Physiology Base Of Calcium-regulation Effect On Malus Baccata Borkh. Seedlings And Differential Proteome In Response To Violently Changing Temperature

In cold-climate zones of China, the early-spring air temperature changes in a rapid and violent-fluctuation mode. This kind of extreme temperature is very harmful to the recovery growth of roots, germinating and leafing in plants at early spring, severely impeded the growth of trees at early growing season. Therefore, in this experiment, Malus baccata Borkh. seedlings after treated with distilled water, calcium and calcium inhibitors were used as materials to study the effects of rapid changes in temperature on respiratory metabolism, nitrogen metabolism, antioxidant enzyme, mitochondrial function and calcium signaling transduction factors in Malus baccata Borkh. roots. Plants were exposed to rapid changes in temperature (5℃â†'20℃â†'10℃â†'0℃) that mimiced changing air temperature in early spring of Shenyang city. Meanwhile, we used iTRAQ labeling technique to study the expression of differential protein in Malus baccata Borkh. roots and leaves under rapid changes in temperature. The main results are as follows:1. Effects of calcium on carbon and nitrogen metabolism in Malus baccata Borkh. roots: During the temperature changes, root vitality significantly decreased.2% CaCl2 and CaAc2 treatment significantly increased root vitality, while 5% CaCl2 treatment had the opposite effects. Root vitalities were depressed by calcium inhibitors. Among them, the most obviously effect was found in TFP treatment.During the temperature changes, total respiration rate decreased first and then increased. Different concentration and types of calcium treatment increased the total respiration rate at the heating stage, but had different effects on total respiration rate at cooling phase. In the cooling stage, only 2% CaCl2 treatment effectively enhance the total respiration rate by increasing the activities of TCA and PPP. On the contrary,5% CaCl2 treatment significantly reduced the total respiration rate at cooling phase. Compared with the control, different calcium inhibitors had different effects on total respiration rate at different temperatures, but showed obviously inhibiting effects at 0℃, and the most obviously effect was found in LaCl3 and TFP treatment.During the temperature changes, the activities of key enzymes in nitrogen metabolism showed a decreasing tendency during the temperature stress.2% CaCl2 treatment enhanced the activities of key enzymes in nitrogen metabolism to some extent. In contrast,5% CaCl2 treatment displayed the opposite trend. CaAc2 treatment significantly increased the acitivity of NR at 20℃ and 10℃ as well as the activity of GDH at 20℃ and 0℃. Calcium inhibitors reversed the effects of 2% CaCl2 treatment. Among them, the most obviously inhibitory effect was found in EGTA and TFP treatment.2. Effects of calcium on mitochondrial function in Malus baccata Borkh. roots:Rapid changes in temperature led to a rapid increase in mitochondrial membrane perameability (MPT) and superoxide anion (O2·-) generation rate. In contrast,1%CaCl2,2%CaCl2 and CaAc2 treatment reduced MPT and O2·- production rate to some extent, but only 2% CaCl2 treatment significantly increased cytochrome c/a as compared with that of control during the whole temperature treatment.5% CaCl2 treatment had the opposite effects. Calcium inhibitors further reduced △Ψm and cytochrome c/a, which enhaced the oxidative stress induced by temperature stress via increasing the production rate of O2·-, except for SO treatment.3. Effects of calcium on calcium signaling factors in Malus baccata Borkh. roots:CaM increased gradually during the temperature stress, whereas CaN content decreased first and then increased.1% CaCl2 treatment increased the content of CaM during the whole temperature stress, but had no effect on CaN content.2% CaCl2 treatment increased CaM content at 10℃ and 0℃. CaAc2 treatment improved the content of CaM and CaN at 20℃ and 10℃, but the opposite effect was found at 0℃.5% CaCl2 treatment reduced the content of CaM and CaN. To some extent, calcium inhibitors reduced the content of CaM and CaN.4. Effects of calcium on antioxidant enzyme activities and membrane lipid peroxidation in Malus baccata Borkh. roots:The activities of SOD, POD, CAT, and APX significantly decreased during the temperature changes.1% CaCl2,2% CaCl2 and CaAc2 treatment increased the activities of SOD, POD, CAT and APX to some extent. In contrast,5% CaCl2 treatment inhibited the activities of SOD and POD. The inhibitory effect of temperature stress on antioxidnats were exacerbated by calcium inhibitors, especially in TFP treatement.Rapid changes in temperature induced the accumulation of H2O2 and MDA.1% CaCl2,2% CaCl2 and CaAc2 treatment signigicanlty reduced the content of H2O2 and MDA. However,5% CaCl2 treatment had the opposite effect. In addition, calcium inhibitors exacerbated the peroxidation of membrane lipid.5. Effects of calcium on the function of Malus baccata Borkh. leaves:Rapid changes in temperature resulted in the decrease of membrane integrity, PS II maximum photochemical efficiency ((pp0), performance index on absorption basis (PIABX), density of PS II reaction centers per excited cross-section (RCQA) and quantum yield for electron transport (cpEo), caused the damage to oxygen evolving complex of PS â…¡.2% CaCl2 treatment significantly improved membrane integrity, φpo and PIABS, alleviated the damage to oxygen evolving complex of PS â…¡.6. Effects of calcium on the expression of proteins in Malus baccata Borkh. roots and leaves:Compared with the proteins in plants harvested at 5℃,339 proteins were significantly changed after the entire experiment.206 of these proteins were up-regulated and 133 of these proteins wer down-regulated. Comparing with control plants harvested at 20℃,72 proteins were significantly changed in calcium-treated plants harvested at 20℃ (51 up-regulated and 21 down-regulated). Comparing with control plants harvested at 0℃,997 proteins were significantly changed in calcium-treated plants harvested at 0℃ (692 up-regulated and 305 down-regulated). The majority of these differential proteins were related to 3 biological processes involved in cellular process, metabolism process and response to stimulus. The proteins related to heat shock protein and dehydrins were up-regulated during the whole temperature stress. Moreover, compared with control plants, calcium treatment (2% CaCl2) has helped to promote the expression of heat shock protein and dehydrins.Compared with the proteins in plants harvested at 5℃,34 proteins were significantly changed after the entire experiment.10 of these proteins were up-regulated and 24 of these proteins wer down-regulated. Comparing with control plants harvested at 20℃,9 proteins were significantly changed in calcium-treated plants harvested at 20℃ (3 up-regulated and 6 down-regulated). Comparing with control plants harvested at 0℃,15 proteins were significantly changed in calcium-treated plants harvested at 0℃ (8 up-regulated and 7 down-regulated). The majority of these differential proteins were related to 4 biological processes involved in cellular process, metabolism process, single-organism process and response to stimulus. The proteins related to photosynthesis, such as CPO, CP43 and D1, were down-regulated during the whole temperature stress.