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Methyl Jasmonate Responsive Proteins in Brassica napus Guard Cells Revealed by iTRAQ-Based Quantitative Proteomics Mengmeng Zhu,†,# Shaojun Dai,†,‡,# Ning Zhu,† Aaron Booy,§ Brigitte Simons,§ Sarah Yi,† and Sixue Chen*,†,∥ †

Department of Biology, Genetics Institute, and Plant Molecular & Cellular Biology Program, University of Florida, Gainesville, Florida 32611, United States ‡ Alkali Soil Natural Environmental Science Center, Northeast Forestry University, Key Laboratory of Saline-alkali Vegetation Ecology Restoration in Oil Field, Ministry of Education, Harbin 150040, China § MDS Analytical Technologies (SCIEX), Ontario, Canada L4K 4V8 ∥ Proteomics Division, Interdisciplinary Center for Biotechnology Research, University of Florida, Gainesville, Florida 32610, United States S Supporting Information *

ABSTRACT: Stomata on leaf epidermis formed by pairs of guard cells control CO2 intake and water transpiration, and respond to different environmental conditions. Stress-induced stomatal closure is mediated via an intricate hormone network in guard cells. Although methyl jasmonate (MeJA) has been intensively studied for its function in plant defense, the molecular mechanisms underlying its function in stomatal movement are not fully understood. Here we report the effects of MeJA on Brassica napus stomatal movement and H2O2 production. Using the isobaric tags for relative and absolute quantitation (iTRAQ) approach, we have identified 84 MeJAresponsive proteins in B. napus guard cells. Most of the genes encoding these proteins contain jasmonate-responsive elements in the promoters, indicating that they are potentially regulated at the transcriptional level. Among the identified proteins, five protein changes after MeJA treatment were validated using Western blot analysis. The identification of the MeJA-responsive proteins has revealed interesting molecular mechanisms underlying MeJA function in guard cells, which include homeostasis of H2O2 production and scavenging, signaling through calcium oscillation and protein (de)phosphorylation, gene transcription, protein modification, energy balance, osmoregulation, and cell shape modulation. The knowledge of the MeJA-responsive proteins has improved our understanding of MeJA signaling in stomatal movement, and it may be applied to crop engineering for enhanced yield and stress tolerance. KEYWORDS: Brassica napus, guard cells, methyl jasmonate, iTRAQ, proteomics, stomatal movement

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INTRODUCTION Stomata are highly specialized structures on leaf epidermis, which are not only responsible for gas exchange and water transpiration but also important for external signal perception and transduction.1 A stomate is a pore composed of a pair of guard cells. These cells can change shape by swelling or shrinking brought about by the influx or efflux of ions, for example, K+, Cl−, and malate.2 Such capability is important for adjusting stomatal aperture in response to a variety of environmental stimuli, such as drought, pathogens, CO2, and light. Other guard cell features, for example, low photosynthetic capacity, high respiration rate, and high abundance of proteins for transport and signaling, also contribute to the guard cell function in response to different environmental factors.3 Because of the importance of stomatal movement, guard cell signal transduction has been a focal area of plant biology © 2012 American Chemical Society

research for decades. As a phytohormone related to dehydration, abscisic acid (ABA) can trigger stomatal closure in a dosedependent manner.2,4 Many key components in this process have been discovered using a variety of strategies.4 Guard cell ABA signaling is one of the best studied signal transduction processes in plants. In addition to ABA, many other hormones have also been found to participate in the regulation of stomatal function, and the interactions between different hormone pathways are important for the regulation.5 Methyl jasmonate (MeJA) and jasmonic acid (JA) regulate a spectrum of developmental processes and plant defense against insects and herbivores.6 Since the first report of MeJA-induced stomatal closure,7 MeJA signal transduction has become a Received: March 5, 2012 Published: May 29, 2012 3728

dx.doi.org/10.1021/pr300213k | J. Proteome Res. 2012, 11, 3728−3742

Journal of Proteome Research

Article

20, 50, and 100 μM) were added, and the stomatal aperture was measured using a Zeiss Axiostar Plus microscope (Carl Zeiss Inc., USA). For ROS scavenging experiment, 20 μM diphenyleneiodonium (DPI), 200 U/mL catalase and 10 mM ascorbic acid were incubated with the epidermal strips for 20 min, respectively, before the addition of 50 μM MeJA. Sixty stomata were analyzed in each experiment and three replicate experiments were conducted. For the proteomic analysis, 50 μM MeJA was added in the second enzyme incubation step during the guard cell preparation. The treatment time was 2 h. All results were presented as means ± standard errors of three replicates. Data were analyzed using one-way ANOVA in the statistical software SPSS 17.0 (SPSS Inc., Chicago, USA). A p value less than 0.05 was considered statistically significant.

new direction in guard cell research. Some overlapping components have been identified in ABA- and MeJA-induced stomatal closure, including production of reactive oxygen species (ROS)8 and nitric oxide (NO),9 activation of K+ efflux channels and slow anion channels,7 as well as myrosinases.10 Therefore, a crosstalk hypothesis was proposed between ABA and MeJA pathways in guard cells.8 The hypothesis is supported by the observations of MeJA hyposensitivity of stomatal closure in the ost1 (ABA hyposensitive) mutant, reduced ABA-mediated stomatal closure in the jar1 (MeJA insensitive) mutant,11 and the involvement of ABA receptor PYL4 in the JA signaling.12 The aforementioned hormoneresponsive components are only a few known to date. These shared components are not only present in the ABA and MeJA pathways, but also may exist in other stress-responsive pathways, allowing plants to develop cross-tolerance, that is, plant acclimation to other stresses after exposure to a specific stress. To better understand plant stress responses, explore the guard cell proteome, and discover more hormone-responsive components, we have employed quantitative proteomics approaches in analyzing B. napus guard cells. Using the iTRAQ approach, a total of 427 distinct proteins have been identified in B. napus guard cells and mesophyll cells.3 Among them, 74 proteins were highly expressed in guard cells compared with mesophyll cells. The preferentially expressed proteins in guard cells are mainly involved in respiration, transport, transcription, cell structure, and signaling processes.3 In addition, 104 ABA-responsive proteins have been identified in the guard cells. Sixty-six ABA-induced proteins are mainly involved in stress and defense, and 38 ABA decreased proteins function in metabolism and protein synthesis.13 However, our knowledge of MeJA-responsive proteins in guard cells is lacking. Here we aim to fill the knowledge gap and complement the ABA proteomic study13 by identifying MeJA-responsive proteins in guard cells. We have discovered 49 MeJA-induced proteins and 35 MeJA decreased proteins. They are involved in photosynthesis, energy/respiration, metabolism, transcription, protein synthesis and fate, signaling, transport, stress and defense, as well as cell structure dynamics. This study represents the most extensive analysis of the MeJA-responsive proteins in guard cells. The results provide further evidence for the crosstalk hypothesis and have revealed several interesting proteins for further investigation. Our work has contributed to a better understanding of the molecular mechanisms underlying of hormone signaling in guard cells.

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Protein Digestion, iTRAQ Labeling and Strong Cation Exchange Fractionation

Three independent guard cell preparations were pooled to yield one biological replicate. Four control replicates and four MeJA replicates, each containing 75 μg protein, were precipitated with cold acetone. The pellet was dissolved in 1% SDS, 100 mM triethylammonium bicarbonate, pH 8.5, followed by reduction, alkylation, trypsin digestion, and labeling using 8-plex iTRAQ reagent kits according to the manufacturer’s instructions (AB Sciex Inc., USA). The control samples were labeled with iTRAQ tags 113, 114, 115, and 116, and the MeJA-treated samples were labeled with tags 117, 118, 119, and 121, respectively. After labeling, the samples were combined and lyophilized. The peptide mixture was dissolved in strong cation exchange (SCX) solvent A (25% v/v acetonitrile, 10 mM ammonium formate, pH 2.8). The peptides were fractionated on an Agilent HPLC system 1100 with a polysulfethyl A column (2.1 × 100 mm, 5 μm, 300 Å, PolyLC, Columbia, USA). Peptides were eluted at a flow rate of 200 μL/min with a linear gradient of 0−20% solvent B (25% v/v acetonitrile, 500 mM ammonium formate) over 50 min, followed by ramping up to 100% solvent B in 5 min and holding for 10 min. The absorbance at 214 nm was monitored and a total of 12 fractions were collected. Reverse Phase Nanoflow HPLC and Tandem Mass Spectrometry

Each SCX fraction was lyophilized and dissolved in solvent A (3% acetonitrile v/v, 0.1% formic acid v/v). An aliquot from each fraction was submitted to different mass spectrometer platforms, QSTAR Elite, TOF/TOF 5800 and TripleTOF 5600 (AB Sciex Inc., USA). The QSTAR Elite analysis was done as previously described.13 Briefly, the peptides were separated using an Eksigent Classic nanoflow HPLC system. Peptides were eluted by application of a linear gradient from 3% solvent B (96.9% acetonitrile v/v, 0.1% acetic acid v/v in water) to 60% solvent B in 1.5 h, followed by ramping up to 90% solvent B in 10 min and equilibrating in solvent A for 20 min (3% acetonitrile v/v, 0.1% acetic acid v/v in water). Peptides were sprayed into the orifice of the QSTAR Elite MS/ MS system, which was operated in an information dependent data acquisition (IDA) mode where a TOF MS scan (m/z 300−1800, 0.25 s) followed by four MS/MS scans (m/z 50− 2000, 30−2000 ms) of the highest abundance peptide ions (with charges of 2−5) were acquired in each cycle. Former target ions were excluded for 60 s. IDA features of Analyst QS software, such as automatic collision energy (smart CE), automatic MS/MS accumulation (smart exit), and dynamic exclusion were selected. The source nebulizing gas and curtain

