-dehydroshikimate
5-dehydroshikimate
delphinidin
3-deoxy-D-arabino-heptulosonate 7- phosphate
deoxyarbutin
deoxypodophyllotoxin
deoxypodophyllotoxin
meta-depside bonds
dhurrin
dianin
dihydrochalcones
dihydroconiferyl alcohol
dihydrokaempferol
dihydromyricetin
dihydroquercetin
dihydroxyflavone
dihydroxyacetone-phosphate
dihydroxychalcone
dihydroxyphenylalanine
1.34
7.9
1.62
1.89
2.42
7.11
3.19
3.20
3.46
1.48
3.18
2.47
3.83
3.85
1.87
6.9
2.14
1.24
1.76
3.52
3.55
3.54
6.17
3.4
2.21
2.41
260
Appendix
DIMBOA
2,6-di-tert-butyl-4-methylphenol
6.3
7.15
E
ellagic acid
emodin
5-enolpyruvylshikimate 3-phosphate
epicatechin
epicatechin-3-gallate
epigallocatechin
epigallocatechin-3-gallate
erythrose-4-phosphate
1.92; 2.23; 4.2
1.65
3.23
7.18
7.18
7.19
7.19
3.17
F
ferulic acic
feruoyl-CoA
flavanone
flavanonol
3-flaven-2,3-diol
2-flaven-3,4-diol
flavone
fructose-1,6-bisphosphate
fructose-6-phosphate
1.15; 3.33
3.37; 3.75
1.29; 6.16;
6.18
2.4
3.64
3.63
2.3
3.3
3.2
G
gallic acid
gallocatechin
ginkgetin
–glucogallin
gluconate-6-phosphate
gluconolactone-6-phosphate
-D-glucopyranose, 1,2,3,4,6-penta-O-galloyl-D-glucopyranose, 2-O-digalloyl-1,3,4,6-tetra-O-galloylD-glucose
glucose-6-phosphate
-D-glucuronide, 4-methylumbelliferyl
1.5; 2.22; 3.47
1.40
1.57
3.98
3.12
3.11
3.102
1.89
2.31
3.1
1.22
Appendix
261
glyceraldehyde-3-phosphate
glycerate-1,3-bisphosphate
glycerate-3-phosphate
glycerate-2-phosphate
guaiacol
3.5
3.6
3.7; 3.21
3.8
4.34
H
heptulosonate 7- phosphate, 3-deoxy-D-arabino3,4,5,3′,4′,5′-hexahydroxydiphenoyl (HHDP) residues
4-homogeranyl-2,3',4',5'-tetrahydroxystilbene
2-hydroxyacetophenone
o-hydroxyacetophenone
p-hydroxybenzaldehyde
p-hydroxybenzoic acid
5-hydroxyconiferyl alcohol
5-hydroxyconiferyl aldehyde
5-hydroxyferulic acid
5-hydroxyferuoyl-CoA
2-hydroxyphenylacetic acid
p-hydroxyphenylethanol
3.18
3.97
7.23
1.10
2.1
4.13
1.4; 4.16
1.77;3.80
3.77
1.16; 3.34
3.38
1.11
7.12
I
indicaxanthin
indole-3-acetic acid
iridoskyrim
isochorismate
isoflavone
isoorientin
1.67
2.49
2.39
3.43
1.33
6.21
J
juglone
1.64
K
kaempferol
1.42; 3.53
L
L-(+)-lariciresinol
3.87
262
Appendix
leucoanthocyanidin
leucocyanidin
leucocyanidin
leucodelphinidin
leucopelargonidin
lignin
luteoferol
luteolinidin
3.61
1.37
3.59
1.38; 3.60
3.58
4.8; 4.11; 4.19;
4.26
1.97
1.52
M
malonyl-CoA
malvidin
maysin
menthylanthranilate
methoxybenzene
7-methoxyapigeninidin
5-methoxyluteolinidin
methoxymaysin
5-methoxypodophyllotoxin
3,4-methylenedioxycinnamic acid
3-methylene 2-oxindole
4-methylumbelliferyl -D-glucuronide
4-methylphenol, 2,6-di-tert-butylmyricetin
3.48
1.50
6.17a
7.6
2.3
1.53
1.54
6.17c
3.89
3.90
2,54
1.22
7.15
1.44; 3.57
N
naringenin
nifedipine
octylmethylcinnamate
octylsalicylate
oleuropein
oxalate
oxybenzone
1.35; 3.51
7.13
7.3
7.2
7.14
2,55
7.5
P
p-aminobenzoic acid
7.1
Appendix
263
pelargonidin
pentagalloylglucose
1,2,3,4,6-penta-O-galloyl- -D-glucopyranose
peonidin
peroxyl radical
petanin
petunidin
phenol
phenol
phenylalanine
phenylethanol, 3,4-dihydroxy
phlobaphene
phloretin
phloridzin
phloridzin
phloroglucinol
phloroglucinol
phloroglucinol
phosphoenolpyruvate
(+)-pinoresinol
pisatin
(–)-plicatic acid
podophyllotoxin
prephenate
procyanidin B2
protocatechuic acid
protocyanin
protoleucomelone
putrescine
pyranose
pyrogallol
pyruvate
∆1-pyrroline
1.45
1.88; 3.99
3.102
1.47
2.52
1.56
1.49
1.1
2.5
3.27
7.11
1.98
6.2
1.27
6.1
1.3
2.13
4.30
3.9
1.72; 3.82
6.23
1.74
3.86
3.25
1.86
1.6; 3.45, 6.14
2.15
2.29
2.56
2.32
4.35
3.101
2.58
Q
quercetin
quinines
1.43; 2.8; 3.56
2.37
264
Appendix
p-quinone
2.45
P
pinosylvin
protocatechuic acid
1.61
3.45; 6.16
R
resorcinol
resveratrol
resveratrol cis-dehydrodimer
resveratrol trans-dehydrodimer
rhamnosyl-isoorientin
rhodanine
ribose-5-phosphate
ribulose-5-phosphate
1.2; 2.12
1.60; 6.24
6.27
6.26
6.22
4.1
3.14
3.13
S
salicylic acid
(–)-secoisolariciresionol
sedoheptulose-7-phosphate
(+)-sesamin
sinapaldehyde
sinapic acid
sinapoyl-CoA
sinapoyl choline
1-O-sinapoyl glucose
sinapoyl malate
sinapyl alcohol
sinapyl aldehyde
skatolyl radical
stilbene, 4-homogeranyl-2,3',4',5'-tetrahydroxy
syringaldazine
syringaldehyde
syringic acid
1.7; 3.41; 6.28
3.88
3.16
1.73
3.78
1.17; 3.35
3.39
1.20; 3.93
3.91
1.19; 3.92
1.70; 3.81
3.78
2.51
7.23
4.36
4.15
4.18
T
tannins, condensed
3.68
Appendix
265
taxifolin
Tellimagrandin II
4,6,3′,4′-tetrahydroxyaurone
tetrahydroxybiphenyls
4,2′,4′,6′ tetrahydroxychalcone
2,3',4',5'-tetrahydroxystilbene, 4-homogeranyltheaflavin
thearubigin
thioglycolic acid
tocopherol
1,3,5-trihydroxybenzene
trihydroxycinnamic acid
2,4,6-trinitrophenol
tuliposide A
tuliposide B
tyrosine
1.36
3.104
3.50
2.36
3.49
7.23
7.20
7.21
4.7
7.10
4.30
3.44
2.6
6.10
6.11
2.40; 3.28
U
ubiquinone
umbelliferone
urolithin B
1.63
1.21
7.22
V
valoneoyl unit
vanillic acid
vanillin
vitamin C
vitamin E
trans- -viniferin
1.93
1.8; 4.17
1.9; 4.14
7.8
7.10
6.25
X
xanthone
xylulose 5-phosphate
1.59
3.15
Y
yatein
3.84
Index
267
Index
(–)-plicatic acid 19
(–)-secoisolariciresionol 109
(+)-pinoresinol 19, 107–109
(+)-sesamin 19
1,2,3,4,6-penta-O-galloyl- -Dglucopyranose 25,130, 131
1,3,5-trihydroxybenzene 3
1,4-dihydroxybenzene 51
1-O-sinapoylglucose 127
2,3',4',5'-tetrahydroxystilbene,
4-homogeranyl- 254
2,4,6-trinitrophenol 39
2,6-dimethoxybenzoquinone 17
2,6-di-tert-butyl-4-methylphenol 244
2-flavene-3,4-diol 93
2-hydroxyacetophenone 4
2-hydroxyphenylacetic acid 4
2-O-digalloyl-1,3,4,6-tetra-Ogalloyl- -D-glucopyranose 25
3,4,5,3′,4′,5′-hexahydroxydiphenoyl
(HHDP) residues 132
3,4-dihydroxyphenyl-ethanol 243
3,4-methylenedioxycinnamic acid
112
3-dehydroquinate 82
3-dehydroquinate dehydratase 82
3-dehydroquinate synthase 82
3-dehydroshikimate 82
3-deoxyanthocyanidins 224, 225
3-deoxy-D-arabino-heptulosonate
7-phosphate 82
3-flavene-2,3-diol 93
3-methylene 2-oxindole 54, 55
4,2′,4′,6′ tetrahydroxychalcone
91, 92
4,6,3′,4′-tetrahydroxyaurone 91
4’6 diamino 2-phenylindole (DAPI)
183
4-aminobutanal 56
4CL 84, 113, 114, 126, 230
4-coumaric acid:CoA ligase 84
4-homogeranyl-2,3',4',5'tetrahydroxystilbene 254
