ARTICLE IN PRESS

Journal of Plant Physiology 166 (2009) 333—343

www.elsevier.de/jplph

Fatty acid a-dioxygenase from Pisum sativum:

Temporal and spatial regulation during germination
and plant development
Anke K. Meisnera,1, Alexander Safferta,2, Peter Schreiera,, Astrid Schönb

a
Institute of Pharmacy and Food Chemistry, University of Würzburg, Am Hubland, 97074 Würzburg, Germany
b
University of Leipzig, Molecular Cell Therapy, Center of Biotechnology and Biomedicine, Deutscher Platz 5, 04103
Leipzig, Germany

Received 20 March 2008; received in revised form 29 May 2008; accepted 29 May 2008

KEYWORDS Summary
a-Oxidation;
a-Dioxygenases are expressed in plants in response to biotic and abiotic stress. They
Fatty acid
catalyze the enantioselective 2-hydroperoxidation of long-chain fatty acids, the
metabolism;
initial step of the a-oxidation pathway of fatty acids in plants. In this study, the
Germination;
complete cDNA of an a-dioxygenase from germinating pea seeds (Pisum sativum) is
Phyto-oxylipins;
presented. The deduced amino acid sequence establishes that the enzyme belongs
Plant defense
to the recently characterized family of a-dioxygenating enzymes in plants. We also
mechanism
present the first systematic study on the expression of a-dioxygenase in germinating
and developing pea plants. During germination, a-dioxygenase mRNA accumulates in
the cotyledons and the embryonic axis of pea seeds de novo. In developing pea
plants, the transcript is detected almost exclusively in roots. The accumulation of
a-dioxygenase protein parallels transcript accumulation in that it is abundant in
germinating as well as young plant tissue, and correlates with loss of mRNA during
plant maturation. a-Dioxygenase enzymatic activity in plant extracts is highest in
cotyledons during imbibition. In the embryonic axis and roots of developing plants
comparable activity levels are observed, whereas in shoots little a-oxidation activity
is detected. With this contribution, we present information on the temporal and
spatial expression of a-dioxygenase during plant germination and development,
supporting the hypothesis that the a-oxidation pathway of fatty acids plays a role
during plant developmental processes.
& 2008 Published by Elsevier GmbH.

Abbreviations: a-DOX, a-dioxygenase; LOX, lipoxygenase.

Corresponding author. Tel.: +49 931 8885480; fax: +49 931 8885484.
E-mail address: schreier@pzlc.uni-wuerzburg.de (P. Schreier).
1
Present address: Bundesamt für Verbraucherschutz und Lebensmittelsicherheit, Mauerstr. 39-42, 10117 Berlin, Germany.
2
Present address: Tetra Pak GmbH, International Packaging Research Laboratories, Untere Waldplätze 27, 70569 Stuttgart, Germany.

0176-1617/$ - see front matter & 2008 Published by Elsevier GmbH.

doi:10.1016/j.jplph.2008.05.009
 ARTICLE IN PRESS
334 A.K. Meisner et al.

Introduction et al., 2001), high-salinity stress (Wei et al., 2000),

wounding (Tirajoh et al., 2005) and heavy metal
Phyto-oxylipins, a group of oxygenated fatty acid stress (Koeduka et al., 2005). Apart from the
derivatives, play a vital role in plant biology. They expression of a-DOXs during the plant response to
are best known for their impact during plant stress, the a-oxidation of fatty acids is detected
defense reactions; compounds generated by fatty during developmental processes, such as plant
acid oxygenation possess signaling activity, e.g. germination. We reported previously that a-DOX
jasmonic acid and C-6 aldehydes, or antibacterial activity is markedly elevated in germinating pea
and antifungal activity. Oxylipins are also known to seeds compared to leaves of mature plants (Saffert
be involved in plant developmental processes. et al., 2000), suggesting that the a-oxidation
(Blée, 2002; Wasternack, 2007). pathway plays a role during the plant germination
The a-oxidation of fatty acids is the pathway by process.
which 2-hydroxy fatty acids, a group of oxylipins, In this contribution, we present the complete
are synthesized. The a-oxidation of fatty acids was cDNA sequence of a-DOX from germinating pea
first discovered in germinating peanut seeds (New- seeds, as well as our results on a-DOX transcript and
comb and Stumpf, 1952) and has later been protein accumulation, and the time-dependent
observed in several other higher plants such as changes in fatty acid a-dioxygenating activity in
pea leaves (Hitchcock and James, 1966), cucum- pea seedlings and young pea plants. Taking our
bers (Galliard and Matthew, 1976) and potatoes results into consideration, the role a-dioxygenation
(Laties and Hoelle, 1967), but also in simple might play in different tissues during plant germi-
organisms like the marine green algae Ulva pertusa nation is discussed.
(Kajiwara et al., 1988). Several stable reaction
products of the a-oxidation pathway have been
detected, i.e. the respective 2-hydroxy fatty acid, Materials and methods
CO2 and a Cn1 aldehyde as well as the correspond-
ing chain-shortened fatty acid. Evidence for the Plant material
intermediary formation of an unstable 2-hydroper-
oxy acid has been provided by the detection of (R)- All studies were performed with P. sativum L. cv.
2-hydroperoxypalmitic acid as the first reaction ‘‘Kleine Rheinländerin’’. Dry seeds were surface ster-
product of the a-oxidation pathway (Akakabe ilized in 4% sodium hypochlorite solution and grown
et al., 1999). The 2-hydroperoxy fatty acids were hydroponically in sterile expanded clay under a 14 h
light/10 h dark photoperiod at 22 1C in a growth chamber.
shown to decarboxylate spontaneously, leading to
Plants were irrigated with a half-strength solution of
the formation of the chain-shortened Cn1 alde- Hoagland’s no. 2 Basal Salt Mixture (Sigma). Plant age
hyde (Hamberg et al., 1999). was defined according to the time of growth after the
The characterization of the family of enzymes start of imbibition. At 6, 12 and 24 h of imbibition the
that catalyze the initial a-dioxygenation step of the embryonic axis and the cotyledons were collected
a-oxidation pathway, i.e. the enantioselective separately. At 3, 6, 9, 12, 16, 20, 24 and 28 d after the
introduction of O2 at the C-2 position of fatty acids start of hydration the growth axis was separated into
leading to the formation of unstable (R)-2-hydro- roots and shoots. All samples were shock frozen in liquid
peroxy fatty acids, was accomplished in 1998 by the nitrogen immediately after collection and stored at
identification of a pathogen inducible oxygenase 80 1C.
(PIOX, later renamed a-DOX for a-dioxygenase)
from tobacco leaves and a homologous enzyme Amino acid sequencing
from Arabidopsis thaliana, both of which are
capable of catalyzing the conversion of fatty acids Amino acid sequencing of internal fragments of
purified a-DOX from germinating pea seeds was per-
to their corresponding 2-hydroperoxides (Sanz
formed as described previously (Saffert et al., 2000).
et al., 1998; Hamberg et al., 1999). In the mean-
time, the occurrence of fatty acid a-dioxygenating
cDNA synthesis
enzymes has been shown for other higher plants,
e.g. Pisum sativum (Saffert et al., 2000) and Oryza
Total RNA was isolated from pea seeds after 3 d
sativa (Koeduka et al., 2002). Expression of a-DOX
of germination according to the method of Logemann
genes in plants has been shown to be induced in et al. (1987). Poly(A)+ RNA was isolated with
response to abiotic and biotic stress, including oligo(dT) cellulose. Complementary DNA was synthesized
bacterial infection (Sanz et al., 1998; Kim et al., using a mix of anchored oligo(dT) primers (dT16A,
2002), cellular signals mediating cell death (Ponce dT16G, dT16C). Two degenerate oligonucleotide primers
de León et al., 2002), herbivore attack (Hermsmeier (DOX1A 50 -ACT-AAG-CAG-GTT-GAR-CTT-AGY-GC-30 and
 ARTICLE IN PRESS
Expression of a-dioxygenase in pea plants 335