EXPERIMENTAL PROCEDURES

Plant Growth and Guard Cell Preparation

Brassica napus var. Global seeds were obtained from Svalöv Weibull AB (Svalöv, Sweden). Seeds were germinated in MetroMix 500 potting mixture (The Scotts Co., USA), and plants were grown under a photosynthetic flux of 160 μmol photons m−2 s−1 with a photoperiod of eight hours at 22 °C light and 20 °C dark. Fully expanded leaves from eight-week-old plants were used for guard cell preparation as previously described.3 MeJA Treatment, Stomatal Aperture and ROS Measurement

Stomatal aperture and ROS measurements were carried out as previously described.13 Briefly, freshly prepared epidermal strips were incubated in a degassed medium (50 μM CaCl2, 10 mM KCl, 10 mM MES-KOH, pH 6.2) for 3 h under light to promote stomatal opening. Different MeJA concentrations (2, 10, 3729



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FASTA database (5 222 402 entries). Search parameters included iTRAQ 8-plex quantification, cysteine modified with methyl methanethiosulfonate, trypsin digestion, thorough searching mode and minimum protein threshold of 95% confidence (unused protein score > 1.3). For quantitative changes, ratios with p-values less than 0.05 present in at least two replicates were considered significant. Only the significant ratios from the replicates were used to calculate the average ratio for the protein. It should be noted that each p-value was generated based on quantitative information derived from at least three independent peptides in each replicate. For a protein to be determined as differentially expressed, it must have been identified and quantified with at least three unique peptides.

gas were set at 12 and 20, respectively. Ion spray voltage was 2200 V and the temperature was 80 °C. For the TOF/TOF analysis, samples were separated using a Pepmap C18 column (Dionex, USA) following a 30 min 5−35% organic gradient and spotted onto target plates using a Tempo spotting system (AB Sciex Inc., USA). A matrix solution of 6 mg/mL α-cyano-4-hydroxycinnamic acid (Sigma Aldrich, USA) in acetonitrile/0.1% trifluoroacetic acid (TFA) (75:25), and 10 mM ammonium phosphate was added postUV in a mixing tee at 1 μL/min using a Harvard syringe pump (Holliston, MA, USA). Spotting interval was 24 s and the plate voltage applied during each spotting event was 2.8 kV. Peptidecontaining LC spots were submitted to the TOF/TOF 5800 analyzer with a 200 Hz repetition rate. MS full scan spectra were acquired from 800 to 4000 m/z. A total of either 800 or 1000 (sample dependent) laser shots were accumulated for each TOF-MS spectrum at an optimized laser setting. Tandem MS mode was operated with a 2 kV collision energy and a CID gas (air) over a range of 10 m/z to 95% of the precursor mass. Precursor mass window was 250 ppm in relative mode. A minimum of 800 and a maximum of 4000 laser shots were accumulated with laser stop conditions set at six product ions of signal-to-noise ratio (S/N) > 60. Data-dependent MS settings included acquisition of up to 20 most intense ions per spot. If two or more consecutive spots were within 200 ppm tolerance, the spot with the maximum S/N was subjected to tandem MS. For the TripleTOF analysis, the TripleTOF 5600 system was coupled to an Ultra 2D Plus HPLC with a cHiPLC Nanoflex microchip system (Eksigent, Dublin, USA). The online trapping, desalting, and analytical separation were conducted using the microfluidic traps and columns packed with ChromXP C18 (3 μm, 120 Å) of the Nanoflex system. Solvents were composed of water/acetonitrile/formic acid (A, 98/ 2/0.2%; B, 2/98/0.2%). After peptide loading, trapping and desalting were carried out at 2 μL/min for 10 min with 100% solvent A. At a flow rate of 350 nL/min, the analytical separation was established by maintaining at 2% solvent B for 5 min, ramping up to 10% solvent B in 2 min, and a linear gradient to 60% solvent B in 60 min. Then the gradient was increased to 90% solvent B for 10 min. Initial chromatographic conditions were restored in 2 min and maintained for 5 min. Data were acquired using an ion spray voltage of 2.2 kV, curtain gas of 20, nebulizer gas of 6, and an interface heater temperature of 150 °C. The MS was operated with a resolution of 30 000fwhm for TOF MS scans. For IDA, survey scans were acquired in 250 ms and as many as 8, 20, or 50 product ion scans were collected if they exceeded a threshold of 125 counts per second (counts/s) and with a 2+ to 5+ charge state. The total cycle time was fixed to either 1.25 s, 1.3 s, or 1.5 s. Four time bins were summed for each scan at a pulser frequency value of 11 kHz through monitoring the 40 GHz multichannel detector with four-anode/channel detection. A sweeping collision energy setting of 35 (15 eV was applied to all precursor ions for collision-induced dissociation. Dynamic exclusion was set for 1/2 of peak width (∼ 8 s), and then the precursor was refreshed off the exclusion list.

JA/MeJA Responsive Element Analysis

Homologous genes encoding the MeJA-responsive proteins were identified in the Arabidopsis genome database (http://www.ncbi. nlm.nih.gov/). The motif analysis tool (http://www.arabidopsis. org/tools/bulk/motiffinder/index.jsp) compares the frequencies of 6 bp elements in the query set with the frequencies of the elements in the current genomic set of 33 518 sequences.14 It was used to calculate the percentage of the motif occurrence and p-value. The p-value is the probability of the occurrence of specific nucleotide combination in the selected genes. Immunoblotting

Proteins from guard cells were extracted in 50 mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.1% Triton X-100, 0.5% β-mercaptoethanol, and 1 mM PMSF with vigorous vortexing. After centrifugation at 20000g, 4 °C for 15 min, the supernatant was collected. Protein concentration was quantified using Bradford assay according to the manufacturer's instructions (Bio-Rad Laboratories, Inc., USA). Guard cell protein extracts (10 μg each sample) were separated on 12% SDS−PAGE and transferred to nitrocellulose membrane using a MiniPROTEAN system (Bio-Rad Laboratories Inc., USA). The membrane was blocked for 1 h at room temperature in phosphate buffered saline (PBS) buffer containing 5% milk and washed at least three times with PBS buffer containing 0.5% Tween 20 (PBST), each for 5 min. Primary antibodies (Agrisera, Sweden) were prepared in the PBS buffer containing 1% BSA (elongation factor 1-alpha EF1A at 1:1000; glutathione-S-tranferase GST class-phi at 1:1000; ADP-glucose pyrophosphorylase ADGP at 1:1000; beta subunit of ATP synthase AtpB at 1:4000; L-ascorbate peroxidase APX at 1:2000), and then incubated with the membrane at 4 °C for overnight. After removing unbound antibodies by washing with PBST, the blots were incubated with goat antirabbit IgG secondary antibody (horseradish peroxidase conjugates, Thermo Fisher Scientific, USA) in the PBST buffer at a dilution of 1:10000, and visualized using a SuperSignal West Femto chemiluminescent kit (Thermo Fisher Scientific, USA). Signal intensity was quantitated using Quantity One software (Bio-Rad Laboratories, Inc., USA). Significant difference (p < 0.05) was assessed using Student’s t test.

Protein Identification and Quantification

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The MS/MS data were analyzed for protein identification and quantification using ProteinPilot Software 3.0.1 (AB Sciex Inc., USA). The false discovery rate was estimated with the integrated PSPEP tool in the ProteinPilot Software to be 1.0% after searching against a decoy concatenated NCBI nonredundant

Exogenous jasmonate has been reported to induce stomatal closure in Paphiopedilum7 and Arabidopsis.10 However, such an effect is not universal in the plant kingdom with the exception of soybean and barley.15,16 To test whether this effect can be extended to B. napus, we analyzed the stomatal movement

RESULTS

MeJA Induces Stomatal Closure and ROS Production

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in MeJA-treated B. napus epidermal peels using the concentration range which has been applied to a genetic relative, A. thaliana.8−11 Our results showed that MeJA induced stomatal closure in a dose- and time-dependent manner (Figure 1). Application of

epidermal tissues were treated with both MeJA and ROS scavengers, catalase, diphenylene iodonium (DPI), and ascorbic acid (ASC), the guard cell H2O2 levels were similar to or lower than control samples (Figure 2). As a result, the stomatal apertures appeared to be similar to those in the control samples. These results showed that MeJA can induce ROS production and stomatal closure, and ROS scavengers could reverse the MeJA-induced stomatal closure, suggesting the importance of MeJA-induced ROS production in stomatal movement. The MeJA and/or ROS scavenger reponsive H2O2 changes in guard cells are similar to those oberved in the ABAtreated guard cells.13 Identification of MeJA Responsive Proteins in Guard Cells