4-methylphenol, 2,6-di-tert-butyl
244
4-methylumbelliferyl -D-glucuronide 6
5-dehydroshikimate 88–90
5-dehydroshikimate dehydrogenase
89
5-enolpyruvylshikimate 3-phosphate
82
5-enolpyruvylshikimate 3-phosphate
synthase 82
5-hydroxyconiferyl alcohol 20, 102,
105, 106
5-hydroxyconiferyl aldehyde 105,
106
5-hydroxyferulic acid 5, 84, 125
5-methoxyluteolinidin 14
5-methoxypodophyllotoxin 109,
111, 112
7-methoxyapigeninidin 14
acetophenones 2, 4
acetyl bromide 164–166, 176
acid constant 38
acid detergent fiber (ADF) 161, 162
acid detergent lignin (ADL) 160,
161, 166, 182
activator (Ac) 46, 70, 93, 98,
100–102, 113, 114, 116
Acutissimin A 29
267
268
Agrobacterium tumefaciens 70
ammonium sulphate 65
aniline sulphate 183
anthocyanidin synthase 92, 97
anthocyanidins 2, 12–15, 24, 92, 93,
97, 168, 203, 204
anthocyanin 2, 15, 17, 43, 92, 93,
97, 98, 100, 102, 151, 153, 167,
168, 201, 206–208, 221, 245
anthocyanin 3-glycosyl transferase 97
Anthocyaninless1 (A1) 92, 98, 99, 102
anthocyanins 151, 153, 167, 168,
201, 202, 206–208, 221, 245
antioxidants 48, 58, 60, 238–240,
242–249
Antirrhinium majus 70, 91
apigeninidin 14
apimaysin 219–222
apoptosis 250–252
apple scab 216
Arabidopsis 70, 72, 81, 88, 90–93,
96, 100, 101, 105–107, 114–117,
126–128, 172, 182, 184, 230
arbutin 51
arogenate 82
arogenate dehydratase 82
arogenate dehydrogenase 82
aromatic 1, 29, 36, 37, 39, 43, 48,
60, 78, 81, 82, 88, 172, 237, 245,
246
aspen 106, 107, 117
atherosclerosis 241, 242, 250
atomic number 35
aureusidin synthase 91, 92, 94
aurones 2, 8, 43, 91, 92
auto-oxidation 48, 49
Avena sativa 72, 106, 246
avenanthramide A, B, D, G 246, 247
avenanthramide L 246
-D-glucopyranose, 1,2,3,4,6-pentaO-galloyl- 25, 130, 161
-D-glucopyranose, 2-O-digalloyl1,3,4,6-tetra-O-galloyl- 25
Index
-D-glucuronide,
4-methylumbelliferyl 6
–glucogallin 130, 131
-glucuronidase (GUS) 6, 7
-oxidation 86, 87
Banyuls 93, 97
beet army worm 215
benzene 1–3, 7, 35–38, 47,
112, 160
benzene, 1,3,5-trihydroxy 3
benzene, 1,4-dihydroxy 3
benzidine 188
benzodioxane 23, 123
benzoic acid 86, 87, 254
benzoic acid 2-hydroxylase 86, 87
benzophenones 2, 16, 237, 239
benzoquinone, 2,6-dimethoxy 17
bergenin 7
betacyanins 2, 17
betaine 206, 209, 210
betanidin 17
biflavonyls 2, 15, 48
bio-affinity chromatography 66
bisphenol A 239
black tea 250, 252, 253
Blumeria graminis 186
Booster 98
boron trifluoride etherate 173
Botrytis cinerea 218, 236
Botrytis tulipae 218
Brassicaceae 6, 126
Bronze1 93
brown midrib 106, 112, 115, 123, 124
butanal, 4-amino 56
butanol-HCl assay 153, 154
butein 8
butyrolactones 218
C3’H 103, 114
C4H 84, 114, 115
caffeate O-methyltransferase 84
caffeic acid 5, 14, 15, 23, 45, 84, 88,
89, 103, 106, 123, 125, 170, 254
caffeoyl-CoA 103, 105
Index
caffeoyl-CoA O-methyltransferase
84, 104, 105, 126
caffeoyl-CoA, D-quinate ester 103
caffeoyl-CoA, shikimate ester 105
callose 186
Camellia sinensis 250
cancer 30, 109, 238, 241, 246,
248–253
candidate gene 72–74, 127, 128, 220
capillary column 201
carcinogens 250, 252
cardiovascular disease 241–243,
