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DOI 10.1002/pmic.201400453
Proteomics 2015, 15, 1706–1716
RESEARCH ARTICLE
Comparative investigation of seed coats of brownversus yellow-colored soybean seeds using an integrated proteomics and metabolomics approach Ravi Gupta1 , Chul Woo Min1 , So Wun Kim1 , Yiming Wang2 , Ganesh Kumar Agrawal3,4 , Randeep Rakwal3,4,5,6 , Sang Gon Kim7 , Byong Won Lee8 , Jong Min Ko8 , In Yeol Baek8 , Dong Won Bae9 and Sun Tae Kim1 1
Department of Plant Bioscience, College of Natural Resources and Life Sciences, Pusan National University, Miryang, South Korea 2 Department of Plant Microbe Interaction, Max Planck Institute for Plant Breeding Research, Cologne, Germany 3 Research Laboratory for Biotechnology and Biochemistry (RLABB), Kathmandu, Nepal 4 GRADE Academy Private Limited, Birgunj, Nepal 5 Organization for Educational Initiatives, University of Tsukuba, Tsukuba, Ibaraki, Japan 6 Department of Anatomy I, Showa University School of Medicine, Shinagawa, Tokyo, Japan 7 Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University, Jinju, South Korea 8 Department of Functional Crops, NICS, RDA, Miryang, South Korea 9 Central Laboratory, Gyeongsang National University, Jinju, South Korea Seed coat color is an important attribute determining consumption of soybean seeds. Soybean cultivar Mallikong (M) has yellow seed coat while its naturally mutated cultivar Mallikong mutant (MM), has brown colored seed coat. We used integrated proteomics and metabolomics approach to investigate the differences between seed coats of M and MM during different stages of seed development (4, 5, and 6 weeks after flowering). 2DE profiling of total seed coat proteins from three stages showed 178 differentially expressed spots between M and MM of which 172 were identified by MALDI-TOF/TOF. Of these, 62 were upregulated and 105 were downregulated in MM compared with M, while five spots were detected only in MM. Proteins involved in primary metabolism showed downregulation in MM suggesting energy in MM might be utilized for proanthocyanidin biosynthesis via secondary metabolic pathways that leads to the development of brown seed coat color. Besides, downregulation of two isoforms of isoflavone reductase indicated reduced isoflavones in seed coat of MM that was confirmed by quantitative estimation of total and individual isoflavones using HPLC. We propose that low isoflavones level in MM may offer a high substrate for proanthocyanidin production that results in the development of brown seed coat in MM.
Received: September 25, 2014 Revised: November 9, 2014 Accepted: December 17, 2014
Keywords: Isoflavone / Mallikong mutant / Phenylpropanoid pathway / Plant proteomics / Seed storage proteins / Soybean seed coat
Additional supporting information may be found in the online version of this article at the publisher’s web-site
Correspondence: Dr. Sun Tae Kim, Department of Plant Bioscience, Pusan National University, Miryang, 627–706, South Korea E-mail:
[email protected] Fax: +82-55-350-5509
C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Abbreviations: FAME, fatty acid methyl ester; IFR, isoflavone reductase; LEA, late embryogenesis abundant; M, Mallikong; MM, Mallikong mutant; SSP, seed storage protein; SBP, sucrosebinding proteins
Colour Online: See the article online to view Figs. 1–5 in colour.