DOX4B 50 -CCA-CAI-GCT-TGG-TGA-CCC-ATT-GA-30 ) were (w/v) SDS for 15 min at room temperature and consecu-
constructed on the basis of the partial amino acid tively at 40, 50 and 60 1C, and finally once with 1  SSC,
sequences of the 70 kDa subunit of purified pea a-DOX, 0.5% (w/v) SDS for 15 min at 60 1C. Membranes were
taking into account highly conserved regions of plant a- exposed to X-ray Retina XBD with an intensifying screen
DOX sequences, and used for PCR amplification. The at 60 1C and subsequently to PhosphoImager plates
resulting PCR product with a length of 830 bp (DOX1A/4B) (Molecular Dynamics).
was cloned into the Sma I restriction site of pUC19 to give
plasmid pDOX1A/4B and sequenced. The fragment was
Antibody production and immunoblot analysis
recovered and labeled radioactively for use as a probe for
cDNA library screening and RNA gel blot hybridization.
Polyclonal antibodies against a-DOX from germinating
pea seeds were raised in rabbits by injecting the 70 kDa
cDNA library construction and screening subunit of the pea a-oxidation enzyme. Following
purification carried out as described previously (Saffert
Poly(A)+ RNA was prepared from total RNA isolated et al., 2000), fractions from Mono-P HR5/20 were
from germinating pea seedlings as described above. separated by native polyacrylamide gel electrophoresis.
Complementary DNA was synthesized and cloned into A single protein band of 230 kDa was detected by staining
the EcoR I-Xho I sites of the Uni-ZAPs XR vector with Coomassie Brilliant Blue. The protein band of
(Stratagene), following the manufacturer’s instructions. 230 kDa was excised from the gel and the respective
The ligation was packaged using a ZAP-cDNAs Gigapacks protein was electroeluted from the gel matrix. The
III Gold Cloning kit (Stratagene). Approximately 109 eluted protein was subsequently separated by SDS-PAGE,
clones were screened with the 32P-labelled DNA probe and the resulting 70 kDa subunit was used for antibody
of pea a-DOX generated from pDOX1A/4B. Transfer of production.
bacteriophage l plaques to nitrocellulose filters and Rabbit antisera often contain immunoglobulins that
subsequent immobilization was performed according to recognize bacterial antigens and thus may lead to
Sambrook et al. (1989). Nitrocellulose filters were nonspecific background signals in immunoblots. To reduce
hybridized overnight at 50 1C in 50% (v/v) formamide, the amount of immunoglobulins recognizing bacterial
5  SSC, 20 mM sodium pyrophosphate, 0.1% (w/v) SDS, antigens, the antiserum was incubated with insoluble
1  Denhardt’s solution (0.02% (w/v) Ficoll, 0.02% (w/v) material of inactivated Escherichia coli cells and used for
polyvinylpyrrolidone, 0.02% (w/v) bovine serum albu- immunoblots after removal of E. coli material and bound
mine, 100 mg mL1 denatured salmon DNA). After hybri- immunoglobulins by centrifugation as described by
dization, blots were washed twice with 5  SSC, 0.1% Gruber and Zingales (1995).
(w/v) SDS for 10 min at room temperature, once with Plant samples were collected at the indicated times of
1  SSC, 0.1% (w/v) SDS for 10 min at room temperature, growth and frozen in liquid nitrogen. Plant material was
once with 0.1  SSC, 0.1% (w/v) SDS at room tempera- ground to a fine powder in liquid nitrogen. Extraction
ture; the washing temperature subsequently was raised buffer (0.1 M Tris–HCl, pH 7.5) was added at a ratio of
step-wise up to 50 1C. Filters were exposed to sensitized 2 mL g1 fresh weight and samples were homogenized.
X-ray Retina XBD with an intensifying screen at 60 1C. The suspensions were clarified by centrifugation for
Positive clones were picked and the pBluescript SK(+/) 10 min at 3000g. A total of 50 mg of total protein were
phagemid excised with the helper phage and recircular- separated on 10% (w/v) SDS-PAGE gels and transferred to
ized. The clone with the longest cDNA insertion covering nitrocellulose membranes by electroblotting. After the
the full length open reading frame was sequenced transfer, membranes were washed in TBS (pH 8.0) and
completely (clone DOX8-7/A). blocked overnight at 4 1C in a solution of 0.2% (w/v)
I-Block (Tropix) in TBS. Subsequently blots were incu-
bated for 5 h at room temperature in blocking solution
Analysis of gene expression
with anti-dioxygenase antiserum (1:1000, purified against
E. coli antigens as described above). Thereafter, mem-
Total RNA was isolated from plant samples with the
branes were washed twice (10 min at room temperature)
RNeasy Plant Mini Kit (Qiagen) according to the manu-
in blocking solution and incubated with a horseradish
facturer’s instructions. For the isolation of total RNA from
peroxidase-conjugated secondary antibody (Sigma) di-
dry seeds and cotyledons the buffer RFC was used, and
luted in blocking solution (1:5000) for 1 h at room
for all other applications the buffer RPE was used. On-
temperature. Antigen–antibody complexes were visua-
column digestion of DNA was performed with RNase-free
lized with Luminol as chemiluminescent substrate.
DNase I. Total RNA (10 mg) was separated electrophor-
etically on 1% (w/v) agarose-formaldehyde gels and
transferred to a positively charged nylon membrane by Enzyme assay
upward capillary transfer with 10  SSC as transfer
buffer. The amount of loaded RNA was documented by Plant samples were harvested at the times indicated
ethidium bromide staining. DNA probes were generated and frozen in liquid nitrogen. Protein extraction was
as described above. Blots were hybridized at 65 1C in performed as described above. After centrifugation
Rapid-hyb Buffer (Amersham Biosciences). After hybridi- protein extracts were used for the determination of
zation, membranes were washed twice with 2  SSC, 1% a-DOX activity. Aliquots were incubated with 1 mM
 ARTICLE IN PRESS
336 A.K. Meisner et al.