246, 249, 253
Castanea 23
catalases 60
catechin 11, 12, 24, 25, 29, 41, 96,
153, 242, 243, 245, 249, 250
catechin, gallic acid ester 12
catechol 41, 45, 48, 49
catechol oxidase 50, 53
CcoA-OMT 84, 85, 106, 114, 115
cDNA 68, 69, 74, 75, 91, 92,
98–100, 102, 103, 105, 106,
113–116, 128, 221, 230
Cercospora nicotianae 228, 229
C-glycosyl flavones 219–221
chalcone isomerase 92, 94, 221, 253
chalcone synthase 91, 94, 98, 129,
221
chalcones 2, 8, 92
chemical ionization 198, 200, 202
chestnut 23
chlorogenic acid 5, 45, 103, 153,
215, 216, 220–222
chlorogenoquinone 215, 216
chlorophorin 254
cholesterol 241, 242
chlorine sulfite 183
chorismate 81, 82, 88
chorismate mutase 82, 87
chorismate synthase 82, 87, 88
chromones 2
cinnamic acid 4-hydroxylase 84
cinnamic acids 2, 5, 84, 86, 88
269
cinnamoyl-CoA reductase 102, 104,
105, 114, 176, 230
cinnamyl alcohol dehydrogenase
(CAD) 105–107, 113, 114,
122–124, 229, 230
cinnamyl alcohols 2, 104, 105, 123,
174
cinnamyl aldehydes 2
cloning 64, 67–74, 84, 100, 101,
103, 127
Colletotrichum circinans 219
Colletotrichum graminicola
186–188
Colorless1 (C1) 99, 100
Colorless2 (C2) 86, 91, 93, 98,
99, 102
complex tannins 23, 24, 29
COMT 84, 106, 114, 122, 123, 124
condensed tannins 11, 23–25, 90,
93, 96, 97, 101, 153–155
coniferaldehyde 20, 104–107, 123,
124, 126, 177, 184, 185
coniferaldehyde/coniferyl alcohol
O-methyltransferase 104
coniferyl alcohol 18, 20, 21, 52, 54,
55, 57, 102–109, 115–118, 122,
163, 184
coniferyl aldehyde 104, 105
coniferyl aldehyde/coniferyl alcohol
5-hydroxylase 104, 105
conjugated 36, 38, 59, 60, 252
copper amine oxidase 56
Cornusiin E 132, 133
coumaric acid 46, 86, 129
coumarins 2, 6, 7, 46, 84, 129
CuZn-superoxide dismutase 53, 54
cyanidin 12, 43, 154, 169, 204,
206, 207
cyanogenic glycosides 217
cyclo-oxgenases 248
cytochrome P450 86, 103, 106, 226
∆1-pyrroline 56
dalbergin 10
270
DAHP synthase 82
defective suppressor mutator
(dSpm) 70
dehydrogenation polymers (DHPs)
52, 82, 118, 163
delphinidin 12, 169, 204, 206
deoxyarbutin 51
deoxypodophyllotoxin 109, 112
derivatization followed by
reductive cleavage (DFRC)
176, 177
detector 169–171, 198, 199
D-glucose 25, 28, 47
dhurrin 217
diabetes 246
dianin 43
dibenzodioxocin 21
diethylsulfate (DES) 67
dihydrochalcones 2, 8
dihydroconiferyl alcohol 20
dihydroflavonol 4-reductase 92, 95,
98, 221
dihydrokaempferol 92
dihydromyricetin 92
dihydroquercetin 92
dihydroxyacetone-phosphate 77
dihydroxychalcone 45
dihydroxyflavonol 10
dihydroxyphenylalanine 50
DIMBOA 214
diphenoloxidase 50
direct injection 200, 204
dirigent protein 20, 21, 107, 118, 119
dissociation (Ds) 70, 93
double-focusing sector mass
spectrometer 199
electron impact 197–200, 202
electrospray ionization (ESI)
200–202, 204–206, 245
ellagic acid 27, 45, 157–159, 252
ellagitannins 24, 26–28, 86, 130,
132, 133, 156, 157, 252, 253
emodin 17
Index
enolase phosphopyruvate hydratase
77
epicatechin 24, 154, 249, 250
epicatechin-3-gallate 250
epigallocatechin 250
epigallocatechin-3-gallate 250
