Environmental and Experimental Botany 130 (2016) 33–41

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Environmental and Experimental Botany

journal homepage: www.elsevier.com/locate/envexpbot

Proteomic and metabolomic profiles in transgenic tobacco (N. tabacum

xanthi nc) to CchGLP from Capsicum chinense BG-3821 resistant to
biotic and abiotic stresses
G. Cardenas-Manríqueza,1, I. Vega-Muñoza,1, A.L. Villagómez-Arandaa,1,
M.F. León-Galvanb , A. Cruz-Hernandeza , I. Torres-Pachecoa , R.M Rangel-Canoc,
R.F. Rivera-Bustamantec, R.G Guevara-Gonzaleza,*
a
Biosystems Engineering Group, School of Engineering, Autonomous University of Queretaro, C.U Cerro de las Campanas, S/N, col. Las Campanas, C.P 76010
Santiago de Queretaro, Queretaro, Mexico
b
Agricultural biotechnology Group, Food Science Department, Life Sciences Division, University of Guanajuato, Ex-Hacienda El Copal, Km 9, Carretera
Irapuato-Silao, C.P 36500, Irapuato, Guanajuato, Mexico
c
Depto. Ingeniería Genética, Centro de Investigación y de Estudios Avanzados-Unidad Irapuato, Km. 9.6, Libramiento Norte, C.P 36821 Irapuato, Guanajuato,
Mexico

A R T I C L E I N F O A B S T R A C T

Article history:
Received 2 January 2016 Plants continuously are challenging biotic and abiotic stresses to survive. Reactive oxygen species (ROS)
Received in revised form 27 April 2016 as hydrogen peroxide are a key signal to transduce environmental stimuli in plants response to stresses.
Accepted 7 May 2016 Transgenic tobacco to CchGLP gene from C. chinense BG-3821, produce constitutive levels of hydrogen
Available online 14 May 2016 peroxide which correlates with tolerance to geminiviruses infections. In the present work, it was
demonstrated that these transgenic tobaccos are also tolerant to drought stress. Moreover proteomic and
Keywords: metabolomic studies showed that both Omic profiles in tobacco tolerant to both biotic and abiotic
Geminiviruses stresses, displayed components and impact on biochemical pathways that likely explain the observed
Drought
phenotype of tolerance in regards to no-tolerant lines and wild type tobaccos. Our results suggested that
Plant defense
hydrogen peroxide levels within these studied plants, correlates with induction of proteins and
Downstream genes
Hydrogen peroxide metabolites related to plant defense to stress, opening the possibility that controlling somehow (i.e.
applying elicitors in a controlled way) hydrogen peroxide or ROS levels within plants, might help to
protect plants in an scenario of global warming for future agriculture.
ã 2016 Elsevier B.V. All rights reserved.

1. Introduction regulating the signaling cascades during biotic and abiotic stresses
(Bari and Jones, 2009). In this sense, an important component of
Plants as sessile organisms are continuously challenged by biotic stress response in plants are the reactive oxygen species (ROS), which
and abiotic factors that cause a physiological and biochemical stress initially were recognized as toxic by-products of aerobic metabolism,
in the organism. In response, plants must be able to adopt survival removed by means of antioxidants and anti-oxidative enzymes
strategies which evolved in diverse and complex mechanisms of (Mejía-Teniente et al., 2013). Currently it is recognized a “ROS wave”
perception and reaction to struggle the adverse conditions of their as an important signaling role in plants controlling processes such as
environment. In order to resist the stress, plants are able to efficiently growth, development, response to biotic and abiotic environmental
balance growth, reproduction and defense response (Prasch and stimuli, and programmed death cell (Mittler et al., 2011). On the
Sonnewald, 2015). Plant hormones as auxins, gibberellins, cytoki- other hand, Germin-Like Proteins (GLPs) are recognized as a
nins, salicylic acid (SA), jasmonic acid (JA) and ethylene (ET) play component in the ROS wave transmission in plants as implicated
important roles in diverse growth, developmental processes and as plant cell defenders, as well as playing a role in the reinforcement
of plant cell wall (Guevara-Olvera et al., 2012; Mejía-Teniente et al.,
2015). Significant expression of GLPs has been shown in salt and
drought stresses, as well as in response to fungal, bacteria and virus
* Corresponding author.
E-mail address: ramon.guevara@uaq.mx (R.G. Guevara-Gonzalez). pathogens in plants (Breen and Bellgard, 2010; Knecht et al., 2010;
1
These authors contributed equal to this work. Park et al., 2004). GLPs have been classified based on their main

http://dx.doi.org/10.1016/j.envexpbot.2016.05.005
0098-8472/ã 2016 Elsevier B.V. All rights reserved.
34 G. Cardenas-Manríquez et al. / Environmental and Experimental Botany 130 (2016) 33–41