On the basis of the physiological data (Figures 1 and 2), 50 μM MeJA was used to ensure cellular responses in B. napus guard cells. Proteins were extracted from control and treated samples, and processed with our iTRAQ LC-MS workflow.13 To compare the performance of the different MS platforms, and most importantly to obtain high coverage of the protein identification and quantification, AB SCIEX QSTAR Elite, TOF/ TOF 5800, and TripleTOF 5600 were utilized to analyze the iTRAQ samples. A total of 491, 892, and 1137 proteins were identified using the different platforms with an unused score threshold of 1.3 (Supplemental Figure 1, Supplemental Table 1, Supporting Information). Compared to QSTAR Elite, more proteins are identified on the TripleTOF 5600 and TOF/TOF 5800 platforms. Within the 271 overlapping IDs from different mass spectrometers, TOF/TOF 5800 and Triple TOF 5600 results show higher unused scores, indicating better significance for identification. In addition, the quantitation results from the three platforms are complementary as well. These results showed the utility of employing different MS platforms in achieving high qualitative and quantitative proteome coverage. The protein identities represent 1220 nonredundant proteins in the B. napus guard cells. Among them, 84 proteins displayed significant changes in expression after MeJA treatment (based on significant changes in at least two out of the biological replicates as defined in the method). With a threshold of fold change (cutoff of 0.6 for decreased expression and 1.5 for increased expression) and p value (p < 0.05), 49 proteins were increased and 35 proteins decreased in expression after MeJA treatment (Tables 1 and 2). On the basis of the Gene Ontology, BLAST alignment, and information from the literature, these MeJA-responsive proteins were classified into 11 functional categories: photosynthesis, energy, metabolism, protein synthesis, protein folding/degradation, signaling, transport,

Figure 1. Time course of stomatal aperture changes in response to MeJA treatment. Different concentrations of MeJA were added to freshly prepared epidermal strips. The stomatal aperture was measured at each time point under a microscope (n = 60).

different concentrations of MeJA (from 2 μM to 100 μM) could reduce stomatal aperture and cause stomatal closure within 2 h. After 30 min treatment, 2 μM MeJA had no obvious effect on stomatal aperture, but 10, 20, 50, and 100 μM MeJA reduced stomatal apertures by about 20%, 30%, 35%, and 47%, respectively. After 1 h, the stomatal apertures were reduced by about 40−50%. The mock control did not exhibit significant stomatal movement during the treatment (Figure 1). The MeJA-induced stomatal closure is similar to what happened in B. napus under ABA treatment.13 ROS are known as essential second messengers in guard cell ABA and MeJA signaling pathways.8,11 To evaluate ROS production in B. napus guard cells under MeJA treatment, we monitored the H2O2 levels in guard cells using oxidation sensitive fluorophore dichlorofluorescein (Figure 2). The H2O2 levels in guard cells were significantly increased in MeJA-treated samples compared to control samples. The relative fluorecence intensity indicative of the relative H2O2 levels in MeJA-treated guard cells was increased more than 1.5 fold than in the control. The percentage of MeJA-responsive cells was estimated to be over 80%. The intracellular localization of H2O2 was observed from chloroplasts and other intracellular locations, which could not be discerned using the light microscope. When the

Figure 2. MeJA-induced ROS production in guard cells. Three dimensional views of ROS levels (as indicated by the fluorescent signals of dichlorofluorescein) are presented. Percentage of signal intensity relative to untreated control stomata was calculated and listed at the bottom. (A) Control. (B) 50 μM MeJA. (C) 50 μM MeJA and 20 mM diphenylene iodonium (DPI). (D) 50 μM MeJA and 200 U/mL catalase (CAT). (E) 50 μM MeJA and 10 mM ascorbic acid (ASC). The pseudocolor key indicates increase of the fluorescence levels from bottom to top. 3731


unused score

3.70 5.61 6.00 4.55 10.65 39.70

10.10 75.65 49.77 10.08 10.39 51.95 13.55 14.32 4.01 33.75 19.20

18.46 27.98 27.93

6.62 17.72 15.75

14.90 22.67 38.89

2.00

8.27

4.18 12.39 31.09

Nb

1. 2. 3. 4. 5. 6.

7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.

18. 19. 20.

3732

21. 22. 23.

24. 25. 26.

27.

28.

29. 30. 31.

gi|157849720 gi|75283326 gi|85700445

gi|167117

gi|15238142

gi|21263610 gi|75249348 gi|75312290

gi|17827 gi|15236129 gi|118595573

gi|75315930 gi|12585448 gi|17865468

gi|25089786 gi|75333362 gi|262400757 gi|461550 gi|119720766 gi|12644156 gi|18391442 gi|12643432 gi|743641 gi|75246084 gi|75311627

gi|15232249 gi|18266039 gi|7267731 gi|405617 gi|75250014 gi|1351030

accession

60-kDa beta-polypeptide of plastid chaperonin-60 precursor (CPN 60) heat shock protein 81-4 (HSP 81-4) protein disulfide isomerase (PDI)-like protein probable mitochondrial-processing peptidase subunit beta (MPPB)

40S ribsomal protein S6

UDP-D-apiose/UDP-D-xylose synthase UDP-glucose pyrophosphorylase (UGP) ADP-glucose pyrophosphorylase, small subunit, chloroplastic (AGP) 3-isopropylmalate dehydrogenase (IPMDH) aspartate aminotransferase 5 (AspAT) aldehyde dehydrogenase family 2 member B7, mitochondrial (ALDH2B7) formate dehydrogenase (FDH), mitochondrial enoyl-acyl-carrier protein reductase (ENR) glycerol kinase NHO1

ATP synthase subunit d, mitochondrial ATP synthase subunit beta-3, mitochondrial ATP synthase subunit beta ATP synthase gamma chain 1, chloroplastic hydrogen-transporting ATP synthase plasma membrane-type ATPase 1 (PMA1) vacuolar H+- ATPase subunit C V-type proton ATPase subunit E1 phosphoenolpyruvate carboxylase (PEPC) phosphoglycerate kinase (PGK) NADP-dependent malate dehydrogenase (MDH)

putative chlorophyll a/b binding protein 2.3 chlorophyll a/b binding protein chlorophyll a/b-binding protein-like LHCII Type III chlorophyll a/b binding protein photosystem II subunit S (PS II) RuBisCO large subunit-binding protein subunit alpha, chloroplastic

name

Table 1. MeJA-Induced Proteins in Guard Cells Identified by iTRAQa

2.40 1.77 2.23

0.04 0.04 0.02

0.00 0.01 0.00

0.03 0.00 0.00 0.00 0.05 0.00 0.02 0.01 0.01 0.00 0.01

0.03 0.01 0.03 0.01 0.01 0.00

P-val 117:113

At5g56030 At2g47470 At3g02090

1.56 1.91 3.05

0.04 0.03 0.00

At5g14780 1.69 0.08 At2g05990 1.67 0.03 At1g80460 1.63 0.02 Protein synthesis (1) At5g10360 6.14 0.03 Protein folding and degradation (4) At1g55490 1.66 0.03

At5g14200 At4g31990 At1g23800

Energy (11) At3g52300 2.54 At5g08680 2.33 AtCg00480 1.64 At4g04640 3.80 At5g13450 4.17 At2g18960 1.87 At1g12840 2.05 At4g11150 2.05 At2g42600 2.47 At1g79550 1.67 At5g58330 1.84 Metabolism (9) At2g27860 1.84 At5g17310 1.54 At5g48300 2.29

(6) 2.88 3.94 3.22 3.63 2.27 1.94

117:113

Photosynthesis At3g27700 At2g34430 At4g10340 At5g54270 At1g44575 At2g28000

homologue in A. thaliana

1.61 1.63 3.13

1.37

3.10

1.82 1.56 1.49

2.29 1.91 2.36

1.49 1.57 2.33

3.66 2.23 1.58 2.73 4.09 1.07 1.89 1.87 1.28 1.56 1.71

3.19 6.37 2.86 2.94 2.25 1.92

118:114

0.04 0.07 0.00

0.11

0.03

0.02 0.10 0.03

0.04 0.04 0.02

0.01 0.01 0.00

0.01 0.00 0.00 0.01 0.06 0.64 0.03 0.02 0.22 0.01 0.01

0.03 0.01 0.05 0.02 0.02 0.00

P-val 118:114

1.26 1.92 2.40

1.53

3.16

1.50 1.82 1.53

2.44 1.77 2.15

1.63 1.49 2.11

1.98 2.03 1.57 2.68 4.33 1.38 1.82 1.94 1.58 1.56 1.72

2.07 3.63 1.87 4.53 2.40 1.96

119:115

0.23 0.03 0.00

0.03

0.17

0.04 0.02 0.01

0.04 0.03 0.04

0.02 0.01 0.00

0.05 0.00 0.00 0.02 0.05 0.03 0.04 0.01 0.04 0.00 0.01

0.08 0.01 0.08 0.04 0.02 0.00

P-val 119:115

1.67 1.21 1.36

1.36

0.30

0.69 1.64 1.61

2.31 1.36 2.70

1.54 1.42 1.24

1.38 1.27 1.45 1.60 1.15 1.53 1.12 1.50 0.69 1.85 1.15

8.09 3.31 3.70 3.84 2.75 1.85

121:116

0.02 0.35 0.88

0.11

0.48

0.64 0.19 0.01

0.05 0.11 0.02

0.01 0.03 0.61

0.22 0.04 0.01 0.14 0.79 0.01 0.88 0.13 0.19 0.00 0.07

0.01 0.03 0.18 0.19 0.02 0.00

P-val 121:116

1.62 1.91 2.86

1.59

4.62

1.66 1.75 1.56

2.38 1.82 2.36

1.62 1.50 2.24

3.10 1.97 1.56 3.07 4.25 1.59 1.92 1.95 2.03 1.66 1.75

4.72 4.31 3.04 3.70 2.42 1.92

3 2 3

2

2

2 2 4

3 3 4

4 4 3

2 4 4 3 2 3 3 3 2 4 3

3 4 2 3 4 4

average R no.c

Journal of Proteome Research Article


3733

4.00 17.39 13.24 15.04 10.69

4.11

29.18 7.70 14.24 18.89 5.03 26.06 10.65 12.15 30.06

5.64 22.90 11.14

32. 33. 34. 35. 36.