palmitic acid for 30 min at 30 1C. After addition of a Sequences corresponding to the remaining clones
saturated acidic solution of 2,4-dinitrophenylhydrazine in were shorter, but identical in regions present in all
ethanol and subsequent extraction with hexane, penta- clones. The complete cDNA sequence (sequence
decanal formed during the incubation was analyzed as its data have been deposited at GenBank under
2,4-dinitrophenylhydrazone by reversed.
accession number AJ784963) comprises an open
Phase HPLC analysis and UV detection at 350 nm. 2,4-
reading frame of 1929 bp in length. The deduced
Dinitrophenylhydrazones were eluted using a binary
gradient system with a flow rate of 1 mL min1. The polypeptide of 643 amino acids has a calculated
gradient proceeded from 95% (v/v) water acidified with molecular mass of 73.3 kDa, with its sequence being
0.05% (v/v) formic acid and 5% (v/v) acetonitrile to 100% in agreement with the partial amino acid sequences
acetonitrile in 20 min and from 100% acetonitrile to 30% of the 70 kDa subunit of the a-DOX purified from
(v/v) acetonitrile and 70% (v/v) 2-propanol in 10 min. For pea (Figure 1). Sequence alignments with known
the quantification of pentadecanal, calibration curves plant fatty acid a-DOXs revealed high similarity on
were generated with the authentic reference synthesized the amino acid level. The highest sequence identity
as described earlier (Saffert et al., 2000). (75% of identical amino acids) was found with the
a-DOX1 from N. tabacum (GenBank accession
number AJ007630), and sequence alignments with
Results the A. thaliana a-DOX1 (accession number
AF334402) revealed 72% identity at the amino acid
Amino acid sequencing of a-DOX purified
from pea and cDNA isolation

As described earlier, the a-oxidation enzyme

purified from germinating pea seeds has a mole-
cular mass of about 230 kDa with subunits of 70 and
50 kDa, a-DOX activity being associated with the
70 kDa subunit. The amino acid sequences of two
internal fragments of the 70 kDa subunit, generated
by cyanogen bromide cleavage, showed highly
significant similarity to the amino acid sequence
of the a-DOX from Nicotiana tabacum (Saffert
et al., 2000). Sequencing of further internal
fragments of the purified enzyme from pea sup-
ported the finding that it is a member of the family
of a-DOX enzymes in plants. For the isolation of the
a-DOX cDNA from pea, poly(A)+ RNA was isolated
from germinating seeds after 3 d of growth, at a
time when a-oxidation activity has been shown to
be high (Saffert et al., 2000) and, in consequence,
the amount of a-DOX transcript could be expected
to reach high levels. After reverse transcription of
poly(A)+ RNA with oligo(dT) primers, a part of the
sequence encoding the pea a-DOX was isolated by
PCR. Degenerate oligonucleotide primers for the
amplification were constructed on the basis of the
amino acid sequences of internal fragments of
the purified pea a-DOX, taking into account highly Figure 1. Sequence alignment of the amino acid
conserved regions in plant a-DOX sequences. A PCR sequences of internal fragments of the 70 kDa subunit
product with a length of 830 bp (DOX1A/4B) was of purified a-dioxygenase from germinating pea seeds (Ps
obtained. This fragment of the pea a-DOX cDNA pep; X indicating amino acids that could not be identified
due to technical problems) and the translated sequence
served as a probe to screen approximately 109
of the complete cDNA of P. sativum a-dioxygenase (Ps
clones of a cDNA library prepared with poly(A)+ RNA DOX). Identical amino acids are shaded in black. Black
isolated from germinating pea seeds. Eventually, 11 bars 1A and 4B indicate the positions of the PCR
positive clones were isolated and analyzed by amplification primers DOX1A and DOX4B. Amino acid
sequencing. The clone with the longest cDNA residues that have been shown to be catalytically
insertion, providing the full length open reading relevant in other plant a-dioxygenases are marked by
frame, was sequenced completely (clone DOX8-7/A). an asterisk (His-168, Tyr-390 and His-393).
 ARTICLE IN PRESS
Expression of a-dioxygenase in pea plants 337

level. Comparison of the pea a-DOX to the feebly--

like protein of A. thaliana (accession number
AY128743), designated as a-DOX2 (Hamberg et al.,
2002), revealed 62% identical amino acid residues.

