Quantitative Proteomics-Based Analysis Supports a Significant Role of

Article pubs.acs.org/jpr

Quantitative Proteomics-Based Analysis Supports a Significant Role of GTG Proteins in Regulation of ABA Response in Arabidopsis Roots Sophie Alvarez,† Swarup Roy Choudhury,† Leslie M. Hicks, and Sona Pandey* Donald Danforth Plant Science Center, 975 North Warson Road, St. Louis, Missouri 63132, United States S Supporting Information *

ABSTRACT: Abscisic acid (ABA) is proposed to be perceived by multiple receptors in plants. We have previously reported on the role of two GPCR-type G-proteins (GTG proteins) as plasma membrane-localized ABA receptors in Arabidopsis thaliana. However, due to the presence of multiple transmembrane domains, detailed structural and biochemical characterization of GTG proteins remains limited. Since ABA induces substantial changes in the proteome of plants, a labeling LC-based quantitative proteomics approach was applied to elucidate the global effects and possible downstream targets of GTG1/GTG2 proteins. Quantitative differences in protein abundance between wild-type and gtg1gtg2 were analyzed for evaluation of the effect of ABA on the root proteome and its dependence on the presence of functional GTG1/GTG2 proteins. The results presented in this study reveal the most comprehensive ABA-responsive root proteome reported to date in Arabidopsis. Notably, the majority of ABA-responsive proteins required the presence of GTG proteins, supporting their key role in ABA signaling. These observations were further confirmed by additional experiments. Overall, comparison of the ABA-dependent protein abundance changes in wild-type versus gtg1gtg2 provides clues to their possible links with some of the well-established effectors of the ABA signaling pathways and their role in mediating phytohormone cross-talk. KEYWORDS: GTG proteins, ABA, quantitative proteomics, iTRAQ, Arabidopsis

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INTRODUCTION The phytohormone abscisic acid (ABA) is a key regulator of many aspects of plant growth and development.1−3 The signal transduction mechanisms of ABA have been studied in great detail over the years, and a number of distinct signaling modules and interactions have been identified.3−5 Multiple ABA receptors, both intracellular and cell surface-localized, have been reported in the past few years. Fourteen PYR/PYL/ RCAR proteins and a chloroplast-localized Mg-chelatase, ABAR, represent the soluble receptors, and two GPCR-type G-proteins, GTG1 and GTG2; represent membrane-localized ABA receptors in Arabidopsis.6−9 An elegant intracellular signal perception and transduction mechanism has been established for the PYL/PYR/RCAR family of soluble ABA receptors that operates via a phosphorylation/dephosphorylation cascade involving the SnRK2 family of protein kinases and PP2C family of protein phosphatases4,10−12 that form the core of ABA signaling pathways. Additionally, a signal transduction mechanism dependent on a class of WRKY transcription factors has been proposed for the chloroplast-localized ABAR/CHLH protein.13 We have previously reported on the role of GTG1 and GTG2 as plasma membrane-localized, G-protein-coupled ABA receptors.9 The GTG proteins exhibit the inherent GTPbinding and -hydrolytic activities of signaling G-proteins but have a topology similar to that of GPCRs. The purified GTG proteins bind ABA, and the plants lacking both GTG genes (gtg1gtg2 mutants) show reduced sensitivity to ABA in all © 2013 American Chemical Society

classic responses. The presence of nine transmembrane domains in the GTG proteins, however, has made it difficult to purify reasonable quantities of fully active proteins for further biochemical and structural analysis. Consequently, in contrast to the soluble ABA receptors, a major gap remains in our understanding of how the GTG proteins affect the ABA signal transduction pathways downstream of signal perception. ABA generates global changes in the genome and proteome of plants. The ABA-regulated transcriptome has been analyzed in great detail, and a core set of ABA-induced and -repressed genes have been identified in Arabidopsis.14 The effect of ABA on plant proteome is only beginning to be investigated. Gelbased proteomics approaches have detected a handful of proteins that change in response to ABA;15−17 however, advances in gel-free quantitative proteomics approaches have been more successful in identifying comparatively larger sets of ABA-responsive proteins. Two recent studies using iTRAQ followed by LC−MS/MS in Arabidopsis guard cells and in Arabidopsis T87 suspension culture cells have identified 8 and 50 cell-specific, ABA-regulated proteins, respectively.18,19 Our previous work to characterize ABA- and/or G-protein-dependent changes in the Arabidopsis root proteome identified 720 proteins of which ∼10% (74 proteins) exhibited changes in response to ABA.20 A clear difference in the protein abundance changes in response to ABA and a lesion in the Arabidopsis GReceived: December 10, 2012 Published: January 21, 2013 1487

dx.doi.org/10.1021/pr301159u | J. Proteome Res. 2013, 12, 1487−1501

Journal of Proteome Research

Article

protein α subunit, GPA1, helped delineate ABA-dependent and -independent pathways regulated by GPA1. These results also corroborated the data obtained previously from multiple molecular-genetic studies and showcased the potential of global proteomic profiling in discovering protein abundance changes relevant to a signal and/or specific gene mutation and in revealing potential downstream pathways.20 The current study was designed to evaluate how the loss of GTG proteins would affect ABA-regulated global changes in the proteome. Toward this end we used 4-plex iTRAQ that allowed simultaneous labeling and detection of the four samples used for this comparison: Ws-2 (WT)-control labeled with reagent 114, WT-ABA-treated labeled with reagent 115, gtg1gtg2-control labeled with reagent 116, and gtg1gtg2-ABAtreated labeled with reagent 117. Quantitative differences in the protein abundance were analyzed for (i) the effect of ABA on the Arabidopsis root proteome and its dependence on the presence of functional GTG1/GTG2 proteins; (ii) the interaction, if any, of signaling pathways dependent on the GTG proteins with the well-established downstream components of ABA signaling; and (iii) the involvement of GTG proteins in mediating plant hormone cross-talk. The results reported in this study reveal the most elaborate ABA-responsive root proteome of Arabidopsis identified to date. Comparative analysis of the ABA-dependent protein abundance changes in the WT versus gtg1gtg2 plants reveals that more than 70% of the ABA-responsive proteins required the presence of functional GTG proteins, supporting their major role in ABA response. Analysis of proteins altered in response to ABA in WT plants but not in gtg1gtg2 provides clues to the downstream signaling components of the GTG receptors and their possible link(s) to some of the well-established effectors of the ABA signaling pathways.

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iTRAQ Labeling and Strong Cation Exchange (SCX)

Protein samples (100 μg each) were reduced and alkylated using iTRAQ kit reagents (Applied Biosystems/MDS Sciex, CA, USA) as per the manufacturer’s protocol. Samples were digested for 16 h at 37 °C with 10 μg trypsin, dried, resuspended in 30 μL of iTRAQ dissolution buffer (500 mM triethyl ammonium bicarbonate), and labeled with iTRAQ isobaric reagents according to the manufacturer’s instructions. WT-control, ABA-treated WT, gtg1gtg2-control, and ABAtreated gtg1gtg2 samples were labeled with reagent 114, 115, 116, and 117, respectively. Three biological replicates were processed at the same time. The 12 labeled digests were pooled into one sample for each replicate. The samples were lyophilized and dissolved in 100 μL of 20% ACN, 5 mM ammonium formate (pH 2.7) for separation by strong cation exchange (SCX). For SCX, 100 μL injections of iTRAQ labeled pooled peptides were fractionated as previously described.20 nanoLC−MS/MS

iTRAQ labeled samples were analyzed using an LTQOrbitrapVelos mass spectrometer (Thermo Fisher Scientific, Rockford, USA) coupled with a nanoLC Ultra (Eksigent, Dublin, USA). Each sample (5 μL) was injected onto the LC− MS/MS system as previously described.21 The samples were first loaded onto a trap column (C18 PepMap100, 300 μm × 1 mm, 5 μm, 100 Å, Dionex, Sunnyvale, USA) at a flow rate of 4 μL/min for 5 min. Peptide separation was carried out on a C18 column (Acclaim PepMap C18, 15 cm × 75 μm × 3 μm, 100 Å, Dionex) at a flow rate of 0.26 μL/min. Peptides from iTRAQ samples were separated using a 80 min linear gradient ranging from 2% to 40% B (mobile phase A, 0.1% formic acid in water; mobile phase B, 0.1% formic acid in ACN). The mass spectrometer was operated in positive ionization mode. The MS survey scan was performed in the FT cell from a mass range of 300 to 1700 m/z. The resolution was set to 60,000 at 400 m/ z and the automatic gain control (AGC) was set to 500,000 ions. HCD fragmentation was used for MS/MS, and the 10 most intense signals in the survey scan were fragmented. A resolution of 7500 was used in the Orbitrap with an isolation window of 2 m/z, a target value of 100,000 ions, and a maximum accumulation time of 1 s. Fragmentation was performed with normalized collision energies of 45% and activation times of 0.1 ms. Dynamic exclusion was performed with a repeat count of 1 and exclusion duration of 75 s. A minimum MS signal for triggering MS/MS was set to 5000 counts.

MATERIALS AND METHODS

Plant Growth and Treatment Conditions and Phenotypic Assays

Arabidopsis wild-type (WT) and gtg1-1gtg2-1 (gtg1gtg2) seeds9 were surface-sterilized and plated on 150 mm plates with filter paper placed on the top of media containing 0.5X MS, 1% sucrose and 1% agar. Plant growth and ABA treatment were as previously described.20 Three biological replicates were performed, each with 1200 plants per genotype per treatment. For evaluation of the effect of jasmonic acid (JA), brassinosteroid, and ethylene, seeds were grown on 0.5X MS, 1% sucrose and 1% agar media containing 1 μM JA, 10 nM BL, or 50 μM ACC, respectively. After stratification at 4 °C for 2 days, the seeds were transferred in light for 1 day, followed by growth in darkness for 5 days. For evaluation of the effect of MG132, seeds were grown in the presence of 25 μM MG132 for 7 days in light.

Data Analysis

Data was processed using Mascot Distiller v2.3 and searched using Mascot Daemon (Matrix Science, London, U.K.). For data processing, the MS/MS settings included single peak window selection from m/z 113.5 to m/z 117.5. All searches were performed against the NCBInr A. thaliana protein database subset (August 2011, 222,009 sequences) using the following settings: trypsin as cleavage enzyme; two missed cleavages; methylthio modification of cysteines; iTRAQ (N terminal) and iTRAQ (K) were fixed modifications; and protein N-terminal acetylation, methionine oxidation and iTRAQ (Y) were selected as variable modifications. The mass error tolerance for precursor ions was set to 15 ppm and 0.8 Da for fragment ions. Proteins are grouped into same set and subset families on the basis of shared/common peptide matches to remove protein inference (proteins identified with the same set of peptides) and identify intersection (proteins identified

Protein Extraction

Total protein was extracted from control and ABA-treated root tissue of WT and gtg1gtg2 as previously described.20 Briefly, the protein pellet was air-dried and resuspended in 200 μL of 1 M urea/0.5 M bicine/0.09% SDS. Protein concentration was determined using the CB-X protein assay (Genotech, St. Louis, MO, USA) according to the manufacturer’s protocol. Three biological replicates were performed for each sample. 1488



Article

Table 1. List of Proteins That Respond to ABA in WT (115/114) and/or gtg1gtg2 Mutant (117/116) Rootsa protein accession no. gi|7529742 gi|4455213 gi|3157944 gi|7269954 gi|693688 gi|17529036 gi|531555 gi|2911042 gi|11228579 gi|3668097 gi|5668814 gi|9758328 gi|7572929 gi|7329685 gi|30689983 gi|4220528 gi|3929649 gi|2827713 gi|10086511 gi|4803926 gi|17381238 gi|1402904 gi|10177041 gi|5734749 gi|6016702 gi|4704730 gi|15242870 gi|166807 gi|1022797 gi|3249084 gi|1546698 gi|16226737 gi|10092266 gi|1402908 gi|3273753 gi|10178147 gi|3395433 gi|135406 gi|166898 gi|166914 gi|1353763 gi|2632059 gi|166666 gi|2286069 gi|21554103 gi|9502167 gi|887390 gi|5902402 gi|2244762 gi|4454036

protein description

115/114

Amino Acid Metabolism 3-Isopropylmalate dehydratase-like protein (small subunit) Glutamine amidotransferase/cyclase Very strong similarity to aminomethyltransferase precursor gb|U79769 from Mesembryanthemum crystallinum Class I glutamine amidotransferase domain-containing protein Aspartate aminotransferase Putative argininosuccinate synthase Aspartate aminotransferase Phosphoglycerate dehydrogenase-like protein Aspartate-semialdehyde dehydrogenase precursor Glycine decarboxylase complex H-protein Carbohydrate Metabolism ESTs gb|H36253 and gb|AA04251 come from this gene Xylose isomerase α-Galactosidase-like protein Transketolase-like protein Transketolase Glucose-6-phosphate isomerase Mitochondrial NAD-dependent malate dehydrogenase Pyridoxal-phosphate-dependent aminotransferase-like protein Similar to transaldolase Putative triosephosphate isomerase AT4g16260/β 1,3 glucanase class I precursor Cell Redox Peroxidase 1-Aminocyclopropane-1-carboxylic acid oxidase-like protein/flavanol synthase like (FLS5) Putative glutathione transferase Putative [Mn] superoxide dismutase Peroxiredoxin TPx1/thioredoxin-dependent peroxidase Purple acid phosphatase 26 Peroxidase Glutathione reductase Similar to red-1 (related to thioredoxin) gene gb|X92750 from Mus musculus Peroxidase ATP3a homologue AT5g03630/F17C15_50 Monodehydroascorbate reductase (NADH)-like protein GSH-dependent dehydroascorbate reductase 1, putative Peroxidase Copper/zinc superoxide dismutase Glutaredoxin-like protein Peroxidase Cytoskeleton Organization RecName: Full=Tubulin α-3/α-5 chain β-2 Tubulin α-2 Tubulin Profilin 1 Defense Response Patatin-like protein Basic endochitinase β-Glucosidase 21 Major latex protein, putative Contains similarity to Pfam PF00232 (Glycosyl hydrolase family 1) Osmotin MLP-43 like protein Major latex protein like Putative major latex protein

1489

SD

NS NS NS

117/116

SD

0.85 0.89 1.12

0.04 0.02 0.10

0.80 0.89 0.82 0.85 0.85 1.14 1.33

0.14 0.02 0.06 0.05 0.02 0.02 0.24

0.81 0.94 NS NS NS NS NS

0.09 0.01

1.10 0.88 1.22 0.73 0.80 0.82 0.83 0.83 0.93 1.09 1.14

0.03 0.04 0.18 0.07 0.03 0.04 0.10 0.03 0.00 0.07 0.11

0.88 0.91 1.11 NS NS NS NS NS NS NS NS

0.03 0.05 0.07

0.57 0.81 0.72 1.27 1.06 1.17 1.18 1.23 0.78 0.85 0.91 0.95 1.13 1.23 1.23 1.33

0.12 0.01 0.02 0.03 0.03 0.18 0.02 0.08 0.07 0.04 0.08 0.01 0.05 0.11 0.20 0.15

0.58 0.74 0.77 0.84 0.89 1.08 1.13 1.31 NS NS NS NS NS NS NS NS

0.11 0.06 0.06 0.10 0.09 0.04 0.04 0.09

0.78 0.78 0.81 1.38

0.03 0.01 0.13 0.08

NS NS NS NS

0.03 0.13 0.69 0.15 0.16 0.01 0.35

1.23 1.29 0.90 1.23 3.11 NS NS NS NS

NS NS 0.87 1.46 2.56 0.79 0.79 1.26 1.28

0.09 0.08 0.07 0.23 0.31



Article

Table 1. continued protein accession no.

