Study On The Physiological Change And The Regulation Mechanism Of GABA Accumulation In Germinating Soybean Under NaCl And NaCl-Ca²⁺ Treatment

Soybeans (Glycine max L.) is rich in dietary proteins, carbohydrates, vitamins and minerals. During the germination of soybean, endogenenous enzymes of seeds are produced or activated, which degrade major components such as protein and starch, etc. to small molecule components which are easy absorbed and utilized. At the same time, anti-nutrition factors are degraded and some new functional compounds such as y-aminobutyric acid (GABA) are accumulated. There are two pathways for GABA synthesis in higher plant, one is GABA shunt, and the other is polyamine degradation pathway, where glutamate decarboxylase (GAD, EC 4.1.1.15), diamine oxidase (DAO, EC 1.4.3.6), polyamine oxidase (PAO, EC 1.5.3.3), and aminoaldehyde dehydrogenase (AMADH, EC 1.2.1.19) are the key enzymes in these two ways. Abiotic stress conditions could strongly increase the GABA-related enzymes activity resulting in the accumulation of GABA. Ca2+ has been proved to alleviate numerous environmental stresses and play an important role in GABA accumulation. In the present study, the mechanism of GABA accumulation in germinating soybeans under NaCl stress and NaCl combining Ca2+ treatment were explored. The main results were as follows:1. The effect of NaCl stress on the physiological metabolism and GABA accumulation in germinating soybeans during germination were investigated. Under NaCl stress, the growth of germinating soybeans was suppressed, the sprout length, respiratory rate, dry matter content, and dry weight and fresh weight of the germinating soybean cotyledon and embryo decreased significantly, while the MDA and H2O2 content increased significantly in the cotyledon and embryo. Meanwhile, soluble protein content increased while free amino acids content decreased. Compared with the control, there were no significantly difference in SOD and CAT activities, however, POD and GPX activities increased significantly in embryo under NaCl stress. There were significantly difference in the osmotic adjustment substances content such as soluble sugar and proline. Under NaCl stress, activities of GAD, DAO, PAO, and AMADH increased. The activities of GAD and DAO in embryo were significant higher than that in cotyledon. After germinating for 4 days under NaCl stress, GABA content was 1.54- and 1.70-fold of the control in cotyledon and embryo, respectively. The Glu and fPAs content in both organs were regulated by NaCl stress. In cotyledon and embryo, supplemental aminoguanidine (AG, inhibitor of amine oxidase) led a significant decrease in DAO and AMADH activities and complete inhibite DAO activity but there was no significantly change in GAD activity, the polyamine degradation pathway was inhibited, then the GABA content in the cotyledon and embryo decreased by 31.5% and 33.5%, respectively. The Glu and fPAs content in the cotyledon increased significantly, and the Glu content decreased significantly while fPAs content increased significantly in the embryo. More than 30% of GABA accumulation was supplied by the polyamine degradation pathway under NaCl stress.2. The effect of NaCl combining Ca2+ treatment on the physiological metabolism and GABA accumulation in germinating soybeans were investigated. Under NaCl stress, CaCl2 led a significant increase in SOD, CAT, POD and GPX activities, and the sprout length, respiratory rate, dry matter content, dry weight and fresh weight of the germinating soybean cotyledon and embryo increased significantly, while the MDA and H2O2 contents decreased significantly. Instead, when treated with LaCl3 (inhibitor of Ca2+ channels), the above matter content showed an opposite character. The soluble sugar content decreased significantly in both organs under CaCl2 or LaCl3 treatment, but the proline content decreased significantly in embryo. Meanwhile, when treated with CaCl2, GAD activity was 1.45- and 1.74-fold of the NaCl stress in cotyledon and embryo, respectively. Moreover, DAO, PAO, and AMADH activities in both organs decreased significantly under NaCl plus CaCl2 treatment, the GABA content in cotyledon and embryo of per germinating soybean was 1.12- and 2.14-fold of the NaCl stress, respectively. GAD, DAO, PAO, and AMADH activities decreased significantly under NaCl plus LaCl3 treatment, resulted in the GABA content decreased significantly in both organs. The fPAs content in cotyledon and embryo and Glu content in cotyledon were decreased under Cacl2 or LaCl3 treatment. Simultaneously, when AG was added under NaCl plus CaCl2 treatment, GABA content of 4-d sprout in the cotyledon and embryo was decreased by 17.0% and 11.7% respectively compared with NaCl treatment. In addition, AG added under NaCl combined with LaCl3 treatment resulted in a decrease in GABA content in the cotyledon and embryo by 20.5% and 7.8% respectively compared with NaCl treatment. In the present study, exogenous CaCl2 mitigated the negative effect of NaCl stress, increased bio mass and promoted GABA content of per germinating soybean. The enzymes activities in GABA shunt increased while in polyamine degradation pathway decreased under NaCl plus CaCl2 treatment and thus decreased the contribution ratio of polyamine