37.

38. 39. 40. 41. 42. 43. 44. 45. 46.

47. 48. 49.

gi|914911 gi|75299507 gi|75274048

gi|122216331 gi|54043095 gi|157849698 gi|169244541 gi|15229806 gi|75248680 gi|75263009 gi|122178786 gi|75099813

gi|125557716

gi|15222248 gi|187936039 gi|75332066 gi|122215093 gi|75331830

accession

glycolate oxidase (GO) glycolate oxidase (GO) superoxide dismutase (SOD) superoxide dismutase (SOD) putative 2-cys peroxiredoxin BAS1 precursor ascorbate peroxidase (APX) glutathione S-transferase (GST) gamma-glutamylcysteine synthetase (GCS) late embryogenesis abundant 2 (LEA 2) protein/dehydrin (DHN) germin-like protein (GLP) lipoxygenase (LOX1) polygalacturonase inhibitor-like protein (PGIP)

hypothetical protein OsI_024484, containing Arf1-Arf5like subfamily domain

protein phosphatase 2A regulatory subunit (PP2A) ADP-ribosylation factor (ARF) probable calcium-binding protein CML13 calmodulin 5 open stomata 1 (OST1)

name

At5g20630 At3g45140 At3g20820

2.01 1.77 1.69

0.04 0.02 0.05

0.00 0.04 0.00 0.02 0.03 0.02 0.04 0.02 0.00

Stress and defense (12) At3g14415 2.40 At3g14415 1.61 At4g25100 2.81 At3g10920 1.91 At3g11630 2.56 At1g07890 3.22 At1g10370 2.11 At4g23100 2.44 At2g44060 2.31

0.03 0.05 0.04 0.01 0.05

1.09 1.98 1.75

2.07 1.57 2.47 2.01 2.81 2.83 1.92 2.27 2.31

1.53

12.59 2.44 1.80 2.11 1.53

P-val 117:113 118:114

0.03

117:113

Signaling (5) At3g25800 8.02 At1g23490 4.74 At1g12310 1.71 At2g27030 2.05 At4g33950 1.50 Membrane and transport (1) At2g47170 1.50


0.11 0.03 0.06

0.00 0.03 0.00 0.02 0.02 0.02 0.05 0.03 0.00

0.00

0.00 0.05 0.03 0.03 0.04

1.45 1.67 1.94

2.01 1.25 3.05 1.94 2.31 2.65 2.13 2.33 2.61

1.80

5.75 1.67 2.01 1.79 1.34

P-val 118:114 119:115

0.12 0.04 0.05

0.00 0.16 0.00 0.02 0.04 0.02 0.04 0.02 0.00

0.00

0.01 0.78 0.01 0.06 0.13

11.48 2.44 2.00

1.92 1.58 1.89 1.32 1.32 1.14 1.66 1.46 1.60

1.27

4.25 2.42 1.56 2.94 1.29

P-val 119:115 121:116

0.02 0.54 0.04

0.00 0.03 0.03 0.07 0.67 0.64 0.07 0.26 0.00

0.00

0.08 0.18 0.05 0.01 0.21

P-val 121:116

6.75 1.81 1.97

2.10 1.59 2.55 1.95 2.56 2.90 2.05 2.35 2.21

1.53

8.79 2.44 1.77 2.37 1.51

2 3 2

4 3 4 3 3 3 3 3 4

4

3 2 4 3 2

average R no.c

a MeJA (50 μM) was applied for 2 h during guard cell isolation and the data represent all significantly induced proteins. The proteins were classified according to their functions. Protein identification confidence (unused score), gene bank index, protein name, protein ratios and p-values given by the ProteinPilot software are presented. bNumberical list of MeJA-responsive proteins. cThe number of significant replicates out of a total of four replicates.

unused score

Nb

Table 1. continued



3734

3.13 3.38 2.00

29 30 31

2.19 3.31

18 19

7.69 7.82 2.01 6.95

5.31

17

25 26 27 28

2.00 8.18 11.96 19.46

13 14 15 16

36.77

4.52 7.19

11 12

24

25.25 2.77 3.52 6.08

7 8 9 10

2.19 3.91 3.79 3.30

12.27 16.26 19.44 10.59 15.78 83.07

1 2 3 4 5 6

20 21 22 23

unused score

Nb

gi|15218215 gi|15220684 gi|1199503

gi|15222111 gi|15225924 gi|4324971 gi|158523427

gi|75328787

gi|15218011 gi|22326646 gi|18401305 gi|15224796

gi|15226055 gi|157849770

gi|15240075

gi|75707983 gi|121550795 gi|15221119 gi|75180270

gi|4033349 gi|15239128

gi|15219234 gi|399091 gi|22329337 gi|15235730

gi|81301541 gi|262400730 gi|262400743 gi|262400775 gi|262400774 gi|75294948

accession

coatomer protein complex subunit beta 2 putative coatomer protein complex, subunit alpha, putative transmembrane channel protein

putative calcium-binding protein, calreticulin putative mitogen-activated protein kinase (MAPK) phospholipase D2 (PLD2) myrosinase

elongation factor 1-alpha (EF1 α)

high mobility group protein (HMG1) tudor domain-containing protein (TDRD) argonaute 4 (AGO4) putative WD-40 repeat protein MSI4

2-isopropylmalate synthase 1 nitrilase 1 aminomethyltransferase-like precursor protein putative alanine-2-oxoglutarate aminotransferase (OGAT) succinate dehydrogenase flavoprotein subunit (SDH) putative fatty acid elongase allene oxide cyclase 1

ATPase 70 kDa subunit vacuolar membrane proton pump 1 (AVP1) sucrose-phosphate synthase/transferase (SPS) putative phosphoenolpyruvate carboxykinase (PEPCK) phosphoenolpyrovate carboxylase (PEPC) 2-oxoglutarate dehydrogenase, E1 component (OGDH)

photosystem II protein D1 photosystem II CP43 chlorophyll apoprotein photosystem II CP47 chlorophyll apoprotein photosystem I P700 chlorophyll a apoprotein A2 photosystem I P700 apoprotein A1 ribulose bisphosphate carboxylase large chain (RuBisCO LSU)

name

117:113

0.74 0.28

Energy (6) 0.63 0.26 0.34 0.66

0.28

0.00

0.01 0.02 0.00 0.05

0.28 0.00

0.06 0.05 0.01 0.13

0.01 0.00 0.00 0.01 0.00 0.01

P-Val 117:113

At2g15090 0.29 0.02 At1g13280 0.69 0.02 Transcription related (4) At1g20690 0.24 0.02 At5g07350 0.22 0.05 At2g27040 0.34 0.05 At2g19520 0.53 0.04 Protein synthesis (1) At5g60390 0.75 0.18 Signaling (4) At1g12900 0.64 0.01 At2g46070 0.57 0.05 At3g15730 0.44 0.02 At5g26000 0.39 0.02 Membrane and transport (3) At1g52360 0.40 0.01 At1g62020 0.39 0.00 At2g45960 0.39 0.05

At5g66760

Metabolism (7) At1g18500 0.44 At3g44310 0.29 At1g11860 0.20 At1g23310 0.47

At1g53310 At5g65750

At1g78900 At1g15690 At1g04920 At4g37870

Photosynthesis (6) AtCg00020 0.31 AtCg00280 0.31 AtCg00680 0.28 AtCg00340 0.41 AtCg00350 0.14 AtCg00490 0.53


