Yongqi Yin

Comparative proteomic and physiological analyses reveal the protective effect of exogenous calcium on the germinating soybean response to salt stress.

Calcium enhances salt stress tolerance of soybeans. Nevertheless, the molecular mechanism of calciums involvement in resistance to salt stress is unclear. A comparative proteomic approach was used to investigate protein profiles in germinating soybeans under NaCl-CaCl2 and NaCl-LaCl3 treatments. A total of 80 proteins affected by calcium in 4-day-old germinating soybean cotyledons and 71 in embryos were confidently identified. The clustering analysis showed proteins were subdivided into 5 and 6 clusters in cotyledon and embryo, respectively. Among them, proteins involved in signal transduction and energy pathways, in transportation, and in protein biosynthesis were largely enriched while those involved in proteolysis were decreased. Abundance of nucleoside diphosphate kinase and three antioxidant enzymes were visibly increased by calcium. Accumulation of gamma-aminobutyric acid and polyamines was also detected after application of exogenous calcium. This was consistent with proteomic results, which showed that proteins involved in the glutamate and methionine metabolism were mediated by calcium. Calcium could increase the salt stress tolerance of germinating soybeans via enriching signal transduction, energy pathway and transportation, promoting protein biosynthesis, inhibiting proteolysis, redistributing storage proteins, regulating protein processing in endoplasmic reticulum, enriching antioxidant enzymes and activating their activities, accumulating secondary metabolites and osmolytes, and other adaptive responses. Biological significance Soybean (Glycine max L.), as a traditional edible legume, is being targeted for designing functional foods. During soybean germination under stressful conditions especially salt stress, newly discovered functional components such as gamma-aminobutyric acid (GABA) are rapidly accumulated. However, soybean plants are relatively salt-sensitive and the growth, development and biomass of germinating soybeans are significantly suppressed under salt stress condition. According to previous studies, exogenous calcium counters the harmful effect of salt stress and increases the biomass and GABA content of germinating soybeans. Nevertheless, the precise molecular mechanism underlying the role of calcium in resistance to salt stress is still unknown. This paper is the first study employing comparative proteomic and physiological analyses to reveal the protective effect of exogenous calcium in the germinating soybean response to salt stress. Our study links the biological events with proteomic information and provides detailed peptide information on all identified proteins. The functions of those significantly changed proteins are also analyzed. The physiological and comparative proteomic analyses revealed the putative molecular mechanism of exogenous calcium treatment induced salt stress responses. The findings from this paper are beneficial to high GABA-rich germinating soybean biomass. Additionally, these findings also might be applicable to the genetic engineering of soybean plants to improve stress tolerance.

Purification, properties and cDNA cloning of glutamate decarboxylase in germinated faba bean (Vicia faba L.).

Gamma-aminobutyric acid (GABA) is a non-protein amino acid with bioactive functions in humans. In this work, glutamate decarboxylase (EC 4.1.1.15, GAD) which is key in the GABA bioformation was purified from 5-day germinated faba beans and characterized. A single band was observed at 58 kDa using sodium dodecyl sulphate gel electrophoresis. GAD optimal activity was at pH 6.0 at 40°C with a K(m) value for glutamic acid (Glu) of 2.63 mM. The enzyme was inhibited significantly by Cu(2+), Fe(3+), Mg(2+), Ba(2+), aminoxyacetate, EGTA, Na(2)EDTA, l-cysteine and beta-mercaptoethanol; and activated at low Ca(2+) 0.2mM. Using RT-PCR, the GAD cDNA was sequenced which indicated 1787 bp long, containing a 1527 bp open reading frame (ORF) that encoded 509 amino-acid peptides with a calculated molecular weight of 57.74 kDa and a pI of 5.41 (GenBank accession number: JX444699).

