Front. Plant Sci. Frontiers in Plant Science Front. Plant Sci. 1664-462X Frontiers Media S.A. 10.3389/fpls.2017.00534 Plant Science Original Research Membrane Proteomics of Arabidopsis Glucosinolate Mutants cyp79B2/B3 and myb28/29 Mostafa Islam 1 2 3 Yoo Mi-Jeong 1 2 Zhu Ning 1 2 Geng Sisi 1 2 4 Dufresne Craig 5 Abou-Hashem Maged 3 El-Domiaty Maher 3 Chen Sixue 1 2 4 6 * 1Department of Biology, University of Florida Gainesville, FL, USA 2Genetics Institute, University of Florida Gainesville, FL, USA 3Department of Pharmacognosy, Faculty of Pharmacy, Zagazig University Zagazig, Egypt 4Plant Molecular and Cellular Biology Program, University of Florida Gainesville, FL, USA 5Thermo Fisher Scientific West Palm Beach, FL, USA 6Interdisciplinary Center for Biotechnology Research, University of Florida Gainesville, FL, USA

Edited by: Wei Wang, Henan Agricultural University, China

Reviewed by: Xiuli Hu, Henan Agricultural University, China; Omar Pantoja, National Autonomous University of Mexico, Mexico

*Correspondence: Sixue Chen schen@ufl.edu

This article was submitted to Plant Proteomics, a section of the journal Frontiers in Plant Science

 11 04 2017 2017 8 534 02 02 2017 24 03 2017 Copyright © 2017 Mostafa, Yoo, Zhu, Geng, Dufresne, Abou-Hashem, El-Domiaty and Chen. 2017 Mostafa, Yoo, Zhu, Geng, Dufresne, Abou-Hashem, El-Domiaty and Chen

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Glucosinolates (Gls) constitute a major group of natural metabolites represented by three major classes (aliphatic, indolic and aromatic) of more than 120 chemical structures. In our previous work, soluble proteins and metabolites in Arabidopsis mutants deficient of aliphatic (myb28/29) and indolic Gls (cyp79B2B3) were analyzed. Here we focus on investigating the changes at the level of membrane proteins in these mutants. Our LC/MS-MS analyses of tandem mass tag (TMT) labeled peptides derived from the cyp79B2/B3 and myb28/29 relative to wild type resulted in the identification of 4,673 proteins, from which 2,171 are membrane proteins. Fold changes and statistical analysis showed 64 increased and 74 decreased in cyp79B2/B3, while 28 increased and 17 decreased in myb28/29. As to the shared protein changes between the mutants, one protein was increased and eight were decreased. Bioinformatics analysis of the changed proteins led to the discovery of three cytochromes in glucosinolate molecular network (GMN): cytochrome P450 86A7 (At1g63710), cytochrome P450 71B26 (At3g26290), and probable cytochrome c (At1g22840). CYP86A7 and CYP71B26 may play a role in hydroxyl-indolic Gls production. In addition, flavone 3′-O-methyltransferase 1 represents an interesting finding as it is likely to participate in the methylation process of the hydroxyl-indolic Gls to form methoxy-indolic Gls. The analysis also revealed additional new nodes in the GMN related to stress and defense activity, transport, photosynthesis, and translation processes. Gene expression and protein levels were found to be correlated in the cyp79B2/B3, but not in the myb28/29.

 Arabidopsis membrane proteome glucosinolate stress and defense molecular networks 0845162 National Science Foundation10.13039/100000001 University of Florida10.13039/100007698 Introduction

Glucosinolates (Gls) as natural anticancer compounds are represented by three major classes of chemical structures (aliphatic, indolic, and aromatic; Yan and Chen, 2007; Sønderby et al., 2010). In addition to their anti-carcinogenic activities, they have a distinct role in plant defense against herbivores (Halkier and Gershenzon, 2006; Yan and Chen, 2007) and pathogens (Kissen et al., 2009). The activities are attributed to their hydrolysis products, such as isothiocyanates, thiocyanates, and nitriles (Halkier and Gershenzon, 2006). Gls biosynthesis starts from methionine, tryptophan or phenylalanine to produce aliphatic, indolic, or aromatic Gls, respectively (Yan and Chen, 2007; Sønderby et al., 2010). Briefly, the substrate amino acid is converted to aldoxime, then to aci-nitro compounds, thiohydroximate, and desulfoglucosinolate. After sulfation, the core Gls structure is formed. In aliphatic Gls biosynthesis, the methionine chain-elongation and the core structure biosynthesis are under the control of three transcriptional factors MYB28, MYB29, and MYB76 (Yan and Chen, 2007; Frerigmann et al., 2012). In the core pathway, the formation of aldoximes is catalyzed by cytochrome P450s CYP79F1 and CYP79F2, and that of the aci-nitro compounds by CYP83A1 (Grubb and Abel, 2006). Then glutathione S-transferase U20 forms thiohydroximates, which are in turn rearranged to desulfoglucosinolate by UGT74B1 (Sønderby et al., 2010), followed by sulfation by SOT17 and SOT18 to produce intact Gls (Sønderby et al., 2010; Mostafa et al., 2016). Similar for indolic Gls, CYP79B2, CYP79B3, and CYP83B1 are responsible for aldoximes and aci-nitro compounds formation, followed by conversion to thiohydroximates by glutathione S-transferase F10, rearrangement to desulfoglucosinolates and sulfation to indolic Gls by SOT16 (Grubb and Abel, 2006; Mostafa et al., 2016). It is clear that the cytochrome P450s play a central role in the Gls biosynthesis, and these proteins are membrane localized (Neve and Ingelman-Sundberg, 2010).

Several studies have reported the relationship between the Gls biosynthetic pathway and other biological pathways in plants, e.g., amino acid and carbohydrate pathways using CYP79F1 RNAi lines (Chen et al., 2012), auxin biosynthesis using cyp79B2/B3 mutant (Zhao et al., 2002) and stress response pathways through environmental perturbation (Martínez-Ballesta et al., 2013). In our previous work, we used Arabidopsis double mutants (cyp79B2/B3 deficient in indolic Gls production and myb28/29 deficient in aliphatic Gls production), and discovered new nodes in the glucosinolate molecular network (GMN) that include stress and defense related proteins like glucan endo-1,3-beta-glucosidase, glutathione S-transferase F7 and glutathione S-transferase F2 and the electron carriers cytochrome B5 isoform C and cytochrome c oxidase subunit 5b-2 (Mostafa et al., 2016). To date, no studies have reported the glucosinolate molecular networks in the membrane proteome context.

Since many known glucosinolate proteins such as the cytochrome P450s are membrane or membrane associated proteins, here we investigated how perturbation of Gls metabolism using the aforementioned mutants affects the Arabidopsis membrane proteome using Tandem Mass Tag (TMT) labeling LC-MS/MS based quantitative proteomics. Analyses of protein interaction networks using STRING and functional enrichment of the identified proteins using agriGO allowed us to discover new nodes and edges in the GMN. With qRT-PCR, we were able to determine the correlation between gene transcripts and membrane proteins in the two mutants. Together with our published soluble proteomics work (Mostafa et al., 2016), this study enables a comprehensive understanding of the Arabidopsis GMNs.

 Materials and methods Plant genotyping, growth, and sample collection

Arabidopsis thaliana (L.) Heynh ecotype Columbia (Col-0) seeds were obtained from the Arabidopsis Biological Resource Center (Columbus, OH, USA). The seeds of cyp79B2/B3 and myb28/29 were kindly provided by Dr. John Celenza (Boston University, Boston, MA, USA) and Dr. Masami Hirai (RIKEN Plant Science Center, Yokohama, Japan), respectively. The mutant genotyping and chemotyping were reported in our previous study (Mostafa et al., 2016). Seed germination and seedling growth were conducted as previously described (Mostafa et al., 2016). Leaves from 5-week old wild type (WT), cyp79B2/B3 and myb28/29 were collected, frozen in liquid nitrogen and stored at −80°C. Four replicates were included per genotype, and each replicate contains 2 g leaves pooled from 12 plants.

 Protein extraction and peptide TMT labeling

Protein was extracted according to Pang et al. (2010) by grinding the leaf tissues in liquid nitrogen and then homogenizing on ice in 10 mM Tris-HCl (pH 7.4), 10 mM KCl, 1.5 mM MgCl2, 10 mM dithiothritol (DTT), 0.5 M sucrose, and 10 mM phenylmethylsulfonyl fluoride (PMSF). The protein extracts were filtered through cheesecloth and centrifuged at 800 g for 10 min at 4°C. The supernatant was transferred to ultracentrifuge tubes and centrifuged again at 100,000 g for 1.5 h at 4°C. The formed microsomal membrane was washed with 100 mM sodium carbonate using a glass dounce homogenizer, followed by centrifugation at 100,000 g for 1.5 h at 4°C. The microsome pellets were rinsed with 500 μl resuspension buffer containing 100 mM HEPES (pH 7), 1% triton X-100 and 0.5 M sucrose, and centrifuged at 800 g for 10 min at 4°C. Protein was precipitated using 5 volumes ice cold 90% acetone overnight at −20°C, followed by washing the pellets once with ice cold 90% acetone and twice with ice cold acetone before solubilizing in 7 M urea, 2 M thiourea, 4% CHAPS, and 0.25% Triton X-100. The protein amount was assayed using an EZQ assay kit (Invitrogen Inc., Eugene, OR, USA).