protein description

115/114

SD

117/116

SD

0.93

0.01 0.43

Ethylene Biosynthesis gi|4883604

Putative S-adenosylmethionine synthetase

NS Glucosinolate Metabolism

gi|4432856 gi|30694982 gi|3402676

Putative dioxygenase Myrosinase 5/BGLU35 Putative myrosinase-binding protein/JAL 22

gi|1389699

Nitrilase 1

1.70 0.80 1.11

0.10 0.08 0.04

1.54 NS NS

0.81

0.17

NS 0.10 0.07

0.00 0.06 0.20 0.19

0.84 0.87 NS NS NS NS

NS NS NS 1.22 0.77 1.18 1.27 1.34 1.43

0.14 0.16 0.14 0.04 0.04 0.17

0.87 0.91 0.93 1.24 NS NS NS NS NS

0.07 0.02 0.04 0.15

0.87

0.00

NS

IAA Biosynthesis Lipid Metabolism gi|11908116 gi|1066348 gi|6851091 gi|9759532 gi|9758577 gi|2739381 gi|9757880 gi|18420117 gi|14334734 gi|3080402 gi|10177435 gi|2462840 gi|4240120 gi|7543910 gi|4314392 gi|1208408 gi|5123562 gi|1754983 gi|5454199 gi|9294041 gi|3212849 gi|4056467 gi|3763918 gi|2911039 gi|16604707 gi|8809603 gi|6686821 gi|7413571 gi|3687244 gi|133866 gi|7546687 gi|12321110 gi|6521012 gi|7671489 gi|6899922 gi|4325324 gi|4262232 gi|3687251 gi|9757825 gi|2654122 gi|4753651 gi|2160158 gi|7262674 gi|7594559

Putative Acyl CoA binding protein Acetyl-CoA carboxylase biotin-containing subunit Acetyl-CoA carboxyltransferase α subunit Outer membrane lipoprotein-like/temperature induced lipocalin Sterol carrier protein 2-like Similar to latex allergen from Hevea brasiliensis/patatin-like protein 2/PLA 2A Mitochondrial Electron Transport NADH dehydrogenase [ubiquinone] fragment S subunit 4 (FRO1) NADH-cytochrome b5 reductase-like protein Putative NADH-ubiquinone oxireductase Putative NADPH quinone oxidoreductase NADH-ubiquinone reductase 75-kd subnit Cytochrome c Cytochrome b5 Ubiquinol−cytochrome c reductase-like protein Putative ferredoxin Nitrogen Metabolism Nitrite reductase Nucleotide Metabolism Nucleotide pyrophosphatase-like protein Other Secondary Metabolism Strictosidine synthase T3P18.13/similar to glutamate synthase Aldehyde dehydrogenase Putative methyl chloride transferase Strong similarity to gb|AB006693 spermidine synthase from Arabidopsis thaliana 3-Isopropylmalate dehydratase, small subunit Phenylpropanoid Metabolism Cinnamyl alcohol dehydrogenase-like protein Putative alcohol dehydrogenase Protein Biosynthesis Unnamed protein product Elongation factor 1B α-subunit 40S ribosomal protein S23 60S ribosomal protein L31e 40S ribosomal protein S11-1 Ribosomal protein S4 40S ribosomal protein S7 homologue, putative Cytoplasmic ribosomal protein S13 40S ribosomal protein-like S9 Multifunctional aminoacyl-tRNA ligase-like protein Lysyl-tRNA synthetase Putative 60S ribosomal protein L7 Putative ribosomal protein L28 60S ribosomal protein L10a Ribosomal protein L23a Ribosomal protein L13a like protein Similar to elongation factor 1-γ (gb|EF1G_XENLA) Strong similarity, practically identical, to a 60S Ribosomal Protein L10 (Wilm’s Tumor Suppressor Protein Homologue) 40S ribosomal S29-like protein 1490

NS NS 0.93 1.25 1.44 1.49

NS

1.09

0.04

1.33 2.46 0.80 0.90 0.92 1.10

0.01 0.32 0.03 0.01 0.02 0.01

1.22 2.54 NS NS NS NS

0.01 0.22

1.26 1.14

0.07 0.02

1.28 1.32

0.04 0.34

NS NS 0.80 0.82 0.62 0.71 0.73 0.75 0.76 0.76 0.76 0.76 0.76 0.80 0.80 0.81 0.83 0.86

0.09 0.09 0.36 0.06 0.21 0.01 0.19 0.12 0.22 0.12 0.03 0.05 0.02 0.03 0.09 0.08

0.89 0.91 0.86 0.88 NS NS NS NS NS NS NS NS NS NS NS NS NS NS

0.01 0.01 0.05 0.14

0.89

0.02

NS



Article


protein description

115/114

SD

117/116

SD

0.89 0.90 0.94 1.06 1.13 1.18 1.30 1.68

0.03 0.04 0.01 0.04 0.02 0.17 0.18 0.39

NS NS NS NS NS NS NS NS

0.84 1.44 1.41 0.77 0.86 1.14 1.35

0.09 0.23 0.03 0.01 0.07 0.00 0.24

0.86 1.30 1.47 NS NS NS NS

0.06 0.03 0.03

0.90 1.20 1.34 1.35 1.40 0.82 0.83 0.87 0.88

0.09 0.02 0.02 0.13 0.01 0.09 0.01 0.09 0.04

1.08 1.15 1.23 1.28 1.32 NS NS NS NS

0.04 0.06 0.02 0.26 0.19

0.90 1.11 1.16 1.19 1.24 1.25 1.52 1.71

0.03 0.02 0.04 0.18 0.28 0.17 0.03 0.11

NS NS NS NS NS NS NS NS

NS 1.09

1.28 NS

0.28

0.01

1.25 0.70 0.81 0.86 0.88 0.89 0.90 1.11 1.18 1.45

0.18 0.13 0.13 0.02 0.07 0.02 0.03 0.03 0.10 0.33

1.21 NS NS NS NS NS NS NS NS NS

0.23

0.68 1.12 1.15 2.87 3.65 5.20 4.95 0.90 4.13

0.13 0.08 0.02 0.13 0.12 0.94 1.27 0.08 1.80

0.77 1.07 1.18 2.96 3.03 3.71 5.22 NS NS

0.06 0.03 0.02 0.82 0.18 2.21 2.01

Protein Biosynthesis gi|3860261 gi|9665161 gi|6714454 gi|8777362 gi|304109 gi|6587799 gi|145348229 gi|4263712 gi|15242093 gi|2062164 gi|21553993 gi|5051761 gi|2446981 gi|16211 gi|2062156 gi|6652888 gi|11994380 gi|3859606 gi|2160296 gi|15724197 gi|12324950 gi|2511584 gi|2511594 gi|9665064 gi|2511590 gi|11994461 gi|9757832 gi|14532526 gi|4038039 gi|435619 gi|435618 gi|12324312 gi|4972075 gi|4467138 gi|5103831 gi|166734 gi|836940 gi|5080776 gi|531379 gi|5802794 gi|4587514 gi|4115934 gi|7671424 gi|3168840 gi|7670026 gi|8778294 gi|6625953 gi|4140257 gi|19698871 gi|9759304 gi|1592675 gi|259445 gi|16390

