Studies On Pysiological Responses Of Vegetable Soybean Seeds To Salt Stress

Soybean [Glycine max (L.) Merr.] with large seed size harvested at immature green seed stage (R6-R7) are consumed as a vegetable. In recent years, with the increasing of market demand, open field and protected cultivation area of vegetable soybean has increased year by year due to the improvement of people standard of living. But with the constant exacerbation of soil salinization and secondary salinization, vegetable soybean yield and quality have been seriously affected. In this study, we chose two salt-tolerance cultivars and two salt-sensitive cultivars from 15 vegetable soybean cultivars collected from home and abroad by identification of their salt tolerance, and determined the 100 mmol-L-1 as their appropriate concentration of NaCl treatment by experiment. Subsequently, using vermiculite culture, difference in seed filling and physiological and biochemical characteristics of two vegetable soybean cultivars with different salt tolerance, 'Lulingtezao'(salt-tolerance cultivar, one of the two salt-tolerance cultivars) and 'Lixianggaochan 95-1'(salt-sensitive cultivar, one of the two salt-sensitive cultivars), were studied, and protein expression of vegetable soybean seeds were studied too under 100 mmol-L-1 NaCl stress. This study aimed at laying foundation for completing and further studying of vegetable soybean salt-tolerant mechanism, and at providing theoretical basis for breeding salt-tolerant cultivars of vegetable soybean. The main results are as follows:During salt stress period, the pod fresh and dry mass, seed size, seed fresh and dry mass of'Lulingtezao'resistant to salt stress were not influenced significantly, while those of'Lixianggaochan 95-1'susceptible to salt stress decreased remarkably at later stages of salinity (the 12th and 15th days) in comparison with its control. Seeds of'Lulingtezao' maintained higher contents of soluble sugar, starch, free aminophenol, soluble protein and oil in comparison with those of'Lixianggaochan 95-1'. These results further showed higher salt tolerance of'Lulingtezao'.Under salt stress, superoxide dismutase (SOD) and peroxidase (POD) in seeds of 'Lulingtezao'maintained higher activity for prolonged time, while the changes of SOD and POD activity in seeds of 'Lixianggaochan 95-1'were opposite with those of'Lulingtezao'. The catalase (CAT) activity in seeds of'Lulingtezao'was not influenced significantly except for the 12th day of salinity when its activity increased remarkably, however, that of 'Lixianggaochan 95-1'was significantly decreased at the 6th and 15th days of salinization. Salt stress enhanced superoxide anion radicals (O2·-) producing rate and resulted in accumulation of hydrogen peroxide (H2O2) in seeds of the two cultivars, but the O2·-producing rate and H2O2 content in seeds of'Lulingtezao' were lower than those of 'Lixianggaochan 95-1'. The malondialdehyde (MDA) content in seeds of'Lulingtezao' increased significantly at later stages of salinity (the 12th and 15th days), while that of 'Lixianggaochan 95-1'increased remarkable at all salt stress stages. These results suggested that higher activities of antioxidant enzymes played an important role in effectively protecting cell membrane.The ascorbate peroxidase (APX) activity in seeds of'Lulingtezao'was induced significantly during 3～12 d of salinity, and that of 'Lixianggaochan 95-1'increased remarkably during 3-9 d of salt stress and decreased significantly at later stage of salinity (the 12th and 15th days). But the increasing extent of APX activity in seeds of 'Lulingtezao'was higher than that of'Lixianggaochan 95-1'. With the increase of APX activity, the activities of dehydroascorbate reductase (DHAR) and glutathione reductase (GR) in seeds of'Lulingtezao'increased significantly during 3-12 d and 6-12 d of salinity respectively, however, the DHAR activity in seeds of'Lixianggaochan 95-1'increased only at the 3rd day of salt stress and its GR activity decreased significantly during 6-15 d of salinity. Meanwhile, the seeds of'Lulingtezao'maintained higher contents of ascorbic acid (AsA) and reduced glutathione (GSH) and lower contents of dehydroascorbic acid (DHA) and oxidized glutathione (GSSG), and they also maintained higher AsA/DHA and GSH/GSSG