A total of 50 μg protein from each replicate was precipitated with ice cold 90% acetone at −20°C overnight, followed by 20,000 g centrifugation at 4°C for 15 min. After washing with ice cold 90% acetone, the pellets were solubilized, reduced, alkylated and digested with modified trypsin (Promega, Madison, WI, USA) at a 1:25 (w/w) ratio for 16 h at 37°C, followed by TMT labeling according to the TMT 6-plex kit manual (Thermo Scientific Inc., San Jose, CA, USA). The WT replicates were labeled with 126 and 127 tags, cyp79B2/B3 replicates with 128 and 129 tags and myb28/29 replicates with 130 and 131 tags at room temperature for 2 h. After quenching with 8 μl 5% hydroxylamine for 30 min, the labeled samples were combined and lyophilized. Two independent experiments and four biological replicates each sample were performed.

 Peptide desalting, strong cation exchange fractionation, and LC-MS/MS analysis

The TMT labeled peptides were desalted on Macrospin C-18 reverse phase mini-column (The Nestgroup Inc., Southborough, MA, USA) and fractionated using an Agilent HPLC 1260 strong cation exchange system as previously described (Mostafa et al., 2016). A total of 12 fractions were collected from each experiment. Each fraction was lyophilized, solubilized in solvent A (0.1% formic acid and 3% acetonitrile), and analyzed using an Easy-nLC 1000 system coupled to a Q-Exactive Orbitrap Plus MS (Thermo Fisher Scientific, Bremen, Germany) according to Mostafa et al. (2016) with minor modifications: The mobile phase gradient was ramped from 2 to 30% of solvent B (0.1% formic acid and 99.9% acetonitrile) in 57 min, then to 98% of solvent B in 6 min and maintained for 12 min. Mass analysis was performed in positive ion mode with high collision dissociation energy. The scan range was 400–2,000 m/z with full MS resolution of 70,000 and 200–2,000 m/z with MS2 resolution of 17,500. The first mass was fixed at 115 m/z, and 445.12003 m/z (polysiloxane ion mass) was used for real-time mass calibration.

 Protein identification and quantification

The MS data were searched using Proteome Discoverer 1.4 (Thermo Scientific, Bremen, Germany) against the Arabidopsis TAIR10 database with 35,386 entries. The searching parameters were set to 300 and 5,000 Da as minimum and maximum precursor mass filters, digestion with trypsin with two missed cleavages, Carbamidomethylation of cysteine was set as a static modification, and TMT6plex of N terminus, TMT6plex of lysine, phosphorylation of STY (serine, threonine, and tyrosine) and methionine oxidation were set as dynamic modifications. Precursor mass tolerance was 10 ppm, fragment mass tolerance was 0.01 Da, spectrum grouping maximum retention time difference was 1.1 and false discovery rate was 0.01 at the peptide level. Proteins quantification based on labeled unique peptides intensities and statistical analyses were performed as previously described (Chen et al., 2012; Mostafa et al., 2016; Sun et al., 2017). The proteomics data were deposited to ProteomeXchange repository (accession number: PXD005781).

 String bioinformatics analysis and gene ontology enrichment

The relationship between the significantly changed proteins and Gls metabolic pathways (Chen et al., 2011; Mostafa et al., 2016) was analyzed using STRING bioinformatics tool (Baldrianová et al., 2015; Ji et al., 2016; Lim et al., 2017). The resulted networks were visualized in the confidence view relying on gene neighborhood, fusion, co-occurrence, co-expression, literature, and available data. To determine the enriched pathways, we performed Singular Enrichment Analysis (SEA) for the changed proteins and the results were compared using a cross comparison of SEA (SEACOMPARE) in the agriGO database (Silva-Sanchez et al., 2013).

 Quantitative real-time polymerase chain reaction (qRT-PCR)

To determine whether protein expression levels were correlated with transcript levels, we conducted qRT-PCR of 44 genes selected based on the proteomics data (32 for cyp79B2/B3 and 22 for myb28/28). This list of primers used in qRT-PCR is provided in Supplementary Table 1. Total RNA was extracted using a RNeasy Plant Mini Kit (Qiagen, Valencia, CA, USA) and cDNA was synthesized with ProtoScript® II Reverse Transcriptase (New England BioLabs, Ipswich, MA, USA). qRT-PCR was performed with VeriQuest SyBr and a fluorescein kit (Affymetrix, Santa Clara, CA, USA) using CFX96 (Bio-Rad, Hercules, CA, USA) as described previously (Koh et al., 2012). For each reaction, three technical and three biological replicates were included. Relative expression of the target genes was calculated using the comparative Ct method (Applied Biosystems, Framingham, USA). The differences in Ct values (ΔCt) between the target gene and two internal controls (AT4G34270 and AT5G44200) were calculated to normalize differences in the starting materials. The expression ratios of cyp79B2/B3 and myb28/29 to WT were calculated and compared to the ratios from the protein data using Pearson's r.

 Results <italic>cyp79B2/B3</italic> and <italic>myb28/29</italic> membrane proteomes

Based on the MS/MS spectra of high confidence peptides derived from the WT, cyp79B2/B3 and myb28/29, we identified 4673 proteins in two independent experiments using Proteome Discover (Supplementary Table 2). Out of these proteins, 3,132 were identified in both experiments, while 1,076 and 465 were unique to experiments 1 and 2, respectively (Figure 1A). A total of 4,655 proteins were available for quantification based on unique TMT labeled peptides, highlighting the high efficiency of labeling. PD enrichment analysis (based on TAIR and Uniprot annotations) of the identified proteins showed 2,171 to be membrane proteins (Figure 1B and Supplementary Table 2). Comparative analysis of the protein expression changes between the mutants and WT at a fold change cutoff (>1.2 and <0.8), a p < 0.05 and transmembrane domain analysis revealed 93 proteins to be increased (Figure 1C) and 99 to be decreased (Figure 1D). Transmembrane domain analysis revealed that 175 out of the 192 differentially expressed proteins contained at least one transmembrane domains. The rest deemed to be membrane associated proteins (Supplementary Table 3). Correlating the changed proteins to those involved in Gls metabolism using STRING showed new nodes and edges (Figures 2, 3). The new nodes can be categorized according to their positions in the network as directly correlated or indirectly correlated to Gls metabolism. They can also be classified according to their biological roles as secondary (stress related) and tertiary (other biological process) connections (Detailed in next sections).

Venn diagrams showing numbers of identified proteins, membrane proteins, changed proteins, and their distributions. (A) Number of identified proteins in two independent TMT experiments at high peptide confidence. (B) Number of identified membrane proteins. (C) Number of significantly increased membrane proteins in cyp79B2/B3 and myb28/29 relative to WT at p < 0.05 and fold change >1.2. (D) Number of significantly decreased membrane proteins in cyp79B2/B3 and myb28/29 relative to WT at p < 0.05 and fold change < 0.8.

STRING analysis of cyp79B2/B3 changed proteins in relation to known proteins in Gls biosynthesis. Known Gls biosynthetic proteins are indicated by red balls, new proteins in the GMN are indicated by gray balls, proteins changed in both mutants are indicated by italic labeling, and uniquely changed proteins in cyp79B2/B3 are indicated by non-italic labeling. Proteins involved in Gls biosynthesis, stress and defense, and other processes are labeled with green, brown, and violet labels, respectively. Connections strength are proportional to edges thickness as derived from neighborhood, gene fusion, co-occurrence, co-expression, previous experiments, and text-mining information at medium confidence score. Asterisk (*) indicates manual connections based on literature. Double asterisk (**) indicates known nodes in both mutants (Mostafa et al., 2016), and triple asterisk (***) indicates known nodes in cyp79B2/B3 (Mostafa et al., 2016). Full names of the mapped proteins can be found in the abbreviation and protein name columns in Tables 1, 2.

STRING analysis of myb28/29 changed proteins in relation to known proteins in Gls biosynthesis. Known Gls biosynthetic proteins are indicated by red balls, new proteins in GMN are indicated by gray balls, proteins changed in both mutants are indicated by italic labeling, and uniquely changed proteins in myb28/29 are indicated by non-italic labeling. Proteins involved in Gls biosynthesis, stress and defense, and other processes are labeled with green, brown, and violet labels, respectively. Connections strength are proportional to edges thickness as derived from neighborhood, gene fusion, co-occurrence, co-expression, previous experiments, and text-mining information at medium confidence score. Asterisk (*) indicates manual connections based on literature. Double asterisk (**) indicates known nodes in both mutants (Mostafa et al., 2016). Full names of the mapped proteins can be found in the abbreviation and protein name columns in Tables 1, 2.

 Common changes of membrane proteins between the <italic>cyp79B2/B3</italic> and <italic>myb28/29</italic>

Nine membrane proteins showed common changes between the two mutants relative to WT, with only one protein increased while the other eight decreased (Table 1). By STRING mapping of the significantly changed proteins (Figures 2, 3), we found seven of the nine proteins represented new connections with the glucosinolate metabolic network (GMN). The role of probable cytochrome c (CYC2) and plastocyanin minor isoform (PETE) in electron transport process (Pesaresi et al., 2009; Welchen et al., 2012) makes them biologically relevant tertiary connections in GMN in a way similar to cytochrome B5 isoform C and cytochrome c oxidase subunit 5b-2 (Mostafa et al., 2016). Photosystem I reaction center subunit IV B (PSAE2), 14-3-3-like protein GF14 nu (GRF7), adenine phosphoribosyltransferase 1 (APT1), alba DNA/RNA-binding protein (F28N24.7) and triose phosphate/phosphate translocator (APE2) form other tertiary nodes. Out of this group, APT1 was the only protein directly connected to the GMN (Figures 2, 3).

List of common membrane proteins showing significant changes in cyp79B2/B3 and myb28/29 mutants relative to WT and their biological functions.

