Studies On Ca²⁺,NO Signal Response Of Pear Callus Under NaCl Stress

In the process of world agricultural production,soil salinization has become one of the important environmental factors that limiting its high-quality development.Different species or different varieties of the same species vary in resistance-related reactions facing the halophytic environment,so there are different adaptability among them.Therefore,one of the utmost concerns of solving the salt damage problem in pear production is in-depth study of the salt tolerance mechanism of pear cells.In this study,callus induced from stem segments of Pyrus betulaefolia Bunge.and P.bretschneideri Rehd.（‘Xuehua’）were used as experimental materials to compare the differences of growth and antioxidant enzymes when exposed to salt medium.Then,the effects of calcium ion donor and nitric oxide donor on cell adaptability of pear were studied.The accumulation of ROS,the expression of ROS-related genes,the difference of calcium ion signal and nitric oxide signal response and the source of each signal under short-term salt stress were analyzed.The relationship between calcium ion signal and nitric oxide signal in pear cell response to salt stress was preliminarily discussed.The experimental results are as follows:1.The callus induced by the stem segments were used as the experimental materials to compare the growth of the two in the salt environment.The results showed that NaCl treatment significantly reduced the relative growth of pear callus.Under the same concentration of NaCl treatment,’Xuehua’ pear was more damaged than Pyrus betulaefolia Bunge.In short,the salt resistance of pear callus is the same as that of tree itself.Exogenous CaCl₂ and SNP could alleviate the inhibitory effect of NaCl stress on the growth of pear callus.2.Under NaCl stress,the activities of SOD,POD and APX in protective enzymes in Pyrus betulaefolia Bunge.increased first and then decreased.The activities of CAT decreased at 6 h,but then increased rapidly.The activities of four antioxidant enzymes in ‘Xuehua’ pear showed a downward trend.Exogenous CaCl₂ and SNP treatment could increase significantly SOD,POD and CAT activities of ‘Xuehua’ pear under NaCl stress,but had no significant effect on APX activities.3.Under NaCl stress,the accumulation of ROS in protoplasts of Pyrus betulaefolia Bunge was 1.2 times as much as that of control,and ‘Xuehua’ pear was 1.39 times as much as that of control.The results showed that salt stress could induce not only the relative expression of Pb Rboh F1 in Pyrus betulaefolia Bunge,but also the relative expression of ROS-related genes such as Pb CAT,Pb POD and Pb Mn SOD.In Xuehua pear,only the relative expression of Pb CAT was increased significantly after 30 minutes of treatment.Exogenous calcium and nitric oxide could activate the expression of Pb Rboh F1 and Pb CAT,Pb POD and Pb Mn SOD.Therefore,this study showed that exogenous calcium and nitric oxide could scavenge ROS accumulated in ‘Xuehua’ pear cells induced by salt stress.4.NaCl stress induced an increase in cytoplasmic calcium ion fluorescence intensity in Pyrus betulaefolia Bunge and ‘Xuehua’ pear protoplasts,but the time of cytoplasmic calcium ion signal in Pyrus betulaefolia Bunge was earlier than that in ‘Xuehua’ pear.When treated with 5 m LaCl₃,10 m M EGTA and 10 μM BAPTA/AM for 60 minutes,all inhibitors significantly inhibited the induction of NaCl stress.the effect of extracellular calcium ion inhibitors was significantly higher than that of intracellular calcium ion inhibitors.The relative expression of calcium-related sensor genes in Pyrus betulaefolia Bunge and ‘Xuehua’ pear showed different characteristics at different stages under NaCl stress.In Pyrus betulaefolia Bunge,the up-regulated expressions of Pb CDPKs,Pb CBL10 and Pb CIPKs were observed at 30 minutes of treatment,while in ‘Xuehua’ pear,the relative expressions of Pb Ca Ms,Pb CDPKs,Pb CBL10 and Pb CIPKs were significantly up-regulated at 60 minutes.The results showed that Pyrus betulaefolia Bunge with strong salt tolerance may have stronger calcium ion signal transduction ability and participated in regulating the resistance of pear cells to salt stress.5.NaCl treatment induced the rapid outbreak of endogenous NO in Pyrus betulaefolia Bunge cells,which was 1.2 times as much as that in control cells at 60 min.Compared with control,there was no significant change in endogenous NO in ‘Xuehua’ pear cells treated with NaCl.L-NAME（NOS inhibitor）and sodium tungstate（NR inhibitor）treatment significantly inhibited the endogenous NO outbreak in Pyrus betulaefolia Bunge cells under NaCl stress.Further studies showed that NaCl treatment activated nitrate reductase activity and Pb NR expression in Pyrus betulaefolia Bunge,but had no significant effect on nitrate reductase activity and Pb NR expression in ‘Xuehua’ pear.Therefore,the results showed that the protoplast cytoplasm of Pyrus betulaefolia Bunge could produce NO signal under salt stress,and the synthesis of NO may mainly depend on the synthesis of nitric oxide-like synthase pathway and nitrate reductase pathway.6.L-NAME and sodium tungstate could inhibit the production of cytoplasmic Ca²⁺ signal induced by NaCl stress in Pyrus betulaefolia Bunge,and the inhibition effect of sodium tungstate was stronger than that of L-NAME.Cytoplasmic calcium ion channel inhibitor LaCl₃ and intracellular calcium ion chelator BAPTA/AM also inhibit the production of NO signal induced by NaCl stress in Pyrus betulaefolia Bunge.However,intracellular calcium ion chelator BAPTA/AM could significantly inhibit the increase of nitrate reductase activity in Pyrus betulaefolia Bunge under NaCl stress.