Organ-Specific Proteomic Analysis of NaCl-Stressed Germinating

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Organ-specific proteomic analysis of NaCl-stressed germinating soybeans Yongqi Yin, Runqiang Yang, and Zhenxin Gu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf500851r • Publication Date (Web): 24 Jun 2014 Downloaded from http://pubs.acs.org on June 30, 2014

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Journal of Agricultural and Food Chemistry

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Organ-specific proteomic analysis of NaCl-stressed germinating soybeans

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Yongqi Yin, Runqiang Yang, Zhenxin Gu*

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College of Food Science and Technology, Nanjing Agricultural University, Nanjing, Jiangsu 210095,

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People's Republic of China

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ABSTRACT: A comparative proteomic approach was employed to explore proteome expression

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patterns in germinating soybeans under NaCl stress and NaCl-aminoguanidine treatment. The

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proteins were extracted from 4-day-old germinating soybean cotyledons and non-cotyledons

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(hypocotyl and radicle), and were separated using two-dimensional polyacrylamide gel

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electrophoresis. A total of 63 and 72 differentially expressed proteins were confidently identified by

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MALDI-TOF/TOF in the non-cotyledons and cotyledons, respectively. These identified proteins

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were divided into ten functional groups and most of them were predicted to be cytoplasm proteins

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in non-cotyledons. Moreover, gamma-aminobutyric acid was accumulated while the major allergen

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(Bd 30K protein) was reduced in the germinating soybeans. The proteins involved in energy

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metabolism and in protein processing in endoplasmic reticulum were enriched under NaCl stress.

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Meanwhile, the negative effect of stress was aggravated once the polyamine degradation was

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inhibited. Redistribution of storage proteins under stress indicated that storage proteins might not

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only function as seed storage reserves but also have additional roles in plant defense.

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KEY WORDS: soybean; proteomics; NaCl stress; polyamines; germination; gamma-aminobutyric

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acid


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INTRODUCTION

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Nowadays, improvement of conventional foods is desirable since functional foods are playing

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positive role in disease prevention.1 Disease control and prevention through diet is considered to be

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the most efficient way to improve health and bring down medical costs for the expanding global

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population.2 Hence, bean sprouts, as one of the vegetables traditionally and popularly consumed in

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China, Korea, Japan, and Southeast Asian countries, are being targeted for designing functional

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foods. Functional bean sprouts contain health-promoting compounds such as gamma-aminobutyric

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acid (GABA), phenolics, phytoalexin and other antioxidants. Their content are largely enhanced

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when sprouts are under various stimuli including biotic stress3 and abiotic stress such as

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light-mediated, ultra violet irradiation, hypoxia,4 mechanical stimulation, and salt stress especially.5,

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6

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diseases according to clinical researches.7 GABA and plant phenolic induced by salt stress in

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sprouts can be harnessed as a source of therapeutic and health-supporting functional ingredients,

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especially for disease-linked diet and environmental-influenced diseases. Previous morphological,

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physiological and genomic studies revealed much knowledge of metabolic process of these

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functional components in cellular and physiological aspects,8 but a systematic understanding of the

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metabolic process of functional components and the parallel molecular processes of plant under

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stressful conditions, especially under salt stress, is still in its infancy.

GABA and plant phenolics prevent certain forms of cancer, and reduce the risk of cardiovascular

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Salt is commonly known to have negative effects on plant growth and development.9 Polyamines

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(PAs) can enhance plant tolerance to various environmental stresses. Exogenous Pas raise stress

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tolerance of plant via stabilizing the signal transduction system, increasing cell membrane stability,

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maintaining cell osmotic balance, improving antioxidant enzymes system dynamic, and meliorating

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photosynthesis of plant.10 The content of PAs is increased under stressful conditions. However, it is 3 ACS Paragon Plus Environment


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unclear if the enhanced endogenous PAs play a similar role to that of exogenous PAs. Additionally,

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researches show that GABA form through the polyamine degradation pathway.8 In plant, putrescine

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is firstly converted to γ-aminobutyraldehyde by diamine oxidase (DAO, EC 1.4.3.6), and then

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γ-aminobutyraldehyde is converted to GABA by aminoaldehyde dehydrogenase (AMADH, EC

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1.2.1.19). Studies demonstrate that at least 30% of GABA accumulation is supplied by the

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polyamine degradation pathway under stressful conditions.4, 11 Therefore, further studies on the

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effect of endogenous PAs on responses to stress by high throughput analysis will provide a new

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understanding of the roles of endogenous PAs and the relationship between GABA and PAs.

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Aminoguanidine (AG) is a specific inhibitor of DAO, blocking the polyamine degradation pathway.

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It is convenient to study the action of endogenous PAs through inhibition of DAO activity; DAO is

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the rate-limiting enzyme of PAs catabolism.