Table 2. MeJA-Reduced Proteins in Guard Cells Identified by iTRAQa

0.44 0.40 0.19

0.63 0.44 0.60 0.35

0.46

0.13 0.14 0.31 0.47

0.39 0.61

0.30

0.57 0.61 0.50 0.61

0.36 0.36

0.52 0.17 0.28 0.19

0.37 0.28 0.21 0.41 0.12 0.44

118:114

0.02 0.01 0.01

0.01 0.01 0.07 0.03

0.00

0.31 0.02 0.03 0.03

0.03 0.31

0.02

0.02 0.02 0.03 0.16

0.02 0.00

0.02 0.02 0.03 0.00

0.00 0.00 0.00 0.01 0.00 0.01

P-Val 118:114

0.43 0.34 0.47

0.66 0.51 0.38 0.36

0.90

0.20 0.35 0.41 0.60

0.33 0.50

0.48

0.42 0.43 0.31 0.91

0.42 0.36

0.55 0.22 0.60 0.54

0.45 0.39 0.27 0.34 0.15 0.43

119:115

0.01 0.00 0.07

0.01 0.02 0.01 0.00

0.63

0.05 0.00 0.09 0.14

0.02 0.08

0.01

0.00 0.02 0.02 0.83

0.01 0.01

0.04 0.04 0.19 0.08

0.03 0.01 0.00 0.01 0.00 0.18

P-Val 119:115

0.65 0.28 0.95

0.52 0.34 0.65 0.15

0.50

0.12 0.31 0.30 0.45

0.52 0.21

0.57

0.52 1.57 0.32 0.37

0.49 0.35

0.67 0.70 1.32 0.11

1.47 1.22 0.34 0.36 0.20 0.42

121:116

0.16 0.00 0.93

0.03 0.00 0.15 0.04

0.00

0.11 0.04 0.03 0.08

0.09 0.01

0.03

0.01 0.85 0.04 0.02

0.02 0.01

0.34 0.55 0.63 0.03

0.29 0.50 0.01 0.16 0.00 0.00

P-Val 121:116

0.43 0.35 0.29

0.61 0.46 0.41 0.31

0.48

0.22 0.26 0.32 0.50

0.34 0.45

0.41

0.49 0.44 0.33 0.42

0.43 0.34

0.54 0.22 0.31 0.15

0.38 0.33 0.28 0.38 0.15 0.46

3 4 2

4 4 2 4

2

2 4 3 2

3 2

4

4 3 4 2

3 4

2 3 2 2

3 3 4 3 4 3

average R No.c



2 0.18 0.03 0.21 0.04 0.15 0.08 0.35 0.06 unknown protein, containing CBS domain gi|37999993 2.16 35

stress and defense, cell division and fate, cell structure, and unknown.

MeJA (50 μM) was applied for 2 h during guard cell isolation and the data represent all significantly reduced proteins. The proteins were classified according to their functions. Protein identification confidence (unused score), gene bank index, protein name, protein ratios and p-values given by the ProteinPilot software are presented. bNumberical list of MeJA-responsive proteins. cThe number of significant replicates out of a total of four replicates.

actin 2 gi|15230191 2.00 34

Article

Characteristics of Guard Cell Proteomic Changes in Response to MeJA

We identified 12 MeJA-responsive photosynthetic proteins in guard cells, including eight photosystem (PS) II proteins, two PS I proteins, and two CO2 assimilation-related proteins (Tables 1 and 2). Among them, four chlorophyll a/b binding proteins, PSII subunit S, and RuBisCO large subunit-binding protein (RuBisCO LSU BP) were induced by MeJA, while three photosystem II proteins (D1, CP43, and CP47), two PS I P700 proteins (A1 and A2), and RuBisCO LSU were reduced in levels. Seventeen MeJA-responsive proteins were involved in carbohydrate and energy metabolism (Tables 1 and 2). Ten of them are H+ transporting energy pumps, of which eight were increased in levels (five ATP synthases in mitochondria or chloroplasts, one plasma membrane ATPase 1 (PMA1), and two vacuolar H+-ATPases (subunit C and E1)) and two were decreased by MeJA (vacuolar ATPase 70 kDa subunit and vacuolar H+-pyrophosphatase 1 (AVP1)). In addition, six starch and sucrose metabolism enzymes were affected by MeJA treatment, including sucrose-phosphate synthase (SPS), phosphoglycerate kinase (PGK), NADP-dependent malate dehydrogenase (NADP-MDH), phosphoenolpyruvate carboxykinase (PEPCK), and two isoforms of phosphoenolpyruvate carboxylase (PEPC). Furthermore, a citric acid cycle-related enzyme, 2-oxoglutarate dehydrogenase (OGDH), was reduced in level by MeJA. Three cell wall enzymes were induced by the MeJA treatment (Tables 1 and 2). They are UDP-D-apiose/UDP-Dxylose synthase (UDP-DA/DX-S), UDP-glucose pyrophosphorylase (UGP) and ADP-glucose pyrophosphorylase (AGP). In addition, three amino acid metabolism-related enzymes, gamma-glutamylcysteine synthetase (GCS), 3-isopropylmalate dehydrogenase (IPMDH), and aspartate aminotransferase 5 (AspAT) were increased by MeJA. Furthermore, we found that MeJA induced some enzymes involved in the dehydrogenation process, such as aldehyde dehydrogenase family 2 member B7 (ALDH2-B7) and formate dehydrogenase (FDH). In contrast, other enzymes such as alanine-2-oxoglutarate aminotransferase, succinate dehydrogenase flavoprotein subunit, 2-isopropylmalate synthase 1, nitrilase 1, and aminomethyltransferase were decreased under MeJA treatment. Moreover, two fatty acid related enzymes, enoyl-acyl-carrier protein reductase (ENR) and fatty acid elongase were affected by MeJA. Interestingly, a key jasmonate biosynthesis enzyme, allene oxide cyclase 1 (AOC1), was decreased by MeJA. MeJA treatment affected gene transcription and protein metabolism in the guard cells. Four proteins (high mobility group protein (HMG1), tudor domain-containing protein (TDCP), WD-40 repeat protein MSI4 (MSI4), and argonaute 4 (AGO4)) involved in transcription were decreased in levels (Tables 1 and 2). Of the two protein synthesis-related proteins, 40S ribosomal protein S6 (40SRP) was induced, but elongation factor 1-alpha (EF1 α) was decreased after MeJA treatment. In addition, four proteins involved in protein folding and degradation were induced by MeJA, that is, 60-kDa betapolypeptide of plastid chaperonin-60 precursor (CPN 60), heat shock protein 81-4 (HSP81-4), protein disulfide isomerase (PDI)-like protein, and mitochondrial-processing peptidase subunit beta (MPPB).

a

2 0.55 0.30 0.73 0.04 0.57 0.11 0.65 0.03

2 4 0.46 0.04 0.11 0.01 0.50 0.04 0.09 0.01 0.62 0.07 0.03 0.00 0.48 0.03 0.01 0.00

Stress and defense (2) At2g33150 0.44 At1g54410 0.03 Cell structure (1) At3g18780 0.53 Unknown (1) At5g10860 0.52 3-ketoacyl-CoA thiolase 2 (KAT 2) water stress induced protein gi|15225798 gi|75127356 2.05 6.24 32 33

accession unused score Nb

Table 2. continued

name


117:113

P-Val 117:113

118:114

P-Val 118:114

119:115

P-Val 119:115

121:116

P-Val 121:116

average R No.c


3735



Article

Table 3. Motif Analysis of Genes Encoding MeJA Responsive Proteins (upstream 500 bp)a motif sequence

absolute absolute no. in no. in genomic selected observed in set genes selected genes

AGGCCC

27

21/77 (27.3%)

5238

GGGCCT

27

21/77 (27.3%)

5238

GCCCAA

36

25/77 (32.5%)

8666

TAAGCC

23

19/77 (24.7%)

4911

GGCCCA

39

23/77 (29.9%)

8914

TGGGCC

39

23/77 (29.9%)

8914

GCCAAA

11

10/77 (13.0%)

9731

CGCCAT

12

12/77 (15.6%)

2625

AAGGCC

19

16/77 (20.8%)

4531

GGCCTT

19

16/77 (20.8%)

4531

GCCCAG

9

9/77 (11.7%)

1712

GCCCAT

24

21/77 (27.3%)

7924

AAGCCT

20

18/77 (23.4%)

5176

GCCTAA

20

17/77 (22.1%)

4814

GGGCCA

12

12/77 (15.6%)

2920

TGGCCC

12

12/77 (15.6%)

2920

TGCCTT

4

4/77 (5.2%)

4957

GGCCCC

5

5/77 (6.5%)

666

GGGGCC

5

5/77 (6.5%)

666

a

observed in genomic set 4180/33518 (12.5%) 4180/33518 (12.5%) 6384/33518 (19.0%) 4426/33518 (13.2%) 5930/33518 (17.7%) 5930/33518 (17.7%) 8218/33518 (24.5%) 2456/33518 (7.3%) 3830/33518 (11.4%) 3830/33518 (11.4%) 1590/33518 (4.7%) 5749/33518 (17.2%) 4661/33518 (13.9%) 4372/33518 (13.0%) 2663/33518 (7.9%) 2663/33518 (7.9%) 4518/33518 (13.5%) 640/33518 (1.9%) 640/33518 (1.9%)

absolute no. in selected motif genes sequence

P-value

observed in selected genes

absolute no. in genomic observed in set genomic set

2.30 × 10−4

AGCCGT

11

11/77 (14.3%)

2787

2.30 × 10−4

TGAGCC

12

12/77 (15.6%)

3032

1.88 × 10−3

GCCAAT

20

17/77 (22.1%)

5342

2.64 × 10−3

ATAGCC

13

13/77 (16.9%)

3680

3.21 × 10−3

AGCCGC

7

7/77 (9.1%)

1451

3.21 × 10−3

ACGGCC

9

7/77 (9.1%)

1512

5.80 × 10−3

GGCCGT

9

7/77 (9.1%)

1512

6.15 × 10−3

TAGCCG

9

9/77 (11.7%)

2187

6.90 × 10−3

TCAGCC

11

11/77 (14.3%)

3234

6.90 × 10−3

ACGCCA

9

9/77 (11.7%)

