| Salinity is an serious problem that threaten agricultural production in in the world. Due to the rapid development of protected cultivation, the secondary salinization of the protected horticulture is becoming increasingly severe in present years, which seriously affects plant growth and development and may lead to a drastic reduction in economic yield. It is important to take a variety of effective means to improve plant salt tolerance. Researches on calcium improving plant tolerance to stress have been made, particularly in salt tolerance. Related researches have been done within physiology, growth, metabolism, gene expression to illustrate the how calcium improve plant salt tolerance, but the research through proteomics to reveal the mechanism is very limited.A hydroponic experiment was conducted to study the effects of exogenous calcium on proteome of salt sensitive cucumber (Cucumis sativus L.) cultivar’Jinchun2’under salt stress. According to the mass spectrometric identification results, combined with related experiments such as mRNA transcription level, photosynthesis gas exchange parameters, chlorophyll fluorescence parameters and carbohydrate metabolism, we discussed the mechanism of calcium on alleviating salt stress of cucumber seedlings. The main results are as follows:Different concentrations of Ca2+were spayed on leaves of cucumber to study the effect of calcium on alleviate salt stress. The fresh and dry biomass of seedlings, plant height and stem thickness decrease significantly under75mmol·L-1NaCl treatment. Different concentrations of Ca2+spayed on leaves have a effect on cucumber seedlings growth under salt stress, but with a concentration effect,4mmol·L-1Ca2+plays a significant role in promoting cucumber seedlings growth and elevated the biomass75.4% compare to NaCl treatment.The soluble protein content of cucumber leaf significantly decreased by salt stress, and it also decreased at third day after exerting Ca2+under salt stress, than it increased at the sixth and the ninth day after exerting Ca+under salt stress. The soluble protein content of cucumber root was increased first and than decreased under salt stress, and it was decreased first and than increased after exerting Ca2+. The results of SDS-PAGE analysis showed that12proteins with relative molecular weight (×103) of87.5,77.9,75.7,74.1,65.1,54.3,50.5,44.7,40.7,31.4,26.0and21.5changed obviously in cucumber leaves, and7proteins with relative molecular weight (×0103) of81.1,76.6,46.9,45.2,39.5,23.8and16.8also changed in roots under salt stress.Proteomic analysis showed that50spots to response to salt stress and/or calcium. These proteins were involved in photosynthesis and energy metabolism, amino acid and lipid metabolism, stress defense, protein and nucleic acid biosynthesis, hormone synthesis, signal transduction and other function, NaCl and/or Ca2+effect on photosynthesis and energy metabolism most obviously. Exogenous CaCl2treatment enhanced Calvin cycle, regulation of glycolysis and pentose phosphate pathway key enzymes, as well as effect amino acids and lipid metabolism, improve stress defense-related proteins, to promote hormone synthesis, and signal transduction. Exogenous calcium show a positive effect on protein level.We picked10gene which encoding key enzyme of photosynthesis and chlorophyll synthesis, such as Rubisco large subunit (rbcL), Rubisco small subunit (rbcS), rubisco activase (RCA), Oxygen-evolving enhancer protein (psbO) and Porphobilinogen deaminase (PBGD). Take research of salt and calcium response at the level of gene transcription to study the relationship between protein express and transcriptional level. The result showed that the transcript accumulation patterns of PRK, Ald,petC, PBGD and Gsa were consistent with protein accumulation patterns. Those gene which closely related to Calvin cycle, the photosynthetic electron transport and chlorophyll biosynthesis such as RbcL, PRK, petC, petH, PBGD, Gsa was down regulated under salt stress, while RbcS, psbO, Ald induced by salinity show the opposite trend. Compare to salt stress, RCA, PRK, petH, Gsa significantly increased after application of exogenous CaCl2. it indicates that calcium play an important role in regulate the key enzyme of photosynthesis and chlorophyll metabolism of cucumber plants under salt stress.75mmol·L-1NaCl inhibited the synthesis of photosynthetic pigments in leaves of cucumber seedlings, damaged the structure of PS Ⅱ and the net photosynthesis was reduced by46.9%; Furthermore, salt stress induced an increase in contents of sucrose, starch and total soluble sugar and the activities of phosphate sucrose synthase(SPS) and sucrose synthase(SS) in cucumber leaves. However, the amylase activity in leaves of cucumber was reduced by salt stress. Compared with cucumber seedling treated with salt stress alone, exogenous calcium application make the net photosynthesis of cucumber seedlings increased by18.5%, and significantly alleviated the decreased photosynthetic pigments contents, photosynthesis activities and the increased carbohydrates contents as well as SS and SPS activities in salt-stressed cucumber seedlings. In contrast, exogenous calcium increased amylase activity in cucumber leaves under salt stress. These results suggested that exogenous calcium could alleviated the damage on photosynthesis induced by salt stress and enhanced salt tolerance of cucumber seedlings by increasing photosynthetic pigments, regulating the plant stomatal opening and key enzymes activities related to carbohydrate accumulation, maintaining the PS Ⅱ activity and reducing the negative feedback inhibition to photosynthesis caused by carbohydrate accumulation in leaves under stress condition. |
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