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Proteomics is the best available molecular tool for analyzing the complete proteome in tissues or

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at organelle level. It also describes the expression patterns of proteome changes in response to

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stressful conditions. Proteomics has been applied to researches on different abiotic stresses for

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soybean seedlings including salt,12,

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seedlings absorb nutrients from external environment and, especially, synthesize organic

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compounds via photosynthesis. However, the functional soybean sprouts should be germinated in

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artificial dark environment and without any external nutrients except its storage material.3, 4 Up to

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now, little information is available on the stress responses of soybeans germinating under dark

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conditions and their response at the proteome level in particular. Identification of distinct proteins

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has contributed to understanding of the molecular mechanism adopted by soybeans in response to

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stress, but the regulatory mechanisms of that operation in divergent organs are still unknown

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without comprehensive organ-specific proteome analyses.

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flooding,14 and drought.15 In these researches, soybean


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Based on the aforementioned issues, in this study, proteomic responses of dark germinating

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soybean non-cotyledons (hypocotyl and radicle) and cotyledons to NaCl stress, combined with

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NaCl and AG treatments, was investigated. The objective was to reveal the organ-specific defence

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strategy, the role of endogenous PAs, and the adaptive mechanisms of dark germinating soybeans

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against salt stress via identification of the differentially salt-induced, endogenous PAs-mediated and

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organ-specific proteins.

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MATERIALS AND METHODS

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Plant Growth Condition. Dry soybean seeds (Glycine max L. cultivar Yunhe) were sterilized,

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washed and steeped with distilled water at 30 ± 1 °C for 4 h. The soaked seeds were then placed in a

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bean sprouting machine to germinate in a dark incubator at 30 ± 1 °C with water containing the

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following additives: (A) CK: deionized water; (B) NaCl: 50 mM NaCl; and (C) NaCl+AG: 50 mM

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NaCl+2.5 mM AG. The culture solution was replaced every 1 d. Three independent biological

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experiments were performed, and 4 d after treatment, the cotyledons and non-cotyledons (hypocotyl

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and radicle) were carefully collected and frozen in liquid nitrogen until analysis.

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Malondialdehyde (MDA) Content Determination. The extent of lipid peroxidation in terms of MDA formation was measured following the method of Madhava and Sresty.16 GABA and Free PAs (fPAs) Contents Determination. GABA and fPAs contents were analyzed as described by Yang et al.11

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Protein Extraction and Two-Dimensional Polyacrylamide Gel Electrophoresis (2-DE). To

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minimize errors, ten cotyledons and non-cotyledons (hypocotyl and radicle) from ten germinated

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soybeans, respectively, were pooled for each biological repeat sample. Protein extraction was

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performed using trichloroacetic acid (TCA)/acetone precipitation with some modifications.17 Briefly,

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sample powder was suspended in 10% (w/v) TCA in acetone containing 1 mM dithiothreitol (DTT), 5 ACS Paragon Plus Environment


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and held at −20 °C for 2 h. After centrifugation and rinse, the pellets were air dried at room

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temperature and dissolved at 4 °C in lysis buffer containing 7 M urea, 2 M thiourea, 4% (w/v)

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3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate, 40 mM DTT and 2% (v/v)

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pharmalyte. Insoluble residue was removed through centrifugation and the protein content was

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determined according to the Bradford method.18

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800 µg of cotyledon protein was applied onto a 24 cm, pH 4-7 linear gradient immobilized pH

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gradient (IPG) strip (GE Healthcare, Buckinghamshire, UK), and was subjected to electrophoresis

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at 20 °C for a total of 94850 V·h. Similarly, 800 µg of non-cotyledon (hypocotyl and radicle)

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protein was applied onto an 18 cm pH, 4-7 linear gradient IPG strip, and was subjected to

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electrophoresis for a total of 90850 V·h. Subsequently, the strips were equilibrated19 and the second

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electrophoretic dimension was performed by 12% sodium dodecyl sulfate-polyacrylamide gel

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electrophoresis (SDS-PAGE) and was run on the Ettan Six vertical set (GE Healthcare) in

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electrophoretic buffer at 15 °C with a cooling device (GE Healthcare). Finally, the gels were stained

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with Coomassie brilliant blue (CBB) G-250.

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Gel Image Analysis. The stained 2-DE gels were digitalized with a gel scanner (Imagescanner

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III, GE Healthcare), and analyzed with PDQuest™ software package (Version 7.2.0, BIO-RAD,

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California, USA). Spots were detected, matched, and normalized on the basis of total quantity of

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valid spots with the parameter of percent volume according to the software guide. For each protein

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spot, triplicate gels were used for each sample and the mean relative volume was used to designate

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the significant and differentially expressed proteins (changed by more than 1.5 fold or less than 0.66

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fold and statistically significant as calculated by one-way ANOVA, P 0.05) between them compared with NaCl-AG treatment (Figure 1A).

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The MDA content increased significantly (P < 0.05) after 4 days in both organs of the NaCl-stressed

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and NaCl-AG treated sprouts (Figure 1B). Figure 1C shows that the growth and development of

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untreated sprouts were better than those under NaCl stress and NaCl-AG treatments. The growth

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was inhibited and membrane integrity was lost after 4 days in both the NaCl-stressed and the

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NaCl-AG treated sprouts.