2250

7.10 × 10−3

CAGGCC

8

7/77 (9.1%)

1661

8.63 × 10−3

GCCATT

20

17/77 (22.1%)

5888

8.82 × 10−3

GGCCTG

8

7/77 (9.1%)

1661

1.00 × 10−2

GCCACG

11

10/77 (13.0%)

2914

1.05 × 10−2

TGCCAC

14

12/77 (15.6%)

3796

1.05 × 10−2

AATGCC

4

4/77 (5.2%)

3908

1.17 × 10−2

TAGCCT

3

3/77 (3.9%)

3284

1.24 × 10−2

TAGCCA

16

14/77 (18.2%)

4709

1.24 × 10−2

CACGTG

20

10/77 (13.0%)

7766

2453/33518 (7.3%) 2805/33518 (8.4%) 4853/33518 (14.5%) 3411/33518 (10.2%) 1377/33518 (4.1%) 1388/33518 (4.1%) 1388/33518 (4.1%) 2051/33518 (6.1%) 2769/33518 (8.3%) 2149/33518 (6.4%) 1530/33518 (4.6%) 5280/33518 (15.8%) 1530/33518 (4.6%) 2594/33518 (7.7%) 3482/33518 (10.4%) 3588/33518 (10.7%) 3065/33518 (9.1%) 4292/33518 (12.8%) 3253/33518 (9.7%)

P-value 1.41 × 10−2 1.46 × 10−2 2.19 × 10−2 2.36 × 10−2 2.49 × 10−2 2.58 × 10−2 2.58 × 10−2 2.62 × 10−2 2.72 × 10−2 3.23 × 10−2 3.74 × 10−2 3.74 × 10−2 3.74 × 10−2 3.79 × 10−2 4.60 × 10−2 4.63 × 10−2 4.68 × 10−2 4.71 × 10−2 8.64 × 10−2

Note: 68 IDs out of 77 contain either GCC motif (p < 0.05) or G box (CACGTG) in the 500 bp upstream of the start codon of each gene.

Promoter Element Analysis of Genes Encoding MeJA Responsive Proteins

Nine MeJA signaling components were identified, including three Ca2+ signaling proteins (calmodulin 5, calcium-binding protein CML13, and calreticulin), a GTP-binding protein of the Ras superfamily (ADP-ribosylation factor (ARF)), two protein kinases (open stomata 1 (OST1) and mitogen-activated protein kinase (MAPK)), phospholipase D2 (PLD2), protein phosphatase 2A regulatory subunit (PP2A), and myrosinase (Tables 1 and 2). Thirteen MeJA-responsive proteins were involved in stress and defense (Tables 1 and 2). Six of them were ROS scavenging enzymes induced by MeJA, that is, two isoforms of Fe-superoxide dismutase (SOD), glycolate oxidase (GO), 2-cys peroxiredoxin BAS1 (Prx), ascorbate peroxidase (APX), and glutathione S-transferase (GST). Three ROS scavenging-related proteins (late embryogenesis abundant family protein (LEA), germin-like protein (GLP) and lipoxygenase (LOX1)) were also induced by MeJA. In addition, MeJA induced the polygalacturonase inhibitor-like protein (PGIP) involved in bacterial and fungal defense. In contrast, two defense-related proteins (3-ketoacyl-CoA thiolase (KAT 2) and water stress induced protein) were decreased after MeJA treatment.

The upstream 500 bp of the genes encoding the MeJAresponsive proteins (Tables 1 and 2) were examined for the common cis-acting elements. Different (Me)JA-responsive elements (JREs) have been identified in several plant species, for example, potato, soybean, tobacco, and Arabidopsis. The most common JREs include a GCC motif and a G box CACGTG.17 All 6 bp elements containing the GCC core sequence and the G box present in the genes are listed in Table 3. The majority of the genes (68 out of 77 with genomic homologues) contain either the GCC motif or a G box sequence in the upstream 500 bp regions. Our result of JRE analysis not only supports the proteomics findings of the MeJAresponsive proteins but also indicates that most of the genes are potentially regulated at the transcriptional level by (Me)JA. Immunoblot Validation of Proteins Identified from Proteomic Analysis

In order to validate the observed abundance changes in proteomic studies, a few proteins of interest were selected for further analysis using Western blotting based on the availability of antibodies. The results confirmed that MeJA treatment of 3736



Article

Figure 3. Western blot of selected MeJA-responsive proteins in B. napus guard cells. (A) Coomassie stained gel image showing equal loading of protein samples (10 μg each lane) from control and MeJA-treated guard cells (MW, molecular weight marker). (B) Representative Western blot images of different proteins in guard cells (ADGP, ADP-glucose pyrophosphorylase small and large subunits; ATP synthase B, subunit B of ATP synthase; EF1α, elongation factor 1-alpha; cAPX|pAPX, cytoplasmic and peroxisomal L-ascorbate peroxidase; GST, glutathione S-tranferase). Arrow indicates the isoform identified by mass spectrometry. (C) iTRAQ ratios of the proteins shown in (B). (D) Quantitative changes of protein levels shown in (B) determined from three Western experiments (***: p < 0.001; **: p < 0.01; *: p < 0.05).

guard cells significantly induced the levels of ADP-glucose pyrophosphorylase (ADGP), beta subunit of ATP synthase (ATPase B), L-ascorbate peroxidase (APX), and glutathione-Stranferase (GST), and decreased the elongation factor alpha subunit (EF1α) (Figure 3). ADGP with two small and two large subunits (L2S2) catalyzes the first committed step in the synthesis of starch in chloroplasts.18 Here we confirmed the existence of multiple subunits of B. napus ADGP since the antibody reacted with one small subunit and two large subunits of ADGP. ATPase B is one of the subunits of mitochondrial ATP synthase, which is involved in ATP synthesis from ADP and phosphate. Whether the increased abundance of this subunit correlates with guard cell ATP production is not known. APX plays a key role in the plant antioxidant system by reducing hydrogen peroxide to water in the chloroplast (tAPX and sAPX), cytosol (cAPX), and peroxisome (pAPX). Here the elevated levels of cAPX (25 kDa) and pAPX (31 kDa) in MeJAtreated guard cells were confirmed (Figure 3). Another component in the ROS scavenging system is GST, which catalyzes conjugation of reduced glutathione to a range of hydrophobic compounds. A 1.4-fold change of the GST abundance in MeJA-treated guard cells compared to the untreated samples showed the same trend found in the proteomic analysis (Figure 3 and Table 1). As a representative of the decreased proteins in response to MeJA, EF1α showed

about 70% decrease at the protein level based on Western blot analysis, compared to a 52% decrease detected using iTRAQ (Figure 3 and Table 2). EF1α is an essential enzyme in elongation phase of protein synthesis and is localized in the cytoplasm. The decrease indicates retarded protein synthesis or protein degradation might serve as a protective mechanism in guard cells in response to MeJA. Although the fold changes determined by Western blotting do not match exactly to the ratios found in the proteomic analysis, the same trend of changes were observed. The consistency and correlation between the immunoblotting and proteomic results have shown the reliability of the iTRAQ-based proteomic analysis.

■

DISCUSSION

ROS Homeostasis during MeJA-Induced Stomatal Closure

Stress conditions induce the generation of ROS (e.g., H2O2), which can serve as secondary messengers or cause oxidative damage to biomolecules and their functions.19 MeJA can induce H2O2 accumulation through plasma membrane NAD(P)H oxidases in guard cells.11,20 H2O2 levels are essential to regulate Ca2+ channels, protein phosphorylation/dephosphorylation, cellular redox state, and ROS-responsive gene expression.20−22 The activities of guard cell protein phosphatase (e.g., ABI1 and ABI2) are sensitive to cellular redox state.22,23 ABI1 and ABI2, belonging to the PP2C protein family, interact with SnRK2 and 3737