Analysis of gene expression during

germination and development

To gain information on the role fatty acid a-

dioxygenation might play during plant germination,
we examined the expression of P. sativum a-DOX
during the germination process. For this purpose,
the amount of a-DOX mRNA in different tissues of
pea plants was determined at various time points
during the germination of pea seeds and during the
growth of young pea plants. To demonstrate
differences in temporal and spatial distribution,
the expression was analyzed differentially in dry
seeds, the embryonic axis and the cotyledons
during the first day after the start of imbibition as
well as in root and shoot tissue of healthy plants on
the third and following days of growth. RNA gel blot
analyses were performed with equal amounts of
total RNA isolated from plant samples collected at
defined time points. Probing with the P. sativum a- Figure 3. RNA gel blot analysis of a-dioxygenase mRNA
DOX cDNA revealed that a-DOX transcript was expression in developing pea plants. Samples obtained
abundant during germination. However, the pat- from roots and shoots were analyzed. Analysis of P.
tern of mRNA accumulation differed considerably in sativum total RNA was performed as in Figure 2.
the analyzed tissues. As can be seen in Figure 2,
compared to dry seeds, a-DOX mRNA steadily of a-DOX mRNA was found at 12 and 24 h after the
accumulated in the embryonic axis as well as in start of hydration in the embryonic axis. After
the cotyledons on the first day after the start of the first day of growth, we exclusively examined the
imbibition. During the first day, the maximum level growth axis and analyzed roots and shoots sepa-
rately. The differentiation of the growth axis into
roots and shoots revealed that a-DOX transcript was
present mainly in roots and to a considerably lower
extent in shoots (Figure 3). The maximum level of
pea a-DOX mRNA in root material was found on the
third day of growth, and the transcript level
decreased continuously afterwards.

Immunoblot analysis of protein accumulation

during germination and development

To determine whether the pattern of transcript

accumulation of P. sativum a-DOX during seed
germination correlates with the level of a-DOX
protein in the respective plant tissues, we exam-
Figure 2. RNA gel blot analysis of a-dioxygenase mRNA
ined the abundance of the protein responsive to the
expression in tissues of germinating pea seeds during the
first day of germination. Samples from dry seeds, antiserum raised against the 70 kDa subunit of
embryonic axes and cotyledons were analyzed. Total purified a-DOX from germinating pea seeds. Protein
RNA (10 mg) was hybridized with a 32P-labelled probe gel blot analyses were performed with equal
derived from the pea a-dioxygenase cDNA. Ethidium amounts of protein (50 mg of total protein) isolated
bromide stained gel before blotting is shown (bottom from plant samples harvested at defined time
panel). points. a-DOX protein was detected in all analyzed
 ARTICLE IN PRESS
338 A.K. Meisner et al.

plant tissues. Similar to the pattern of mRNA findings from the RNA gel blot analyses, where the
accumulation, a-DOX protein was abundant in time course of transcript accumulation showed a
tissues of young plants, with decreasing amounts pronounced distinction between the transcript
in samples derived from developing plants. The levels in root and in shoot tissue, a similar time
highest amounts of a-DOX protein were detected in course was observed for root and shoot tissue with
dry seeds as well as in cotyledons during the first regard to protein accumulation (Figure 4B). After
24 h of imbibition; a weaker signal was detected in the third day of growth, the amount of detected
protein samples from the embryonic axis on the a-DOX protein in shoots and roots decreased to
first day of germination (Figure 4A). After the first reach a consistently low level around day 12 after
day of germination, we examined the growth axis, the start of imbibition.
which was separated into root and shoot. The
maximum amount of a-DOX protein was detected
on the third day of growth. But in contrast to the Time-dependence of a-DOX activity in
germinating and developing pea plants