Accession number Locus Protein name Abbreviation* FC cypa p-value cypb FC myba p-value mybb Function TMDs tool References
Q9S714 At2g20260 Photosystem I reaction center subunit IV B PSAE2 1.640 0.016 1.359 0.044 Photosynthesis D, H, T Tair
F4I6B4 At1g30470 SIT4 phosphatase-associated family protein AT1G30470 0.782 0.030 0.781 0.049 Phosphatase reactions D, H, T Tair
Q96300 At3g02520 14-3-3-like protein GF14 nu GRF7 0.755 0.006 0.677 0.002 Binding of protein with phosphor ylated amino acids D, T Tair
Q9LJX0 At3g28860 ABC transporter B family member 19 ABCB19 0.720 0.019 0.753 0.009 Auxin transport D, H, S, T, M Lin and Wang, 2005
P31166 At1g27450 Adenine phosphoribosyltransferase 1 APT1 0.716 0.016 0.689 0.043 Adenine phosphorylation D, H, T Allen et al., 2002
Q9LP53 At1g29250 Alba DNA/RNA-binding protein F28N24.7 0.680 0.027 0.527 0.008 Binding of nucleic acid D, H, S, T Tair
F4KG20 At5g46110 Triose phosphate/phosphate translocator TPT APE2 0.608 0.005 0.784 0.043 Transport of triose phosphate D, H, S, T, M Tair
O23138 At1g22840 Probable cytochrome c CYC2 0.356 0.000 0.517 0.037 Electron transport Integral/peripheral membrane protein Birchmeier et al., 1976; Welchen et al., 2012
P11490 At1g76100 Plastocyanin minor isoform PETE 0.186 0.031 0.185 0.028 Electron transport D, H, S, T Pesaresi et al., 2009

Abbreviations for the shared proteins in Figures 2, 3.

Fold change at cut-off point >1.2 or < 0.8.

p < 0.05.

TMDs, transmembrane domains; D, Das; H, HMMTOP; S, SOSUI; T, TMPred; M, TMHMM.

 Specific changes of <italic>cyp79B2/B3</italic> membrane proteins

Sixty-four and 74 membrane proteins showed unique increases and decreases, respectively, in the cyp79B2/B3 mutant (Table 2). Seventy-seven new nodes were discovered by the STRING mapping of these cyp79B2/B3 proteins to the GMN (Figure 2). It was obvious that perturbation of the indolic Gls metabolism affected a group of stress-related membrane proteins forming new secondary nodes. Representative examples from this group are calmodulin-like protein 12 (CML12; Cazzonelli et al., 2014), mediator of RNA polymerase II transcription subunit 37c (MED37C; Lee et al., 2009), SNAP25 homologous protein (SNAP33; Eschen-Lippold et al., 2012), dynamin-related protein 1E (DRP1E; Minami et al., 2015), protein ILITYHIA (ILA; Monaghan and Li, 2010), glyceraldehyde-3-phosphate dehydrogenase (GAPC2; Guo et al., 2012), L-ascorbate peroxidase 3 (APX3; Narendra et al., 2006), Ras-related protein (RABA4B; Antignani et al., 2015), annexin D1 (ANN1; Gorecka et al., 2005; Jia et al., 2015), hypoxia-responsive family protein (At5g27760), and malate dehydrogenase 2 (mMDH2; Jones et al., 2006).

List of membrane proteins in the cyp79B2/B3 mutant showing significant changes relative to WT and their biological functions.