60S acidic ribosomal protein P2 Putative 60S ribosomal protein L9 Putative 60S ribosomal protein L22-2 60S acidic ribosomal protein P3 poly(A)-binding protein Strong similarity to gb|AF067732 ribosomal protein S12 from Hordeum vulgare Phosphorylase-like protein protein 40S ribosomal protein S12 Protein Folding TCP-1/cpn60 chaperonin family protein Jasmonate inducible protein isolog Contains similarity to jasmonate inducible protein HSP90-like protein/SHD endoplasmin-like protein AtGDI2/Rab GDP dissociation inhibitor Calnexin homologue Jasmonate inducible protein isolog/myrosinase-binding-like protein Proteolysis 26S proteasome AAA-ATPase subunit RPT6a Cucumisin-like serine protease; subtilisin-like protease Cathepsin b like cysteine protease γ-VPE At2g39050/T7F6.22/hydroxyproline rich glycoprotein Putative aminopeptidase; 4537−10989 Multicatalytic endopeptidase/PAC1 Multicatalytic endopeptidase complex, proteasome precursor, β subunit Contains similarity to a tumor-related protein from Nicotiana tabacum gb|U66263 and contains a trypsin and protease inhibitor Multicatalytic endopeptidase complex, proteasome component, β subunit type 2b Cysteine proteinase Cysteine protease component of protease-inhibitor complex RD21A Cathepsin b-like cysteine protease Protease inhibitor II/defensin like protein Thiol protease/Rd21A Thiol protease/Rd19A Putative trypsin inhibitor; 19671−20297 Proton Transport Proton pump interactor Probable V-type H+-transporting ATPase subunit B2 Signal Transduction ESTs gb|H37032, gb|R6425, gb|Z34651, gb|N37268, gb|AA713172, and gb|Z34241 come from this gene GTP-binding protein Sar1A Calcium-dependent protein kinase (CDPK3/6) Similar to protein kinases/VH1-interacting kinase 14−3−3-like protein GF14 λ 14−3−3 protein GF14 κ Similar to WO8E3.3 gi|3880615 putative GTP-binding protein from C. elegans cosmid gb|Z92773 Contains similarity to Methanobacterium thermoautotrophicum transcriptional regulator (GB:AE000850) Inorganic pyrophosphatase-like protein Copper homeostasis factor Stress Response Universal stress family protein Germin-like protein subfamily T member 2 NAD-dependent formate dehydrogenase 1A LEA-like protein Low-temperature-induced 65 kD protein/CAP160 protein Late embryogenesis abundant protein LEA like LEA76 homologue type 1 Glycine-rich protein Lti78 1491



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protein description

115/114

SD

117/116

SD

NS 0.76 1.25

0.13 0.17

0.90 0.93 1.07

0.02 0.03 0.02

0.77 1.33

0.06 0.30

NS NS

1.34

0.03

NS

NS 0.64 0.81 1.16 0.95 1.15 0.69 0.79 0.80 0.82 0.88 0.88 1.18 1.22 1.24 1.25 1.27 1.31 1.44 1.44

0.09 0.04 0.03 0.02 0.07 0.18 0.11 0.16 0.14 0.07 0.08 0.08 0.02 0.12 0.10 0.20 0.38 0.25 0.31

1.15 0.64 0.86 1.11 1.13 1.21 NS NS NS NS NS NS NS NS NS NS NS NS NS NS

Transport gi|2058282 gi|16160 gi|3834309 gi|472877 gi|3850579 gi|9758458 gi|8778615 gi|11127601 gi|18072743 gi|3319350 gi|6587836 gi|5263319 gi|21537388 gi|4337027 gi|3319349 gi|6094555 gi|13877685 gi|240254562 gi|6523100 gi|3080438 gi|2088662 gi|6041858 gi|8778650 gi|6671934 gi|10176842 gi|10176849

Ran binding protein/AtRanbp1a Adenosine nucleotide translocator Strong similarity to glycoprotein EP1 gb|L16983 Daucus carota and a member of S locus glycoprotein family PF|00954 Plasma membrane intrinsic protein 2a Strong similarity to gb|D14550 extracellular dermal glycoprotein (EDGP) precursor from Daucus carota/ aspartyl protease-like protein Nuclear transport factor 2 Unknown Similar to small nuclear ribonucleoprotein SM D3 Dormancy-associated protein homologue Methylthioalkylmalate synthase-like protein F9D12.8 gene product PATL2 patellin-4 DPP6 N-terminal domain-like protein Unknown Glyoxysomal fatty acid β-oxidation multifunctional protein MFP-a F9D12.7 gene product Hypersensitive-induced response protein 3 Heme-binding-like protein Uncharacterized protein Putative protein putative protein Expressed protein/clathrin light chain protein Unknown protein F5O11.4 Unknown protein/MD-2-related lipid recognition domain-containing protein Unnamed protein product/remorin family protein Unnamed protein product/putative lipid binding cavity

0.10 0.11 0.02 0.02 0.13 0.02

a

Proteins identified as significantly changing in abundance in either of the comparison are classified according to their functional categories. The average and standard deviation (SD) of ratio 115/114 and 117/116 are indicated when the ratio was significant in at least 2 biological replicates. If the change was not significantly reproducible in at least 2 biological replicates, it was marked as NS (not significant).

with same subset of peptides). An additional filtering of at least 2 unique/distinct peptides (excluding same peptides with different charge state or modification states) with an expectation value p < 0.05 was also used. An automatic decoy database search was also performed to assess the false positive rate of protein identification. The settings used to process the quantification results are previously described.21 Briefly the protein ratio type was the “weighted” geometric mean, normalization was “summed intensity”, outlier removal was “automatic” (Dixon’s method up to 25 data points, Rosner’s method above 25 data points), the peptide threshold was “at least homology” (i.e., peptide score does not exceed absolute threshold but is an outlier from the quasi-normal distribution of random scores), the minimum number of peptides was two (Supplemental Table 1), and the expected value of 0.05 was used as threshold for peptide confidence. The ratio reported in Supplemental Table 2 is the geometric mean and the standard deviation is the geometric standard deviation. The confidence interval is obtained by dividing and multiplying the average by the standard deviation, which is never less than 1.0. A ratio indicated as significant by an asterisk means it is significantly different from 1 at a 95% confidence level. Only the proteins identified and quantified with a ratio marked significant in at least two biological replicates were used for further analysis. The average and standard deviation were calculated and reported in Table 1.

RNA Extraction, cDNA Synthesis, and Quantitative Real-Time PCR (qRT-PCR)

Root tissue obtained from control and ABA-treated seedlings was used as a source of total RNA. Growth and treatment conditions were the same as those for seedlings grown for proteomics experiments. RNA isolation and qRT-PCR reactions were performed as described.22 Sequences of genespecific primers used in this study are available in Supplemental Table 4. Western Blotting and Quantification

Protein extractions and Western blotting were performed as previously described.20 ABI5 antibody (Abcam) was used at 1:2000 dilution and anti-rabbit IgG (secondary antibodies, Promega Corp.) were used at 1:10000 dilution. The blots were scanned and band intensities were quantified using Image J (http://rsbweb.nih.gov/ij/).

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RESULTS

Comprehensive Inventory of Arabidopsis WT and gtg1gtg2 Root Proteins

WT and gtg1gtg2 Arabidopsis seedlings were grown side by side and treated with either ABA or ethanol (solvent control). Roots were harvested after treatment, and the proteome was analyzed as previously described.21 The schematic of the experimental design is shown in Figure 1. A total of 2086 proteins were 1492



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Figure 1. Schematics of the experimental design for the iTRAQ proteomics analysis. WT, wild-type Ws plants; gtg, gtg1gtg2 mutant plants.