Erysiphe graminis 186
erythrose-4-phosphate 79
esterification 27, 45, 46, 105, 127,
129, 130, 218
ethanethiol 173, 174
ethanol 42, 124, 125, 153, 160, 164,
176, 183–185
ethers 41, 47
ethidium bromide 183
ethyl methanesulfonate (EMS)
67, 91
eucalyps 102
Eucalyptus grandis 114
Eucalyptus gunnii 102
expressed sequence tag (EST) 96
F5H 84, 105, 106, 114, 115, 122, 184
FAH1 105, 184
fast atom bombardment (FAB) 202,
204
fast protein liquid chromatography
(FPLC) 66
Fenton reaction 58, 60
Fenugreek 243
ferric chloride (FeCl3) 183
ferulate 5-hydroxylase 84, 184
ferulic acid 5, 15, 84, 105, 106,
112, 125, 126, 172, 174, 246,
247, 254
feruoyl-CoA 105
flavanone 3-hydroxylase 92, 94
flavanones 2, 9, 10, 92, 94, 222
flavanonols 2, 10, 11, 38, 92
flavans 2, 11
flavone synthase 92, 94, 222, 223
flavones 2, 10, 12, 15, 92, 220–223
flavonoid 3’5-hydroxylase 92, 94,
221
Index
flavonoid 3’-hydroxylase 92, 94, 221
Folin-Ciocalteu reagent 152, 153, 155
forage 123, 161, 162, 166, 178
Forestal solvents 168
formaldehyde 155
Forsythia intermedia 107
Fourier-transform infrared
spectroscopy (FT-IR) 181
French paradox 241
fructose biphosphate aldolase 77, 79
fructose-1,6-bisphosphate 77
fructose-6-phosphate 77, 79, 81
fumonisin 254
Fusarium graminearum 229
fusarium head blight 229
Fusarium oxysporum 218
Fusarium verticillioides 254
271
grains 125, 246
grapes 29, 206, 207, 226, 241,
248–250
green tea 250, 251
guaiacol 53, 188
guaiacyl 21, 117, 172, 184
H2O2 53, 58, 60, 117, 243, see also
hydrogen peroxide
HCT 103, 105, 114, 176
Heliothis zea, 215 see also tomato
fruit worm
Helminthosporium maydis 187, 188
hemolysis 243
heptulosonate 7- phosphate,
3-deoxy-D-arabino- 82
hexose phosphate isomerase 77,
79, 81
high-performance liquid
chromatography (HPLC) 66,
105, 169, 170, 172, 245, 249
Himalayan mayapple 109
histochemical stains 52, 53, 183,
185, 229
hybridization 35, 56, 99, 115
hydrogen bond 40–43
hydrogen cyanide (HCN) 217
hydrogen peroxide 53, 58, 163, 227,
242, 243
hydrophobic interaction
chromatography 66
hydroxycinnamic acids 45, 46, 84,
86, 88, 102, 116, 125, 126, 158,
160, 206
hydroxycinnamoyl-CoA
shikimate/quinate hydroxycinnamoyl transferase 103, 104,
176, see also HCT
hydroxyl radical 58, 59
hypersensitive response 186, 230
hypochlorite 58, 183
gallic acid 4, 12, 25, 45, 86, 88–90,
130–132, 153–158, 243
gallocatechin 11, 12
gallotannins 24–26, 86, 130, 131,
155, 156
GAP-dehydrogenase 77, 79
gas chromatography (GC) 162, 167,
170–174, 176, 178, 200, 202
gel filtration 66
gene chips 15
general phenylpropanoid pathway
84, 86, 89, 102, 114, 125, 127
germin 55
ginkgetin 15
gluconate-6-phosphate 79, 81
gluconolactone-6-phosphate 79
glucose-6-phosphate 77, 79, 81
glyceraldehyde-3-phosphate 77,
79, 81
glycerate phosphate mutase 77, 79
glycerate-1,3-bisphosphate 77
glycerate-2-phosphate 77
glycerate-3-phosphate 77
glycerate-3-phosphate kinase 77, 79
glycolysis 76, 77, 79, 81, 82
indicaxanthin 17
glycosides 2, 15, 17, 47, 151, 185, 217 indole-3-acetic acid 54
272