enzymatic activity, oxalate oxidase (OXO), superoxide dismutase Mejía-Teniente et al., (2013), using samples at day 0 and 15 after
(SOD) or ADP glucose pyrophosphatase/phosphodiesterase (AGP- drought stress treatments. Phenylalanine ammonia lyase (pal) and
Pase) (Breen and Bellgard, 2010; Dunwell et al., 2008). OXO and SOD Catalase-1 (cat-1) gene expression in N. tabacum was carried out by
activities result in hydrogen peroxide production, an important ROS. RT-PCR essentially as described in Guevara-Olvera et al. (2012).
Hydrogen peroxide has a recognized role in plant stress signaling
either as a local signal for hypersensitive cell death or as a diffusible 2.3. Protein extracts
signal of induction of defense response genes in adjacent cells
(Mejía-Teniente et al., 2013). A germin-like protein gene (hencefor- Plant leaf total soluble protein in each case, was extracted with
ward named CchGLP) highly induced during incompatible inter- a 40 mM tris, 10 mM EDTA and 1% PVPP buffer, precipitated with
actions with geminiviruses and applications of SA and ET was acetone and resuspended in rehydration buffer (8 M urea, 2%
previously identified in Capsicum chinense Jacq. accession CHAPS and 0.5% DTT). Determination of protein concentration was
BG-3821 by our group (Barrera-Pacheco et al., 2008). CchGLP gene carried out using the Bradford reagent (Bio-Rad protein assay dye
encodes a protein with Mn-superoxide dismutase activity (SOD) reagent concentrate, Bio-Rad Laboratories, USA) using bovine
when expressed in bacterial systems (León-Galván et al., 2011). serum albumin (BSA) as standard.
When transgenically and constitutively expressed in Nicotiana
tabacum cv. xanthin nc, CchGLP provided resistance to geminivirus 2.4. Separation by two-dimensional gel electrophoresis (2-DE) and
infection (Guevara-Olvera et al., 2012); moreover when CchGLP was identification
silenced in C. chinense accession BG-3821 increased susceptibility to
geminiviruses in this accession (Mejía-Teniente et al., 2015). The Proteins extracts from each sample were cleaned using the
aforementioned transgenic tobacco plants displayed significant ReadyPrep TM 2-D Cleanup Kit (Bio-Rad Laboratories, USA).
increase in ROS levels in comparison to non-transgenic geminivirus Proteins were separated by 2-DE. For first dimension, 7 cm IPG
susceptible plants (Guevara-Olvera et al., 2012). The high ROS levels strips pH 3–10 (Bio-Rad Laboratories, USA) were rehydrated on a
in these transgenic plants, could be signaling several routes in Protean1 i12 TM (Bio-Rad Laboratories, USA) with 80 mg of protein
response to both biotic and abiotic stimuli as suggested in other in 125 ml of rehydration buffer for 16 h, 50 Vh and 20  C. The
plant models (Bailey-Serres and Mittler, 2006). Thus, the aim of this proteins were focused with 50 mA, 20  C, 250 Vh for 15 min, then
study was to assess the response to drought stress in these the voltage was gradually increased up to 4000 Vh, finally the
transgenic tobacco to CchGLP, as well as to evaluate differences at voltage was maintained up to reach 15 000 Vh. IPG strips were
proteomic and metabolomic levels between these transgenic equilibrated with DTT buffer (I) 6 M urea, 0.05 M tris-HCl pH 8.8l,
tobaccos in comparison with an azygote control (harboring but 2% SDS, 20% glycerol, and 2% DTT followed by a an iodoacetamide
not expressing CchGLP) and wild type, trying to correlate them with buffer (II) (DTT replaced by 2.5% iodoacetamide) each for 15 min
the response of these plants to stress. with shaking. Second dimension was performed in 12% SDS
polyacrylamide gels in a vertical Mini-PROTEAN1 chamber (15 mA
2. Materials and methods for 2 h) using a 0.0.25 M Tris, 0.192 M glycine, 0.1% SDS buffer. Gels
were stained with Brilliant blue Coomassie R-250. Protein
2.1. Plant material identification was achieved by mass spectrometry and peptide-
mass fingerprints were analyzed using MASCOT (http://www.
Three highly expressing CchGLP transgenic lines of N. tabacum matrixscience.com), searching in swissprot database and using
xanthi nc (L8, L25 and L26) as well as an azygote line (L1), and wild data available in UniProt database (http://www.uniprot.org). The
type plant seeds (wt) were generated in previous work and seeds software used to identify differential proteins was Melanie
were obtained by self-pollination and testing the presence of (GeneBio, Geneva, Switzerland).
CchGLP expression and ROS levels before experiments according to
Guevara-Olvera et al. (2012). The non-expressing CchGLP trans- 2.5. Metabolites extraction
genic line L1 and wt plants were used as controls. Seeds were
surface sterilized by a 10 min incubation in 2% sodium hypochlorite Plant leaves of 15 days, and 2, 4 and 6 months old of transgenic
and 0.1% Tween 20 solution and three washes with sterile distilled lines were analyzed. Sample tissue was lyophilized for 72 h and
water. Seeds were germinated in flasks with Murashige-Skoog dried tissue was stored at 80  C until extraction. For the total
medium and kept at room temperature under a 16 h light/8 h dark phenolic, flavonoid, condensed tannins, anthocyanin and antioxi-
photoperiod. Six weeks old seedlings were transferred into dant content and antioxidant activity analysis the extraction
individual pots with Peat Moss as substrate, and then used for method were as following: methanol was added to dried tissue
all the experiments. (100 mg ml 1) and shaken for 24 h in the dark. Samples were
centrifuged at 5000 rpm for 5 min and the organic phase was
2.2. Drought stress studies recovered and stored for analysis. Assays were realized in a 96
wells microplate. The extraction procedure for HPLC and GC–MS
Drought studies were carried out essentially as reported by analysis was the following: dried tissue was immersed in methanol
other authors (Kanneganti and Gupta, 2008). Drought studies were (5 mg ml 1 for HPLC and 10 mg ml 1 for GC–MS) and incubated in a
carried out with tobacco plants of 4–6 leaves stage, but sonication bath for 40 min at room temperature. The samples were
maintaining a deficit in water or not during 2 weeks in a centrifuged at 3000g for 20 min at 4  C and organic phase was
greenhouse with a temperature between 25 and 28  C, relative collected. For HPLC analysis the samples were filtered through a
humidity 55–60% and natural illumination (14 h light/10 h dark). 0.45 mm Whatman filter. For GC–MS the samples were evaporated
Plants with water deficit treatment were only watered in the first in a nitrogen stream and derivatized with 50 ml of N,O-Bis
day (day 0) of the experiment and then no more water was added (trimethylsilyl)trifluoroacetamide (BSTFA).
during 15 days, on the other hand, control plants were daily
watered to field capacity during the same time. Experimental 2.6. Total phenolic content assay
unit in these experiments was 30 plants, and data of plant
mortality were collected in the days 7 and 15 of the experiment. Total phenolic content was tested with the Folin-Ciocalteu
Hydrogen peroxide detection was carried out as described by assay described (Feregrino-Perez et al., 2011). The reaction was
 G. Cardenas-Manríquez et al. / Environmental and Experimental Botany 130 (2016) 33–41 35

