Molecular Mechanism Of Receptor-like Kinase RLK610 In Regulating Tomato Saline-alkali Stress Tolerance

In China,the area of saline-alkali soil continues to expand,which seriously inhibits the crop growth.The stress effects of soil saline-alkalization on plants include the effects of both salt stress and alkali stress.However,most studies have focused mainly on salt stress,and little attention has been given to salt-alkali mixed stress,the molecular mechanism of salt-alkali mixed stress is still unclear.Studying and the response mechanisms of plants under salt-alkali stress,and mining new saline-alkali stress response genes,are contribute to the breeding of saline-alkali tolerant varieties and provide an important theoretical basis for improving the utilization rate of saline-alkali land and the sustainable development of agriculture.Plant receptor-like kinases（RLKs）play an important role in sensing environmental signals.As an environmental signal,saline-alkali stress can be sensed by RLKs which located on the plasma membrane and trigger intracellular physiological responses and regulate the growth and development in plant.Therefore,exploring the molecular mechanism of RLKs involved in regulating the response of plants to saline-alkali stress has important theoretical value for the cultivation of saline-alkali-tolerant crops.In this study,through homozygous identification and saline-alkali screening of RLK mutant in Arabidopsis thaliana,four RLK mutants that were insensitive to saline-alkali stress were obtained.Analysis of the saline-alkali tolerant phenotype of mutant rlk6-10,the mutant plant slrlk610-cr of RLK6-10 tomato homologous gene SlRLK610 and its overexpression plant SlRLK610-OE were created by gene editing technology.The molecular mechanism of the receptor-like kinases RLK6-10 and SlRLK610 regulating plant salt-alkali resistance was preliminarily discussed.The main results are as follows:（1）Screening of saline-alkali tolerant RLK mutants.Our preliminary experiments found that 6 mM NaHCO₃ significantly inhibited Arabidopsis root growth.Preliminary work in the laboratory identified a batch of RLK mutant induced by saline and alkali.Taking saline-alkali inhibition of taproot growth as the standard,RLK mutants with abnormal sensitivity to saline-alkali stress were screened,and four RLK mutants with reduced sensitivity to saline-alkali stress were screened:rlk6-10,rlk11-17,rlk12-20 and rlk18-7.（2）rlk6-10 has decreased sensitivity to saline-alkali stress.Under normal conditions,the growth level of rlk6-10 is consistent with that of Col-0;however,under 100 mM NaHCO₃saline-alkali treatment,the shoot growth level of rlk6-10 is significantly better than Col-0,which verifies that rlk6-10 has a certain resistance to saline-alkali stress.（3）A possible mechanism by which RLK6-10 regulates salinity and alkali resistance.To further explore the molecular mechanism of rlk6-10 saline-alkali tolerance by q RT-PCR analysis.The results showed that the regulation of ion balance,the mechanism of ROS scavenging,the accumulation and signal transduction of ABA,DNA damage repair,and regulation of stress-related transcription factors in plants work together or some of them play a role,so as to increase rlk6-10 salt and alkali resistance（4）Tomato SlRLK610 regulates salinity and alkali resistance.Homologous genes SlRLK610 and SlRLK637 were found in tomato by sequence alignment,we constructed overexpression vector and gene editing（CRISPR/Cas9）vector,and genetic transformation was carried out in M82,and homozygous mutant slrlk610-cr has been obtained.Under normal circumstances,slrlk610-cr were dwarf and their biomass was lower than that of M82,indicating that SlRLK610 also played an important role in tomato growth and development.To verify the function of SlRLK610 in tomato response to saline-alkali stress,we observed the growth level of slrlk610-cr under 100 mM NaHCO₃ saline-alkaline treatment and determined physiological indicators,found that the inhibition of slrlk610-cr under saline-alkali stress was significantly lower than that of M82.Under saline-alkali stress,the accumulation of ROS in slrlk610-cr was significantly lower than that of M82,the content of anthocyanin was significantly increased,and the level of biomass and chlorophyll was not significantly decreased.This indicated that slrlk610-cr has a certain tolerance to saline-alkali stress.（5）Subcellular localization of SlRLK610/SlRLK637.Through transient transformation of tobacco,the subcellular localization of SlRLK610/SlRLK637 was observed,and it was found that SlRLK610/SlRLK637 proteins were localized in the plasma membrane.It indicated that SlRLK610/SlRLK637 may play a role in the process of plasma membrane sensing or transmission of saline-alkali stress signals.（6）SlRLK610 is selected during tomato domestication.The domestication analysis was performed,we found that SlRLK610 was changed during tomato domestication process.The difference in saline-alkali-induced expression level of SlRLK610 in the origin species LA1589and cultivar M82 may be due to the variation in the promoter region of SlRLK610.In this study,we discovered a receptor-like kinase RLK6-10 in Arabidopsis thaliana that can sense salt-base signals and regulate plant growth and development,and verified that SlRLK610 also has the same function in tomato as in Arabidopsis thaliana.It provides a reference for the following research on plant response to saline-alkali stress.