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1
Introduction
The seed coat or testa is a protective outer covering of the ovule and consist of two integuments or outer layers of cells [1, 2]. In case of soybean, the seed coat matures after the development of endosperm and embryo [3]. Besides acting as a physical barrier, the seed coat has other multifunctional roles majorly in the metabolic control of seed development and dormancy [3], disease resistance [4, 5] and in metabolism of nutrients from parent plant [1, 3]. Soybean seed coat accounts for 8–10% of total seed mass [6] and comprises of cellulose (14–25%), hemicellulose (14–20%), pectin (10–12%), protein (9–12%), uronic acid (7–11%), ash (4–5%), lipid (4–5%), and lignin (3–4%) on the basis of dry weight [7]. In addition to these, soybean seed coats also contain large amount of secondary metabolites, including phenolic acid derivatives (flavonoids/isoflavonoids/anthocyanidins/ proanthocyanidin), alkaloids, terpenoids, and steroids [8]. Seed color in soybean is determined by the levels of anthocyanins and proanthocyanidins in the seed coats [9]. It was shown that yellow colored soybean seeds neither contain anthocyanins nor proanthocyanidins while black and imperfectblack seeds accumulate both anthocyanins and proanthocyanidins in their seed coats. However, in case of brown and buff colored seed coats, only proanthocyanidins were detected suggesting that the seed coat color in brown and buff colored soybean is determined by levels of proanthocyanidins only [9]. There are ample of studies on the characterization of soybean seed coats at the biochemical, genetic, and genomic levels because of anti-microbial and anti-fungal roles of its secondary metabolites [10, 11]. In addition, numerous studies have also revealed that the beneficial health effects of colored soybean are due to its diverse phytochemicals contents, such as isoflavones, saponins, proanthocyanidins, and anthocyanins [12–15]. Previous reports on analysis of soybean seed coat proteins led to the identification of an array of enzymes including chitinase [16], peroxidase [17], invertase [3], subtilisin-like serine protease [18], and a BURP domain containing protein [19], suggesting that soybean seed coat is metabolically active [20]. However, the overall proteome of the seed coat has remained largely elusive. To date, comparative proteomic analysis of total seed proteins involved in seed development have been conducted to understand evolutionary relationships or metabolic changes among soybean accessions [20–22]. In case of soybean seed coat, only one study has been conducted till date where a shotgun proteomic approach was used to identify total seed coat proteins, resulting in the identification of a total of 1372 proteins majorly involved in primary and secondary metabolism, cellular structure, stress responses, nucleic acid metabolism, protein synthesis, folding and targeting, hormones, signaling, and seed storage proteins (SSPs) [20]. However, there is no report on comparative quantitative proteome analysis of seed coats in soybean cultivars differing in seed coat colors. Such study is of prime interest to understand the metabolic pathways that result in the C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
development and accumulation of different pigments in the seed coats. In the present study, an integrative proteomics and metabolomics analyses was carried out in order to identify the differentially expressed proteins and metabolites between two contrasting yellow and brown-colored soybean seed cultivars. For this purpose, we selected Mallikong (M) and Mallikong mutant (MM) cultivars with yellow versus brown seed coat colors, respectively. MM is a naturally derived mutant of M and thus it is presumed that there would not be much differences in the other metabolic pathways in these two cultivars except for those responsible for the development of seed coat color.
2
Materials and methods
2.1 Plant material and growth M and MM cultivars were grown at the experimental field of the Department of Functional Crop, National Institute of Crop Science, RDA at Miryang, South Korea (latitude 35⬚N) in June and seeds were harvested in the October 2012 (average temperature 23.5 ± 3.5⬚C, average day length 12 h 17 min). Filling seeds of M and MM cultivars were harvested at 4, 5, and 6 week-after-flowering (WAF). Seed coats were dissected from the collected seeds and stored at –70⬚C until used.
2.2 Protein extraction and 2DE analysis Total proteins from seed coats were isolated as described previously [23] and subjected to 2DE analysis according to the previously published protocol [24, 25]. Briefly, seed coat proteins from three growth stages from M and MM seeds were isolated using Tris-Mg-NP-40 buffer followed by TCA precipitation. Pellets obtained after precipitation were dissolved in the rehydration buffer containing 7 M Urea, 2 M Thiourea, 4% v/v CHAPS, 2 M DTT, and 0.5% v/v IPG buffer pH 4–7 (GE Healthcare, Waukesha, WI, USA). Protein concentrations in each fraction were determined using 2D-Quant kit (GE Healthcare). Proteins (600 g) were loaded on the 24 cm IPG strips, pH 4–7 by passive rehydration loading overnight at 20⬚C. Iso-electric focusing was carried out using following protocol: 50 V for 4 h, 100 V for 1 h, 500 V for 1 h, 1000 V for 1 h, 2000 V for 1 h, 4000 V for 2 h, 8000 V for 5 h, 8000 V for 9 h, and 50 V for 6 h on IPGphore II platform (GE Healthcare). After IEF, the strips were reduced in an equilibration buffer [6 M urea, 30% v/v glycerol, 2% w/v SDS, 50 mM TrisHCl (pH 6.8), and 0.1 mg/mL bromophenol blue] containing 1% DTT as the first step and then alkylated by 2.5% iodoacetamide as the second step. The second dimension analysis was carried out on 13% SDS-polyacrylamide gels using EttanDalt twelve (GE Healthcare), after which the gels were stained with colloidal Coomassie Brilliant Blue (CBB) [24, 25]. A total of three biological replicates were performed for each dataset. www.proteomics-journal.com
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2.3 Image acquisition and data analysis Images of the colloidal CBB stained 2DE gels were acquired using a transmissive scanner (PowerLook 1120, UMAX) with a 32 bit pixel depth, 300 dpi resolution, and brightness and contrast set to default. For the analysis of the 2DE gels, raw tiff image files were imported in the ImageMaster 2D Platinum software (ver. 6.0, GE Healthcare) and spots were detected from three biological replicates and averaged. For the quantitative analysis, the volume of each spot was normalized as an average of the volume of spots on the gel and then spot volumes were calculated to determine the relative abundance of proteins in the experimental samples. Statistical analyses of spot volumes were performed using the one way-ANOVA to determine statistically significant values (p ࣘ 0.05) using MeV software (Supporting Information Table 1).
2.4 MALDI-TOF/TOF MS identification of differential protein spots Differential protein spots were excised from the 2DE gels and were subjected to in-gel digestion as described previously [24]. Briefly, in gel reduction was carried out using 10 mM DTT in 100 mM ammonium bicarbonate at 56⬚C for 30 min. Alkylation of reduced proteins was done using 50 mM iodoacetamide in 100 mM ammonium bicarbonate for 30 min in dark. Gel pieces were washed with 1:1 ammonium bicarbonate and ACN solution and dehydrated using 100% ACN for 5 min. Gel pieces were digested with 5 L of trypsin solution (20 ng/L, Gold Mass Spectroscopy Grade, Promega, Madison, USA) in 50 mM ammonium bicarbonate pH 7.8 for 16 h at 37⬚C. Tryptic digested peptides were extracted twice with 0.1% TFA. Each sample was mixed with same volume of matrix (10 mg/mL ␣cynohydroxycinnamic acid, 0.1% TFA, 50% ACN), loaded on a MALDI target plate and allowed to dry at 25⬚C. Prepared samples of tryptic peptides were subjected to MALDI-TOF/TOF MS analysis using ABI 4800 Plus TOF-TOF Mass Spectrometer (Applied Biosystems, Framingham, MA, USA) [26]. Spectra were calibrated with the peptide calibration standard (Mass Standard Kit for the 4700 Proteomics Analyzer; calibration Mixture 1), prepared in the same way. The ten most and least intense ions per MALDI spot with signal/noise ratios >25 were selected for subsequent MS/MS analysis in 1 kV mode using 800–1000 consecutive laser shots. MS/MS spectra were searched against the Uniprot/Swiss-Prot database (14,926,175 sequences; 5,299,740,401 residues) and soybean peptide database obtained from the soybean genome database (Phytozome ver. 8.0, http://www.phytozome.net/soybean) by Protein Pilot v.3.0 software (AB Sciex, Framingham, MA, USA) using MASCOT as search engine (ver. 2.3.0, Matrix Science, London, UK). The search parameters used for the protein identification were as follows: fixed modificationscarbamidomethylation of cysteines, variable modificationmethionine oxidation, peptide, and fragment ion mass C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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tolerances-50 ppm, maximum trypsin missed cleavage-1 and instrument type-MALDI-TOF/TOF. Only significant hits, as identified by the MASCOT probability analysis (p < 0.05) were accepted.
2.5 Data processing and statistical analysis Functional annotations of the identified proteins were carried out using GO database using UFO web server (http://ufo.gobics.de/) and GO term enrichment analysis was carried out using agriGO database (http://bioinfo.cau.edu.cn/agriGO/). For hierarchical clustering and PCA, spot volumes of the differential protein spots were log2 transformed and used for clustering using multiexperimental viewer (MeV) software and for PCA using XLSTAT software.
2.6 Total isoflavone and individual isoflavone profiling For quantitation of isoflavones in seed coats of M and MM, samples (1 g) were pulverized and incubated with 20 mL of 50% methanol at room temperature in a rotary shaker at 200 rpm for 24 h. Samples were filtered with 0.45 m syringe filter and separated (20 L) using a HPLC (Agilent 1100 series, Agilent Techologies, Inc., USA) equipped with Lichrospher 100 RP 18e column (5 m) at 30⬚C. The mobile phase was 0.1% acetic acid and acetonitrile with a flow rate of 1.0 mL/min. The isoflavones such as daidzin, glycitin, genistin, mal-glycitin, mal-daidzin, mal-genistin, and daidzein were detected at 260 nm and quantified based on comparisons with retention times and peak areas of standards [27].
2.7 Total proteins and amino acid analysis Total protein content in M and MM seeds was analyzed using protein analyzer (rapid N cube, Germany). Samples were powdered using a high speed vibrating sample mill (CMT T1– 100, Japan). The samples (50 mg) were wrapped in nitrogenfree paper and pressed to pellets with the forming tool. Default parameters were used for the analysis. Glutamic acid (9.52% N) was used as test standard and a protein factor of 6.25 was used. All samples were analyzed in duplicates [27]. The amino acid profiling was carried out using an amino acid analyzer (Biochrom 30, Biochrom Ltd., Cambridge, UK). Briefly, the samples were hydrolyzed with 6 N HCl in sealed glass tubes filled with nitrogen at 110⬚C for 24 h. The HCl was removed from the hydrolyzed sample on a rotary evaporator and the sample volume was brought to 10 mL with 0.2 M sodium citrated buffer, pH 2.2. Amino acids were determined using nihydrin as color reactant and on a cation exchange resin column [27]. www.proteomics-journal.com
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Figure 1. Graphical representation of the experimental workflow utilized in the current study. Upper panel shows morphological charaterstics of Mallikong (M) and Mallikong mutant (MM) seeds at 4, 5, and 6 weeks after flowering (WAF).
1709 of seed development and resolved on high-resolution 2DE gels. A total of 18 gels consisting of three biological replicates for each datasets were performed (Supporting Information Fig. 1). Analysis of 2DE gels using ImageMaster2DPlatinum software showed 867, 850, and 788 reproducible spots at 4, 5, and 6 WAF, respectively (Fig. 2). A total of 178 protein spots showed differential abundance in MM compared with M of which 68, 54, and 56 spots were from 4, 5, and 6 WAF, respectively. MALDI-TOF/TOF MS analysis successfully identified and confidently assigned 172 protein spots out of the 178. Of these 172 identified spots, protein abundance of 62 (blue, broken arrows) and 105 (red, solid arrows) was increased and decreased respectively in MM than M (Fig. 2). In addition, five protein spots were present only in MM (marked with black arrows in Fig. 2). These five proteins were identified as GroES-like zinc-binding alcohol dehydrogenase family protein (spot 243), caffeoyl coenzyme A 3-O-methyltransferase 2 (spot 252), Xaa-Pro aminopeptidase 2 (spot 253), proteasome subunit alpha type-6 (spot 254), and proteasome subunit alpha type (spot 255) (Supporting Information Table 3).
3.3 Hierarchical clustering and principal component analysis of the identified proteins 2.8 Total oil and fatty acid profiling Total lipids were extracted and fatty acid methyl esters (FAMEs) were prepared by acid-catalyzed transesterification of total lipid as described previously [28] consisting following steps: Soxhlet extraction, saponification, followed by acidcatalyzed transesterification, and finally extraction of FAMEs in hexane. FAMEs were subsequently analyzed by capillary GC (Agilent 7890A) with a HP-FFAP capillary column (25 m × 0.32 mm, id × 0.5 m). The percentage of fatty acid was calculated by standard values of peak areas of C16:0, C18:0, C18:1, C18:2, C20:0, C20:1, and C22:0 methyl esters [28].
3
Results and discussion
3.1 Morphological characteristics of M and MM Phenotypic analysis of M and MM plants showed similar morphological characteristics like flower color, leaf shape, leaf color, pubescence color, and pod color, except for the seed coat and hilum colors (Supporting Information Table 2). Seed coat color of M is light yellow while that of MM is light brown (Fig. 1). Hilum color of M is yellow while MM has white colored hilum (Supporting Information Table 2).
3.2 Proteome profiling of M and MM seed coats confidently assign 172 differential protein spots In order to find out the differences in the seed coats of M and MM, total seed coat proteins were isolated from three stages C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
The GO annotation of the 172 identified proteins clustered these into 16 functional categories including primary metabolism (20%), SSPs (20%), stress response (14%), unknown (9%), ROS detoxification (6%), gene regulation (6%), protein folding (6%), protein synthesis and regulation (5%), protease inhibitor (3%), cell structure (2%), secondary metabolism (2%), energy metabolism (2%), photosynthesis (2%), xenobiotic detoxification (2%), signaling (1%) and transport (1%) (Supporting Information Fig. 2). A comparison of this study with the previously published report on soybean seed coat proteome [20] showed that out of the total 172 identified proteins, 118 (68.6%) were novel to this study with only 54 (31.39%) proteins common to both studies (Supporting Information Fig. 3). The novel proteins from this study were mainly SSPs, different isoforms of sucrosebinding proteins (SBPs) and isoforms of Ran-binding proteins. These 118 novel proteins might be specific to the soybean cultivar as the previous study was performed with Jack cultivar of soybean [20]. In addition, there is a high variability in MS results therefore; it might be possible that some proteins just escape the analysis in one run or in another in previous or current study. In order to understand the interdependence of protein profiles across the three seed developmental stages in M and MM, hierarchical clustering was performed. After spot normalization, the datasets were log-transformed to base 2 to normalize the scale of abundance and subjected to the clustering using MeV software. A total of six clusters were grouped based on the similar abundance profiles of the identified proteins (Fig. 3A–C). Cluster 4 was the most abundant group with 39 proteins while cluster 1 was the www.proteomics-journal.com
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Figure 2. Representative 2DE gel maps of seed coats of M and MM from three seed development stages. Approximately 600 g of the proteins were resolved on 24 cm IPG strips pH 4–7 on first dimension and on 13% SDS-PAGE on second dimension. Spots were visualized by using colloidal CBB staining and gels were compared using ImageMaster 2D Platinum software (ver. 6.0). Spots with red arrows showed downregulation while blue arrows showed up-regulation in MM in comparision with M. Spots marked with black arrows were unique to MM.
least abundant group containing only 11 proteins. Proteins of cluster 1 showed almost similar abundance patterns at 4 WAF (stage 1) and 5 WAF (stage 2) compared to a higher abundance at 6 WAF (stage 3). The proteins of cluster 1 were associated with primary metabolism, protein synthesis and regulation, stress response, and SSPs. Besides, 27% of the proteins were either hypothetical or with unknown functions. C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Clusters 2 and 3 exhibited 33 and 30 proteins respectively and contained proteins that showed a gradual decrease in the abundance pattern from 4 to 6 WAF. The proteins of these clusters were mainly associated with primary metabolism, energy metabolism, photosynthesis, protein synthesis and regulation, gene regulation, and protease inhibitor activity. The downregulation of these protein groups during seed www.proteomics-journal.com
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Figure 3. Hierarchical clustering analysis of the identified proteins based on their similar expression profiles. All 178 differentially expressed proteins were grouped into six clusters. (A) Heat map is shown on the top. (B) The expression profiles of the clustered proteins are shown below for M and MM in the three growth stages (1–3). The x-axis of the graph represents seed development stages while the y-axis represents log-transformed value of protein expression. (C) Functional groups in each cluster are depicted by pie chart below the expression graphs of the protein. (D) PCA analysis of the identified proteins.
developmental stages might be associated with the seed maturation as matured seed is almost quiescent with negligible or no metabolic activities. Proteins of cluster 4 showed almost no changes in their abundance pattern at 4 and 5 WAF followed by sudden decrease at 6 WAF. The major functional groups in this cluster were primary metabolism, ROSdetoxification, SSPs, etc. Proteins in clusters 5 and 6 showed increase abundance at 6 WAF. Proteins of these clusters C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
were mainly associated with the stress response, SSPs, and primary metabolism. As soybean seeds undergo excessive dehydration during maturation, an increased accumulation of stress-related proteins and SSPs at 6 WAF was expected. PCA was also carried out to identify the protein clusters responsible for correlated variance. PCA results showed that the major differences in the spot volumes between M and MM seed coats were at 4 and 5 WAF with no significant www.proteomics-journal.com
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Figure 4. GO term enrichment analysis of the differentially expressed proteins identified in M and MM seed coats using AgriGO database.(A) Analysis of upregulated proteins (A) and downregulated proteins (B) in MM within the “Biological Process” category.
differences at 6 WAF (datasets of M and MM overlapped each other) (Fig. 3D–E). These results can be explained in terms of seed development process. At 4 and 5 WAF stage, seed is metabolically active as this is the period when seed filling takes place in soybean, therefore differences in the M and MM proteins are expected at these stages. However, at 6 WAF stage, seeds are completely matured and no or negligible metabolic activities takes place in it, therefore no major differences in PCA results were observed at this stage. The GO term enrichment analysis was also carried out on all identified proteins using agriGO database [29]. Results showed decrease in abundance of proteins involved in biological processes (such as primary metabolism, catabolic process, etc.) in MM, supporting the results of hierarchical clustering (Fig. 4A). Proteins with increased accumulation in MM were mainly associated with the reproductive, developmental, and embryonic developmental processes (Fig. 4B).
3.4 Functional significance of the identified proteins 3.4.1 Seed storage proteins SSPs accounted for 20% of the total identified proteins (Supporting Information Fig. 2 and Supporting Information Table 3). These SSPs were different isoforms of beta-conglycinin ␣ C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
subunit (spots 2, 3, 10, and 163), beta-conglycinin ␣’ subunit (spots 8, 12, and 142), beta-conglycinin  subunit (spots 48, 49, 161, 162, 260, and 262), glycinin (spots 73 and 74), glycinin 1 (spots 37, 41, 148, and 154), glycinin 2 (spots 31, 32, 33, 177, 179, and 181), G5 protein (spots 175 and 182), and mutant glycinin subunit A1aB1b (spots 193 and 194). Of these, abundance of 57% of SSPs was higher in M compared to MM. SSPs are important constituent of soybean seeds and comprise of up to 70–80% of the total proteins [30, 31]. In soybean, glycinin consists of five subunits (G1–G5) that are encoded by five nonallelic genes. In this study, only G1, G2, and G5 subunits were identified, suggesting that either other subunits of glycinin are not present in the seed coat and are specific to seeds only or the abundance pattern of other subunits were similar in both M and MM and hence not identified in this study. -Conglycinin is composed of three subunits, ␣-subunit, ␣’-subunit, and -subunit. The first two subunits are encoded by same mRNA group while the -subunit of -conglycinin is encoded by another mRNA group [30–32]. In this study, all the three subunits of -conglycinin were detected, suggesting that in addition to their high abundance in seeds, these are also present in the seed coats of soybean. Surprisingly, no isoforms of either glycinin or -conglycinin were identified in the previous study [20]. This difference in identified proteins might be due to differences in soybean cultivars used as experimental materials in this and the other www.proteomics-journal.com
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Figure 5. A schematic diagram of the differentially modulated proteins involved in primary and secondary metabolism in the seed coat of M and MM. Numbers indicate the spot numbers corresponding to Supporting Information Table 1. Numbers in red indicate upregulated spots, while numbers in green represent downregulated spots in MM compared with M.
study [20]. It is likely that SSPs could serve as biomarkers for classification of soybean cultivars.
3.4.2 Proteins involved in primary metabolism Soybean seeds are rich in proteins and fatty acids, which are accumulated during seed development. In addition, it is also quite established that seeds act as a major sink and accumulate lots of carbohydrates during development [3]. Since the current study utilized seed coats from different stages of seed development, we identified a plethora of enzymes involved in primary metabolism (20% of total identified proteins) (Supporting Information Fig. 2). Sugars are transported in the plants in the form of sucrose through the phloem. From phloem, sucrose accumulates in the apoplast and finally enters in the cytoplasm where it is catabolized by the glycolytic pathway [33]. In this study, seven isoforms of SBPs were identified (spots 55, 147, 149, 150, 152, 153, and 160). SBPs are involved in the transportation of sucrose from apoplast to cytoplasm, where it is catabolized to produce energy. Interestingly, all the isoforms of SBPs showed increased protein abundance in the MM, suggesting a higher requirement of sucrose or energy in the seed coats of mutant during seed development. In addition to SBPs, various enzymes of glycolysis including enolase, fructose-bisphosphate aldolase, glyceraldehyde3-phosphate dehydrogenase, and four isoforms of phosphoglycerate kinase were also identified; however protein abundance of all these enzymes were decreased in the MM C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
(Fig. 5). These results showed that although MM seed coats accumulate a lot of sucrose in the cytoplasm, it is not catabolized through the glycolytic pathway. Either the amount of sucrose remains higher in the MM seed coats or sucrose might be converted into glucose-6-phosphate and degraded by other sugar catabolizing pathways like pentose-phosphate pathway. UDP-glucose pyrophosphorylase is involved in the conversion of glucose-1-phosphate into UDP-glucose which ultimately leads to the production of sucrose or cell wall polysaccharides [34]. In this study, two isoforms of UDPglucose pyrophosphorylase were identified and both of them were downregulated in MM. In addition to the sugar metabolizing enzymes, other enzymes related to amino acid metabolism were also identified. Glutamine synthetases are involved in the formation of glutamine from glutamate and ammonia. Three isoforms of glutamine synthetase were found to be down-regulated in MM. Iso-citrate dehydrogenase is a key enzyme of citric acid cycle, involved in the conversion of isocitrate to ␣-ketogluteric acid. Protein abundance of iso-citrate dehydrogenase was also decreased in MM, suggesting a downregulation of citric acid cycle in this cultivar. Formate dehydrogenase catalyzes the conversion of formate to carbon dioxide and yield energy in the form of NADH. Four isoforms of formate dehydrogenase were identified of which three showed increased protein abundance in MM, suggesting a high requirement of energy during seed development in MM compared to M. These findings reveal overall downregulation of proteins involved in the key metabolic pathways in MM than M indicating utilization of alternative pathways for energy production in MM (Fig. 5). www.proteomics-journal.com
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Figure 6. (A) Zoom-gel regions of M and MM 2DE gels showing expression pattern of IFR isoforms during the seed development stages. (B) Measurement of individual and total isoflavone content in M and MM seed coats using HPLC.
3.4.3 Stress-related proteins During maturation, seeds undergo excessive dehydration therefore, a large number of proteins involved in desiccation stress, were identified in this study. Late embryogenesis abundant (LEA) proteins are a group of hydrophilic proteins which accumulate to a high level during seed dehydration [35]. LEA isoforms protect the proteins from denaturation and inactivation during dehydration of the seeds [36]. Besides, some of the LEA isoforms also have membrane stabilization function. Nine isoforms of LEA and three isoforms of dehydrins were identified in this study of which all the isoforms of dehydrins and eight isoforms of LEA showed upregulation in MM compared to M, indicating a higher degree of dehydration of MM seeds compared to M. In the previous study [20], 27 isoforms of LEA were identified in seed coat of soybean cultivar Jack suggesting their vast abundance and requirement in soybean seed coats at the time of their maturation.
[36]. Protein spots 75 and 76 were identified as the isoforms of isoflavone reductase (IFR), and showed a similar trend of their abundance during seed development in M and MM. Proteomic analysis clearly showed decreased protein abundance of IFR isoforms at 4–6 WAF in MM than M, suggesting that it could be one of the key factor involved in color development in the seed coats of M and MM. The protein abundance of IFR homologues was low in MM at 4 and 5 WAF during which major changes in seed physiology takes place. At 6 WAF or matured stage, the amount of IFR was either equal (spot 76) or higher (spot 75) in MM in comparison with the M (Fig. 6A). IFRs are involved in the biosynthesis of isoflavones, which are important secondary metabolites for plants [37]. Isoflavones act as defense molecules for plants as these have antimicrobial, antifungal, and feeding deterrent properties [38]. Lower levels of IFR hinted toward low levels of isoflavones in MM.
3.5 Isoflavone profiling of soybean seeds 3.4.4 Proteins of phenylpropanoid pathway Phenylpropanoid pathway is a source of coumarins, lignin, flavones, isoflavones, flavonols, anthocyanins, and proanthocyanidin that are the important weapons for plant defense C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
As decreased abundance of IFR was observed in the MM seed coats during seed development (Fig. 6A), total isoflavone content, and isoflavone profiling were carried out in M and MM using HPLC. Interestingly, total isoflavone content was found significantly lower in the MM and was calculated as www.proteomics-journal.com
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1398 ± 63 g/g in comparison with 1794 ± 85 g/g, calculated in M (Fig. 6B). In addition to the total isoflavones, the concentration of individual isoflavones including genistin, mal-genistin, daidzein, daidzin, mal-daidzin, glycitein, glycitin, and mal-glycitin, were also assayed. Similar to the total isoflavone content, concentrations of all individual eight isoflavones were found to be lower in MM in comparison to M (Fig. 6B). In order to investigate if any other biochemical differences also exist between the M and MM, other biochemical parameters like total protein, total oil, individual amino acids and individual fatty acids concentrations were also measured in the seed coats of M and MM. Results showed that M contained 20.3% oil and 36.1% protein while the MM contained 20.5% oil and 36.5% proteins, suggesting that protein and oil contents of seed coats of M and MM are almost similar (Supporting Information Tables 4 and 5). Fatty acid profiling of M and MM seed coats showed no differences in the amount of ␣-linolenic acid (C18:3); however, a slight difference in the content of palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1), and linoleic acid (C18:2) was observed (Supporting Information Table 4). Amino acids profiling did not show any differences between M and MM except for the cysteine content. The amount of cysteine in M was 1.02 mg/g, while it was calculated as 2.84 mg/g in MM (Supporting Information Table 5). Cysteine is a sulfur containing amino acid and participates in the formation of glutathione, which is an important antioxidant. Interestingly, one protein spot specific to MM (spot no. 252) was identified as caffeoyl coenzyme A 3-O-methyltransferase 2. This enzyme catalyzes the conversion of S-adenosyl-L-methionine to S-adenosyl-Lhomocysteine, which is a precursor for both adenosine and cysteine biosynthesis, thus partial explaining high levels of cysteine in MM in comparison with M. Overall, except for the changes in isoflavone concentrations, no other major changes in the biochemical composition of seed coats of M and MM were observed.
strate for the proanthocyanidin production in MM, leading to development of brown seed coat. The pathways for the biosynthesis of isoflavones and proanthocyanidin are similar in which one flavone “naringenin” is a key regulator. Naringenin is a precursor for both isoflavone and proanthocyanidin production. Since low levels of isoflavones were observed in MM, it can be expected that naringenin in MM seeds would be utilized for the synthesis of proanthocyanidins, which results in the production of brown-colored seed coats. This work was supported by grants from the Next-Generation BioGreen 21 Program (SSAC, grant#:PJ009571), Cooperative Research Program for Agriculture Science & Technology Development (Project No. PJ007155) of Department of Functional Crop, National Institute of Crop Science and National Agenda Programs for Agricultural R&D (PJ01004602201401), Rural Development Administration (RDA), Republic of Korea. RG acknowledge financial support from RDA. The authors have declared no conflict of interest.
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Concluding remarks
The aim of this study was to unravel the major differences in the M and MM seed coats that lead to the development of yellow and brown seed coat colors, respectively, using an integrated proteomics and metabolomics approach. This study identified 172 differentially expressed proteins between M and MM, enriching our current knowledge on seed coat proteomics and opening a door for improving the nutrient value of soybean seeds for better human life. In this direction, this study proposes that low level of IFR in MM compared with M might be one reason for differences in their seed coat color. The seed coat color in soybeans is determined by the levels of proanthocyanins [9]. As both isoflavones and proanthocyanidins are synthesized via phenylpropanoid pathway in plants, reduced concentration of isoflavones may offer a high sub C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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