Figure 2. Distribution of ABA-responsive proteins in different functional categories. The plot shows percentage of proteins decreasing and increasing in abundance in each functional category identified only in ABA-treated WT Arabidopsis roots.

study.20 More than 64% of these proteins were identified in at least two biological replicates and were used for further analysis. Proteins were categorized according to their biological process

confidently identified with at least 2 unique peptides among the 3 replicates (Supplemental Table 1), resulting in almost 3-fold higher coverage of the root proteome compared to our previous 1493



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reactions (11.7%) associated with the increase in proteins involved in cell redox homeostasis (4.1%). Many proteins related to primary and secondary metabolism were also differentially regulated in response to ABA in the WT root proteome. Effect of Loss of GTG Proteins on ABA-Responsive Proteome. The protein abundance changes in response to ABA specifically dependent on the presence of functional GTG1/GTG2 proteins were evaluated by comparing the ABAregulated proteome in gtg1gtg2 (117/116) with the ABAregulated proteome in WT (115/114). Of the total 161 ABAresponsive proteins in WT, the abundance of 114 proteins (∼70%) did not change in the gtg1gtg2 in response to ABA (Figure 3), signifying that more than two-thirds of the ABA-

using blast2GO program (http://www.blast2go.org/) and assigned to 68 categories (Supplemental Figure 1). The major categories include response to stress, oxidation reduction, defense response, translation, and protein folding and transport. Interestingly, even at this increased coverage level, only 68 out of 198 (34%) root biomarkers previously identified23 overlapped with the proteins identified in this study. Similarly, 43% of the proteins identified in our report are unique and do not overlap with the proteins identified in a recent study that found 4454 proteins in Arabidopsis roots using a similar proteomics approach.24 These observations suggest that even though the coverage of root proteins has improved significantly in the past few years, the list is still far from complete and novel, uncharacterized proteins and protein isoforms remain to be identified. ABA-Responsive Proteome of Arabidopsis Roots and the Role of GTG Proteins

To evaluate the role of GTG1 and GTG2 proteins in regulating ABA-dependent proteomic changes, the following protein abundance changes were compared: (1) WT-ABA/WTControl (ratio115/114) for identification of the ABA responsive proteome in wild-type Arabidopsis roots; (2) WTABA/WT-Control (ratio 115/114) with gtg1gtg2-ABA/ gtg1gtg2-Control (ratio 117/116) for protein abundance compromised in response to ABA in gtg1gtg2 roots; and (3) gtg1gtg2-Control/WT-Control (116/114) for proteins affected by gtg1gtg2 mutation in the absence of exogenous ABA. A total of 204 proteins showed a significant difference in at least one of the comparisons of protein abundance (Supplemental Table 2). The average and standard deviation were calculated from the replicates only if the ratio was significantly different in at least two of the replicates. A total of 34 functional categories were identified among proteins that exhibit changes in at least one of the comparisons described above. Many proteins were affected in more than one comparison. Effect of ABA on the WT Arabidopsis Root Proteome. Analysis of the ratio 115/114 revealed a total of 161 proteins that showed a significant change in protein abundance with 81 decreasing and 80 increasing, making it the most elaborate ABA responsive proteome reported to date (Table 1). These proteins were assigned to 32 different functional categories (Figure 2). Proteins representing the categories “response to stress” and “defense response” constituted 25% and 10%, respectively, of total proteins that changed in response to ABA. Many well-established ABA-marker proteins such as late embryogenesis abundant-like (LEA-like) proteins, low temperature induced protein (LTI protein), and response-todesiccation (Rd) proteins25−27 were identified in these categories (Table 1), validating the efficacy of ABA treatment used in our experiments. The protein biosynthesis (translation) functional category was also highly represented in the ABA-dependent proteome with 26 proteins changing in response to ABA, including 17 ribosomal proteins. This category made up the second largest percentage (19%) of the total proteins that decreased in abundance in response to ABA. The decrease in protein biosynthesis in combination with the increase in abundance of proteins involved in proteolysis (12% of the total number of proteins that increased in abundance) supports a major role of ABA in modulating overall protein biosynthesis and protein stability.28,29 Another noticeable change was the increase in protein abundance of enzymes involved in oxidation−reduction

Figure 3. Number of ABA-regulated proteins in WT and gtg1gtg2 plants. The Venn diagram shows overlap between the changes observed with the ratios 115/114 and 117/116 and consequently the number of proteins involved in the different categories of ABA- and GTG1/GTG2-dependent responsiveness. Both ABA up-regulated and ABA down-regulated proteins are included in the analysis.

responsive proteome in WT Arabidopsis roots was directly dependent on the presence of functional GTG1/GTG2 proteins (Table 1). Forty-seven proteins were common to both WT and the gtg1gtg2, suggesting that ABA-regulated expression changes for these proteins are independent of GTG1/GTG2 proteins (Figure 3; Table 1) The GTG-dependent, ABA-responsive proteins identified in our study represent a wide range of functional categories. Significant dependence on the GTG proteins was observed in protein biosynthesis and proteolysis, two major categories identified in the ABA responsive proteome. Eighty-five percent of the proteins in the protein biosynthesis category, and 70% of the proteins in the proteolysis category were affected by ABA in WT plants but showed no change in abundance in the gtg1gtg2. Interestingly, the proteolysis category also includes different cysteine and thiol proteases, many of which are bona f ide ABA/ stress/drought marker proteins such as Rd19 and Rd21.25−27 It should be noted that even though all proteases identified in this study were upregulated by ABA in WT roots, a subset of them were independent of the GTG proteins. A similar trend was observed for a number of other ABA marker proteins belonging to LEA and Lti protein families where all proteins exhibited ABA-dependent expression changes, but only a subset of them were also dependent on the GTG proteins, e.g., Lti78. These observations suggest that both GTG protein-dependent and -independent ABA perception and signaling mechanisms exist in parallel in Arabidopsis roots. Of the 10 proteins classified under the category “signal transduction”, nine changed in response to ABA in WT plants but not in gtg1gtg2. This category includes the two GTP1494



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Table 2. Proteins Affected by the Absence of GTG1/GTG2 Proteins in the Absence of ABAa protein accession no. gi|404166 gi| 12407662 gi|6723436 gi|6006863 gi|22656 gi|1906830 gi|1336084 gi|3176668 gi|6587847 gi| 10120450 gi|3775985 gi| 15028193 gi|6522567 gi|3399769 gi|7800199 gi|6652882 gi|2599092 gi|1022778 gi|1255987 gi|4056502 gi|6671950 gi|3201620 gi|7406447 gi| 11079484 gi|3717948 gi|1755160

116/114

SD

60s ribosomal protein L13-1 Translation initiation factor 3 subunit b

protein description translation translation

0.63 0.78

0.14 0.09

60s ribosomal protein L26 Methionine synthase Nitrilase 1

response to stress; translation response to stress; amino acid metabolic process; methylation response to stress; glucosinolate metabolic process; auxin metabolic process response to stress; protein folding and transport response to stress; oxidation reduction; amino acid metabolic process translation amino acid metabolic process