achieved as follows: 40 ml of methanolic extract was diluted with and a Phenomex ODS-C18 reverse phase capillary column
460 ml of distillate water and 250 ml of the Folin-Ciocalteu reagent (250  4.6 mm, 5 mm). The mobile phase consisted in formic acid
from Sigma. After 5 min the reaction was stopped with 1250 ml of 1:99 v/v (A) and acetonitrile (B), the initial A/B proportion was
2% Na2CO3 and incubated for 60 min at room temperature. The 98/2, 68/32 after 30 min, 45/55 at 48 min, 5/95 at 53 min and 98/2
absorbance of the samples was measured at 765 nm on a UV–vis at 57 min 1 ml of sample was analyzed at 1 ml/min constant flow
spectrophotometer. Gallic acid was used as phenolic standard and rate. Chromatograms were recorded at 280 nm. Chlorogenic acid,
the total phenolic content was expressed as mg gallic acid epicatechin, coumaric acid, hesperidin, caffeic acid, epigallocate-
equivalents g-1 sample. chin gallate, rutin, sinapic acid, ellagic acid, and vanillin were used
as standards on calibration curves for quantitative analyses.
Samples were from 2 weeks post-germination plants.
2.7. Total flavonoid content assay
2.12. Metabolomic profile by GC–MS
Total flavonoids content was determined by the 2-amino-
ethyldiphenylborate spectrophotometric assay described(Fere-
The samples were analyzed using an Agilent 7890-5975C
grino-Perez et al., 2011). It consisted of mixing 50 ml of the
GC–MS instrument. The samples were injected onto a HP-5MS
methanolic extract with 180 ml of distillate water and 20 ml of 1% 2-
capillary column (30 m  0.25 mm, 0.25 mm). The analysis was
aminoethyldiphenylborate solution in a 96 wells microplate. The
done in fragmentation mode at 250  C. The initial oven tempera-
absorbance of the solution was read at 404 nm on a UV–vis
ture was 100  C and increased at 40  C/ min to 310  C, where was
spectrophotometer. Rutin was used as flavonoid standard and the
held constant for 7.5 min. Helium was used as carrier gas at a flow
total flavonoid content was expressed as mg rutin equivalents g 1
rate of 1 ml/min. The ChemStation software from Agilent was used
sample.
to process the ion chromatogram and the metabolites was
identified using the ChemStation software which was linked
2.8. Total condensed tannins assay
to the NIST mass spectral library. Peaks with match above 50%
of identity were considered. Samples were from 2 weeks
Total condensed tannins content was accomplished with the
post-germination plants.
vanillin assay (Hagerman, 2011) with modifications. The method
consisted in mixing 50 ml of the methanolic extract with 100 ml of
2.13. Statistical analysis
1% vanillin and 100 ml 8% HCl. After 20 min of incubation at room
temperature, the absorbance was read at 492 nm on a UV–vis
The statistical significance of the resulting data was confirmed
spectrophotometer. Catechin was used as tannin standard.
with one-way ANOVA (P < 0.01), and Fisher LSD test to determine
Condensed tannins content was expressed as mg catechin
the significant different levels of metabolites among tobacco lines.
equivalents g 1 sample.
To visualize metabolite profile heatmaps were established using
the package gplots in R and the web-based pipeline MetaboAnalyst
2.9. Total anthocyanin assay version 2.0.