interleukin 242, 243
ion exchange chromatography 65
ion trap mass analyzer 198
ionization 119, 171, 197, 198, 200,
202–208, 245
iridoskyrim 48
isochorismate 88
isochorismate synthase 87, 88
isocoumarins 2, 7
isoflavone 10
isoorientin 222
jasmonic acid 229
juglone 17, 51
kaempferol 92
Klason lignin 160–162, 166
L-(+)-lariciresinol 109
Laccases 50–52, 107, 117, 132,
133, 226
L-ascorbate 240, 241
L-dehydroascorbate 240
Leucoanthocyanidin 10, 11, 92, 93
leucocyanidin 1, 92, 96
leucodelphinidin 11, 92
leucopelargonidin 92
lignans 2, 18, 19, 20, 53, 102, 107,
108, 118, 120, 246
lignin 2, 20, 21, 52, 53, 57, 102,
105–107, 112–126, 159–166,
171–178, 180–188, 219,
229–231
Linum album 109, 112
Linum flavum 109, 111, 112
lipid peroxidation 59, 240, 243
lipoxygenases 248
liquid chromatography (LC) 169,
170, 200–202, 204, 252
loblolly pine 114, 117
low-density lipoprotein (LDL)
242, 253
luteoferol 30
luteolinidin 14
Index
Lycopersicon esculentum 70, 215,
see also tomato
maize 15, 30, 70, 84, 91–93,
98–101, 106, 115, 118, 123,
124, 171, 182, 186–188, 220,
221, 223, 230
malonyl-CoA 9, 91, 130
malvidin 12, 206, 207
map-based cloning 72, 100, 127
matrix-assisted laser desorption
ionization (MALDI) 200, 202,
204, 206–208
Mäule reagent 184
mayapple 109
maysin 219–223
melanin 51
menthylanthranilate 237
messenger RNA (mRNA) 55, 63,
68, 229
meta-depside bonds 25
metal complexes 43, 45
methoxybenzene 47
methoxymaysin 219–222
methylene Blue 184
microarray 115
molecular ion 199
monolignols 18–20, 52, 54, 57,
84, 102, 104, 107, 112,
116–119, 176
monooxygenases 53
monophenol monooxygenase 50, 53
multigene family genes 68, 106, 112
multivariate statistics 182
mutator (Mu) 70, 93
myricetin 12, 92
N 75, 92, 101, 162, 165, 169, 209
NaNO2/HCl assay 158
naringenin 10, 30, 92
near infrared reflectance (NIR)
spectroscopy 181, 182
Nectria haematococca 226
neolignans 2, 19
Index
273
p-coumaroyl-CoA, D-quinate ester
103, 105
p-coumaryl alcohol 18, 20, 21, 102,
117, 118
p-coumaryl aldehyde 102, 105
p-diphenoloxidase 50
pearl millet 123, 124
pectinases 217
pelargonidin 12, 204
Pennisetum glaucum 123
pentagalloylglucose 25, 26, 130–133
oak 23, 29, 130, 169, 183, 252
pentagalloylglucose: O2
oat 246, 247
obesity 246
oxidoreductase 133
octylmethylcinnamate 237
pentose phosphate pathway 76, 77,
octylsalicylate 237
79, 81, 82
o-diphenolase 50
peonidin 12, 206, 207
o-hydroxyacetophenone 41
peroxidases 18, 50, 53–57, 60, 107,
oleuropein 244
117, 118, 163, 187–189, 226, 230
olives 243–245
peroxide 58
orbital 35–38, 40
peroxyl radical 55, 59
Oryza sativa 72, 106
Pestalotia palmarum 230
oxalate 55–57
petanin 15
oxalate oxidase 55, 56
Petunia 91
oxidation 48–51, 53–58, 60, 64, 79, petunidin 12, 206, 207
86, 117, 126, 127, 157, 168, 172, phenol 1, 2, 4, 18, 23, 38–40, 42,
216, 240, 242, 243, 250, 253
45, 47, 48, 50, 103, 152, 158,
oxybenzone 237
199–201, 237, 239, 243, 244
phenolase 50, 53
PAL, see phenylalanine ammonia
phenolic acids 2, 4, 86, 244, 245
lyase
phenoloxidase 50
PAL-box 113, 116
phenylacetic acids 2, 4