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GABA and fPAs Contents in Germinating Soybeans. The GABA and fPAs contents in the

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cotyledons and non-cotyledons (hypocotyl and radicle) of 4-day germinating soybeans under NaCl

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stress and NaCl-AG treatment were analyzed. After germinating for 4 days under NaCl stress,

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GABA content was 1.54- and 1.70-fold of the control in the cotyledons and non-cotyledons,

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respectively (Figure 2A). The GABA content in the non-cotyledons of 4-day germinating soybeans

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treated with NaCl combined AG were not significantly (P > 0.05) different compared with the

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soybeans under NaCl stress, while the GABA content in cotyledons was significantly (P < 0.05)

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lower than in those under NaCl stress (Figure 2A). The fPAs content decreased significantly in both

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organs of the 4-day germinating soybeans under NaCl stress compared with the control (Figure 2B).

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After treated with AG, the DAO activity, as the rate limiting enzyme of PAs catabolism, was

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completely inhibited (Supplementary Figure 1), and the content of fPAs in 4-day germinating 8 ACS Paragon Plus Environment

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sprouts were all significantly (P < 0.05) higher than those of the sprouts under NaCl stress (Figure

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2B).

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Identification of Differentially Expressed Proteins Induced in Non-cotyledons of 4-Day

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Germinating Soybeans under NaCl Stress and NaCl-AG Treatment. Comparative proteomic

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approach was used to determine the changes in protein abundance in non-cotyledons of germinating

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soybeans exposed to NaCl stress and NaCl-AG treatment. The extracted proteins in the

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non-cotyledons of 4-day germinating soybeans were separated using 2-DE and the

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isoelectric focusing (IEF) on 18 cm linear IPG gels stained with CBB (Figure 3A). Three

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independent experiments were performed as biological replicates (Supplementary Figure 2). The

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relative intensities of all the spots were analyzed using the PDQuest software. In total, more than

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950 protein spots in germinating soybean non-cotyledons were reproducibly detected on the 2-DE

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gels. A total of 67 proteins showed a less than 0.66-fold or more than 1.5-fold difference (P < 0.05)

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in relative volume in non-cotyledons under different treatments. These proteins were excised from

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the gels for MALDI-TOF/TOF mass spectrometry. Finally, 63 proteins displayed in Table 1 were

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confidently identified according to the NCBI database (Supplementary Figure 3, and Supplementary

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Table 1).

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In the NaCl-treated group, 62 differentially expressed proteins were discovered compared with

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CK, including 37 (59.7 %) up-regulated and 25 (40.3 %) down-regulated (Figure 4A, 5A). These

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proteins were divided into 10 functional classes according to KEGG and literatures (Table 1, Figure

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5A). Nine identified proteins were annotated as uncharacterized proteins, and six of them were

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searched for their homologs using BLAST (http://www.ncbi.nih.gov/BLAST/). At the amino acid

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level, the six proteins showed more than 65 % positives, indicating that they might have similar

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functions.

All

of

the

energy-related,

synthesis-related 9

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proteins

and

most

of

the


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disease/defense-related non-cotyledon proteins increased significantly under NaCl treatment, but all

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the transporters-related, signal transduction-related, secondary metabolism-related and most

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uncharacterized non-cotyledon proteins decreased under NaCl treatment (Figure 4A, 5A). Then, the

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subcellular localization information of these identified proteins was also analyzed using WOLF

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PSORT prediction, which revealed that 36 (58 %) differentially expressed proteins identified were

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predicted to be cytoplasm proteins. All of the endoplasmic reticulum proteins, vacuole proteins and

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mitochondria proteins showed an increased abundance while half of the cytoplasm proteins and

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chloroplast proteins showed a decreased abundance under NaCl treatment (Figure 5C).

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A total of 51 non-cotyledon proteins showed differential responses in the NaCl-AG treated group

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compared with the group under NaCl stress. Among these identified proteins, 22 were up-regulated

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proteins and 29 were down-regulated proteins (Figure 4A, 5B). For disease/defense-related proteins,

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the number of proteins that increased or decreased was equal. In addition, the majority of

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metabolism-related, energy-related and protein synthesis-related proteins showed an increased

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abundance. However, the majority of the proteins involved in secondary metabolism, protein

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destination/storage and metabolism showed a decreased abundance (Figure 4A, 5B). Most of the

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increased non-cotyledon proteins were predicted to localize in the chloroplast, vacuole and

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endoplasmic reticulum, while the cytoplasm proteins and mitochondria proteins were nearly equally

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increased or decreased (Figure 5D).

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Identification of Differentially Expressed Proteins Induced in Cotyledons of 4-Day

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Germinating Soybeans under NaCl Stress and NaCl-AG Treatment. To analyze the extracted

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proteins in 4-day germinating soybean cotyledons, 24 cm linear IPG gels were used. Approximately

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1150 protein spots were observed in all the three biological replicates of stained gels

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(Supplementary Figure 4). Among them, 72 protein spots which showed a more than 1.5-fold 10 ACS Paragon Plus Environment

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difference (P

Organ-Specific Proteomic Analysis of NaCl-Stressed Germinating

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