Total anthocyanin content was determined with the differential 3. Results

pH method described elsewhere (Sutharut and Sudarat, 2012).
Briefly, two dilutions were prepared as follows, 200 ml of the 3.1. Plant mortality under drought stress
methanolic extract were mixed with NaCl (0.025 M, pH 1.0) for the
first dilution, and sodium acetate (0.4 M, pH 4.5) for the second. Table 1 displays results of mortality in transgenic and wt plants
After 15 min, the absorbance was measured at 510 and 700 nm for under drought stress during 15 days. It was observed that for the
both dilutions, against a blank cell with distilled water on the azygote L1 and wt plants, plant mortality was significantly higher
spectrophotometer. Total anthocyanin content was expressed as than those observed in highly expressing CchGLP lines L8, L25 and
mg cyaniding-3-glucoside equivalents g 1 sample. L26 at days 7 and 15 of the experiments. Line 8 (L8), showed the
significant lowest mortality in these experiments, and based on
2.10. Antioxidant activity assay this criteria this line was chosen for further omic analysis in this
work. In the case of well-watered plants, no dead plants were
Antioxidant activity was evaluated by the DPPH method
described elsewhere (Feregrino-Perez et al., 2011). 20 ml of the
methanolic extract were mixed with DPPH (150 mM, 80% v/v Table 1
Percentage of plant mortality in transgenic tobacco to CchGLP under drought stress
aqueous methanol). The plate was covered and left in dark at room experiments.
temperature. After 30, 60, 75, 90 and 120 min, absorbance was
measured at 520 nm on the spectrophotometer. Antioxidant Drought stress Day 7 Day 15***

activity and total antioxidants content results were expressed as L1 3.3 (1/30)*,a 93.3 (28/30)a
mM Trolox equivalents g 1 sample. Antiradical activity (ARA) was L8 0b 10.0 (3/30)c
L25 0b 16.7 (5/30)b
determined by the Burda and Oleszek equation:
L26 0b 16.7 (5/30)b
Wt 3.3 (1/30)a 100 (30/30)a
% Inhibition = [Ac(o) AA (t)/Ac(o)]  100
Well-watered Day 7 Day 15
where Ac(o) is the absorbance of control at t = 0 min and AA (t) is L1 0** 0
L8 0 0
the absorbance of antioxidant at t = 1 h.
L25 0 0
L26 0 0
2.11. Analysis of phenolic compounds by HPLC Wt 0 0
*
(x/y) denotes number of death plants/total plants).
Phenolic identification and quantification were conducted on a **
0 indicates that no plants die during the experiment.
Waters 600 HPLC-DAD system equipped with a Waters 717 plus ***
In days 7 and 15 in drought experiments, different letters in this column
autosampler, a Waters UV/vis 2487 detector (Milford, MA, USA) indicates significant difference (P < 0.01).
36 G. Cardenas-Manríquez et al. / Environmental and Experimental Botany 130 (2016) 33–41

detected during the experiments (Table 1). Typical phenotype of of 19 compounds showed an identical pattern in all the lines. The
these plants at 0 and 15 days after drought-stress is shown in Fig. 1. non-expressing CchGLP transgenic line L1 displayed a similar
pattern of metabolites to wt. L1 showed few alterations in some
3.2. Differential proteomic profiles in L1 and L8 transgenic tobacco metabolites like a fewer presence in tyrosine, valine, phenylalanine
and leucine amino acids, as well as phosphoric acid, linoleic acid,
2D SDS-PAGE and mass spectrometry allowed detection of 58 malic acid and glucose, and a slight increase in inositol and uridine.
spots significantly detected as differential between lines 1 and 8 A significant alteration in contents in 32 compounds was shown in
(Table 2). Several proteins related with stress, metabolism, cell the transgenic line 8 (Fig. 2).
growth-differentiation and protein metabolism were detected
(Table 2). Detailed information of identified proteins is shown in 3.5. Detection of pal and cat-1 gene expression during drought stress
supplementary file 1. Data of peptide sequence, theoretical and
experimental pI, molecular mass, etc is shown. In addition, Both proteomic and metabolomic profiles suggested that
supplementary file 2 displays proteins profile images observed phenylpropanoids pathways must be induced during drought
during the proteomic studies. studies. However, proteomic profile did not detect up-regulated
proteins of this pathway (i.e Phenylalanine ammonia lyase, “PAL”).
3.3. Total phenolics, flavonoids, tannins, anthocyanins and antioxidant Based on this aspect, an RT-PCR gene expression assay for pal and
activity in L1 and L8 transgenic tobacco cat-1 gene expression was carried out (Fig. 5). As shown, pal
expression was induced in wt, L1 and L8, showing the highest
As first metabolites analysis approach in the transgenic tobacco expression L8. the case of cat-1, was not detected significantly
plants evaluated in this work, the levels of total phenolics, induced at 15 days after drought-stress (Fig. 5).
flavonoids, tannins and anthocyanins at 2, 4 and 6 months after
germination were determined in transgenic and wt tobacco leaves 4. Discussion
(Table 3). In transgenic lines expressing CchGLP it was observed a
higher content in bioactive compounds in comparison to the wt in Results regarding drought tolerance obtained with wt and
all the phenological stages evaluated (Table 3). Moreover, the older transgenic tobaccos, could be explained by the high CchGLP
the plant the higher the amount of bioactive compounds detected. transcript expression, which promote an increase in ROS levels, in
The antioxidant capacity displayed a stable pattern during plant lines 8, 25 and 26. On the contrary, the null CchGLP expression in
growth and a similar content between the lines (Table 3). Line 1 and wt plants, contribute to their low ROS levels reported by
Guevara-Olvera et al., 2012 and tested in this work. Interestingly,
3.4. Differential metabolomic profiles between lines 1 and 8 after drought stress treatments (day 15) in all cases ROS (H2O2)
levels significantly increased, however wt and L1 H2O2 levels were
Samples of 2 weeks post-germination of lines 1, 8 and wt plants, significantly lower than those in L8 (Fig. 2). Moreover, Guevara-
were used to evaluate specific metabolites by HPLC and GC–MS as Olvera et al. (2012), also reported L8 as showing the highest
mentioned in materials and methods. The reason to have chosen 2 resistance to geminivirus infections.
weeks was because in that time lines 1 and 8 displayed contrasting On the other hand, in the proteomic study, proteins detected
phenotypes to geminivirus (Guevara-Olvera et al., 2012) and could explain the phenotype in regards to tolerance to gemini-
drought tolerance (present work, see Table 1). Thus, four phenolic viruses and drought stresses in line 8 in comparison to line 1. For
compounds including chlorogenic acid, caffeic acid, rutin and instance, To keep the levels of reactive oxygen species (ROS) under
quercetin, were detected in this work (Table 4). A total of 51 control, plants have non-enzymatic and enzymatic antioxidant
metabolites were identified based on their mass spectra profile by systems to protect cells from oxidative damage caused by drought
the GC–MS assay, which included amino acids, glucosides, fatty stress. Among drought stress-related proteins detected, plant
acids, organic acids, terpenes and alcohols (Fig. 2). In Fig. 2, a total peroxidases are associated with the regulation of ROS levels and

Fig. 1. Typical phenotype during drought-stress experiments at 0 and 15 days of wt and CchGLP-transgenic lines L1 and L8.
 G. Cardenas-Manríquez et al. / Environmental and Experimental Botany 130 (2016) 33–41 37

Table 2
Differential proteins detected between transgenic tobacco lines 1 and 8.

Accesion Identified protein Biological Process Ocurrente

No. frequency
Stress-related proteins
Q9XIV8 Peroxidase N1 ROS scavenging and production 2.93
Q9FM96 Glucosidase 2 subunit beta Stress response (perception of bacterial elongation factor Tu) PAMPs 2.3
O65719 70 kDa heat shock protein Stress response (heat shock, cold, virus infection) 2.1
QBLNYS Stromal ascorbate peroxidase Response to oxidative stress 1.89
Q14TB1 90 kDa heat shock protein Stress response (virus) 1.87
Q9SZ15 Serine hydroxymethyltransferase Abiotic stress resistance (high light, salt, HR) 1.8
P21240 Chaperonin 60 subunit beta 3, chloroplastic Rubisco folding, possible heat stress tolerance 1.8
Q8RVF8 Thioredoxin peroxidase Oxidative stress response 1.77
Q03250 Glycine-rich RNA-binding protein RNA transcription or processing during stress (hydric stress, cold stress) 1.68
induced by hydrogen peroxide
P23547 Glucan endo-1,3-B-glucosidase Defense against pathogens 1.5
Q9ZP16 Enoyl-CoA hydratase Required for wound-induced jasmonate biosynthesis 1.3
P46309 Glutamate-cysteine ligase A, chloroplastic Defense against pathogens, Glutathione biosynthesis 1.2
Q9ZP50 Fish-Like protein Ptf Possible protein metabolism induced in stress 1.2
P13046 Pathogenesis-related protein R major form Stress response 1.12
Q96528 Catalase Protection from hydrogen peroxide 1.1
BCECN9 Aminoaldehyde dehydrogenase 2 Stress response 1.1
B9A6I8 Germin-Like protein Stress response 1.06
Q96321 Importin subunit alpha Protein transport, drought stress tolerance 1.01
QM9651 RAN GTPase-activating protein 2 Response to salt stress 1.01
Q7G8G4 Os10g0125700 protein Cold stress response, secretory pathways 0.8
P92947 Monodehydroascobate reductase Redox homeostasis 0.7
H3JU07 SGT1 Defense response 0.6