degradation pathway for GABA formation. The GAD, DAO, and PAO activities decreased result in reduced the GABA content and the effect of Ca2+ was inhibited then the growth of germinating soybeans was suppressed under NaCl plus LaCl3 treatment.3. Designed degenerate primers, outer and inner primers for 3’and 5’Race. Then using RACE technology, soybean AMADH cDNAs were sequenced. The 1679 bp long of soybean AMADH cDNA was obtained which containing a 1515 bp open reading frame (ORF) that encoded 505 amino-acid peptides (GenBank accession number:KC478661). The predicted molecular weight of soybean AMADH was 54.79 kDa and its predicted isoelectric point was 5.16.4. The effect of NaCl stress and NaCl plus Ca2+treatment on the GABA synthesis related enzymes gene expression in germinating soybeans were investigated. Under NaCl stress, GAD, DAO, and AMADH expression levels increased significantly. AG treatment decreased DAO and AMADH expression levels in cotyledon while there were no significantly difference in GAD expression level in both organs under NaCl stress. Meanwhile, NaCl plus CaCl2 treatment decreased DAO and AMADH expression levels in cotyledon and GAD, DAO, and AMADH expression levels in embryo while there was no significantly difference in GAD expression level in cotyledon. NaCl plus LaCl3 treatment inhibited GAD and DAO expression levels in both organs. The expression levels of GAD, DAO, and AMADH had no significantly change compared with NaCl stress under AG treatment. NaCl stress enhanced the GABA synthesis related enzymes activities and their gene expression levels to accumulated GABA content. Additionally, the increasement of GAD activity was caused by the effect of exogenous Ca2+ instead of its gene expression. The decreased contribution ratio of polyamine degradation pathway for GABA formation was resulted from the inhibited GABA synthesis related enzymes activities and their gene expression levels. Moreover, LaCl3 decreasd the accumulation of GABA through inhibited the GAD and DAO activities and their gene expression levels. The gene expression levels of GAB A synthesis related enzymes were not be affected by AG.5. A comparative proteomic approach was employed to explore proteome expression patterns in germinating soybean under NaCl stress. A total of 72 and 63 differentially expressed proteins were successful identified in cotyledon and embryo under NaCl stress and NaCl plus AG treatment, respectively. Compared with the control,62.5% of the differentially expressed proteins in germinating soybean cotyledon represent various kinds of seed storage protein and the remaining proteins involved in metabolism, energy, cell growth/division, proteolysis, transporters and disease/defense. These differentially expressed proteins were localized in cytoplasm, nucleus, chloroplast, mitochondria, and vacuole. The differentially expressed proteins in embryo were divided into 9 functional classes, including metabolism, energy, cell growth/division, protein synthesis, protein destination/storage, transporters, signal transduction, disease/defense and secondary metabolism, and were predicted to be localized in cytoplasm, nucleus, chloroplast, mitochondria, vacuole, endoplasmic reticulum, and cytoplasm skleton. The Glu and PAs metabolic pathway were affected by NaCl stress, compared with the control, glutamine synthetase PR-2 and glutamine synthetase precursor abundant decreased significantly under NaCl stress while methionine synthase and 5-methyltetrahydropteroyltriglutamate homocysteine methyltransferase abundant increased significantly. Moreover, under NaCl stress, the abundance of AMADH higher than the control, which is beneficial to promote GABA formation in germinating soybean.6. Proteome expression patterns in germinating soybean under NaCl combining Ca2+ treatment were investigated. A total of 80 and 71 differentially expressed proteins were successful identified in cotyledon and embryo under NaCl plus CaCl2 or LaCl3 treatment, respectively. These differentially expressed proteins involved in metabolism, energy, cell growth/division, synthesis, protein destination/storage, transporters, signal transduction, disease/defense and secondary metabolism, and were predicted to be localized in cytoplasm, nucleus, chloroplast, mitochondria, vacuole, endoplasmic reticulum, and cytoplasm skleton. Exogenous CaCl2 was found to redistribute storage proteins, regulate protein processing in the endoplasmic reticulum, enrich the signal transduction pathway, facilitate the energy pathway and transportation, promote protein biosynthesis, inhibit proteolysis, strengthen the antioxidant enzyme system, accumulation of the secondary metabolites and regulate other physiological changes and modulate global proteomic responses so increasing the tolerance of germinating soybeans to NaCl stress. Glutamine synthctasc PR-2 and glutamine synthetase precursor which involved in Glu metabolic decreased significantly under NaCl plus CaCl2 treatment, however, glutamine synthetase PR-2 could not observed and glutamine synthetase precursor increased significantly under NaCl plus LaCl3 treatment. Meanwhile, CaCl2 treatment increased the abundance of S-adenosylmethionine synthetase, methionine synthase and AMADH while LaCl3 treatment decreased their abundances.