Accession Locus tag Protein name Abbreviation* FCa p-valueb Function TMDs References
Q42545 At5g55280 Cell division protein FtsZ homolog 1 FTSZ1 2.225 0.016 Division of chloroplast and protein binding D, T Osteryoung et al., 1998
P25071 At2g41100 Calmodulin-like protein 12 CML12 2.074 0.012 Stimuli response D, T Cazzonelli et al., 2014
Q9S726 At3g04790 Probable ribose-5-phosphate isomerase 3 RPI3 2.034 0.005 Bacterial response and management of pentose phosphate cycle D, H, T Jones et al., 2006
O82533 At2g36250 Cell division FtsZ homolog 2-1 FTSZ2-1 2.032 0.012 Division of chloroplast and protein binding D, H, T Osteryoung et al., 1998
Q9LXJ0 At3g52750 Cell division FtsZ homolog 2-2 FTSZ2-2 1.823 0.031 Division of chloroplast and protein binding D, H, T McAndrew et al., 2008
O82660 At5g23120 Photosystem II stability/assembly factor HCF136 HCF136 1.770 0.010 Photosynthesis process D, H, T Meurer et al., 1998
Q94K76 At5g18470 Curculin-like (Mannose-binding) lectin family protein AT5G18470 1.769 0.035 Binding of carbohydrate D, H, S, T, M Tair
Q93VK7 At5g14910 At5g14910/F2G14_30 AT5G14910 1.752 0.036 Metal transport Memebrane associated Tair, Friso et al., 2004
P53492 At5g09810 Actin-7 ACT7 1.752 0.004 Stress response and growth D, T Jelenska et al., 2014
P56757 AtCg00120 ATP synthase subunit alpha ATPA1 1.750 0.044 Bacterial and cold response and synthesis of ATP T Jones et al., 2006
F4JRH9 At4g12880 Early nodulin-like protein 19 ENODL19 1.715 0.010 Stimuli response and electron carrier D, H, S, T, M Tair
P22954 At5g02490 Probable mediator of RNA polymerase II subunit 37c MED37D 1.683 0.017 Bacterial, viral and heat response and transcription control D, H, T Uniprot
P0CJ46 At2g37620 Actin-1 ACT1 1.676 0.003 Growth and ATP binding D, T Kandasamy et al., 2002; tair
P19366 AtCg00480 ATP synthase subunit beta ATPB 1.661 0.050 Fungal and cold response and ATP metabolism D, T Mukherjee et al., 2010; tair
Q9AST9 At1g73110 At1g73110/F3N23_39 AT1G73110 1.614 0.036 Hydrolysis process and binding of ATP D, H, T Tair
Q8LEQ0 At5g47700 60S acidic ribosomal protein P1-3 RPP1C 1.594 0.023 Translation and binding of protein D, H, T Tair
Q9SUS3 At4g11380 Beta-adaptin-like protein B BETAB-AD 1.588 0.010 Transport of protein D, T Tair
F4HR88 At1g33590 Leucine-rich repeat (LRR) protein AT1G33590 1.582 0.004 Defense process D, H, T, M Ascencio-Ibáñez et al., 2008
Q9LPV8 At1g12920 Eukaryotic peptide chain release factor subunit 1-2 ERF1-2 1.577 0.027 Termination of translation D, T Tair
Q8H107-3 At4g26910 Isoform 3 of dihydrolipoyllysine-residue succinyltransferase AT4G26910 1.577 0.024 L-lysine catabolism and a member of tricarboxylic acid cycle D, H, T Tair
P17745 At4g20360 Elongation factor Tu TUFA 1.563 0.006 Translation, binding of GTP and Cys nitrosylation D, T Tair
Q9LHA8 At3g12580 Mediator of RNA polymerase II transcription subunit 37c MED37C 1.543 0.003 Response to stress D, T Lee et al., 2009
Q940B8 At3g16630 Kinesin-13A KINESIN-13A 1.524 0.027 Binding of ATP D, H Tair
F4HW29 At1g08450 Calreticulin-3 CRT3 1.520 0.010 Defense process D, H, S, T, M Sun et al., 2014
P23321 At5g66570 Oxygen-evolving enhancer protein PSBO1 1.514 0.019 Bacterial response and photosynthesis process D, H, T Tair; Murakami et al., 2002
Q8L940 At5g01410 Pyridoxal biosynthesis protein PDX1.3 PDX13 1.467 0.020 Stress response T Czégény et al., 2014
Q96292 At3g18780 Actin-2 ACT2 1.463 0.003 Red light response and root growth D, T Kandasamy et al., 2002; tair
Q8VZC7-2 At5g45510 Isoform 2 of Probable disease resistance protein AT5G45510 1.455 0.018 Defense process D, T Desveaux et al., 2005
Q9LET7 At3g56690 Calmodulin-interacting protein 111 CIP111 1.442 0.042 Hydrolysis and binding of ATP D, T Tair
Q8LCA1 At2g46820 Protein curvature thylakoid 1B CURT1B 1.436 0.035 Photosynthesis and DNA binding D, H, S, T, M Tair
Q9LZF5 At5g03350 Lectin-like protein At5g03350 AT5G03350 1.426 0.010 Salicylic acid and immunological response D, H, T Armijo et al., 2013
O81742 At4g23460 Beta-adaptin-like protein C BETAC-AD 1.417 0.016 Transport of protein D, T Tair
Q9FJH6 At5g60790 ABC transporter F family member 1 ABCF1 1.404 0.002 Transport process and Binding of ATP D, H, T Tair
Q39251 At3g46000 Actin-depolymerizing factor 2 ADF2 1.399 0.001 Depolymerization of actin Membrane Tair and Abe et al., 1996
O22265 At2g47450 Signal recognition particle 43 kDa CAO 1.396 0.020 Response to light D, T Walter et al., 2015
P56753 AtCg01110 NAD(P)H-quinone oxidoreductase subunit H NDHH 1.374 0.034 Photosynthesis and oxidation reduction activities D, T Tair
Q9SRY4 At1g02910 Protein low PSII accumulation 1 LPA1 1.368 0.016 Member of photosystem D, H, S, T, M Peng et al., 2006
Q9SL67 At2g20140 26S proteasome regulatory subunit 4 homolog B RPT2B 1.361 0.007 Hydrolysis of ATP and generation of gametes D, T Tair
Q9FXA1 At1g49750 At1g49750 protein AT1G49750 1.340 0.034 D, H, S, T Tair
A8MS75 At3g54890 Light-harvesting complex I chlorophyll a/b binding protein 1 LHCA1 1.339 0.018 Photosynthetic process D, H, S, T Tair
Q9SR77 At3g10130 Heme-binding-like protein AT3G10130 1.330 0.024 Binding of heme D, T Tair
P22953 At5g02500 Probable mediator of RNA polymerase II subunit 37e MED37E 1.328 0.004 Immunity response D, T Noël et al., 2007
F4ISI7 At2g19480 Nucleosome assembly protein 12 NAP1; 2 1.318 0.027 Repair and binding of DNA D Iglesias et al., 2013
Q9LIK9 At3g22890 ATP sulfurylase 1 APS1 1.305 0.039 Biosynthesis of hydrogen sulfide D, T Tair
Q9SJZ7 At2g22360 Molecular chaperone DnaJ AT2G22360 1.304 0.025 Heat response and binding activity D, T Tair
O65719 At3g09440 Heat shock 70 kDa protein 3 HSP70-3 1.300 0.030 Viral and heat response and binding activity D, T Agudelo-Romero et al., 2008; Palmblad et al., 2008; Tair
B3H5R4 At5g58260 At5g58260 protein AT5G58260 1.298 0.009 Fungal response and oxidation reduction reactions D Mukherjee et al., 2010; Tair
Q9FMA3 At5g56290 Peroxisome biogenesis protein 5 PEX5 1.294 0.017 Movement of proteins to peroxisome D, T Ramón and Bartel, 2010
O80885 At2g32480 Arabidopsis serin protease ARASP 1.274 0.038 Proteolytic action and stress response D, H, S, T, M Sokolenko et al., 2002
Q42044 At2g45180 Bifunctional inhibitor/lipid-transfer protein/seed storage 2S albumin AT2G45180 1.271 0.032 Proteolytic action and lipid transport D, H, S, T, M Tair
F4JTP5 At4g38470 ACT-like protein tyrosine kinase AT4G38470 1.270 0.042 Kinase and binding activities D, H, T Tair
Q9S7P9 At5g61210 SNAP25 homologous protein SNAP33 1.264 0.035 Immunity process D Eschen-Lippold et al., 2012
Q9M0F9 At4g29220 6-phosphofructokinase 1 PFK1 1.262 0.043 Fructose 6 phosphate metabolism and glycolysis D, T Mustroph et al., 2007
Q8L7L0 At5g18570 GTP-binding protein OBGC OBGL 1.250 0.010 Stimuli response D, T Chen et al., 2014
Q9FNX5 At3g60190 Dynamin-related protein 1E DRP1E 1.246 0.005 Cold response D, T Minami et al., 2015; tair
Q8RY46 At1g70610 ABC B family member 26 ABCB26 1.237 0.007 Transport activity D, H, S, T, M Tair
Q39142 At2g34430 Light-harvesting chlorophyll protein complex II subunit B1 Lhb1B1 1.232 0.049 Photosynthetic process D, H, S, T Tair
P92549 AtMg01190 ATP synthase subunit alpha ATPA2 1.227 0.005 Oxidative stress response D, T Sweetlove et al., 2002
Q9SA78 At1g30630 Coatomer subunit epsilon-1 AT1G30630 1.226 0.006 Transport process D, T Tair
F4J0B1 At3g28520 AAA-type ATPase family protein AT3G28520 1.226 0.039 Binding and hydrolysis of ATP D, T Tair
Q9ZPH9 At4g00750 Probable methyltransferase PMT15 AT4G00750 1.218 0.024 Stress response and methylation process D, H, S, T, M Rama Devi et al., 2006; tair
F4I894 At1g64790 Protein ILITYHIA ILA 1.217 0.017 Immunity process D, H, S, T Monaghan and Li, 2010
O49636 At4g22310 At4g22310 AT4G22310 1.213 0.007 Transport of pyruvate D, T Tair
P56754 Atcg00420 NAD(P)H-quinone oxidoreductase subunit J NDHJ 1.207 0.036 Oxidation reduction and response to sulfur deficiency D, T Tair
Q9FX54 At1g13440 Glyceraldehyde-3-phosphate dehydrogenase GAPC2 GAPC2 0.798 0.013 Stress response D, T Guo et al., 2012
P19456 At4g30190 ATPase 2, plasma membrane-type AHA2 0.798 0.048 ATP metabolism D, H, S, T, M Tair
P42761 At2g30870 Glutathione S-transferase F10 GSTF10 0.798 0.025 Indolic glucosinolate biosynthesis D, H, T Mostafa et al., 2016
Q9LNH6 At1g48240 Novel plant SNARE 12 NPSN12 0.793 0.006 Transport of protein D, H, S, T, M Tair
F4ICF5 At1g25290 RHOMBOID-like protein 10 RBL10 0.790 0.001 Root and flower growth D, H, T, M Thompson et al., 2012
O65282 At5g20720 20kDa chaperonin CPN21 0.789 0.021 Defense process Plasma membrane Kawamura and Uemura, 2003, Takáč et al., 2014
Q84MC0 At3g06035 Uncharacterized GPI-anchored protein AT3G06035 0.788 0.035 Precursor for glycoprotein D, H, S, T, M Tair
Q9FK25 At5g54160 Flavone 3'-O-methyltransferase 1 OMT1 0.787 0.003 Flavonoid metabolism D, T Muzac et al., 2000
Q9FJN8 At5g65270 Ras-related protein RABA4a RABA4A 0.786 0.003 Binding of GTP and pollen tube growth D, T Tair; Szumlanski and Nielsen, 2009
P92963 At4g17170 Ras-related protein RABB1c RABB1C 0.785 0.029 Binding of GTP and transport activity D, T Tair
P56759 Atcg00130 ATP synthase subunit b ATPF 0.783 0.012 Respiration process D, H, T, M Tair
P93834 At2g19860 Hexokinase-2 HXK2 0.782 0.047 Phosphorylation of hexoses D, H, S, T, M Jang et al., 1997
Q9CAD6 At1g63710 Cytochrome P450 86A7 CYP86A7 0.781 0.023 Oxidation reduction and metabolism of fatty acid D, H, S, T, M Duan and Schuler, 2005
O81016 At2g26910 ABC transporter G family member 32 ABCG32 0.780 0.037 Transport activity and cuticle formation D, H, S, T, M Tair; Bessire et al., 2011
A8MQG9 At1g73650 Uncharacterized protein AT1G73650 0.779 0.021 Oxidation reduction reactions and lipid metabolism D, H, S, T, M Tair
Q9C6X2 At1g32050 Secretory carrier-associated membrane protein 4 SCAMP4 0.778 0.025 Carrier activity D, H, S, T, M Law et al., 2012
Q96282 At5g49890 Chloride channel protein CLC-c CLC-C 0.777 0.002 Salt stress D, H, S, T, M Jossier et al., 2010
A8MQG9 At1g73650 Uncharacterized protein AT1G73650 0.779 0.021 Oxidation reduction reactions and lipid metabolism D, H, S, T, M Tair
Q9C6X2 At1g32050 Secretory carrier-associated membrane protein 4 SCAMP4 0.778 0.025 Carrier activity D, H, S, T, M Law et al., 2012
Q96282 At5g49890 Chloride channel protein CLC-c CLC-C 0.777 0.002 Salt stress D, H, S, T, M Jossier et al., 2010
Q9STT2 At3g47810 Vacuolar protein sorting-associated protein 29 VPS29 0.776 0.044 Transport activity Membrane associated Jaillais et al., 2007; Zelazny et al., 2013
Q42564 At4g35000 L-ascorbate peroxidase 3 APX3 0.775 0.041 Antioxidant action and stress response D, H, T, M Narendra et al., 2006; tair
Q8VZM7 At5g02940 Putative ion channel POLLUX-like 1 AT5G02940 0.774 0.043 Transport of ions D, H, S, T, M Tair
Q8LB17 At3g58460 Uncharacterized protein AT3G58460 0.774 0.030 Proteolytic action D, H, S, T, M Knopf and Adam, 2012
Q8LCP6 At1g75680 Endoglucanase 10 AT1G75680 0.773 0.000 Cellulase and hydrolase action D, H, S, T, M Tair
Q94CI7 At5g27350 Sugar transporter ERD6-like 17 SFP1 0.772 0.012 Carbohydrate transport D, H, S, T, M Quirino et al., 2001
Q39099 At2g06850 Xyloglucan endotransglucosylase/ hydrolase protein 4 XTH4 0.770 0.027 Stimuli response, cell wall development and hydrolase action D, H, S, T, M Campbell and Braam, 1999; tair
Q944A7 At4g35230 Probable serine/threonine-protein kinase AT4G35230 0.763 0.026 Immunity process and phosphorylation of proteins D, T Shi et al., 2013; tair
Q949R9 At5g20090 Mitochondrial pyruvate carrier 1 AT5G20090 0.763 0.046 Transport of pyruvate D, H, T Li et al., 2014
Q9LTL0 At3g26290 Cytochrome P450 71B26 CYP71B26 0.760 0.028 Binding of oxygen D, H, S, T, M Tair
Q9SMQ6 At4g39990 Ras-related protein RABA4b RABA4B 0.754 0.017 Defense process D, T Antignani et al., 2015
Q9C5M0 At5g19760 Dicarboxylate/tricarboxylate transporter DTC DTC 0.750 0.021 Dicarboxylate transport D, H, T Picault et al., 2002
O82204 At2g19730 60S ribosomal protein L28-1 RPL28A 0.748 0.032 Translation Tair
Q9LFA3 At3g52880 Probable monodehydroascorbate reductase isoform 3 AT3G52880 0.745 0.024 Oxidation reduction D, H, T Lisenbee et al., 2005
Q9SEL6 At5g39510 Vesicle transport v-SNARE 11 VTI11 0.740 0.001 Transport D, H, S, T, M Tair
Q9LPZ3 At1g11410 G-type lectin S-receptor-like serine/threonine-protein kinase AT1G11410 0.733 0.049 Kinase and binding activities D, H, S, T, M Tair
Q8LE26 At2g38480 CASP-like protein At2g38480 AT2G38480 0.729 0.002 D, H, T, M Tair
P43287 At2g37170 Aquaporin PIP2-2 PIP2-2 0.725 0.011 Water deficiency response and transport D, H, S, T, M Javot, 2003; Tournaire-Roux et al., 2003;
Q9SYT0 At1g35720 Annexin D1 ANN1 0.719 0.046 Salt stress, binding and transport activities D Gorecka et al., 2005; Jia et al., 2015
Q9M1E7 At3g45600 Tetraspanin-3 TET3 0.719 0.042 Member of aging process D, H, S, T, M Tair
Q39101 At5g01600 Ferritin-1 FER1 0.713 0.027 Bacterial and stress response and iron homeostasis D, H, T Tair
F4JP88 At4g17615 Calcineurin B-like protein 1 CBL1 0.712 0.029 Stress response Associated with membrane kinase Ren et al., 2013; Feng et al., 2015
F4JIN3 At4g21180 DnaJ / Sec63 Brl domains-containing protein ATERDJ2B 0.712 0.044 Transport of protein D, H, S, T, M Tair
O23482 At4g16370 Oligopeptide transporter 3 OPT3 0.705 0.035 Transport activity D, H, S, T, M Wintz et al., 2003
Q9C8G5 At1g30360 Early-responsive to dehydration stress protein T4K22.4 0.703 0.007 Water deficiency (stress) response and ion transport D, H, S, T, M Rai et al., 2016; tair
Q8L8Z1 At4g15630 CASP-like protein At4g15630 AT4G15630 0.701 0.024 Binding of protein D, H, S, T, M Tair
Q9FYK0 At1g24650 Leucine-rich repeat protein kinase F21J9.31 LRR-RLK 0.701 0.043 Growth process. D, S, T, M Dai et al., 2013
Q8GWP3 At2g26975 Copper transporter 6 COPT6 0.700 0.005 Transport of copper D, H, S, T, M Garcia-Molina et al., 2013
Q8LG60 At5g27760 Hypoxia-responsive family protein AT5G27760 0.698 0.029 Oxygen deficiency response D, H, T Tair
Q9FF88 At5g23920 At5g23920 AT5G23920 0.695 0.038 …… D, H, S, T, M Tair
Q8LAA6 At4g23400 Probable aquaporin PIP1-5 PIP1-5 0.694 0.049 Controls water channels, salt stress response D, H, S, T, M Weig et al., 1997; Tair
Q93XY5 At2g20230 Tetraspanin-18 TOM2AH2 0.690 0.049 ….… D, H, S, T, M Tair
P30302 At2g37180 Aquaporin PIP2-3 PIP2-3 0.685 0.003 Salt stress and water deficiency D, H, S, T, M Daniels et al., 1994
Q8RWZ6 At2g01420 Auxin efflux carrier component 4 PIN4 0.683 0.019 Transport of auxin D, H, S, T, M Zhang et al., 2015
A1XJK0 At1g18320 Mitochondrial inner membrane translocase subunit TIM22-4 TIM22-4 0.668 0.044 Transport of protein D, H, S, T, M Tair
Q39196 At4g00430 Probable aquaporin PIP1-4 PIP1-4 0.667 0.006 Water deficiency response and transport D, H, S, T, M Li et al., 2015; Tair
Q9LZI2 At3g62830 UDP-glucuronic acid decarboxylase 2 UXS2 0.664 0.049 Xylose metabolism D, H, S, T, M Harper and Bar-Peled, 2002
Q9LIL4 At3g22845 Transmembrane emp24 domain-containing protein p24beta3 AT3G22845 0.652 0.038 Transport activity D, H, T, M Tair
Q9CAN1 At1g63120 RHOMBOID-like protein 2 F16M19.4 0.646 0.018 Proteolytic activity D, H, S, T, M Kanaoka et al., 2005
Q9SUV2 At4g32390 Probable sugar phosphate/phosphate translocator AT4G32390 0.640 0.019 Transport activity D, H, S, T, M Tair
Q8GYN5 At3g25070 RPM1-interacting protein 4 RIN4 0.633 0.043 Bacterial response and immunity process D Axtell and Staskawicz, 2003
Q9LVE0 At3g21670 Protein NRT1/ PTR FAMILY 6.4 NPF6.4 0.628 0.031 Transport activity and nitrate synthesis D, H, S, T, M Okamoto et al., 2003
Q9LFS3 At5g16010 3-oxo-5-alpha-steroid 4-dehydrogenase family protein F1N13_150 0.627 0.007 Oxidation reduction reactions and lipid metabolism D, H, S, T, M Tair
Q9FQ24 At3g55005 Protein TONNEAU 1b TON1B 0.617 0.001 Growth process and organization of microtubule D, T Azimzadeh et al., 2008
B9DFR9 At2g45960 Plasma membrane intrinsic protein 1B, At2g45960 protein PIP1B 0.615 0.005 Water deficiency D, H, S, T, M Alexandersson et al., 2005
O23596 At4g17550 Putative glycerol-3-phosphate transporter 4 AT4G17550 0.613 0.009 Transport activity D, H, S, T, M Tair
Q9LVM5 At5g58220 Allantoin synthase/Uric acid degradation bifunctional protein TTL 0.611 0.030 Cell growth control, allantoin biosynthesis and catabolism of urate D, H, T Tair
Q08733 At1g01620 Aquaporin PIP1-3 PIP1-3 0.601 0.020 Water deficiency response and transport D, H, S, T, M Kammerloher et al., 1994
Q8VZQ3 At1g17200 CASP-like protein At1g17200 AT1G17200 0.590 0.016 Binding activity D, H, S, T, M Tair
Q9M386 At3g54200 Late embryogenesis abundant hydroxyproline-rich glycoprotein F24B22.160 0.588 0.004 ….… D, H, S, T, M Tair
F4I082 At1g55260 Glycosylphosphatidylinositol-anchored lipid protein transfer 6 AT1G55260 0.583 0.002 Binding and transport of lipid D, T Edstam and Edqvist, 2014
Q9FN38 At5g53880 Putative uncharacterized protein AT5G53880 0.576 0.012 …… D, T Tair
F4JDN8 At3g26700 Protein kinase family protein AT3G26700 0.544 0.025 Kinase action D, H, S, T, M Tair
Q9ZV07 At2g39010 Probable aquaporin PIP2-6 PIP2-6 0.531 0.022 Controls water channels D, H, S, T, M Alexandersson et al., 2010
P93004 At4g35100 Aquaporin PIP2-7 PIP2-7 0.526 0.032 Salt stress response D, H, S, T, M Weig et al., 1997
F4JY28 At5g18630 Putative class 3 lipase AT5G18630 0.513 0.008 Lipid metabolism D, H, S, T, M Tair
A8MQK3 At3g15020 Malate dehydrogenase 2 mMDH2 0.414 0.028 Bacterial response and carbohydrate metabolism D, H, T Jones et al., 2006, Tair