Metabolism-related proteins
P11605 Nitrate reductase (NADH 1) Nitrate assimilation 3.1
Q1WIQ6 Glyceraldehyde-3-phosphate dehydrogenase A NADPH generation 2.8
Q9FPL3 Phosphoribosyl-amino-imidazole-carboxamide-formil- Purine biosynthesis 2.3
transferase/IMP cyclohydrolase
B3F8G8 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase Terpenoid biosynthetic process 2.14
Q2QMV8 Adenine phosphoribosyl transferase 1, putative Nucleoside metabolic process 2.1
B8AZJ5 Phosphoglycerate kinase Central metabolism 2
Q9LW96 Inositol-3-phosphate synthase Polyol metabolism 1.9
P00826 ATP synthase subunit B Central metabolism 1.7
P10896 Ribulose diphosphate carboxylase/oxygenase activase 2 Central metabolism 1.7
Q84WB8 Putative GMP synthase GMP biosynthetic process 1.7
Q006P9 Chloroplast NADP-malic enzyme precursor Malate metabolic process 1.6
Q50008 Cobalamin-independent methionine synthase 1 Aminoacid metabolism 1.2
Q05758 Ketol-acid reductoisomerase L-isoleucine biosynthesis 1.14
P00823 ATP synthase subunit a Central metabolism 1.1
P09468 ATP synthase subunit epsilon chain Central metabolism 1.1
P00840 ATP synthase subunit 9, mitochondrial Central metabolism 1.1
Q40545 Piruvate kinase isozyme A, chloroplastic Glycolisis 1.09
P39207 Nucleoside diphosphate kinase 1 Nucleoside triphosphate synthesis 0.9

Cell growth and differentiation-related proteins

O81905 Receptor-like serine/threonine-protein kinase Cellular expansion and differentiation 3.4
O22467 Histone-binding protein MSI1 Cell proliferation 3.1
Q9XEN8 Annexin Cell cycle 2.8
Q7DMA9 Peptidyl-prolyl-cis-trans isomerase Cell division/cytokins and auxin control 2.6
P10896 RUBISCO activase Central metabolism 2.01
Q7DMA9 Peptidyl-prolyl-cis-trans isomerase Cell division 1.7
Q9M0Y8 Vesicle-fusing ATPase Vesicle-mediated transport 1.1
Q9LZF6 Cell division control protein 48 homolog E, Transitional Cell division/growth process 0.6
endoplasmic reticulum ATPase
Q9M436 Chloroplast FtsZ-like protein Cell cycle 0.5

Protein metabolism-related proteins

Q40567 Tobacco calretulin Protein folding 3.2
B9RMA5 Peptidyl-prolyl cis-trans isomerase Protein metabolism 2.7
Q9FXT9 26S protease regulatory subunit 7 Protein degradation 2.2
P41382 Eukaryotic initiation factor 4A-10 mRNA binding to ribosomes 1.7
O04450 T-complex protein 1 subunit epsilon Folding proteins 1.3
P68158 Elongation factor Tu Protein biosynthesis 0.7
Q9LS91 Elongation factor EF-2 Protein biosynthesis 0.7
Q9FXG2 Glycine-tRNA ligase Protein biosynthesis 0.6

have the capacity to both scavenge and produce ROS (Nanda et al., conditions and the provision of a strong reductant (O’Brien et al.,
2010). Peroxidase N1 belongs to the Class III peroxidase family. This 2012). Cell wall metabolism is very important for cell develop-
family utilizes H2O2 and other hydroperoxides as a substrate. ment, peroxidases are considered to play a primary role regulating
However, these enzymes can also generate H2O2 under specific cell wall elongation, another defense response against pathogen
38 G. Cardenas-Manríquez et al. / Environmental and Experimental Botany 130 (2016) 33–41

Table 3
Contents of total bioactive compounds and antioxidant activity in 2, 4 and 6 months old CchGLP transgenic tobacco lines and wild type plants.

Line Phenolics Flavonoids Tannins Anthocyanins Antioxidant

(mg GAE g 1
) (mg RE g 1) (mg CE g 1
) (mg TE g 1) activity
(mM ET g 1)
WT
2m 469.82  21.1e 698.903  82.351c 25.52  3.83d 2.00  0.30f 3.35  0.21b
4m 663.94  22.04d 696.23  111.60c 54.63  2.28b 66.61  7.78d 2.81  0.09b
6m 718.39  45.32d 956.21  122.61b 82.70  7.51a 103.53  4.62c 2.91  0.16b
L1
2m 472.22  33.2e 1218.25  80.32a 21.07  4.36d 2.64  0.41f 3.12  0.35b
4m 729.74  10.31d 790.01  120.28c 34.63  1.98c 28.69  11.9 e 3.21  0.19b
6m 1391.09  52.79a 1121.81  140.85b 46.77  9.19b 145.56  15.86b 2.83  0.34b
L8
2m 459.05  70.6e 710.74  30.77b 18.47  2.54 d 1.55  0.19f 3.22  0.31b
4m 891.95  21.01c 605.12  117.72b 41.60  1.12c 79.94  10.65d 2.90  0.13b
6m 1142.24  38.91b 1376.14  123.06a 87.09  9.58a 147.92  10.14b 3.08  0.29b
L25
2m 478.21  30.0 e 562.38  97.82b 21.07  2.71d 5.09  0.21f 3.23  0.17b
4m 842.96  33.81b 799.51  112.27b 34.48  5.56c 60.77  11.28d 3.25  0.15b
6m 1069.11  72.95b 1110.83  158.76b 61.96  8.86a 184.13  25.43a 2.81  0.26b
L26
2m 523.71  15.74a 782.00  92.71b 18.84  3.53d 1.42  0.22f 3.28  0.18b
4m 825.86  9.78c 852.93  104.217b 52.20  4.03b 44.10  8.18c 3.94  0.18a
6m 1493.53  64.48a 1412.94  87.11a 79.50  9.39a 154.07  17.76a 3.99  0.27a