Article

Calcium Oscillation and Protein (De)Phosphorylation in Response to MeJA

PYR/PYL/RCAR to form a protein complex. This protein complex regulates transcription factors, ion channels, and the plasma membrane NADPH oxidase during stomatal movement.22 Thus, ROS levels in guard cells need to balance between oxidative damage and signaling in coping with stress conditions. In this study, MeJA was found to induce H2O2 production in guard cells, and the increased H2O2 levels can be recovered to normal levels in the presence of ROS scavenger activities. Concomitantly, the MeJA-responsive stomatal aperture changes appeared to follow the changes of H2O2 (Figure 2). This suggests that the ROS/cellular redox state is important in the stomatal MeJA signaling process. Here some ROS scavenging enzymes and redox-related proteins were found to be altered in the MeJA signaling process. Seven ROSrelated enzymes (two isoforms of Fe-SOD, GO, Prx, APX, GST, and GLP) were induced by MeJA (Table 1, Figure 4). SOD is the key enzyme catalyzing the dismutation of superoxide into oxygen and H2O2. GO is another enzyme for H2O2 generation by converting glycolate to glycoxylate. GLPs have SOD or oxalate oxidase activity leading to the production of H2O2.24 It is reported that GLPs were induced by MeJA or H2O2 in Nicotiana attenuate,25 barley,26 Capsicum chinense,27 and Arabidopsis.28 The up-regulation of these enzymes by MeJA could increase the H2O2 levels in guard cells, and subsequently activate the H2O2 signaling processes.11 On the other hand, the up-regulation of APX, Prx, GST, and gamma-glutamylcysteine synthetase (GCS) in MeJA-treated guard cells contributes to the removal of excessive H2O2 for ROS homeostasis. APX catalyzes H2O2 to water using ascorbate as an electron donor in a glutathione-ascorbate cycle. Prx employs a thiol-based catalytic mechanism to reduce H2O2 and is regenerated using thioredoxin as an electron donor in the Prx/Trx pathway. GST has GPX activity to reduce organic hydroperoxides of fatty acids and nucleic acids to the corresponding monohydroxyalcohols. The MeJA-induced GST accumulation was found in Arabidopsis leaves in our previous study.6 In addition, GCS is the first enzyme in glutathione (GSH) biosynthesis and is involved in ABA-regulated stomatal closure.29 Recently, catalase (CAT) was reported to be important for ROS scavenging in ABA-induced stomatal closure.30 However, CAT was not detected in this study. Other antioxidants, including LEA2/DHN, LOX1 and PGIP, were found to be induced by MeJA in this study. LEA2/DHN belongs to LEA family, which may have several functions including redox balancing as an antioxidant.31 LEA2/DHN can also function as a chaperone for maintaining protein structure and function32 and can regulate signal transduction or JAresponsive gene expression.33 LOXs catalyze the hydroperoxidation of unsaturated fatty acids to initiate the synthesis of various oxylipins including JA. LOXs were induced by MeJA in Arabidopsis leaves and roots, and soybean suspension culture cells.34−36 Interestingly, another enzyme involved in JA biosynthesis, 3-ketoacyl-CoA thiolase 2 (KAT2), was reduced in guard cells after MeJA treatment. KAT2 catalyzes the last step of β-oxidation of fatty acid to produce JA and ROS as a byproduct.37 Thus, the decreased KAT2 in MeJA-treated B. napus guard cells may promote ROS homeostasis. The direct involvement of the ROS related proteins in stomatal movement deserves further investigation.

MeJA-induced H2O2 production can activate Ca2+ channels that mediate Ca2+ influx to elevate cytosolic calcium concentration ([Ca2+]cyt) in guard cells, which leads to activation of slow anion channels and inactivation of inward-rectifying K+ channels.21 These events cause K+ efflux, guard cell turgor decrease, and subsequent stomatal closure. Calmodulin (CaM) is an abundant intracellular Ca2+ receptor in guard cells. CaM and CaM-regulated protein phosphorylation are necessary for the [Ca2+]cyt signaling in the MeJA signaling pathway.11 In addition, extracellular CaM contributes to stomatal movement by triggering a cascade of intercellular signaling events.38 Although it was reported that [Ca2+]cyt oscillation plays a key role in the guard cell JA signaling,39 the information on MeJAregulated Ca2+-binding proteins remained obscure. Here we identified the up-regulation of two isoforms of CaMs (CaM13 and CaM5) under MeJA treatment. We also found a MeJAdecreased calreticulin, an ER-localized Ca2+-binding protein as a regulator of Ca2+ homeostasis. These results provide evidence for the potential functional involvement of Ca2+-binding proteins in stomatal closure (Figure 4). Various protein phosphatases and kinases are important regulators in the ABA-/MeJA-signaling cascades in stomatal closure. In our proteomics results, we found MeJA-induced PP2A and OST1, as well as a MeJA-reduced MAPK. The PP2A is a ubiquitous and conserved heterotrimeric serine/threonine phosphatase playing a dual regulatory role in the ABA signal transduction. In Arabidopsis guard cells, PP2A (RCN1) is confirmed to be a positive transducer in early ABA/MeJA signal cascade to regulate ABA/MeJA-induced ROS production, NO levels, [Ca2+]cyt increase, and inward K+ channel suppression.9,40,41 Interestingly, the elevation of NO levels induces the generation of lipid second messenger phosphatidic acid (PA) by activating phospholipase D (PLD) and phospholipase C (PLC) during stomatal closure.42 In the PLD family, PLD1 was known to be involved in ABA-induced stomatal closure,43 but the functions of the other family members are not known. Here we identified a MeJA-reduced PLD2 in B. napus guard cells. The role of PLD2 in guard cell MeJA response deserves further investigation. Another ABA/MeJA-induced protein kinase is OST1 in the SnRK2 subfamily. OST1, together with a PP2C phosphotase and the ABA receptor PYR/PYL/RCAR, form the core regulatory complex in guard cell ABA signaling. The binding of ABA to PYR/PYL/RCAR deactivates the PP2C due to the blocking of the PP2C active site, and in consequence keeps the OST1 in the phosphorylated and active form to regulate the downstream targets, including the NADPH oxidase, slow anion channel, K+ inward-rectifying channel, as well as transcription factors.44 In the MeJA signaling pathway, OST1 was also found to be involved in regulation of NADPH oxidase activity and H2O2 production in guard cells.11 Interestingly, a MAPK identified in this study was decreased in response to MeJA. Several MAPKs (e.g., MPK4, MPK9, and MPK12) are preferentially expressed in guard cells and positively regulate ROS-mediated ABA signaling.45,46 Downregulation of the MAPK in B. napus guard cells may be attributed to a feedback inhibitory mechanism in MeJA signaling (Figure 4). In addition, myrosinase was also found to be decreased in response to MeJA. Myrosinase is an abundant protein in guard cells, which hydrolyzes glucosinolates to produce isothiocyanates. Isothiocyanates may regulate ABA/ MeJA-mediated stomatal movement (Figure 4).10,47 However, 3738



Article

Figure 4. Schematic model of MeJA-responsive proteins in B. napus guard cells. The dashed lines represent tentative edges in the network. [Ca2+]cyt, cytosolic calcium concentration; 1,3BPGA, 1,3-bisphosphoglycerate; 2-OA, 2-oxo acids; 2-PGA, 2-phosphoglycerate; 3-MD, 3-malate derivative; 3PGA, 3-phosphoglycerate; 40SRP, 40S ribosomal protein S6; ADP, adenosine diphosphate; ADGP, ADP-glucose pyrophosphorylase; ADPG, ADPglucose; AGO4, argonaute 4; AOC, allene oxide cyclase; APX, ascorbate peroxidase; ARF, ADP-ribosylation factor; Asc, ascorbic acid; ATP, adenosine triphosphate; CaM, calmodulin 5; CML, calcium-binding protein CML13; CP, photosystem II CP43/CP47; CPN, 60-kDa betapolypeptide of plastid chaperonin-60; CRT, calreticulin; D1, photosystem II protein D1; DAG, diacylglycerol; DHA, dehydroascrobate; EF1α, elongation factor 1-alpha; ER, endoplasmic reticulum; F-6P, fructose-6-phosphate; G, glucosinolates; G-1P, Glu-1-phosphate; G3P, glyceraldehyde3-phosphate; G6P, glucose-6-phosphate; GCS, gamma-glutamylcysteine synthetase; GLP, germin-like protein; GO, glycolate oxidase; GPA1, αsubunit of the trimeric G protein; GSH, reduced glutathione; GSSG, oxidized glutathione; GST, glutathione S-transferase; H2O2, hydrogen peroxide; HMG, high mobility group protein; HSP, heat shock protein; IP3, inositol trisphosphate; IPMDH, 3-isopropylmalate dehydrogenase; ITC, isothiocyanates; KAT2, 3-ketoacyl-CoA thiolase 2; LEA, late embryogenesis abundant 2; LHC, light harvesting complex chlorophyll a/b binding protein; LOX1, lipoxygenase; M, myrosinase; MAPK, mitogen-activated protein kinase; MDA, monodehydroascrobate; MDH, NADP-dependent malate dehydrogenase; MeJA, methyl jasmonate; MPPB, mitochondrial-processing peptidase subunit beta; MSI4, WD-40 repeat protein MSI4; NADH, nicotinamide adenine dinucleotide; NADP, nicotinamide adenine dinucleotide phosphate; OGAT, alanine-2-oxoglutarate aminotransferase; OGDH, 2-oxoglutarate dehydrogenase; OST1, open stomata 1; P700, photosystem I P700; PA, phosphatidic acid; PDI, protein disulfide isomeraselike protein; PEP, phosphoenolpyruvate; PEPC, phosphoenolpyrovate carboxylase; PEPCK, phosphoenolpyruvate carboxykinase; PG, polygalacturonase; PGIP, polygalacturonase inhibitor-like protein PGK, phosphoglycerate kinase; PIP2, phosphatidylinositol 4,5-bisphosphate; PLD, phospholipase D; PM ATPase, plasma membrane ATPase 1; PP2A, protein phosphatase 2A; PP2C, protein phosphatase 2C; Prx, 2-cys peroxiredoxin BAS1; PS II, photosystem II subunit S; PYL, pyrabactin resistance-like; PYR, pyrabactin resistance; RCAR, regulatory component of ABA receptor; RLSB, RuBisCO large subunit-binding protein subunit alpha; ROS, reactive oxygen species; RuBisCO, ribulose-1,5-bisphosphate carboxylase/oxygenase; RuBP, ribulose-1,5-bisphosphate; RuBP, ribulose-1,5-bisphosphate; S7P, sedoheptulose-7-phosphate; SBP, sedoheptulose1,7-bisphosphate; SDH, succinate dehydrogenase flavoprotein subunit; SOD, superoxide dismutase; TDRD, tudor domain-containing protein; TF, transcription factor; UDA/UDX Synthase, UDP-D-apiose/UDP-D-xylose synthase; UDP, uridine diphosphate; UDPDA, UDP-D-apiose; UDPDG, UDP-D-glucuronate; UDPDX, UDP-D-xylose; UDPG, UDP-glucose; UGP, UDP-glucose pyrophosphorylase; V-H+-ATPase, vacuolar H+-ATPase.

the physiological significance of myrosinase reduction by MeJA is not known.