Abbreviations for unique proteins in Figure 2.

Fold change at cut-off point >1.2 or < 0.8.

p < 0.05.

TMDs, transmembrane domains; D, Das; H, HMMTOP; S, SOSUI; T, TMPred; M, TMHMM.

The role of cytochromes P450s 86A7 (CYP86A7) and 71B26 (CYP71B26) in oxidation reduction reaction and oxygen binding (Duan and Schuler, 2005) makes them biologically relevant tertiary nodes in the GMN (Figures 2, 4). Other interesting new tertiary nodes related to Gls metabolism include division protein FtsZ homologs 1 (FTSZ1), 2-1 (FTSZ2-1), 2-2 (FTSZ2-2), curculin-like (mannose-binding) lectin family protein (At5g18470), isoform 3 of dihydrolipoyllysine-residue succinyltransferase component of 2-oxoglutarate dehydrogenase complex 2 (At4g26910), elongation factor Tu (TUFA), ATP sulfurylase 1 (APS1), ABC transporter B family member 26 (ABCB26), flavone 3'-O-methyltransferase 1 (OMT1), Ras-related protein (RABA4A), Ras-related protein (RABB1C), endoglucanase 10 (AT1G75680), dicarboxylate/tricarboxylate transporter (DTC), probable monodehydroascorbate reductase isoform 3 (At3g52880), vesicle transport v-SNARE 11 (VTI11), DnaJ/Sec63 Brl domains-containing protein (ATERDJ2B), and glycosylphosphatidylinositol-anchored lipid protein transfer 6 (At1g55260). Myrosin cells (myrosinase storage sites) endocytosis is controlled by SYP22 from SNARE complex and VPS9A (Shirakawa et al., 2016). Here the decrease of VTI11 from this family is in agreement with the reduced myrosinase, nitrile specifier protein and Gls levels in the soluble proteome (Mostafa et al., 2016) and supports the cross talk between Gls and its hydrolyzing enzymes.

Predicted positions of the directly connected nodes and connected cytochrome nodes on the glucosinolate metabolic pathway. Italic indicates proteins changed in both mutants, * indicates proteins changed in cyp79B2/B3, and ** indicates proteins changed in myb28/29. Red color means increased proteins, green color means decreased proteins. Full names of the directly connected proteins can be found in the abbreviation and protein name columns in Tables 1–3.