attacks include cell wall modifications such as lignin deposition, production is increased prominently under drought, heat or
accumulation of phenolic compounds among others (de Marco their combination stress and may play a crucial role in abscisic
et al., 1999; O’Brien et al., 2012). Ascorbate peroxidases are key acid-induced antioxidant defense (Hu et al., 2010). Hsp90 is most
H2O2 scavenging enzymes from the chloroplasts and cytosol of known for managing protein folding but it also plays a key role in
plants cells (Faize et al., 2011). Under non-stress conditions, signal-transduction networks, this is because most of its known
ascorbate peroxidase is expressed at extremely low levels, substrates are signal transduction proteins such as steroid
however, the expression of this enzyme is rapidly induced when hormone receptors and signaling kinases (Wang et al., 2004).
leaves are subjected to light excess, wounding or under salt stress While Hsp70 is up-regulated by cold stress, Hsp90 is
(Aghaei and Komatsu, 2013; Ball et al., 2004). It is thought that the down-regulated by the same stress (Kosová et al., 2011), and both
accumulation of ascorbate peroxidases in plants is a part of the are up-regulated by oligochitosan and oligopectin elicitors (van
cell’s detoxification system during oxidative burst. Aubel et al., 2013). Glutamate cysteine ligase catalyzes the
Glucan endo-1,3-b-glucosidase corresponds to a Pathogenesis- biosynthesis of glutathione, thereby playing an important role
Related (PR) protein induced in tobacco plants infected with in plant metabolism and stress response. It is induced by both
P. tabaci, P. parasitica and TMV (Meins and Ahl, 1989), in tomato abiotic and biotic stresses. Glutathione is involved in detoxification
infested with Bemisia tabaciBemisia tabaci, oligochitosan and against ROS, xenobiotics compounds, heavy metals and may act as
oligopectin elicitors (Park and Ryu, 2014; van Aubel et al., 2013); a signaling molecule (Ball et al., 2004; Hothorn et al., 2006;
in addition, drought stress also induced this protein in wheat Liedschulte et al., 2010). Interestingly, the germin-like protein
(Gregorová et al., 2015). This protein targets pathogen cell walls, (CchGLP) was also detected up-regulated in L8 as expected.
strengthening against virus, bacteria and fungi, thus it is suggested Moreover, several proteins related to primary and secondary
that this polysaccharide modifying enzyme affect accumulation of metabolism (energy production, nucleotides synthesis, terpenoids
soluble sugars under water stress. Heat-shock proteins (HSPs) are synthesis, etc), protein synthesis and cell proliferation (protein
molecular chaperones, contributing to cellular homeostasis under kinases, annexin, histone binding proteins, etc), were also detected
optimal and stress conditions. Its major functions include protein up-regulated in this study (Table 2). Altogether the proteomic
folding, assembly, translocation, stabilization, refolding and profile obtained suggested that L8 up-regulated proteins related
degradation of proteins (Feder and Hofmann, 1999; Wang et al., with plant performance compatible with stress tolerance in
2004). HSPs are not only induced by heat stress but also by other comparison to L1. It is expected that the proteome found correlates
stresses such as cold, drought, inadequate or excessive light (Feder with the metabolomic profile in these plants, as shown in other
and Hofmann, 1999; Kosová et al., 2011; Li et al., 2011). Hydrogen plant models related with networking governing plant metabolism
peroxide production in plants leads to HSPs accumulation, Hsp70 (Gaudinier et al., 2015).
Bioactives detected as well as the antioxidant capacity
associated (Table 3), suggested that antioxidant activity is
associated with homeostasis strategies to keep a redox balance
Table 4 between ROS production and the antioxidants compounds in the
Phenolic compounds detected in transgenic tobacco lines 1 and 8.
organism system (Bakht and Shafi, 2012). Based on the results
Line Chlorogenic acid Caffeic acid Rutin Quercetin shown in Table 3, it is suggested that some total bioactive
wt 527.5b** 4.2a ND* 2.05a compounds were increased in their contents, not withstanding, not
1 530.4b 3.9a 2.09b 2.1a always this increase correlated with phenotype behavior of the
8 546.1a 4.1a 2.3a 2.1a plants. Several previous studies have reported similar results
*
ND = not detected. regarding that total bioactive compounds not always explains
**
Different letter in the same column indicates significant difference (P < 0.01). plant tolerance to stresses (Tierranegra-García et al., 2011). Thus, it
 G. Cardenas-Manríquez et al. / Environmental and Experimental Botany 130 (2016) 33–41 39

Fig. 2. Heatmap of accumulation patterns of metabolites identified in wt, and transgenic L1 and L6 tobacco plants.