Organ-Specific Proteomic Analysis of NaCl-Stressed Germinating Soybeans

A comparative proteomic approach was employed to explore proteome expression patterns in germinating soybeans under NaCl stress and NaCl-aminoguanidine treatment. The proteins were extracted from 4-day-old germinating soybean cotyledons and noncotyledons (hypocotyl and radicle) and were separated using two-dimensional polyacrylamide gel electrophoresis. A total of 63 and 72 differentially expressed proteins were confidently identified by MALDI-TOF/TOF in the noncotyledons and cotyledons, respectively. These identified proteins were divided into ten functional groups and most of them were predicted to be cytoplasmic proteins in noncotyledons. Moreover, γ-aminobutyric acid was accumulated while the major allergen (Bd 30K protein) was reduced in the germinating soybeans. The proteins involved in energy metabolism and in protein processing in endoplasmic reticulum were enriched under NaCl stress. Meanwhile, the negative effect of stress was aggravated once polyamine degradation was inhibited. Redistribution of storage proteins under stress indicated that storage proteins might not only function as seed storage reserves but also have additional roles in plant defense.

Sequence analysis of diamine oxidase gene from fava bean and its expression related to γ-aminobutyric acid accumulation in seeds germinating under hypoxia-NaCl stress.

BACKGROUND γ-Aminobutyric acid (GABA) is synthesized via the polyamine degradation pathway in plants, with diamine oxidase (DAO) being the key enzyme. In this study the cDNA of DAO in fava bean was cloned and its expression in seeds germinating under hypoxia-NaCl stress was investigated. RESULTS Fava bean DAO cDNA is 2199 bp long and contains 2025 bp of open reading frame that encodes 675 amino acid peptides with a calculated molecular weight of 76.31 kDa and a pI of 5.41. Hypoxia and hypoxia-NaCl stress enhanced DAO activity and resulted in GABA accumulation in germinating fava bean. However, DAO gene expression was down-regulated under hypoxia compared with non-stress condition, while its expression in the cotyledon and shoot was up-regulated under hypoxia-NaCl. In addition, DAO expression could be promoted to enhance GABA accumulation after increasing the stress intensity using NaCl. DAO gene expression was significantly inhibited by aminoguanidine treatment under hypoxia but increased under hypoxia-NaCl. CONCLUSION Under hypoxia, GABA accumulation due to NaCl was mainly concentrated in the cotyledon. The GABA content increase under hypoxia did not result from DAO gene expression, but DAO existing in seeds was activated under hypoxia. DAO gene expression was up-regulated to enhance GABA accumulation after increasing the stress intensity.

Hypoxia treatment on germinating faba bean (Vicia faba L.) seeds enhances GABA-related protection against salt stress

The γ-aminobutyric acid (GABA) is a non-protein amino acid with some functional properties for human health. Its content is usually lower in plant seeds. Hypoxia or salt (NaCl) stress is an effective way for accumulating GABA during seed germination. However, NaCl stress on GABA accumulation under hypoxia is currently infrequent. The effect of NaCl on GABA accumulation in germinating faba bean (Vicia faba L.) under hypoxia was therefore investigated in this study. Faba bean seeds were steeped in citric acid buffer (pH 3.5) containing NaCl with a final O2 concentration of 5.5 mg L-1 and germinated for 5 d. Results showed that 60 mmol L-1 NaCl was the optimum concentration for GABA accumulation in germinating faba beans under hypoxia. Germination for 5 d under hypoxia-NaCl stress was less beneficial for GABA accumulation than only hypoxia (control). Polyamine degradation pathway played a more important role for accumulating GABA in germinating faba bean as an adaptive response to NaCl stress. Removing NaCl significantly increased GABA content, while it decreased glutamate decarboxylase (GAD) activity. Simultaneously, polyamine was accumulated, which might be related to the enhancement of physiological activity after recovery. When treated with aminoguanidine (AG) for 3 d, GABA content decreased by 29.82%. These results indicated that the tolerance ability of GABA shunt to NaCl stress was weaker than that of polyamine degradation pathway. The NaCl treatment for 3 d under hypoxia could raise the contribution ratio of polyamine degradation pathway for GABA accumulation. The contribution ratio of polyamine degradation pathway for GABA formation was 29.82% when treated for at least 3 d