D-Xylose is the main content of cell wall, and D-apiose is a plant-specific branched-chain monosaccharide critical for cell wall cross-linking. Thus, AXS is important for cell wall structural integrity and thickening (Figure 4).49 UGPase and AGPase are key enzymes for the synthesis of cell wall contents. They were found to be responsive to drought in potato guard cells.50 Besides, a polygalacturonase inhibitor-like protein (PGIP) was for the first time found to be accumulated in MeJA-treated guard cells. B. napus genome encodes at least 16 PGIPs, most of which were induced by MeJA.51 PGIPs may impede pathogen invasion by inactivating polygalacturonase secreted by pathogens. In this study, PGIPs could be involved in the cell wall modification that enables stomata to respond to MeJA (Figure 4).

MeJA-regulated Cytoskeleton Dynamics and Cell Wall Modification

Cytoskeleton dynamics and cell wall modification contribute to guard cell shape changes during stomatal movement. Actin dynamics is important for stomatal movement, and actin polymerization is regulated by ABA through phosphorylation/ dephosphorylation.48 Here we found that MeJA decreased actin levels in B. napus guard cells, implying actin abundance is regulated by MeJA. In addition, three cell wall modificationrelated proteins (AXS, UGPase, and AGPase) were induced by MeJA in B. napus guard cells. AXS catalyzes the conversion of UDP-D-glucuronate to UDP-D-apiose and UDP-D-xylose. 3739



Article

MeJA-regulated Gene Expression and Protein Modification

MeJA-induced starch synthesis-related enzymes (UDP-DA/DX-S, UGP, ADP, and ADGP) as well as MeJA-induced sucrosephosphate synthase (SPS) are regulatory enzymes for plant starch−sucrose interconversion (Figure 4).59 Higher concentration of starch in closed stomatal guard cells was observed in Vicia faba and Commelina communis.57,58 In addition, the accumulated starch might provide an endogenous reserve for organic anion synthesis in the course of stomatal reopening.57 The MeJA-increased enzymes in our results were proposed to promote starch synthesis and reduce sucrose contents for stomatal closure. Another osmoregulatory metabolite in guard cells is malate. Our proteomics results revealed that some malate metabolism-related enzymes were changed, such as the MeJAincreased NADP-MDH, MeJA-decreased PEPCK, and two MeJA-responsive isoforms of PEPC with opposite expression patterns (Figure 4). PEPC catalyzes a metabolic branching point for malate accumulation, and PEP production is regulated by PEPCK and NADP-MDH.60,61 How these changes affect guard cell malate levels remains to be determined.

We found four proteins involved in the regulation of gene expression, high mobility group protein (HMG1), WD-40 repeat protein MSI4 (MSI4), tudor domain-containing protein (TDCP), and argonaute 4 (AGO4), were decreased by MeJA in guard cells (Figure 4). HMG1 can modulate DNA interaction, chromatin association, and interaction with transcription factors. The expression levels of HMG1 are responsive to stress conditions (e.g., salt and drought) and affect the expression of stress-responsive genes. MSI4 is a chromatin assembly and modification-related protein and it interacts with histone and pre-mRNA to regulate gene expression and pre-mRNA processing.52 AGO4 is a component of the RNA-induced silencing complex that affects DNA methylation and transcriptional gene silencing.53 Taken together, the four MeJA-responsive proteins function to regulate gene expression. Further investigation is needed as to their significance and mode of action in MeJAmediated stomatal movement. Proteins involved in proteins synthesis and folding were induced by MeJA. They are 40S ribosomal protein S6, chaperonin (e.g., plastid chaperonin 60 and HSP 81-4), MPPB peptidase, and PDI. PDI can promote protein disulfide formation, isomerization, and reduction. The activity is dependent on cellular redox potential and polypeptide substrates. PDI has been shown to protect cells against oxidative burst resulting in degradation of thylakoid membrane in rice,54 and to be regulated by JA in tomato.55 The increased expression of PDI in guard cells may protect cellular components against potential damage by elevated ROS after MeJA treatment.

Protein Components Shared between MeJA and ABA Pathways

In stomatal guard cells, MeJA and ABA signaling pathways share some downstream components.8,10,41,44 For example, both hormones regulate protein phosphatase 2A (PP2A), OST1, ROS and NO production, cytosolic calcium elevation, calciumdependent protein kinases (CDPKs), MAPKs, myrosinase, NAD(P)H oxidase activity, and slow anion channels.44 In the present study, we confirmed some of the aforementioned components in the ABA and MeJA signaling pathways in B. napus guard cells (Figure 4, Supplemental Tables 1 and 2). Furthermore, we found other components in response to ABA and MeJA by comparing the MeJA results with our previous ABA proteomics results (Supplemental Tables 2 and 3).13 The analysis showed that 65 proteins were uniquely identified in ABA-treated guard cells and 60 were only identified in MeJAtreated guard cells. A total 13 protein/protein family components were identified in both ABA- and MeJA-treated guard cells. Of the common proteins, 10 proteins were induced by ABA and MeJA in guard cells. They are involved in photosynthesis (chlorophyll a/b binding protein, PS II subunit, RuBisCO binding protein), energy metabolism (ATP synthase and vacuolar H+-ATPase), protein synthesis (40S ribosomal protein), protein modification (PDI), ROS scavenging (GST and LOX), and osmoregulation (MDH). Three proteins were reduced in levels by ABA and MeJA in guard cells, that is, coatomer protein, AGO4, and RuBisCO. Although the number of common proteins in ABA and MeJA samples is small due to incomplete proteome coverage of current technologies, the functional categories of proteins identified in both samples are very similar. This study has revealed many proteins with potential involvement in the MeJA pathway underlying the regulation of stomatal movement. The results provide further evidence for the crosstalk hypothesis of the two pathways. It should be noted that some of the proteins are likely to play other roles in the guard cells, indirectly related or even unrelated to stomatal movement. Further hypothesis testing studies using genetics, biochemistry, and physiological approaches are needed to characterize the functions of the proteins and cellular processes in MeJAmediated stomatal movement. An improved understanding of hormonal signaling in stomatal movement will facilitate efforts in crop engineering for enhanced yield and stress tolerance.

Photosynthesis, Energy Supply and Osmoregulation

Stomatal aperture is controlled by guard cell turgor pressure directly regulated by energy-dependent ion and solute transport across cell membranes.44 As an energy producing process, photosynthesis in guard cells is a subject of debate. Guard cell chloroplasts are supposed to operate light electron transport for ATP and reductant production, blue-light signaling, and carbon assimilation. However, guard cells contain 20−50 fold less chlorophyll than mesophyll cells, and the CO2 assimilation rate could be 2−4% that of the mesophyll cells.44 Guard cell photosynthesis has been proposed to coordinate with mesophyll photosynthesis in the course of stomatal movement.44 Our proteomics results revealed that PS II proteins (e.g., four chlorophyll a/b binding proteins, PS II subunit S, D1 protein, PS II CP43, and CP47), PS I proteins (e.g., PSI P700 A1 and A2), and the Calvin cycle enzymes (e.g., RuBisCO LSU and RuBisCO large subunit-binding protein) were regulated by MeJA (Figure 4). These proteins involved in light harvesting, electron transfer, and carbon assimilation in guard cells may play a role in regulating stomatal movement. Guard cells are known to have a high respiration rate.56 Our proteomic analysis identified PGK catalyzing ADP to form ATP (Figure 4). In addition, MeJA induced the expression of mitochondrial ATP synthase subunits, two subunits of vacuolar H+-ATPases, and a plasma membrane ATPase in guard cells. The up-regulation of these enzymes of ATP synthesis or hydrolysis suggests active energy metabolism and membrane transport activities in guard cells after MeJA treatment leading to stomatal closure. Starch is the main CO2 fixation product in guard cells. It is a old hypothsis in the last century that the interconversion between starch and sucrose can result in osmotic changes and alteration in guard cell turgor.57,58 In our results, four 3740



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ASSOCIATED CONTENT

S Supporting Information *

Supplemental Figure 1. Venn diagrams showing the complementary nature of different mass spectrometry platforms in protein identification and quantification; Supplemental Table 1, List and functional classification of identified proteins and quantitative information; Supplemental Table 2, MeJA- and ABA-induced proteins in guard cells identified by iTRAQ; Supplemental Table 3, MeJA- and ABA-reduced proteins in guard cells identified by iTRAQ. These materials are available free of charge via Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: (352) 273-8330. Fax: (352) 273-8284. E-mail: schen@ ufl.edu. Author Contributions #

These authors contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by funding from the National Science Foundation (MCB 0818051) to S.C. Drs. Sarah M. Assmann and Xiaofen Jin from Pennsylvania State University are thanked for productive collaboration on this project.

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