0.83 0.84 0.86

0.01 0.03 0.01

0.87 0.87 0.88 0.90

0.04 0.04 0.02 0.06

amino acid metabolic process

0.90

0.06

unknown

0.91

0.08

0.93

0.01

4-coumarate- ligase-like protein Myrosinase-binding protein Polygalacturonase inhibiting protein 26S protease regulatory subunit 6b homologue Nucleosome chromatin assembly factor group 14−3−3-like protein GF14 ε 14−3−3-like protein GF14 χ 40s ribosomal protein S5 Putative 40s ribosomal protein S5 Thiocyanate methyltransferase 1 Binding to Tom V RNA 1L (long form) protein 1-Aminocyclopropane-1-carboxylic acid oxidase

response to stress; defense response; phenylpropanoid biosynthetic process; cofactor metabolic process; amino acid metabolic process; cell wall metabolic process response to stress; phenylpropanoid biosynthetic process response to stress; defense response; proteolysis defense response; signaling pathway proteolysis histone modification response to stress; brassinosteroid mediated signaling pathway response to stress; brassinosteroid mediated signaling pathway translation translation unknown signaling pathway oxidation reduction; ethylene metabolic process

0.94 1.03 1.05 1.05 1.08 1.09 1.09 1.11 1.11 1.11 1.14 1.16

0.01 0.01 0.03 0.02 0.02 0.08 0.10 0.02 0.02 0.05 0.05 0.03

3-Isopropylmalate small subunit Germin-like protein

response to stress; amino acid metabolic process response to stress; oxidation reduction

1.43 1.46

0.20 0.18

Chaperone protein htpg family protein Glutamate dehydrogenase 2 Expressed protein 2-Dehydro-3-deoxyphosphoheptonate aldolase 3deoxy-d-arabino-heptulosonate 7-phosphate synthetase Ornithine carbamoyltransferase Strong similarity to glycoprotein EP1 gb|L16983 Daucus carota and a member of S locus glycoprotein family PF|00954. Phenylalanine ammonia-lyase

biological pathway

a

The proteins are ranked from the most decreasing to most increasing in abundance. The average and standard deviation (SD) of 116/114 ratio are indicated.

fatty acid β oxidation, MD-2, and an unknown protein with lipid-binding cavity, also exhibited ABA-dependent protein abundance changes in WT but not in gtg1gtg2 roots. Interestingly, all ABA-responsive proteins related to cytoskeleton organization identified in this study were also dependent on the presence of GTG proteins. Our analysis identified 17 proteins that changed in response to ABA in the gtg1gtg2 but not in WT plants (Table 1). This observation suggests that alternative pathways induced by ABA exist in the mutant to compensate for the compromised changes normally induced by ABA through GTG1/GTG2 proteins. The proteins identified were classified into diverse functional categories, a majority of which related to the biological pathways’ response to stress and amino acid and lipid metabolism. ABA-Independent Protein Differences between WildType and gtg1gtg2 Roots. In addition to the ABAdependent protein abundance changes, 26 proteins exhibited differences between WT and gtg1gtg2 roots in the absence of ABA (Table 2). Fourteen of these proteins were present in lower abundance, and 12 were present in higher abundance in the gtg1gtg2 compared to the WT roots. The majority of

binding proteins Sar1A and WO8E3.3, two 14−3−3 proteins, and two protein kinases. One of the protein kinases, CPK3/ CPK6, is a well-known downstream component of multiple ABA signaling pathways.30−32 Many proteins related to phytohormone biosynthesis and signaling pathways were also identified as responding differently to ABA in WT versus gtg1gtg2 roots. It should be noted that we did not identify GPA1 in this proteomic comparison possibly due to its membrane association and low quantity. Even though our previous data comparing the proteome of gpa1 mutant to that of WT roots supported the role of GPA1 in regulation of ABA response and substantiated the previous physiological data, due to the lack of substantial overlap between the two ABAdependent proteome data sets any common targets that might directly link GTG proteins and GPA1 could not be identified in this study. Remarkable differences were also observed in proteins related to lipid metabolism. All proteins identified in this category were found to change only in WT or in gtg1gtg2 plants, with no overlap. Additional proteins related to lipid metabolisms/signaling, classified as “unknown category” according to Blast2Go (Table 1), e.g., proteins related to 1495



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correlation of the proteomics data with transcript data, we first performed qRT-PCR analysis of At3g19390 gene in WT and gtg1gtg2 roots with or without ABA treatment. The expression profile of At3g19390 gene was similar to its protein expression profile: the gene was expressed at a higher level but was not affected by ABA treatment in gtg1gtg2 plants, whereas it was highly ABA-up-regulated in WT plants (Figure 4A). We then

proteins present in lower abundance in the gtg1gtg2 roots were related to amino acid and protein metabolism and biosynthesis. Two enzymes involved in phenylpropanoid metabolism, 4coumaroyl-CoA ligase (4CL) and phenylalanine ammonia lyase, were also less abundant in gtg1gtg2 roots. One of the proteins present at a higher level in the gtg1gtg2 compared to the WT plants was MSI4 (FVE). MSI4 is a key regulator of the autonomous pathway that controls flowering time.33−35 MSI4 represses the expression of FLC (flowering locus C) and mutants lacking the MSI4 gene flower very late under both long day and short day conditions. It is interesting to note that the gtg1gtg2 plants also flower early when grown under either short day or long day conditions,9 potentially due to the presence of high levels of MSI4 protein. A detailed analysis of gtg1gtg2 plants, with respect to the regulation of flowering time by autonomous or environmentally regulated pathways, will provide further insight into the mechanisms that lead to their early flowering phenotype. Two proteins related to phytohormone biosynthesis were differently abundant in WT versus gtg1gtg2 roots. A 1aminocyclopropane-1-carboxylate (ACC) oxidase that catalyzes the last step of ethylene biosynthesis was present at a higher level, and nitrilase II, an enzyme involved in indole acetic acid (IAA) biosynthesis, was present at a lower level in gtg1gtg2 roots. In addition, two signal transduction-related 14−3−3 proteins (GF14 ε and χ) were also present at a higher level in gtg1gtg2 roots. GF14 chi is known to interact with the transcriptional repressor BZR1, which regulates brassinosteroid signaling and biosynthesis.36,37 Differential expression of these hormone biosynthesis/signaling-related proteins in the gtg1gtg2 roots suggests a role for them in plant hormone cross-talk mediated via GTG proteins. To elucidate the functional networks that exist in the ABAregulated proteome, we used all ABA-regulated proteins as baits to identify their predicted or confirmed interaction partners using the AI-1MAIN (Arabidopsis interactome mapping consortium38). Forty-seven proteins from our ABA-regulated protein data set were present in the AI-1MAIN database, which resulted in 253 binary protein−protein interactions. Only eight of these proteins and 18 binary interactions were also present in the gtg1gtg2 mutants (Supplemental Figure 2). Supplemental Table 3 shows the details of all proteins involved in binary interactions.

Figure 4. ABA-dependent expression profile of ABI-interacting proteins in WT and gtg1gtg2. (A) Expression of At3g19390 in WT versus gtg1gtg2 plants. (B) Transcriptional up-regulation of PYL11, PYL12, and PYL13 in gtg1gtg2 plants compared to WT plants in the absence of ABA. (C) Transcriptional down-regulation of PYL11, PYL12, and PYL13 in WT plants compared to gtg1gtg2 plants in the presence of ABA. For each gene, qRT-PCR amplification experiments were performed three times independently and the data were averaged. The expression values were normalized against Actin2/8 gene expression. Error bars represent the standard error of the mean.