Out of these new nodes, 15 formed direct edges with the GMN: FTSZ1, CML12, FTSZ2-2, At5g18470, At4g26910, TUFA, MED37C, APS1, SNAP33, ILA, GAPC2, OMT1, CYP71B26, ANN1, and mMDH2 in addition to the membrane associated protein (20 kDa chaperonin, CPN21). As we detected a side network correlated to indolic GMN (Mostafa et al., 2016), here we also found a side network strongly correlated to indolic Gls metabolism as it contains nine stress-related proteins out of eleven. These proteins are xyloglucan endotransglucosylase/hydrolase protein 4 (XTH4; Campbell and Braam, 1999), aquaporin PIP2-2 (Javot, 2003; Tournaire-Roux et al., 2003), probable aquaporin PIP1-5 (Weig et al., 1997), aquaporin PIP2-3 (Daniels et al., 1994), probable aquaporin PIP1-4 (Li et al., 2015), plasma membrane intrinsic protein 1B (PIP1B; Alexandersson et al., 2005), aquaporin PIP1-3 (Kammerloher et al., 1994), probable aquaporin PIP2-6 (Alexandersson et al., 2010), and aquaporin PIP2-7 (Weig et al., 1997). Other members in this side network are bifunctional inhibitor/lipid-transfer protein (At2g45180; which has a proteolytic action) and a tetraspanin-18 (TOM2AH2) with unknown functions.

 Specific changes of <italic>myb28/29</italic> membrane proteins

Membrane proteomics of the myb28/29 mutant showed 28 and 17 proteins to be significantly increased and decreased, respectively (Table 3). STRING analysis of the increased and decreased myb28/29 specific membrane proteins revealed 21 new nodes in the GMN (Figure 3). Except for the directly connected and stress-related GTP-binding nuclear protein (RAN1; Jiang et al., 2007), other connections including 17 ribosomal proteins [e.g., 60S ribosomal protein L14-2 (RPL14B), 40S ribosomal protein S15-1 (RPS15) and 40S ribosomal protein S15-4 (RPS15D)], and actin-11 (ACT11), ADP/ATP carrier protein 1 (AAC1) and eukaryotic translation initiation factor 3 subunit F (TIF3F1) formed tertiary nodes. These tertiary nodes are connected to the GMN through two bridges (directly connected nodes) which are 60S ribosomal protein L15-1(RPL15A) and 60S ribosomal protein L13-1 (BBC1). The expression changes in ribosomal proteins reflect a correlation between aliphatic Gls perturbation and the translation process in A. thaliana.

List of membrane proteins the myb28/29 mutant showing significant level changes relative to WT and their biological functions.

Accession number Locus tag Protein name Abbreviation* FCa p-valueb Function TMDs References
P51422 At3g55750 60S ribosomal protein L35a-4 RPL35AD 1.972 0.025 Translation and RNA binding Tair
Q9T043 At4g27090 60S ribosomal protein L14-2 RPL14B 1.928 0.040 Translation and RNA binding D Tair
Q9LST0 At5g60160 AT5g60160/f15l12_20 AT5G60160 1.686 0.026 Proteolytic activity D, H, T Tair
Q9LZ57 At5g02450 60S ribosomal protein L36-3 RPL36C 1.653 0.024 Translation D Tair
Q9M0E2 At4g29410 60S ribosomal protein L28-2 RPL28C 1.593 0.009 Translation and RNA binding Tair
F4I472 At1g04270 40S ribosomal protein S15-1 RPS15 1.501 0.025 Translation D, H, T Tair
Q8W463 At4g17560 50S ribosomal protein L19-1 AT4G17560 1.488 0.030 Translation D, H, T Tair
Q9FY64 At5g09510 40S ribosomal protein S15-4 RPS15D 1.461 0.036 Translation D, H, T Tair
O23515 At4g16720 60S ribosomal protein L15-1 RPL15A 1.417 0.016 Translation Tair
Q9LZ41 At5g02610 60S ribosomal protein L35-4 RPL35D 1.411 0.010 Translation D Tair
F4HRB4 At1g45201 Triacylglycerol lipase-like 1 TLL1 1.374 0.025 Hydrolysis of lipids D, H, S, T, M Tair
Q9SUJ1-2 At3g05710 Isoform 2 of Syntaxin-43 SYP43 1.372 0.026 Fungal response and transporter activity D, H, S, T, M Zheng et al., 1999; Nielsen and Thordal-Christensen, 2012
Q93VG5 At5g20290 40S ribosomal protein S8-1 RPS8A 1.359 0.028 Translation D Tair
B9DGY1 At3g07700 ABC1 kinase AT3G07700 1.351 0.012 Oxidative stress response D, H, T, M Yang et al., 2012
A8MQA1 At3g49010 60S ribosomal protein L13-1 BBC1 1.349 0.041 Translation and RNA binding Tair
P49693 At4g02230 60S ribosomal protein L19-3 RPL19C 1.331 0.023 Translation and RNA binding Tair
O22795 At2g33450 50S ribosomal protein L28 RPL28 1.331 0.032 Translation and RNA binding Tair
Q9C514 At1g48830 40S ribosomal protein S7-1 RPS7A 1.327 0.031 Translation D Tair
P49637 At1g70600 60S ribosomal protein L27a-3 RPL27AC 1.274 0.006 Translation and RNA binding Tair
F4IHJ8 At2g21580 40S ribosomal protein S25-2 AT2G21580 1.273 0.032 Translation D Tair
P53496 At3g12110 Actin-11 ACT11 1.267 0.014 Cytoskeleton component, Binding of ATP D, T McDowell et al., 1996; Jia et al., 2013
Q9FH02 At5g42270 ATP-dependent zinc metalloprotease FTSH 5 FTSH5 1.240 0.012 Leaf coloration and photo-inhibition D, H, T Sakamoto et al., 2002
P31167 At3g08580 ADP, ATP carrier protein 1 AAC1 1.238 0.015 Transport activities D, H, T, M Tair
P51418 At2g34480 60S ribosomal protein L18a-2 RPL18AB 1.229 0.028 Translation Tair
Q9LVI9 At3g17810 Putative dehydrogenase PYD1A 1.224 0.016 Oxidation reduction reactions, pyrimidine and uracil metabolism D, T Zrenner et al., 2009
Q8RWA5 At1g25380 Nicotinamide adenine dinucleotide transporter 2 NDT2 1.223 0.043 Transport activities D, H, T Bedhomme et al., 2005
Q8W486 At1g04910 O-fucosyltransferase family protein AT1G04910 1.213 0.042 Glycosyl groups transfer D, H, S, T, M Voxeur et al., 2012
P51427 At3g11940 40S ribosomal protein S5-2 RPS5B 1.206 0.002 Translation and RNA binding D, T Tair
O04202 At2g39990 Eukaryotic translation initiation factor 3 subunit F TIF3F1 0.799 0.029 Translation and development of embryo D, T Xia et al., 2010
Q9FQ25 At3g55000 Protein TONNEAU 1a TON1A 0.782 0.046 Cell division and cytoskeleton organization D, T Azimzadeh et al., 2008
Q84LG4 At3g09800 Coatomer subunit zeta-2 AT3G09800 0.773 0.049 Transport of protein D, H, T Tair
P41916 At5g20010 GTP-binding nuclear protein Ran-1 RAN1 0.770 0.002 Salt stress response and GTP binding D, T Jiang et al., 2007
Q9ZVA2 At1g78830 At1g78830/F9K20_12 F9K20.12 0.767 0.017 Binding of carbohydrate D, H, S, T, M Tair
Q9FIX1 At5g39730 AIG2-like protein AT5G39730 0.740 0.021 Salt stress response D Tair
Q9LS26 At5g46570 At5g46570 BSK2 0.740 0.036 Kinase activity D, T Tair
Q93ZH0-2 At1g21880 Isoform 2 of LysM domain-containing GPI-anchored protein 1 LYM1 0.738 0.005 Immunity and defense activity D, H, S, T, M Willmann et al., 2011
Q94EG6 At5g02240 Uncharacterized protein AT5G02240 0.734 0.040 Abscisic acid response D, T Ghelis et al., 2008
Q0WSY2 At1g19835 Filament-like plant protein 4 FPP4 0.725 0.036 D Tair
Q9C500 At1g47200 WPP domain-containing protein 2 WPP2 0.716 0.049 Growth of lateral roots and mitotic division D Patel, 2004
Q9FIJ2 At5g47890 NADH dehydrogenase 1 alpha subcomplex subunit 2 AT5G47890 0.690 0.050 Oxidation reduction reactions Mitochondrial membrane Michalecka et al., 2003, Murray et al., 2003
Q9FPJ4 At5g47200 Ras-related protein RABD2b RABD2B 0.687 0.013 Binding of GTP D, T Tair
Q94F08 At5g62630 HIPL2 protein HIPL2 0.678 0.004 Binding of carbohydrate and oxidation reduction reaction D, H, S, T Tair
Q9SZ51 At4g31840 Early nodulin-like protein 15 ENODL15 0.666 0.017 Stimuli response and electron carrier D, H, S, T, M Tair
P48421 At4g13770 Cytochrome P450 83A1 CYP83A1 0.664 0.033 Glucosinolate biosynthesis, insect response D, H, S, T, M Mostafa et al., 2016
Q9SK39 At2g24940 Probable steroid-binding protein 3 MP3 0.376 0.031 Binding of steroid and heme Membrane associated Tair, Yang et al., 2005

Abbreviations for unique proteins in Figure 3.

Fold change at cut-off point >1.2 or < 0.8.

p < 0.05.

TMDs, transmembrane domains; D, Das; H, HMMTOP; S, SOSUI; T, TMPred; M, TMHMM.

