The Mechanism Research Of Ethylene Was Involved In Ca²⁺-Induced Adventitious Rooting Of Cucumber Explants Under Salt Stress

In the process of plant growth and development,salt stress,as a severe harmful abiotic stress,has a very serious impact on the process of plant growth and development.Especially in the production process of agricultural products,salt stress will lead to the reduction of the yield and quality of agricultural products,and serious salt stress will even cause the death of crops,resulting in the loss of agricultural products.Calcium and ethylene are important signal molecules in plant signal transduction.Several studies have shown that calcium and ethylene are involved in plant physiology,biochemistry,growth and development.Moreover,calcium and ethylene play a critical role in the process of plants resisting biotic and abiotic stresses.However,it is not clear whether the interaction between calcium and ethylene will affect the adventitious root development of cucumber explants under salt stress.Therefore,based on the background of adventitious root development of cucumber explants under salt stress,the mechanism of calcium-ethylene interaction alleviating the inhibition of adventitious roots induced by salt stress was studied through the determination of endogenous ethylene synthesis and signal transduction,sodium ion transport and other related indexes of cucumber explants,in order to provide a theoretical basis for calcium and ethylene interaction to alleviate plant salt stress.The main results are as follows:1.The effects of calcium and ethylene on adventitious rooting of cucumber explants under salt stress were studied.The results showed that low concentration of NaCl could promote the adventitious root formation of cucumber explants,but with the increase of NaCl concentration,the adventitious rooting of cucumber explants was significantly inhibited.13 mM NaCl was selected as the moderate stress concentration to simulate salt stress,and the effects of calcium and ethylene on adventitious rooting under salt stress were further studied.The results showed that 10?M calcium chloride?CaCl₂?or 0.1?M ethephon?ethylene donor?treatment had the greatest biological effects on promoting adventitious rooting under salt stress.In addition,the removal of endogenous ethylene significantly reduced the effect of exogenous calcium on adventitious rooting under salt stress,indicating that ethylene was involved in exogenous calcium-induced the process of adventitious root formation under salt stress.2.The effect of exogenous calcium on endogenous ethylene synthesis during adventitious root formation of cucumber explants under salt stress was studied.The results showed that exogenous CaCl₂ treatment could significantly increase the activities of ACC synthase?ACS?and ACC oxidase?ACO?in cucumber explants,and promote the biosynthesis of 1-aminocyclopropane-1-carboxylic acid?ACC?,thus increasing ethylene biosynthesis in cucumber explants under salt stress.Moreover,Ca²⁺significantly up-regulated the gene expression of CsACS3,CsACO1,CsACO2,CsEIN2 and CsEIN3 under salt stress,and negatively regulated the gene expression of CsETR1,CsETR2,CsERS and CsCTR1.However,the use of Ca²⁺chelator?EGTA?and calcium channel inhibitor?LaCl₃?could significantly reverse the regulatory effect of Ca²⁺on the expression of these genes.These results suggest that Ca²⁺may affect the adventitious root development of cucumber explants through regulating endogenous ethylene biosynthesis and signal transduction under salt stress.3.The protein interactions during the adventitious rooting of cucumber explants under salt stress were studied.During this process,171 proteins were identified to interact with EIN3 in the ethylene signaling pathway.Among these proteins,glutathione reductase?GR?and methionine synthase?MY?were selected to further study the effect of exogenous calcium on the interaction of EIN3 with GR and MY.The results showed that during the development of adventitious roots in cucumber explants,salt stress reduced the interaction between EIN3 and GR and MY.However,application of exogenous calcium could increase the interaction between EIN3 and GR and MY under salt stress.4.The role of ethylene in exogenous calcium-regulated Na⁺transport during the adventitious roots of cucumber explants under salt stress was studied.The results showed that during adventitious rooting under salt stress,CaCl₂ treatment can significantly reduce the Na⁺content of cucumber explants while increase the K⁺content.However,ethylene inhibitor treatment significantly reversed the effect of Ca²⁺on endogenous Na⁺and K⁺content.These results indicated that during the adventitious root formation of cucumber explants under salt stress,ethylene,as a downstream signaling molecule of Ca²⁺,may participate in the process of regulating the balance of Na⁺and K⁺.Furthermore,ethylene participates in Ca²⁺-regulated Na⁺transporter including plasma membrane Na⁺/H⁺transporters SOS1,vacuolar membrane Na⁺/H⁺reverse transport protein NHX1,plasma membrane proton pump HA3 and vacuolar membrane H⁺-ATPase protein VHA-A,thus affecting the Na⁺transport in cucumber explants.In addition,ethylene also plays a positive role in Ca²⁺-affected cell ultrastructural stability of cucumber explants under salt stress.To sum up,ethylene may be as a downstream signaling molecule for Ca²⁺to maintain a lower Na⁺concentration and a higher K⁺level in cucumber explants by regulating Na⁺transport.Moreover,ethylene was involved in Ca²⁺-affected structural integrity of cucumber explant cells to improve salt tolerance of cucumber explants for regulating the adventitious rooting of cucumber explants under salt stress.