To evaluate the temporal development of the

a-DOX activity and its spatial distribution during the
germination of pea seeds, we analyzed a-DOX
activity by determining the conversion of palmitic
acid to pentadecanal by protein extracts from
different tissues of germinating and young pea
plants.
The pH-optimum of pea a-DOX lies between pH
6.8 and 8, peaking at pH 7.4 (Saffert et al., 2000).
Therefore, a pH of 7.5 was chosen to perform
activity assays. Palmitic acid has been shown to be
a suitable substrate for a-DOX from pea (Saffert,
2001). Taking into account the accessibility of
pentadecanal as reference substance, palmitic acid
was chosen as substrate for standard activity
assays. The incubation of crude homogenates of
young pea leaves and crude extracts of germinating
peas with fatty acids of different chain lengths
leads predominantly to the formation of the
respective chain-shortened aldehydes, the corre-
sponding 2-hydroxy fatty acids being formed to a
lesser extent (Adam et al., 1998). The initial
products of the a-oxidation pathway – the 2-
hydroperoxy fatty acids – are instable compounds.
It is therefore not feasible to quantify them in
order to determine a-DOX activity in standard
activity assays. The chain-shortened aldehyde,
the main product of the reaction, was quantified
to measure a-DOX activity. Pentadecanal formed
during the enzymatic reaction was analyzed by
reversed.
Phase HPLC as its 2,4-dinitrophenylhydrazine
Figure 4. Immunoblot analysis of a-dioxygenase protein derivative. a-DOX activity is expressed as nmol of
accumulation in different plant tissues. P. sativum pentadecanal formed from palmitic acid per min
protein extracts (50 mg of total protein, separated by
either per mg of total protein or per g of sample
SDS-PAGE) were probed with the antiserum raised against
the 70 kDa subunit of a-dioxygenase purified from pea. fresh weight.
The position of molecular mass markers is indicated on Again, we considered the dry seeds, the embryo-
the left. (A) Samples obtained from dry seeds, embryonic nic axis and cotyledons on the first day after the
axes and cotyledons during the first day of germination start of imbibition as well as the growth axis,
were analyzed. (B) Analysis of samples obtained from divided into roots and shoots, from day 3 to 28 of
roots and shoots of pea plants. growth (Figure 5). Of all analyzed plant samples,
 ARTICLE IN PRESS
Expression of a-dioxygenase in pea plants 339

Figure 5. Analysis of a-dioxygenase activity in different tissues of germinating pea seeds and developing pea plants. (A)
a-Dioxygenase activity, analyzed in dry seeds (black bars), the embryonic axis (light gray bars) and in cotyledons (dark
gray bars) of pea seeds on the first day of germination. Activity is expressed as nmol of pentadecanal formed min1 g1
of fresh weight. (B) a-Dioxygenase activity as in A but expressed as nmol of pentadecanal formed min1 mg1 of total
protein. (C) a-Dioxygenase activity, analyzed in roots (black bars) and shoots (light gray bars) of pea plants. Activity is
expressed as nmol of pentadecanal formed min1 g1 of fresh weight. (D) a-Dioxygenase activity as in C but expressed
as nmol of pentadecanal formed min1 mg1 of total protein. Each data point represents the values obtained in four
independent experiments. The mean and standard deviation are given.
 ARTICLE IN PRESS
340 A.K. Meisner et al.