Fig. 3. Impact on metabolic pathways suggested by the metabolites identified in transgenic line 8 in comparison to L1 and wt tobacco plants.
40 G. Cardenas-Manríquez et al. / Environmental and Experimental Botany 130 (2016) 33–41

Fig. 5. Detection of pal and cat-1 gene expression in wt and CchGLP-transgenic lines
L1 and L8 of N. tabacum cv. xanthin nc after 0 and 15 days of drought = stress
experiments.

between L1 and L8, the main altered pathways in the expressing

CchGLP transgenic line corresponded to the aminoacyl-tRNA
Fig. 4. Hydrogen peroxide levels during drought-stress studies in wt and CchGlp-
biosynthesis, the alanine, aspartate and glutamate metabolism,
transgenic L1 and L8 lines of N. tabacum cv, xanthin nc. Different lettters between the nitrogen metabolism, and the valine biosynthesis, leucine and
respective columns of the same color in days 0 and 15 indicates significant isoleucine (Fig. 3).
difference (P < 0.05). Finally, although no up-regulated protein of phenylpropanoids
pathway was detected in the proteome of transgenic tobacco
during the study, pal gene expression was detected and slightly
is expected that detecting and quantifying specific bioactives by induced during drought-stress studies in L8, L1 and wt (Fig. 4).
metabolomic approaches, some compounds might correlate with Interestingly, pal levels were higher, as expected in L8, even before
the proteome and the phenotype of stress tolerance. drought-stress (day 0). L8, is a transgenic line that constitutively it
The results of specific phenolics detected in the present work, is expressing the CchGLP gene encoding a Mn-SOD based on
agree with others elsewhere in tobacco plants (Torras-Claveria experiments in E. coli expression systems (León-Galván et al.,
et al., 2012; Wang et al., 2008). Chlorogenic acid and rutin were 2011). This “constitutive” induction of Mn-SOD in this line (and in
significantly in higher amounts in line 8 in comparison to line 1 the other high expressing CchGLP lines as L25 and L26), likely it is
(Table 4). Chlorogenic acid is the main polyphenol in tobacco leaves maintaining a state of alert of these plants to respond to any biotic
(Wang et al., 2008; Wooten et al., 2009), which agree with our or abiotic stress. Likely, PAL and other phenylpropanoid pathway
results, being the one with higher concentration in our samples. related-proteins were present in a low quantity such that they
Likewise, it is associated with hydroxyl and superoxide radicals scaped the limit of detection in our proteomic study. Currently in
removal. Compounds like chlorogenic acid and rutin can form our laboratory we are finishing the study of deep-transcriptome
conjugates with carbohydrates, polyamines and other phenols as a profile by RNA-Seq of the tobaccos evaluated in the present work,
part of a strategy of storage and detoxification of the phenyl- in order to get a wider scope of the Omic profiles of these plants,
propanoids metabolism intermediaries (Torras-Claveria et al., hoping to help us in unravel in more detail the mechanisms of
2012). The heatmap graphi graphic displayed in Fig. 2 matched tolerance to biotic and abiotic stress observed in CchGLP transgenic
with the type of compounds characterized in metabolomic profiles N. tabacum cv. xanthin nc.
performed in other studies (Broeckling et al., 2004; Mungur et al.,
2005). Line 1 and wt tobacco although displayed similar phenotype 5. Conclusions
towards drought tolerance and previously geminivirus resistance
(Guevara-Olvera et al., 2012) showed some differences in the High ROS production in CchGLP expressing lines generated a
presence and levels of some metabolites detected in the present response by the plant to counteract oxidative stress, causing a slow
work. These fluctuations may be result of natural response to development in the plant. Our results suggested that maintaining
environmental stimulus because as demonstrated by Guevara- certain ROS levels within tobacco plants (and likely in other
Olvera et al. (2012), showing that L1 exhibits the same level species) might be an interesting strategy to help plants to tolerate
infection against geminiviruses as the wt. biotic and abiotic stress conditions, as those predicted to face in
On the other hand, significant higher contents in 32 compounds close future agriculture due to global warming provoking an
was shown in the transgenic line 8 in comparison to L1 and wt, environment adverse for food production. This increase of some
suggested changes in the plant metabolism in this line, likely as an metabolites and proteins associated with plant defense mecha-
adjustment to the transgene expression of CchGLP as an important nisms, should contribute to the protection mentioned for
role in metabolic pathways signaling of the ROS over-expression in agricultural activities the century XXI. It can be speculated that,
L8. This latter results also suggested that the changes in the in challenge with biotic and abiotic stresses, the plants expressing
contents of these metabolites in L8 in regards to L1, was the result higher and controlled ROS levels (likely by controlled elicitation),
of a strategy adapted by the L8 to tolerate the oxidative stress to could respond faster and stronger by activating resistance
which cells are subjected and which in turn, might be closely mechanisms.
related to its capacity to response against biotic and abiotic stress
(Mejía-Teniente et al., 2013; Moller and Sweetlove, 2010). The Acknowledgements
group of metabolites with more variation was the amino acids,
suggesting a greater involvement in metabolism and protein Authors thanks to FORDECYT (193512) and Ciencia basica
synthesis as shown in the metabolic pathways impact graphic SEP-CONACYT (178429) for funding support to the present
(Fig. 3). According with the differential metabolites profile found research.
 G. Cardenas-Manríquez et al. / Environmental and Experimental Botany 130 (2016) 33–41 41

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