Volume 5, Issue 5, Pages 1138-1150 (September 2012) Proteomics and Metabolomics of Arabidopsis Responses to Perturbation of Glucosinolate Biosynthesis  Ya-zhou Chen, Qiu-Ying Pang, Yan He, Ning Zhu, Isabel Branstrom, Xiu-Feng Yan, Sixue Chen  Molecular Plant  Volume 5, Issue 5, Pages 1138-1150 (September 2012) DOI: 10.1093/mp/sss034 Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 1 Morphological and Gene Expression Analysis of Empty Vector (EV) Plants, CYP79F1 RNAi Lines 2–1 and 39–2, and cyp79f1 Mutant Plants. (A) Phenotypes of 3-week-old plants. (B) Phenotypes of 8-week-old plants. (C) Transcript levels of CYP79F1, CYP79F2, CYP79B2, and CYP79B3 in the different plants showing in (A). Actin 1 was used as control for equal loading. Molecular Plant 2012 5, 1138-1150DOI: (10.1093/mp/sss034) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 2 Proteomics and Metabolomics Platforms Used for Analyzing Proteins and Metabolites in Empty Vector (EV) Control, CYP79F1 RNAi Lines 2–1 and 39–2. (A) Complementary approaches in proteomics and metabolomics. (B) Number of differential proteins identified using 2D-DIGE and iTRAQ. (C) Number of differential metabolites identified using GC–MS, LC–MS, and MRM. Molecular Plant 2012 5, 1138-1150DOI: (10.1093/mp/sss034) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 3 Functional Classification of Differentially Expressed Proteins and Metabolites in CYP79F1 RNAi Lines 2–1 and 39–2 Compared to Vector Control. (A) Subcellular localization of differentially expressed proteins. The number next each bar is the number of proteins confidently assigned to each subcellular location. (B) Functional groups of differentially expressed proteins. The table shows the distribution of proteins in each functional category. The number highlighted gray is the total number of proteins in each category. The bar graph shows the distribution of the proteins into their functional classes. Please refer to Supplemental Table 3 for details. (C) Functional groups of differential metabolites. Please refer to Supplemental Table 4 for details. Molecular Plant 2012 5, 1138-1150DOI: (10.1093/mp/sss034) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 4 Changes of Metabolites and Proteins in Central Metabolic Pathways in RNAi Lines 2–1 and 39–2 Compared to Vector Control. (A) Metabolites in red indicate increased levels and in green indicate decreased levels in the RNAi lines compared to vector control. The square boxes beside the metabolites indicate relative fold changes in L2-1 (top) and L39-2 (bottom). The numbers in different metabolic steps highlight enzymes involved in the pathways. (B) Relative changes in the levels of different enzymes numbered as in (A). SBPASE, Sedoheptulose-1,7-bisphosphatase; TST, transketolase; RPI, ribulose-5-phosphate isomerase; RBCL, ribulose bisphosphate carboxylase large chain; RBCS, rubisco small chain; RBA, rubisco activase; GAPA, glyceraldehyde-3-phosphate dehydrogenase A; GAPA2, glyceraldehyde-3-phosphate dehydrogenase A 2; GAPB, glyceraldehyde-3-phosphate dehydrogenase B; GAPC1, glyceraldehyde 3-phosphate dehydrogenase C 1; GAPC2, glyceraldehyde 3-phosphate dehydrogenase C 2; PGM2, phosphoglucomutase 2; PGM3, phosphoglucomutase 3; SHM1, serine hydroxymethyltransferase 1; GPI, glucose-6-phosphate isomerase; A1E, aldose 1-epimerase; FBA, fructose-bisphosphate aldolase; FBA1, fructose-bisphosphate aldolase 1; FBA2, fructose-bisphosphate aldolase 2; PGK, phosphoglycerate kinase; PDC, pyruvate dehydrogenase E1 component alpha subunit; SKL2, shikimate kinase like 2; AOAT2, alanine-2-oxoglutarate aminotransferase 2; ACO1, aconitate hydratase 1; ACO2, aconitate hydratase 2; ICDH, isocitrate dehydrogenase; MDH1, malate dehydrogenase 1; ME2, NADP-malic enzyme 2; ASP5, aspartate aminotransferase 5; SAM2, S-adenosylmethionine synthetase 2; SAM3, S-adenosylmethionine synthetase 3; MHM, 5-methyltetrahydropteroyltriglutamate-homocysteine methyltransferase; BCAT4, branched-chain amino acid transaminase 4; MAM1, methylthioalkylmalate synthase 1; BCAT3, branched-chain amino acid transaminase 3; IPMS1, 2-isopropylmalate synthase 1; ST18, sulfotransferase 18; TSB2, tryptophan synthase beta-subunit 2; NIT1, nitrilase 1; TGG2, thioglucoside glucohydrolase 2; ARG, arginase; GS2, glutamine synthetase 2. Molecular Plant 2012 5, 1138-1150DOI: (10.1093/mp/sss034) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 5 Heat Map of Metabolite Changes in Amino Acid Category and Carbohydrate/Energy Category in Response to Glucosinolate Perturbation. Molecular Plant 2012 5, 1138-1150DOI: (10.1093/mp/sss034) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 6 Hormone Levels in Empty Vector (EV) and CYP79F1 RNAi Plants. The hormone levels were measured using MRM LC–MS. Values (mean ± SD) were obtained from three independent experiments. MeJA, methyl jasmonic acid; JA, jasmonic acid; DiJA, 9,10-dihydrojasmonic acid; SA, salicylic acid; IAA, indole-3-acetic acid; ICA, indole-3-carboxylic acid; MeIAA, methyl indole-3-acetic acid; BA, benzoic acid; ABA, abscisic acid; ZG, trans-zeatin glucoside; ZR, zeatin riboside; ZIN, zeatin. Letters above the columns indicate statistical significance between samples (p < 0.05). Molecular Plant 2012 5, 1138-1150DOI: (10.1093/mp/sss034) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 7 Model of Glucosinolate Network Derived from Proteomics and Metabolomics of Plants with Perturbed Glucosinolate Biosynthesis. Down-regulation of CYP79F1 and CYP79F2 caused accumulation of methionine and SAM, which can lead to changes in the levels of other amino acids and sugars, chloroplast dysfunction, oxidative stress, and changes in other primary and secondary metabolisms, especially hormone metabolism. All these processes combined result in the ultimate plant phenotype. Molecular Plant 2012 5, 1138-1150DOI: (10.1093/mp/sss034) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions