Comparative investigation of seed coats of brown- versus yellow-colored soybean seeds using an integrated proteomics and metabolomics approach

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Received: 25-Sep-2014; Revised: 09-Nov-2014; Accepted: 17-Dec-2014

This article has been accepted for publication and undergone full peer review but has not been through the copyediting,

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Record. Please cite this article as doi: 10.1002/pmic.201400453.

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Title

Comparative investigation of seed coats of brown- versus yellow-colored soybean seeds

using an integrated proteomics and metabolomics approach

Authors

Ravi Gupta1, Chul Woo Min

1, So Wun Kim

1, Yiming Wang

2, Ganesh Kumar Agrawal

3,4,

Randeep Rakwal3,4,5,6

, Sang Gon Kim7, Byong Won Lee

8, Jong Min Ko

8, In Yeol Back

8,

Dong Won Bae9, and Sun Tae Kim

1,*

1Department of Plant Bioscience, College of Natural Resources and Life Sciences, Pusan National

University, Miryang, 627-707, Republic of Korea

2Department of Plant Microbe Interaction, Max Planck Institute for Plant Breeding Research, Cologne,

Germany

3Research Laboratory for Biotechnology and Biochemistry (RLABB), GPO Box 13265, Kathmandu,

Nepal

4GRADE Academy Private Limited, Adarsh Nagar-13, Main Road, Birgunj, Nepal

5Organization for Educational Initiatives, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba, Ibaraki

305-8577, Japan

6Department of Anatomy I, Showa University School of Medicine, 1-5-8 Hatanodai, Shinagawa,

Tokyo 142-8555, Japan

7Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University, Jinju,

South Korea

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8Department of Functional Crops, NICS, RDA, Miryang 627-803, South Korea

9Central Laboratory, Gyeongsang National University, Jinju, South Korea.

Running title: Soybean seed coat proteome and metabolome

Correspondence: Dr. Sun Tae Kim, Department of Plant Bioscience, Pusan National

University, Miryang, 627-706, South Korea.

E-mail: [email protected]

Tel.: +82-55-350-5505

Fax: +82-55-350-5509

SUMMARY

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

up-regulated and 105 were down-regulated in MM compared with M, while 5 spots were

detected only in MM. Proteins involved in primary metabolism showed down-regulation in

MM suggesting energy in MM might be utilized for proanthocyanidin biosynthesis via

secondary metabolic pathways which leads to the development of brown seed coat color.

Besides, down-regulation of two isoforms of isoflavone reductase indicated reduced

isoflavones in seed coat of MM which was confirmed by quantitative estimation of total and

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individual isoflavones using HPLC. We propose that low isoflavones level in MM may offer

a high substrate for proanthocyanidin production which results in the development of brown

seed coat in MM.

Keywords:

Isoflavone / Mallikong mutant / Phenylpropanoid pathway / Proanthocyanidin/ Seed

storage proteins / Soybean seed coat

Abbreviations: IFR, isoflavone reductase; LEA, Late embryogenesis abundant; M,

Mallikong; MM, Mallikong mutant; SSP, Seed storage protein; SBP, Sucrose binding

proteins;

Total Words: 6507

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].

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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 imperfect-black 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

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changes among soybean accessions [20,21,22]. In case of soybean seed coat, only once 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 which result in the 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

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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 µg)

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 hr, 100 V for

1 hr, 500 V for 1 hr, 1000 V for 1 hr, 2000 V for 1 hr, 4000 V for 2 hr, 8000 V for 5 hr, 8000

V for 9 hr, and 50 V for 6 hr 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

Tris-HCl (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 data set.

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

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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 (Supplementary

Table 1).

2.5 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 acetonitrile (ACN) solution and

dehydrated using 100% ACN for 5 min. Gel pieces were digested with 5 µl of trypsin

solution (20 ng/µl, Gold Mass Spectroscopy Grade, Promega, Madison, USA) in 50 mM

ammonium bicarbonate pH 7.8 for 16 hrs at 37 °C. Tryptic digested peptides were extracted

twice with 0.1% trifluroacetic acid (TFA). Each sample was mixed with same volume of

matrix (10 mg/ml α-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

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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 (14926175 sequences; 5299740401 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 modifications-

carbamidomethylation of cysteines, variable modification- methionine oxidation, peptide and

fragment ion mass 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.

2.6 Data processing and statistical analysis

Functional annotations of the identified proteins were carried out using Gene Ontology (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 principal component analysis (PCA), spot intensities of the differential

proteins were log2 transformed and used for clustering using multi-experimental viewer

(MeV) software and for PCA using XLSTAT software.

2.7 Total Isoflavone and individual isoflavone profiling

For quantitation of isoflavones in seed coats of M and MM, samples (1 g) were pulverized

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and incubated with 20 ml of 50% methanol at room temperature in a rotary shaker at 200 rpm

for 24 hrs. Samples were filtered with 0.45 µm syringe filter and separated (20 µL) using a

HPLC (Agilent 1100 series, Agilent Techologies, Inc., USA) equipped with Lichrospher 100

RP 18e column (5 μm) at 30°C. The mobile phase was 0.1% acetic acid and acetonitrile with

a flow rate of 1.0 mL min-1

. 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].

2.8 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 nitrogen free paper and pressed to pellets with

the forming tool. Default parameters are 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 oC for 24 hr. The HCl was removed from the

hydrolyzed sample on a rotary evaporator brought to 10 ml with 0.2 M sodium citrated buffer,

pH 2.2. Amino acids were determined on a Biochrom 30 amino acid analyzer using nihydrin

as colour reactant and on a cation exchange resin column [27].

2.9 Total oil and Fatty acid profiling

Total lipids were extracted and fatty acid methyl esters (FAMEs) were prepared by acid-

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catalyzed transesterification of total lipid as described previously [28] consisting following

steps: Soxhlet extraction, saponification, followed by acid-catalyzed transesterification, and

finally extraction of FAMEs in hexane. FAMEs were subsequently analysed by capillary gas

chromatography (Agilent 7890A) with a HP-FFAP capillary column (25 m × 0.32 mm, i.d. ×

0.5 μm). 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 (Supplementary 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 (Supplementary 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 of seed development and resolved on high-resolution 2DE

gels. A total of 18 gels consisting of 3 biological replicates for each datasets were performed

(Supplementary 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

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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 (broken arrows) and 105 (solid arrows) was

increased and decreased respectively in MM than M (Fig. 2). In addition, five protein spots

were present only in MM. 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) (Supplementary

Table 3).

3.3 Hierarchical clustering and principal component analysis of the identified proteins

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%) (Supplementary 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 (Supplementary Fig. 3).

The novel proteins from this study were mainly SSPs, different isoforms of sucrose binding

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

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soybean [20]. In addition, there is a high variability in mass spectrometry 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 data sets were log-transformed to base 2 to normalize the scale of

abundance and subjected to the clustering using MeV software. A total of 6 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 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. Clusters 2 and 3 exhibited 33 and 30

proteins respectively and contained proteins which 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 down-regulation of these

protein groups during seed 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, ROS-detoxification, SSPs, etc. Proteins in clusters 5 and 6 showed

increase abundance at 6 WAF. Proteins of these clusters were mainly associated with the

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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.

Principal component analysis (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

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)

SSPs accounted for 20% of the total identified proteins (Supplementary Fig. 2 and

Supplementary Table 3). These SSPs were different isoforms of beta-conglycinin α subunit

(spots 2, 3, 10 and 163), beta-conglycinin α’ subunit (spots 8, 12 and 142), beta-conglycinin β

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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) which are encoded by five non-allelic genes. In this study, only the 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. β-conglycinin

is composed of three subunits, α-subunit, α’-subunit and β-subunit. The first two subunits are

encoded by same mRNA group while the β-subunit of β-conglycinin is encoded by another

mRNA group [30-32]. In this study, all the three subunits of β-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 β-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 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) (Supplementary Fig. 2).

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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, glyceraldehyde-3-phosphate dehydrogenase and four isoforms of

phosphoglycerate kinase were also identified; however protein abundance of all these

enzymes were decreased in the MM (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 sucrose or

cell wall polysaccharides [34]. In this study, two isoforms of UDP-glucose

pyrophosphorylase were identified and both of them were down-regulated 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 α-ketogluteric acid. Protein abundance of iso-

citrate dehydrogenase was also decreased in MM, suggesting a down-regulation of citric acid

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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 down-regulation of proteins involved in the key metabolic pathways in MM

than M indicating utilization of alternative pathways for energy production in MM (Fig. 5).

3.4.3 Stress related proteins

During seed 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 8 isoforms of LEA

showed up-regulation in MM compared to M indication 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.

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 [36].

Protein spots 75 and 76 were identified as the isoforms of isoflavone reductase (IFR), and

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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 to 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 and

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 responsive

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

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 1398 63 µg/g in comparison with 1794 85 µg/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 8 isoflavones were found to be lower in MM in comparison to M (Fig. 6B).

In order to investigate if any the other biochemical differences also exist between the

M and MM, other biochemical parameters like total protein, total oil, individual amino acids

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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 (Supplementary Table 4 and 5). Fatty acid profiling of M and MM

seed coats showed no differences in the amount of α-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 (Supplementary 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 (Supplementary

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-L-homocysteine, which

is a precursor for both adenosine and cysteine biosynthesis, thus 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.

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 versus 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

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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 substrate 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.

Acknowledgements

This work was supported by a grant from the Next-Generation BioGreen 21 Program (SSAC,

grant#:PJ009571), Cooperative Research Program for Agriculture Science & Technology

Development (Project No. PJ007155) of Development 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.

Authors declare no conflict of interest.

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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).

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Figure 2. Representative 2D gel maps of seed coats of M and MM from three seed

development stages. Approximately 600 µg of the proteins were resolved on 24 cm IPG strips

pH 4 – 7 on first dimension and on 12 % 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 down-regulation while blue

arrows showed up-regulation in MM in comparision with M. black arrowswere unique to

MM.

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Figure 3. Hierarchical clustering analysis of the identified protein based on their similar

expression profiles. All 178 differentially expressed proteins were grouped into 6 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.

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Figure 4. Gene Ontology term enrichment analysis of the differentially expressed proteins

identified in M and MM seed coats using AgriGO database.(A) Analysis of up-regulated

proteins (A) and down-regulated proteins (B) in MM within the “Biological Process”

category.

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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

corresponding to Supplementary Table 1. Numbers in red indicate up-regulated spots, while

numbers in green represent down-regulated spots in MM compared with M.

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Figure 6. (A) Zoom-gel regions of M and MM 2-D 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.