 Gene ontology analysis of the significantly changed membrane proteins

AgriGO enrichment analysis of the changed proteins was conducted at the biological processes (BP), cellular components (CC), and molecular functions (MF) levels. By annotating 147 changed membrane proteins in the cyp79B2/B3 using SEA, we got 302 enriched GO terms for BP (Supplementary Figure 1), 63 for CC (Supplementary Figure 2), and 47 for MF (Supplementary Figure 3). SEA of 54 changed membrane proteins in the myb28/29 showed 45 enriched GO terms for BP (Supplementary Figure 4), 56 for CC (Supplementary Figure 5) and 2 for MF (Supplementary Figure 6). SEACOMPARE of the mutant revealed 271 BP, 21 CC, and 46 MF GO terms to be enriched specifically in cyp79B2/B3, while 14 BP, 14 CC, and one MF were the specifically enriched GO terms in myb28/29 (Supplementary Table 4). From this BP analysis, it was obvious that responses to stimuli including abiotic, chemical and stress were highly enriched in cyp79B2/B3 in addition to transport, photosynthesis and metabolic processes. In myb28/29, the most enriched BP terms were those related to translation process. This observation supported our results concerning the stimuli and translation-related proteins in the cyp79B2/B3 and myb28/29, respectively (Supplementary Table 4). On the level of CC, the high enrichment of membrane GO terms supported the effectiveness of our membrane preparation procedure (Supplemental Figures 2, 5).

 Comparison of protein expression data with transcription data

To determine whether protein level changes correlated with gene transcription changes, we examined the transcript levels of 32 and 22 genes from cyp79B2/B3 and myb28/28, respectively (Supplementary Table 5). The two mutants exhibited different patterns of correlation. In comparison of cyp79B2/B3 to WT, the genes investigated showed a positive correlation between transcript and protein levels in both direction and degree of expression (r = 0.6579, p = 4.269e−05; Supplementary Figure 7). However, in comparison of myb28/29 to WT, the genes did not show correlation between the transcript and protein levels (r = 0.0887, p = 0.6945; Supplementary Figure 7), only three out of the 22 genes showed similar regulation at both transcript and protein levels. For example, At4g13770 encoding cytochrome P450 83A1, exhibited down-regulation in myb28/29 compared to WT (Supplementary Table 5). The difference in the degree of correlation in these two mutants implies that different regulatory mechanisms are involved in the transcriptional and posttranscriptional processes in different genotypes (Marmagne et al., 2010; Koh et al., 2012).

 Discussion

As a result of Gls metabolism perturbation, many changes in the levels of soluble (Mostafa et al., 2016) and membrane proteins took place. It was interesting to discover new cytochromes to be involved in the GMN. In addition, several groups of stress and defense-related proteins as well as binding and transport activity proteins were related to the indolic and aliphatic GMNs, in addition to a group of ribosomal proteins in the myb28/29 mutant.

Three new cytochromes in the glucosinolate molecular network

Cytochromes play a key role in Gls biosynthesis. In aliphatic Gls biosynthesis, CYP79F1 and CYP79F2 catalyze the conversion of chain-elongated methionines to aldoximes, which are metabolized by another cytochrome (CYP83A1) to aci-nitro compounds, precursors of desulphoglucosinolates (Grubb and Abel, 2006). As to indolic Gls biosynthesis, CYP79B2 and CYP79B3 convert tryptophan to aldoximes, that are metabolized by CYP83B1 to form the aci-nitro compounds (Grubb and Abel, 2006). In addition, there is another CYP81F2 catalyzing the conversion of indolic-3-glucosinolate to 4-hydroxy-indolic-3-glucosinolate (Sønderby et al., 2010). Furthermore, CYP71A12 and CYP71A13 can metabolize indolic aldoximes to indole acetonitrile and subsequently indole acetic acid derivatives (Nafisi et al., 2007). In our previous study, we reported cytochrome B5 isoform C and cytochrome c oxidase subunit 5b-2 to be new nodes in the aliphatic and indolic GMNs, respectively (Mostafa et al., 2016). Here we discovered cytochrome P450 86A7 (CYP86A7) in redox reaction and metabolism of fatty acids (Duan and Schuler, 2005), and cytochrome P450 71B26 (CYP71B26) as new nodes in the indolic GMN. Based on STRING analysis, CYP71B26 is connected to CYP81F2 through a direct edge, while CYP86A7 is connected indirectly to CYP81F2 through lectin family proteins (At5g03350 and At5g18470; Figures 2, 4). Given that their connection to a specific and key enzyme in indolic Gls biosynthetic pathway (CYP81F2) and their expression levels were decreased in the cyp79B2/B3 mutant (Table 2), it is reasonable to hypothesize that CYP86A7 and CYP71B26 play specific roles in 4-hydroxy indolic-3-glucosinolate production (Figure 4). Especially their precursor (indolic-3-glucosinolate) and the product were decreased in cyp79B2/B3 mutant as revealed in our previous study (Mostafa et al., 2016). Also by similarity, we can predict a role for the enzymes in hydroxy indolic-1-glucosinolate production (Figure 5) as its synthesizing enzymes are not known (Sønderby et al., 2010). The third new cytochrome discovered in this study is a probable cytochrome c At1Gg22840 (CYC2), which plays a role in electron transport process (Welchen et al., 2012). CYC2 is in the shared decreased protein category, forming new connections with aliphatic GMN through ADP/ATP carrier protein 1 (AAC1) and 60S ribosomal protein L15-1 (RPL15A), which is connected to GSTF9, GSTF10 and GSTF11, and with indolic GMN through eukaryotic peptide chain release factor subunit 1–2 (ERF1-2), 60S ribosomal protein L28-1 (RPL28A) and adenine phosphoribosyltransferase 1 (APT1). APT1 is connected to GGP1 and SUR1. Although the CYC2 function awaits for further studies, it might play a role in the conversion of aci-nitro compounds to thiohydroximates.

Hypothesized roles of CYP86A7 and CYP71B26 in the hydroxylation of indolic-1-glucosinolate (top panel) and the potential dual functions of flavone 3′-O-methyltransferase in flavonoid and Gls metabolism. Circles indicate chemical modifications to the substrates.

 Stress related membrane protein changes as a secondary result of glucosinolate metabolism perturbation

Plant Gls metabolism is responsive to stress conditions, e.g., temperature and light stress (Martínez-Ballesta et al., 2013), water stress (Khan et al., 2010), salt stress (Guo et al., 2013), and microbial stress (Clay et al., 2009). In our previous study, glucan endo-1,3-beta-glucosidase, glutathione S-transferase F2 and glutathione S-transferase F7 in addition to others as stress-related proteins were found to connect to the Gls pathway (Mostafa et al., 2016). Here we found the levels of 51 stress-related proteins changed significantly in the cyp79B2/B3 mutant and six with changes in the myb28/29 mutant. In the cyp79B2/B3 membrane proteome, a group of general stimuli response-related proteins exhibited significant changes compared to WT (Table 2). Among them, the following are examples to directly connect with Gls enzymes: calmodulin-like protein 12 (CML12; Cazzonelli et al., 2014; connected to the indolic GMN via MYB122 and CYP81F2), mediator of RNA polymerase II transcription subunit 37c (MED37C; Lee et al., 2009; connected via GSTF9 to GMN, with possible role in thiohydroximate formation), and glyceraldehyde-3-phosphate dehydrogenase (GAPC2; Guo et al., 2012; formed edges with GMN through MYB28, MYB29, MYB76, and MYB34, suggesting roles in methionine chain-elongation and tryptophan synthesis; Figures 2, 4). It is known that GAPC2 participates in the oxidation of glyceraldehydes-3-phophate to glycerate from which pyruvate is formed. The pyruvate can be converted to acetylCoA for methionine chain-elongation in aliphatic Gls biosynthesis or for synthesis of tryptophan in indolic Gls pathway (Mann, 1987). Both glucosinolate classes were decreased in the cyp79B2/B3 mutant in our previous study (Mostafa et al., 2016) together with GAPC2 in this study. Therefore, the connection between GAPC2 and MYBs in the STRING maps reflects functional relationship and does not necessarily indicate direct physical interaction. Another stress related group showing expression level changes was the salt stress and water deficiency group represented by chloride channel protein CLC-c (Jossier et al., 2010), aquaporin PIP2-2 (Javot, 2003; Tournaire-Roux et al., 2003), annexin D1 (ANN1; Gorecka et al., 2005; Jia et al., 2015; formed edge with GSTF9), early-responsive to dehydration stress protein (Rai et al., 2016), probable aquaporin PIP1-5 (Weig et al., 1997), aquaporin PIP2-3 (Daniels et al., 1994), probable aquaporin PIP1-4 (Li et al., 2015), plasma membrane intrinsic protein 1B (Alexandersson et al., 2005), aquaporin PIP1-3 (Kammerloher et al., 1994), probable aquaporin PIP2-6 (Alexandersson et al., 2010), and aquaporin PIP2-7 (Weig et al., 1997; Figures 2, 4). The decreased expression of this group of aquaporins (Table 2) confirms crosstalk between indolic Gls production and water deficiency enzymes (Khan et al., 2010). The mechanism underlying such crosstalk is intriguing. The reduction in aquaporins potentiates our observation of retarded growth of Gls mutants (Mostafa et al., 2016). The decreased Gls production resulted in stress status, which led to decreased water uptake and decreased expression of aquaporins, and thus growth retardation.

The immunity and defense process was also affected by Gls perturbation, and it is represented by changes in the directly connected nodes: SNAP25 homologous protein (SNAP33; Eschen-Lippold et al., 2012; connected by MYB51 in tryptophan synthesis and CYP81F2 to GMN), protein ILITYHIA (ILA; Monaghan and Li, 2010; playing a role in methionine chain elongation by forming edges with IMD1, IMD2, and IMD3) and a 20 kDa chaperonin (CPN21; Takáč et al., 2014; connected to GMN by the edge GGP1; Figures 2, 4). Another protein exhibiting expression changes and connected to GMN is malate dehydrogenase 2 (mMDH2), which participates in bacterial defense (Jones et al., 2006; Figures 2, 4). In myb28/29, a GTP-binding nuclear protein Ran-1 (Jiang et al., 2007) was found to connect MYB28, MYB29, MYB76, MYB34, MYB51, and MYB122, suggesting its role in methionine chain-elongation and tryptophan synthesis (Figures 3, 4 and Table 3).