the highest a-DOX activity per g of plant fresh et al. (1998) are conserved in the a-DOX from pea
weight was found in the cotyledons at 24 h after (His-168, Tyr-390 and His-393) (Figure 1). The
the start of imbibition. During the first day of corresponding residues have been shown to be
imbibition the a-DOX activity increased steadily essential for the activity of a-DOX from rice
to the maximum at 24 h (95 nmol of pentadeca- (Koeduka et al., 2002) and A. thaliana (Liu et al.,
nal min1 g1 fresh weight). The activity in the 2004).
embryonic axis was less than half of the activity In our study on a-DOX expression in germinating
in cotyledons and remained constant (37 nmol and young pea plants, we show that germination
of pentadecanal min1 g1 fresh weight). In the leads to a pronounced accumulation of a-DOX mRNA
embryonic axis as well as in the cotyledons, the in pea seeds and that a-DOX protein is abundant in
activity was higher than in dry seeds (Figure 5A). germinating and young pea plants. a-DOX mRNA
The specific activity on the first day of germina- accumulated during imbibition in the embryonic
tion ranged from 0.5 nmol of pentadeca- axis and cotyledons, indicating de novo transcrip-
nal min1 mg1 protein in dry seeds to 4 nmol of tion. In the growth axis, a-DOX transcript was
pentadecanal min1 mg1 protein in cotyledons detected almost exclusively in roots and to a
(Figure 5B). These findings are in accordance with considerably lower extent in shoots. These results
the results of RNA and protein gel blot analyses. are in accordance with the findings in mature
When the growth axis was exclusively considered healthy A. thaliana plants, where a-DOX1 transcript
after the first day of germination, the a-DOX has been detected in the roots, but no transcript
activity with reference to the fresh weight of has been found in leaves and stems (Ponce de León
plant material was found to be generally higher in et al., 2002).
roots than in shoot tissue. Activity levels in roots Similar to transcript accumulation, a protein
with reference to the fresh weight remained immunoresponsive to antiserum raised against the
relatively constant over the observed time course 70 kDa subunit of the purified a-DOX from pea was
(peaking at days 12 and 16). In shoot tissue, the abundant in dry seeds and in imbibed cotyledons as
activity was slightly increased on the first days well as the embryonic axis. Unlike the time course
of growth and decreased subsequently to the of transcript accumulation, that of protein accu-
low level typical for healthy mature plants mulation was comparable in roots and in shoots. It
(Figure 5C). It is noteworthy that, in shoots, no is not known whether this is due to the existence of
significant variation of the a-DOX specific activity different enzyme isoforms, which could possibly
(0.4–1.3 nmol of pentadecanal min1 mg1 pro- have been detected by the antiserum and not the
tein) was observed during the time course. In roots, DNA probe, or to a different regulation of a-DOX
however, an increase in the specific activity was expression in roots and shoots. It has also been
visible with a maximum specific activity of reported that, for some genes, mRNA levels do not
18 nmol of pentadecanal min1 mg1 protein on correlate well with protein levels, making it
day 16 (Figure 5D). This increase in the specific a- difficult to predict protein expression from mRNA
DOX activity in root extracts is not reflected in the levels (Gygi et al., 1999). Although it might be
results on a-DOX mRNA and protein accumulation. conceivable that other pea proteins are recognized
nonspecifically by the antiserum used for the
immunoblots, taking into account the protein
Discussion purification as procedure, it must be considered
highly improbable that the antiserum is not specific
An enzyme capable of a-dioxygenating fatty acid for a-DOX. To provide additional evidence, the
substrates in the C-2 position has previously been antiserum has been shown to recognize a-DOX from
isolated from germinating pea seeds (Saffert et al., pea expressed in E. coli and purified (data not
2000). We isolated the cDNA encoding the P. shown).
sativum a-DOX, the deduced amino acid sequence With respect to the temporal and spatial dis-
homologous to known plant a-DOXs. The P. sativum tribution of fatty acid a-dioxygenating activity
a-DOX shares higher amino acid identity with the during the germination process and the growth of
enzyme a-DOX1 from A. thaliana than with the young pea plants, a-dioxygenation of palmitic acid
putative a-DOX2 from A. thaliana (72% versus 62%), increased markedly during the imbibition process.
indicating that the a-DOX from P. sativum belongs When comparing a-DOX activity in roots and in
to the a-DOX1 branch of the family of fatty acid shoots normalized to the samples’ fresh weight, it
a-DOXs in plants. The amino acid residues sug- is noteworthy that activity levels in roots exceeded
gested to be catalytically relevant in a-DOX1 from those in shoots by 2–8 times. The low activity levels
N. tabacum (His-167, Tyr-389 and His-392) by Sanz in shoots are characteristic for healthy mature
 ARTICLE IN PRESS
Expression of a-dioxygenase in pea plants 341