ratio, while the changes of AsA, GSH, DHA, GSSG, AsA/DHA and GSH/GSSG ratio in seeds of'Lixianggaochan 95-1'were opposite with those of 'Lulingtezao'. These results indicated that higher and relatively balanced activities of APX, GHAR and GR in seeds of'Lulingtezao'promoted the AsA-GSH cycle to operate quickly and efficiently, so that enhanced the regeneration of AsA and GSH and these resulted in the seeds maintained higher level of antioxidants and stronger redox power (AsA/DHA and GSH/GSSG ratio). This might be one of the important reasons for higher salt tolerance of 'Lulingtezao'. Polyamines (PAs) are low-molecular-weight biologically active compounds, playing important roles in plants response to environmental stresses. Under salt stress, seeds of 'Lulingtezao'maintained higher free spermidine (Spd) and free spermine (Spm) content and free (Spd+Spm)/Put ratio, and maintained lower free putrescine (Put) content and free Put/PAs ratio in comparison with those of'Lixianggaochan 95-1'. Meanwhile, seeds of 'Lulingtezao'accumulated higher conjugated Spd and conjugated Spm content and higher bound Put and bound Spd content when compared with those of'Lixianggaochan 95-1'. Moreover, the arginine decarboxylase (ADC) activity in seeds of'Lulingtezao'increased significantly during salt stress period, while that of'Lixianggaochan 95-1'decrease remarkably at later stage of salinity. Under salt stress, the polyamine oxidase (PAO) activity in seeds of'Lulingtezao'enhanced instantaneously (at the 3rd day), however, that of 'Lixianggaochan 95-1'increased significantly at all stages of salinity. These results suggested that to maintain stronger capabilities for polyamine synthesis, for the conversion of free Put to free Spd and free Spm and of free PAs to conjugated PAs and bound PAs are very important for enhanced salt tolerance of vegetable soybean seeds.After separation of proteins from the seeds at the 5th day (15 d after flowering) of salt stress by two-dimensional gel electrophoresis (2-DE), the difference in protein expression was analyzed between NaCl treatment and the control.327 proteins were detected at salt stressed 2-DE map, of which 28 proteins were found to be differentially expressed. Among these salt-stress-changed proteins,16 proteins were up-regulated whereas another 12 proteins were down-regulated significantly. Out of the 28 proteins,6 proteins which abundances changed more than 2.5 fold were subjected to matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) followed by database searching which allowed the identification of 5 spots. The identified proteins were Glycinin G2 precursor, actin, similar to GLYCININ G2 PRECURSOR, alpha-tubulin-like protein and Kunitz trypsin inhibitor. The functions of identified proteins under salt stress were discussed. These results indicated that the protein metabolism at early seed-filling of vegetable soybean was influenced significantly under salt stress, and actin, alpha-tubulin-like protein and Kunitz trypsin inhibitor might be involved in salt stress responses. The results suggested that vegetable soybean seeds made positive responses, in signal transduction and in inhibiton of protein hydrolysis, etc., to salt stress in order to reduce the harm to them caused by salinity.Under NaCl stress, the expressions ofβ-conglycinin subunits and glycinin acidic polypeptides were affected significantly, too. Thise study showed that the abundances of a, a', A1a and A4 were all down-regulated significantly at the 10th day of salinization. At the 15th day of salinity, the abundances of a,βand A4 increased remarkably, while those of A1a and A3 decreased significantly. At the 20th day of salt treatment, the abundance of A1a was up-regulated significantly, while the abundances of a, p, A2, A3 and A4 were down-regulated remarkably. Though the abundances of either subunits or acidic polypeptides were increased significantly at a certain period, the overall expression levels of globulins in the seeds were decreased significantly during salt stress period. These results indicated that the accumulation of seed globulins of vegetable soybean was inhibited remarkably during salt stress period, and the numbers of both subunits and acidic polypeptides with inhibited expressions were increased when the stress time was prolonged.