 Effects of glucosinolate metabolism perturbation on other processes and nodes

Gls biosynthetic pathway is organelle specific and involves transport starting from methionine chain-elongation, sulfate transport, and ending with Gls storage in the seeds (Sønderby et al., 2010; Gigolashvili and Kopriva, 2014; Jørgensen et al., 2015). Here we report a decrease in ABC transporter B family member 19 (Lin and Wang, 2005) in both mutants (Table 1). In addition to their role in sulfate transport, ABC transporters are involved in transporting Gls hydrolysis products (Kang et al., 2011). This result indicates the decrease in glucosinolate levels in the mutants feedback regulate the ABC transporter level. In cyp79B2/B3, a curculin-like (mannose-binding) lectin family protein (At5g18470) involved in carbohydrate binding forms connections with MYB51 and CYP81F2 (Figures 2, 4). How this lectin family protein function is not known. Another biological process affected by the Gls perturbation is photosynthesis as revealed by the increase of photosystem I reaction center subunit IV B in both mutants (Table 1), and increases in cyp79B2/B3 photosystem II stability/assembly factor HCF136 (Meurer et al., 1998), protein curvature thylakoid 1B, NAD(P)H-quinone oxidoreductase subunit H, light-harvesting complex I chlorophyll a/b binding protein 1 and light-harvesting chlorophyll protein complex II subunit B1 (Table 2). The increased activity in the photosynthetic process could be a strategy to compensate for the internal stress in the mutants as indicated by changes of many stress-related proteins (Tables 2, 3; Mostafa et al., 2016). It was obvious that aliphatic Gls metabolism perturbation activated the ribosomal protein expression as reflected by the increased levels of 18 ribosomal proteins in the myb28/29 (Table 3). The biological implication of this change is not known although we can correlate it to the regulation of aliphatic Gls biosynthetic pathway by MYB28 and MYB29 (Li et al., 2013).

In both mutants, adenine phosphoribosyltransferase 1 (APT1) acting on adenine phosphorylation (Allen et al., 2002) showed connections with GGP1 and SUR1, so it might have a role in thiohydroximate formation (Figures 2–4). Its decrease in levels may be a feedback of the decreased Gls production in the mutants. In cyp79B2/B3, FtsZ homolog 1 (FTSZ1) involved in chloroplast division and protein binding (Osteryoung et al., 1998) was found to connect with BCAT3 and GSTF9, suggesting it may affect methionine chain-elongation and thiohydroximate synthesis. Interestingly, another FtsZ homolog 2-2 (FTSZ2-2; McAndrew et al., 2008) was also connected with GSTF9 (Figures 2, 4). Isoform 3 of dihydrolipoyllysine-residue succinyltransferase component of 2-oxoglutarate dehydrogenase complex 2 (At4g26910) is a member of tricarboxylic acid cycle and can affect methionine biosynthesis and its coupling to acetylCoA in the chain elongation process. Interestingly, it was found to form multiple connections with GMN via BAT5, BCAT3, IMD1, IMD2, IMD3, GSTF9, and SUR1 (Figures 2, 4). In addition, ATP sulfurylase 1 (APS1), a hydrogen sulfide biosynthesis enzyme, formed edges with GGP1 and SUR1, suggesting its potential role in thiohydroximate synthesis (Figures 2, 4). The increased levels of the aforementioned proteins may reflect a feedback mechanism to compensate for reduced Gls levels in the cyp79B2/B3. Flavone 3'-O-methyltransferase 1 (OMT1) in flavonoid metabolism (Muzac et al., 2000) was connected with FMO1, so it could participate in sulfinyl Gls formation (Figures 2, 4). This finding provides another line of evidence for the pathway interaction between phenylpropanoids and glucosinolates. Previously, methionine derived aldoximes were shown to directly or indirectly inhibit caffeic acid O-methyltransferase (COMT) and caffeoyl-CoA O-methyltransferase CCoAOMT), leading to low levels of phenylpropanoid metabolites (Hemm et al., 2003). Here the decreased levels of OMT1 in cyp79B2/B3 may contribute to the decreased production of sulfinyl Gls in the mutant. The data support our metabolomics finding concerning the decreased shikimate level (Mostafa et al., 2016). Another possibility of the OMT1 activity is methylation of hydroxy-indolyl Gls to form methylated indolic Gls (unknown before, Sønderby et al., 2010) in a way similar to methylation of quercetin into isorhamnetin (Figure 5). In myb28/29, 60S ribosomal proteins L13-1 (BBC1) and L15-1 (RPL15A) might be a component in thiohydroximate synthesis through the connections with GSTF9, GSTF10 and/or GSTF11. Both proteins were increased, presumably to compensate for the deficiency of aliphatic Gls in the mutant (Mostafa et al., 2016).

 The proteome and transcriptome correlation

In the cyp79B2/B3, the defense and stress-related genes calreticulin 3 (At1g08450; Sun et al., 2014), calmodulin (At2g41100; Cazzonelli et al., 2014), lectin (At5g03350; Armijo et al., 2013), and SNAP25 (At5g61210; Eschen-Lippold et al., 2012) showed significant upregulation in the transcriptome and increases in the proteome. Malate dehydrogenase 2 expression was decreased at both the transcript and protein levels, and it is known to be involved in bacterial defense (Jones et al., 2006). These data have provided additional evidence for the relationship between indolic glucosinolates and stress responses. The overall positive correlation between protein and gene expression levels in the cyp79B2/B3 indicates transcriptional regulation of indole glucosinolates. In myb28/29, although there was no overall correlation between transcript and protein levels, isoform 2 of LysM (At1g21880; Willmann et al., 2011) and AIG2 (avirulence induced gene, At5g39730) exhibited similar downregulation patterns as their corresponding proteins. Both genes are involved in cellular stress responses (Jiang et al., 2007; Willmann et al., 2011). Post-transcriptional and post-translational regulations may contribute to the non-correlation between the expression of some of the genes and their encoded proteins in myb28/29.

 Conclusions

Glucosinolate biosynthetic process is controlled by several cytochrome proteins known to be localized to the membrane, but little is known about how Gls metabolism would affect the membrane proteome. In this study, we aim to address this important question utilizing the TMT labeling based quantitative proteomics of two genetic mutants, i.e., cyp79B2/B3 as the indolic Gls mutant and myb28/29 as the aliphatic Gls mutant. We identified 4,673 proteins, out of which 2,171 were membrane proteins. From these membrane proteins and after transmembrane domain analysis, 192 exhibited different levels relative to WT, with cytochrome P450 86A7, cytochrome P450 71B26 and probable cytochrome c representing new cytochromes potentially involved in GMN. Based on our analyses, the first two might play a role in hydroxyl-indolic Gls production. In addition, a flavone 3′-O-methyltransferase 1 is hypothesized to participate in the methylation process of the hydroxyl-indolic Gls to form methoxy-indolic Gls. GO functional enrichment revealed important processes related to stress response, transport activities and photosynthesis in the cyp79B2/B3 and those related to protein translation in the myb28/29. A transcription profiling of both mutants showed a strong correlation between transcript and protein levels in cyp79B2/B3, and no significant correlation in myb28/29. Overall, the new nodes and edges discovered in the GMNs are useful resources for future hypothesis-testing experiments and ultimately toward engineering and breeding of Gls profiles with positive impacts on human health and plant defense.

 Author contributions

IM performed the experiments, data analysis and paper drafting; MY performed qRT-PCR experiment and data analysis; NZ participated in protein extraction and peptides labeling; SG conducted the statistical analysis; CD contributed in LC/MS analysis of peptides; MA and ME provided supervision and advice, and SC designed the experiments, supervised the work and finalized the manuscript.

Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer XH and handling Editor declared their shared affiliation, and the handling Editor states that the process nevertheless met the standards of a fair and objective review.

We would like to thank Chen laboratory members for their support and co-operation. The US National Science Foundation (NSF CAREER 0845162), University of Florida, and the Egyptian Government represented by the Egyptian Cultural and Educational Bureau at Washington DC are acknowledged for funding this project.

 Supplementary material

The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fpls.2017.00534/full#supplementary-material

Biological process GO enrichment of membrane proteins differentially expressed in cyp79B2/B3 compared to WT.

Cellular component GO enrichment of membrane proteins differentially expressed in cyp79B2/B3 compared to WT.

Molecular function GO enrichment of membrane proteins differentially expressed in cyp79B2/B3 compared to WT.

Biological process GO enrichment of membrane proteins differentially expressed in myb28/29 compared to WT.

Cellular component GO enrichment of membrane proteins differentially expressed in myb28/29 compared to WT.

Molecular function GO enrichment of membrane proteins differentially expressed in myb28/29 compared to WT.

Correlation between transcript and protein levels inferred from 32 to 22 genes for cyp79B2/B3 and myb28/29, respectively. Pearson correlation r = 0.6579 (p = 4.269e−05) for cyp79B2/B3 and r = 0.0887 (p = 0.6945) for myb28/29.

Primer information used in qRT-PCR.

Proteomics data from two independent experiments (the data were generated using Proteome Discoverer 1.4 by searching the raw data against the Arabidopsis tair 10 database).

Transmembrane domains predection analyses.

GO enrichment of proteins differentially expressed in cyp79B2/B3 and myb28/29 mutants relative to WT using AgriGO SEACOMPARE.

Gene expression at transcript and protein levels in cyp79B2/B3 and myb28/29 relative to WT.

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glucosinolate

 GMN

glucosinolate molecular network

 GO

Gene Ontology

 TMT

tandem mass tags

 WT

Arabidopsis thaliana wild type.