p-aminobenzoic acid 237
phenylalanine 27, 82, 84, 86–90, 127
paper 120–122
phenylalanine ammonia lyase (PAL)
papillae 186–188, 219
84, 86, 87, 113, 114, 230
pathogenesis-related (PR) proteins
phenylethanol, 3,4-dihydroxy 243
86, 227
phlobaphenes 2, 30, 31, 101,102
p-coumaric acid 5, 15, 84, 88, 103,
phloretin 214, 216, 217
125, 176, 246
phloridzin 8, 214, 216, 217
p-coumaroyl CoA, shikimate
phloroglucinol 3, 42, 155, 158,
ester 103, 105
184, 185
p-coumaroyl-CoA 9, 91, 102, 103,
phloroglucinol-HCl 184, 187
105, 130
phosphoenolpyruvate 77, 82
phosphofructokinase 77, 79
p-coumaroyl-CoA 3’-hydroxylase
photosynthesis 76, 77
104
neutral detergent fiber (NDF) 161
Nicotiana benthamiana 105
Nicotiana tabacum 70, 113
nifedipine 243
nitrobenzene oxidation 172
nitrous acid oxidation 157
nuclear magnetic resonance (NMR)
88, 123, 178–181, 202
nucleus 35, 40, 100, 179, 180
274
p-hydroxybenzaldehyde 172, 173
p-hydroxybenzoic acid 4, 172
p-hydroxyphenyl 117
p-hydroxyphenylethanol 243, 244
phytate 246
phytoalexins 224, 226, 246
phytoanticipins 213
phyto-oestrogens 246
Phytophthora cinnamomi 186
pinoresinol/lariciresionol reductase
109, 111
pinosylvin 16
Pinus taeda 114, 117
pisatin 226
plasma desorption 202–204
podophyllotoxin 108–110, 112
Podophyllum hexandrum 109,
110, 112
Podophyllum peltatum 109
polymerase chain reaction (PCR)
68, 69, 71, 73, 100
polymerization 21, 25, 30, 52, 93,
102, 116–120
polyphenols 2, 239, 241, 242, 245,
246, 248, 250–252
poplar 52, 72, 86, 117, 122, 126, 170
Populus tremuloides 106
Populus trichocarpa 72, 117
potassium iodate assay 155, 156
p-quinone 51
preformed antimicrobial metabolites
213
prephenate 82
prephenate aminotransferase 82, 87
proanthocyanidins 24, 154, 155,
245, 253
procyanidin B2 24
programmed cell death 251
protocatechuic acid 88, 89, 219
protocyanin 43
protoleucomelone 46
Pseudomonas putida 228
Puccinia graminis 186, 229
Puccinia recondita 229
Index
pulping 121, 123
Purple plant (Pl) 99, 100, 102
putrescine 56
pyranose 48
pyrogallol 188, 189
pyrolysis (Py) 162, 177, 178
pyruvate 77, 79, 87, 88
pyruvate kinase 77, 79
pyruvate lyase 87, 88
quadrupole mass analyzer 198
quantitative trait locus (QTL) 73,
74, 220–222
quercetin 12, 41, 92, 242–244,
249, 250
Quercus 23
quinines 48, 56, 158
quinones 2, 17, 215, 216, 221
radicals 18, 21, 23, 48, 52–54, 58, 59,
107–109, 116–118, 239–241, 248
reactive oxygen species (ROS) 58,
60, 229, 241–243
recombinant protein 74–76, 105,
106, 116, 126
Red color 98
REF1 126
resorcinol 3, 42
resveratrol 16, 226, 248, 249
resveratrol cis-dehydrodimer 226
resveratrol trans-dehydrodimer
226, 248
rhamnosyl-isoorientin 222
rheumatoid arthritis (RA) 241, 250
Rhizoctonia solani 230
rhodanine 156
rhodanine assay 156
Rhus typhina 23, 88, 89, 131
ribose-5-phosphate 79, 81
ribulose-5-phosphate 79, 81
rice 72,106, 177, 230
salicylic acid 4, 86, 87, 227
salmon silk 222, 223
Index
Salmonella typhimurium 249
saturated fat 241
scurvy 240, 241
secoisolariciresinol dehydrogenase
109, 111
sedoheptulose-7-phosphate 79