plants and are in agreement with our previous It has been noted that analogies exist between
finding, that the rate of a-oxidation in pea leaves is processes in the lignifying xylem of Zinnia elegans
distinctly lower than in germinating seeds (Saffert and the oxidative burst observed during the
et al., 2000). The findings for a-dioxygenating hypersensitive plant cell response (Barcelo, 1999).
activity in germinating pea plants differ from the In cotyledons, where a-DOX protein, activity and
results for a-DOX activity during the germination of mRNA are highly abundant, the disorganization of
rice seedlings reported by Koeduka et al. (2002). In cellular structures allows the mobilization of
rice seedlings, a-dioxygenation of fatty acids has nutrients to be transported to the growing parts
not been observed until a week after sowing, and of the plant. For the hypogaeic P. sativum it is well
little activity has thereafter been detected in root known that the cotyledons undergo senescence
extracts, whereas in shoot extracts, the activity during germination as soon as the storage material
levels have been found to rise steadily up to a 10- has been mobilized.
fold increase in comparison to roots. These diver- The reaction products of the a-oxidation pathway
ging results may be attributable to differences in belong to the oxylipins, a family of compounds
the biology of monocotyledonous and dicotyledo- whose members are known to possess antimicrobial
nous plants, as differences in the regulation of activity (Prost et al., 2005). In planta, 2-hydroxy
a-dioxygenating enzymes in rice, tobacco and fatty acids of differing chain lengths have been
Arabidopsis have been reported as well (Koeduka characterized as the main products of the fatty
et al., 2005). When considering our results on acid a-oxidation pathway and have been shown to
a-dioxygenating activity with respect to the act during plant–pathogen interaction (Hamberg
amount of total protein, a pronounced increase in et al., 2003). Since roots are continuously exposed to
the specific activity was observed in roots during soil bacteria, and are therefore prone to infection,
maturation of the plants. This increase was not the preferential occurrence of a-oxidation processes
reflected in the time course of transcript accumu- in roots could indicate a role for a-oxidation as a
lation or in the accumulation of immunoresponsive permanent system of protection against infection in
protein. In Arabidopsis and tomato, the existence roots. It has been shown that healthy transgenic
of additional a-DOX isoforms has been postulated plants expressing defense-related genes prior to
(Hamberg et al., 2002; Tirajoh et al., 2005). Our pathogen infection become more resistant to certain
results suggest that in P. sativum different isoforms pathogens (Cao et al., 1998). Expression of a-DOX
of a-dioxygenating enzymes coexist as well. Possi- during germination could thus contribute to improv-
bly, after the first days of development a shift from ing the defensive potential of the plant during early
the expression of pea a-DOX1 to other isoform(s) growth processes.
takes place. It is also conceivable that the enzyme With the characterization of a-DOXs, a further
activity is modulated by glycosylation or phosphor- class of enzymes in plants capable of dioxygenating
ylation of the protein. We have found that the fatty acids has been described in addition to
pea enzyme is glycosylated in vivo (data not lipoxygenases (LOXs). a-DOXs as well as LOXs not
shown), but nothing is known about other protein only catalyze related reactions, but show also
modifications or their possible regulatory signifi- similarities in their expression pattern. They share
cance in this case. parallels with regard to the expression during
Our observation that a-dioxygenating expression plant–pathogen interaction, but also with regard
was detected predominantly in the roots and to the expression during developmental processes
cotyledons of pea plants during plant germination as senescence and germination. As has been
and development is plausible when we consider postulated for the family of a-DOXs, different
that expression of a-DOX has been reported to isoforms of LOXs exist in most, if not all, plant
occur in plants in response to incidences associated species studied thus far. Young and expanding
with cellular damage such as herbivore attack, tissues usually contain high levels of LOX enzymes,
pathogen infection or senescence (Sanz et al., but increases in senescing tissue have been
1998; Hermsmeier et al., 2001; Obregón et al., reported as well. The actual function of LOXs in
2001). Disruption of the cellular structure and cell the process of germination remains unknown,
death is frequently correlated with the generation although several hypotheses have been put for-
of an oxidative burst which is known to be part of a ward, including nutrient mobilization, membrane
general stress defense pathway up-regulated by degradation or defense against pathogens (reviewed
biotic and abiotic stresses as well as during in Porta and Rocha-Sosa, 2002; Liavonchanka and
different plant developmental processes such as Feussner, 2006).
senescence. Roots consist mainly of lignified cells With the present systematic study we provide
that need to undergo cell death during lignification. evidence that a-DOX mRNA and protein accumulate
 ARTICLE IN PRESS
342 A.K. Meisner et al.

during the germination of pea seeds. We have Hamberg M, Sanz A, Rodriguez MJ, Calvo AP, Castresana
shown specifically that a-dioxygenating activity is C. Activation of the fatty acid a-dioxygenase pathway
highly abundant in the cotyledons of germinating during bacterial infection of tobacco leaves. J Biol
seeds. In healthy developing plants, a-DOX expres- Chem 2003;278:51796–805.
sion is detected preferentially in roots, with the Hermsmeier D, Schittko U, Baldwin IT. Molecular inter-
time course of a-dioxygenating activity suggesting actions between the specialist herbivore Manduca
sexta (Lepitopdera, Sphingidae) and its natural host
the existence of additional isoforms of a-DOX in pea
Nicotiana attenuata. I. Large-scale changes in the
plants. Future work will serve to investigate the
accumulation of growth- and defense-related plant
occurrence of further a-DOX isoforms in pea and mRNAs. Plant Physiol 2001;125:683–700.
the impact of the a-oxidation process and its Hitchcock C, James AT. The mechanism of alpha-oxida-
reaction products on the germination process. tion in leaves. Biochim Biophys Acta 1966;116:413–24.
Kajiwara T, Yoshikawa H, Saruwatari T, Hatanaka A,
Kawai T, Ishihara M, et al. Enzymatic formation of
Acknowledgments unsaturated long chain fatty aldehydes in Ulva
Pertusa. Phytochemistry 1988;27:1643–5.
We are grateful to Prof. H.-J. Gross, Prof. H. Kim Y-C, Yi S-Y, Mang HG, Seo YS, Kim WT, Choi D.
Beier and Prof. U. Fischer for providing laboratory Pathogen-induced expression of cyclo-oxygenase
space and facilities. We thank G. Grimmer for homologue in hot pepper (Capsicum annuum cv.
expert technical assistance and kind support. Pukang). J Exp Bot 2002;53:383–5.
Koeduka T, Matsui K, Akakabe Y, Kajiwara T. Catalytic
properties of rice a-oxygenase. J Biol Chem 2002;277:
22648–55.
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