Analysis of Known ABA Signaling Pathways That Are Compromised in the gtg1gtg2 Mutants

evaluated changes in regulation of PYR/PYL transcripts by qRT-PCR, with each of the 14 PYR/PYL family genes using gene-specific primers with WT and gtg1gtg2 roots with ABA or EtOH (control) treatment. The expression of almost all PYR/ PYL family genes was down-regulated by ABA (Supplemental Figure 3) as has been previously reported.40 Interestingly, the members of one of the clades of the PYR/PYL family, consisting of PYL11, PYL12, and PYL13 genes, exhibited significantly different expression in gtg1gtg2 plants inherently and also in response to ABA. The gtg1gtg2 plants have 4- to 5fold higher expression of PYL11, PYL12, and PYL13 genes compared to WT plants (Figure 4B). More importantly, the ABA-dependent down-regulation of PYL11, PYL12, and PYL13 transcript levels were significantly compromised in gtg1gtg2 compared to the WT roots (Figure 4C). The expression profiles of the other 11 members of the PYR/PYL family were similar in WT and gtg1gtg2 (Supplemental Figure 3), although a

The PP2C phosphatases and SnRK2 family of kinases form the core of many ABA signaling pathways.12 Interestingly, one of the cysteine proteinases, At3g19390 (gi:9757832), identified in our study as an ABA-upregulated and GTG-dependent protein, was previously identified as an ABI1 (PP2C)-interacting protein in the same screen that discovered the PYR/PYL family of ABA receptors as ABI1-interacting proteins.39 This led us to evaluate whether the PYR/PYL family of ABA receptors have differential expression patterns and ABA-dependent regulation in a gtg1gtg2 background. Since the direct assessment of protein quantity of the individual PYR/PYL proteins is not possible at the moment, we evaluated whether changes in the transcript level of different proteins could reliably reflect the changes observed at the protein level in our data set. Specifically, our proteomic data showed that At3g19390 protein was present at a higher level in the gtg1gtg2 compared to the WT roots. To evaluate the 1496



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Figure 5. Phenotypic analysis of WT and gtg1gtg2 plants in the presence of different hormones. Phenotype of WT and gtg1gtg2 plants were compared in the presence of ACC, BL, and JA compared to control media. Root and hypocotyl length were quantified for at least 20 seedlings per genotype per treatment. The experiment was repeated three times, and data were averaged. Error bars represent the standard error of the mean. The pictures show representative images from one experiment. Asterisk (*) represents statistically significant differences between the WT and gtg1gtg2 responses to specific hormones as determined by Student’s t test (P < 0.05).

recent report describes the role of PYL8 in regulation of ABA response in Arabidopsis roots.41

Possible Downstream Targets of ABA Signaling Affected in gtg1gtg2 Plants

Phenotypic Analysis of gtg1gtg2 Plants in the Presence of Different Phytohormones

An over-representation of proteins related to proteasomal degradation and ubiquitination pathways in the ABA-responsive proteome of WT plants and their absence from gtg1gtg2 prompted an evaluation of the effect of well-established, cellpermeable proteasome inhibitor MG13242 on overall growth of WT and gtg1gtg2 plants. The gtg1gtg2 seedlings were significantly less sensitive to MG132 compared to the WT plants in root growth assays (Figure 6A). ABI5 is one of the most well characterized proteins that integrate ABA signaling pathways to ubiquitin-mediated regulation and proteasomal degradation.43−47 ABA causes massive increase in ABI5 transcript and protein levels.43,44 ABI3, another target of ubiquitination pathway, acts upstream of ABI5, and regulates its levels.29,48 We evaluated the level of ABI5 transcript and protein as readout of the proteasomemediated regulation of ABA signaling pathways in the WT and gtg1gtg2 plants. The transcript and protein levels of ABI5 increased significantly in response to ABA in WT plants as expected. Interestingly, the gtg1gtg2 plants accumulated a higher level of ABI5 transcript and protein compared to WT without ABA treatment (Figure 6B,C). The ABI5 transcript level did not change significantly in response to ABA in gtg1gtg2

The proteomics data suggested differential abundance of proteins related to signaling or biosynthesis of other plant hormones especially jasmonic acid, ethylene, and brassinosteroids in gtg1gtg2 compared to the WT roots. To assess the biological significance of these changes, we evaluated the response of WT and gtg1gtg2 plants to different phytohormones. On control media, WT and gtg1gtg2 plants were phenotypically indistinguishable (Figure 5); however, the gtg1gtg2 plants exhibited less sensitivity to ethylene in reduction of hypocotyl length, root length, and ethylene-induced hook angle in the presence of the ethylene precursor ACC compared to WT plants (Figure 5). Similar hyposensitivity was observed for the gtg1gtg2 plants in response to epibrassinolide (BL, the most active brassinosteroid) in the hypocotyl length and in root length inhibition compared to the WT plants (Figure 5). In contrast, the gtg1gtg2 plants were significantly more sensitive to methyl jasmonate (JA) compared to the WT plants (Figure 5). 1497



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potential downstream targets of important proteins where indepth structure/function characterization may not be possible. The experimental design (Figure 1) allowed us to conduct a side-by-side comparison of the four samples used in this study in a 4-plex experiment. The use of root tissue limited the contribution from highly abundant plant proteins such as Rubisco, which resulted in a better overall representation of other proteins. Moreover, the relatively large sample size (1200 plants/sample) circumvented the influence of any possible nonbiological variation which may result from plants grown on different plates. These factors, in combination with three biological replicates and application of stringent filters for peptide identification, resulted in the detection of 2086 proteins (Supplemental Table 1). The proteins represent diverse biological functions with an over-representation of proteins related to stress and defense response, consistent with the use of ABA-treated samples (Supplemental Figure 1). Thirty-one of the 68 categories assigned to the proteins identified in the root proteome represent various metabolic or biosynthetic pathways, including proteins related to primary metabolism, which are known to be highly abundant as well as relatively low abundance proteins related to specialized pathways such as phytohormone and phenylpropanoid biosynthesis. Comparing this data set to our previous root proteome data set20 reveals some but not complete overlap. Similarly, partial overlaps observed with other studies of root proteomes23,24,49 suggest that different protein isolation and analysis methods provide complementary data sets. ABA treatment resulted in widespread changes in the root proteome with ∼10% of total proteins exhibiting a change in abundance. These proteins encompass a variety of functional categories corroborating an effect of ABA on various biosynthetic, metabolic, signaling, and protein modification processes (Figure 2).

Figure 6. Altered regulation of the ubiquitin-proteasome pathway in gtg1gtg2 plants. (A) Effect of proteasomal inhibitor MG132 on root length of WT and gtg1gtg2 plants. The experiment was repeated three times independently with similar results. Image from one representative experiment is shown. (B) ABA-dependent transcript level of ABI5 in WT and gtg1gtg2 plants as determined by qRT-PCR. Amplification experiments were performed three times independently with two biological replicates and the data were averaged. The expression values were normalized against Actin2/8 gene expression. Error bars represent the standard error of the mean. (C) ABA-dependent protein expression level of ABI5 in WT and gtg1gtg2 plants. The top panel shows Western blot with ABI5 antibodies. Bottom panel shows quantification of Western blot data using Image J (http://rsweb.nih. gov/ij/).

Role of GTG Proteins in Regulating ABA-Responsive Proteome

Multiple proteins have been identified as ABA receptors in Arabidopsis, including the PYR/PYL family of cytosolic proteins,6,7 the chloroplast-localized ABAR protein,8 and the GTG proteins.9 The signal transduction pathways for the PYR/ PYL family proteins have been described in elegant details, and crystal structure of many of the PYL receptors has been elucidated in ABA bound and unbound forms.12,50,51 Such structural/biophysical studies are relatively complicated for transmembrane receptors such as GTGs, where purification of sufficient amounts of active protein and a demonstration of conformational changes in ligand-bound versus unbound forms is extremely difficult. Furthermore, a recent report using independently generated mutant alleles of GTG genes suggests a normal ABA sensitivity of the gtg1gtg2 double mutants.52 The extent to which the altered phenotypes of different mutant alleles depend on specific growth conditions and seed quality remains to be ascertained, and a side by side comparison of all three mutants is warranted to explain the observed discrepancies. However, we reasoned that if the GTG proteins constitute a major component of the ABA perception and signaling pathway, the global view of ABA response would differ substantially between plants with or without functional GTG proteins. The results from this proteomic analysis corroborate our hypothesis as more than 70% of the proteins that exhibit abundance changes in response to ABA in WT plants require the presence of GTG proteins (Figure 3). Moreover, the proteins that show abundance changes in

plants (Figure 6B), and although an ABA-dependent increase was observed in ABI5 protein levels (Figure 6C), the response was attenuated compared to the WT plants, suggesting altered regulation of ABI5 synthesis and/or stability in the gtg1gtg2 background.

■

DISCUSSION

Arabidopsis Root Proteome and Its Regulation by ABA

Signal transduction pathways are executed by changes in the abundance, modifications, and stability of proteins. A comprehensive evaluation of the proteome and a systematic knowledge of the changes that occur in the proteome in response to any given signal are therefore crucial to understanding the physiology and underlying mechanisms of any biological response. In this study, we utilized a nontargeted proteomics approach to uncover the diversity of proteins that change in response to ABA and their dependence on the presence of GTG proteins. Our analysis reveals a comprehensive inventory of global and ABA-responsive root proteome and emphasizes the crucial role played by the GTG proteins in the regulation of ABA response, thereby showcasing the effectiveness of such large-scale approaches in pinpointing 1498



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response to ABA in WT plants only (GTG-dependent) were not restricted to specific categories but present in each of the functional categories of the ABA-responsive proteome (Table 1), suggesting that ABA signaling via GTG proteins does not target a specific pathway but utilizes the overall ABA signaling machinery of the cell. An additional 47 proteins whose abundance is not affected by the lack of GTG proteins suggest that parallel signaling pathways are activated in response to ABA and may utilize different ABA receptors.

factor involved in proteasome pathway. Consistent with the observation that many of the ABA-regulated proteins do not respond to or have significantly attenuated response to ABA in the gtg1gtg2 mutant background, the regulation of ABI5 transcript and protein is also highly compromised in gtg1gtg2 plants (Figure 6B,C). Furthermore, the gtg1gtg2 plants accumulate higher levels of ABI5 transcript and protein under nonstressed conditions, possibly due to alteration in ABA signaling pathways involved in maintaining the ABI5 levels in plants. Identification of this vast range of proteins as GTGdependent, even at the limited protein coverage (∼161 ABAresponsive proteins), suggests that major ABA signaling pathways are shared between different receptor subclasses and are compromised in the gtg1gtg2 plants.

Known ABA Signaling Components Altered in the gtg1gtg2 Mutants’ ABA-Responsive Proteome

Our analysis identified several verified components of ABA signaling pathways that showed changes in response to ABA in a GTG protein-dependent manner. Interestingly, one subclass of PYR/PYL receptors themselves exhibited altered regulation by ABA in the absence of GTG proteins (Figure 4). PYL11, PYL12, and PYL13 constitute a small subfamily of PYR/PYL receptors4 and are significantly down-regulated by ABA in WT plants but not in the gtg1gtg2 plants. These receptors are most similar to each other and are smaller in size (159−164 aa) due to shorter N- and C-termini, compared to the other members of the family (183−221 aa). The crystal structure of PYL11, 12, and 13 is not known, although based on the sequence similarity, their ABA-binding “gate” and “latch” regions are well conserved. Since recent structure and modeling based studies have shown that very similar PYL receptor family members may have distinct properties,50,51,53 unique attributes may exist for the regulation of these three receptors. The mechanism by which the lack of GTG proteins affects ABA-dependent transcriptional down-regulation of a PYL receptor subclass remains an open question at this juncture; however, it does indicate the possibility of cross-talk between different receptor subclasses and suggests that the ABA signal perception initiated by the GTG proteins may utilize the same protein kinase/ phosphatase core to transduce the signal as the soluble receptors. This idea is further strengthened by the observation that additional key ABA signaling pathway intermediates are also mis-regulated in the gtg1gtg2 plants. A prime example includes the CPK3/CPK6 protein, which is down-regulated by ABA in WT plants but exhibits no change in gtg1gtg2 (Table 1). CPK3/CPK6 is one of the best characterized members of the CDPK family in Arabidopsis and its involvement has been shown in multiple responses including ABA-dependent ion channel regulation in guard cells, drought and salt stress acclimation, and methyl jasmonate signaling.30−32 CPK3/CPK6 also provides an important link between Ca2+-dependent ABAsignaling mechanisms and PYL/PYR-dependent signaling pathways, which are largely calcium-independent. ABI5 is another central regulator of ABA signaling pathway that shows quantitative difference in gtg1gtg2 mutant background (Figure 6). ABI5 integrates ABA-dependent transcriptional regulation with the ubiquitin 26S proteasome system for protein degradation and is proposed to be a direct downstream target of the SnRK/PP2C core ABA signaling module.29,47 Even though we did not identify ABI5 in the proteomic data set, the following several factors made it the most attractive and easily tractable target in our experiments: (1) the differential regulation of a substantial number of proteins related to proteolysis and ubiquitination pathways in WT versus gtg1gtg2 plants; (2) the lower sensitivity of gtg1gtg2 seedlings to the proteasome inhibitor MG132 (Figure 6A); and (3) the wellestablished role of ABI5 as the key ABA regulated transcription

Role of GTG Protein in Plant Hormone Cross-Talk

Plant hormone cross-talk is an underlying theme of plant signaling.54,55 This notion is emphasized by the fact that many mutants identified in screens for altered sensitivity toward one hormone display altered phenotypes in response to other related hormones. This proteome analysis identified several proteins related to ethylene, jasmonic acid, and brassinosteroid signaling and/or biosynthesis as differently abundant in WT versus gtg1gtg2 plants. Phenotypic analysis demonstrated that the gtg1gtg2 mutants showed significantly reduced sensitivity in response to ACC and BL and hypersensitivity to JA (Figure 5). Analysis of the protein interaction network of ABA-responsive proteins differently regulated in WT versus gtg1gtg2 plants also supports the role of GTG proteins in plant hormone cross-talk. Even with the limited number of proteins present in the interaction data set (∼25% of the ABA-responsive proteins), it is obvious that in many cases entire protein interaction modules related to a particular hormone signal were completely missing from the gtg1gtg2 compared to the WT plants (Supplemental Figure 2). These data provide a glimpse of possible interconnected protein networks that exist in response to a signal and their differential interactions in the context of all protein−protein interactions that exist in a cell. Further improvement of proteomic techniques and enrichment of protein−protein interaction data will help identify the entire protein interaction modules that exist in response to a signal and the pathways which are altered in gtg1gtg2 plants. In conclusion, the data presented in this study show that ABA induces major changes in the protein expression levels, and a majority of these changes require the presence of functional GTG proteins. Differential abundance changes of known components of ABA signaling pathways in the gtg1gtg2 plants support the hypothesis that extensive cross-talk exists between different types of ABA receptors and the key downstream components may be affected by the activity of multiple receptors. Since ABA is both perceived as an extracellular signal as well as produced intracellularly and perceived as a signal, parallel mechanisms must exist in plants to optimize their overall response to unfavorable environmental conditions. Further studies analyzing protein−protein interaction, phosphoprotein profile, and higher order mutants among different receptor families will help unravel the interaction between different components of ABA signaling network. 1499



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ASSOCIATED CONTENT

S Supporting Information *

This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: 314-587-1471. Fax: 314-587-1571. E-mail: spandey@ danforthcenter.org. Author Contributions †

These authors contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Mr. Oleg Todorov for technical support and Ms. Christine Ehret for critical editing of the manuscript. This work was supported by an internal grant from DDPSC to S.P..

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