shikimate dehydrogenase 82, 89
shikimate kinase 82
silage 123
sinapaldehyde 20, 106, 107, 124,
126, 127
sinapic acid 5, 84, 125–128
sinapoyl choline 6, 126
sinapoyl malate 6, 126, 127
sinapoylcholinesterase 127, 128
sinapoylglucose:choline
sinapoyltransferase 127, 128
sinapoylglucose:malate
sinapoyltransferase 127, 128
sinapyl alcohol 18, 20, 21, 102, 103,
106, 107, 115–117, 230
size exclusion chromatography 66
skatolyl radical 55
snapdragon 70, 91, 113
SNG1 127
sodium dodecyl sulfate
polyacrylamide gel
electropherosis (SDS-PAGE) 67
sorghum 30, 106, 113, 124, 217, 224
Sorghum bicolour 30, 123, 225
Spinach 53
Spinacia oleracea 53
Spodoptera exigu 215, see also beet
army worm
stem rust 229, 230
stilbene synthase 129, 130
stilbene, 4-homogeranyl-2,3',4',5'tetrahydroxy 254
stilbenes 2, 16, 84, 129, 130, 226,
249, 254
stover 125
Straphylococcus aureus 237
Streptomyces scabies 125
sumac 23, 88–90, 131
275
superoxide dismutase 53, 54, 60
superoxide radical 53, 54, 58
suppressor mutator (Spm) 70, 91
syringaldazine 187, 188
syringaldehyde 172, 173
syringic acid 172
syringyl 21, 23, 117, 172, 183,
184, 230
systemic acquired resistance (SAR)
86, 88, 227–229
tanning 23
tannins 2, 10, 11, 23–26, 29, 53,
130, 153–157, 183, 185, 239,
253
tannins, condensed 90, 93, 96, 97,
101, 153, 154
taxifolin 10
T-DNA 70, 100
T-DNA tagging 70, 96, 100
tea 250–253
tellimagrandin II 132, 133
tellimagrandin II: O2 oxidoreductase
133
tetrahydroxybiphenyls 48
theaflavin 250
thearubigin 250
thin-layer chromatography (TLC)
45, 64, 127, 128, 166–169
thioacidolysis 173, 174, 176
thioglycolic acid 162, 163, 182
time-of-flight mass analyzer 199,
106
tobacco 70, 86, 105, 113, 114, 170,
176, 228
tobacco mosiac virus (TMV) 228
tocopherol 240, 241, 249
toluidine Blue O 184, 187
tomato 70, 215, 253
tomato fruit worm 215
trans-coumaric acid-2-O-glucoside
129
transcription factor 96, 98–101, 113,
116, 170, 171, 252
276
transgenic 56, 105, 113, 114,
117, 120, 122, 170, 171, 181,
228, 253
transparent testa (tt) 91, 93, 101
transparent testa glabra (ttg) 91
transposable elements 70, 71
transposon tagging 70, 91–93, 98, 99
transposons 70, 71, 101
trans- -viniferin 226
trihydroxycinnamic acid 88
triose-phosphate isomerase 77, 79
tuliposide A 218
tuliposide B 218
tyrosinase 50, 51, 53
tyrosine 50, 51, 81, 82, 84, 88, 152
ubiquinone 17
UDP-glucose:sinapic acid
glucosyltransferase 127, 128
UDP-glucosyltransferases 116
umbelliferone 6, 7
urolithin B 252, 253
Uromyces apendiculatus 189
UV radiation 126, 237, 238, 250, 251
Index
valoneoyl unit 29
vanillic acid 4, 172, 254
vanillin 4, 154, 172, 173, 185
vanillin assay 154, 172
vanillin-HCl 185
Venturia inaequalis 216
Verticillium albo-atrum 215
vitamin C 240, 241
vitamin E 240, 241
white rot fungi 53, 121, 219
Wiesner reaction 184, 185
wine 23, 25, 29, 93, 241, 242, 245,
248, 249, 252, 253
Xanthomonas campestris 230
xanthones 2, 16
xylem 56, 106, 114, 117, 126, 170
xylulose 5-phosphate
yatein 109
Zea mays 39, 70, 123
Zinnia elegans 56, 105, 113