The Biochemical Mechanism Of Receptor-like Cytoplasmic Kinase LRRK1 In Salt Stress Regulation In Rice

Rice is the most important crop in China.Soil salinization is a major factor that threatens rice yield in recent years.The damage of soil salinization to plants is mainly caused by osmotic stress,ionic stress and accompanying oxidative stress brought by high concentration of salt.The problem of using salinized land can be solved by developing and creating new varieties of rice which tolerant to salt stress.Receptor-like protein kinases(RLKs)in plants are a class of enzyme-coupled receptors located in cell membranes,consisting of extracellular domains,transmembrane domains and intracellular kinase domains,which play an important regulatory role in the response to stress in plants.Receptor-like cytoplasmic kinases(RLCKs)are a class of protein kinase without extracellular domains,which are downstream signal transduction components of RLK,and involved in the regulation of plant growth and development,immune and abiotic stress responses.Previous studies have shown that the RLCK in rice is affected by abiotic stresses such as salt stress,but the molecular mechanism in salt stress response of RLCK is still unclear.In this study,RLCK gene LRRK1 that are involved in rice salt tolerance response,and the signal transduction pathway of LRRK1 involved in salt stress response was analyzed.The results are as follows:(1)LRRK1 negatively regulates salt tolerance in rice.Compared with wild-type(WT),overexpressing LRRK1 plants were more sensitive to salt stress,while their knockout mutant LRRK1 was more tolerant to salt stress during seedling and reproductive growth stages.Under salt stress,the chlorophyll content,malondialdehyde(MDA)content of LRRK1 overexpression lines were significantly lower than those of the wild type,while the chlorophyll content,malondialdehyde(MDA)content of LRRK1 mutant were higher than WT.(2)Leucine repeat RLK(LRR-RLK)protein STRK2 is the upstream signal transduction component of LRRK1.A LRR-RLK protein,STRK2,was identified by immunoprecipitation and mass spectrometry(IP-MS)analysis of the LRRK1 complex.LRRK1 can interact with STRK2 in vitro and in vivo which are proved by yeast biheterozygous,GST pull-down and bimolecular fluorescence complementation(Bi FC)experiments.(3)STRK2 negative regulatory response salt tolerance in rice.STRK2 overexpressed transgenic lines and knockout mutant STRK2 were constructed.It was found that the overexpressed STRK2 lines were sensitive to salt stress,while the mutant STRK2 showed strong salt tolerance,and its survival rate was significantly higher than WT under salt stress.STRK2 and LRRK1 were in the same signal transduction pathway and of salt stress signal perception,which lead to t he highly consistent phenotypes to salt stress of LRRK1 lines and STRK2 lines.(4)STRK2 phosphorylated LRRK1.The vitro phosphorylation analysis showed that both LRRK1 and STRK2 had self-phosphorylation activity,and STRK2 could phosphorylate LRRK1 in vitro.Phosphorylation site analysis by mass spectrometry showed that the Ser20 of LRRK1 was the key target site for STRK2 phosphorylat ed LRRK1.(5)CSN5 is the downstream signal transduction component of LRRK1.By IP-MS analysis of LRRK1 complex,CSN5,a component of COP9 complex,was screened and identified.LRRK1 can interact with CSN5 in vitro and in vivo which are proved by yeast biheterozygous,CO-IP and bimolecular fluorescence complementation(Bi FC)experiment.(6)LRRK1 is involved in the regulation of salt stress response by regulating the dynamic balance of reactive oxygen species in rice.After salt stress treatment,compared with the wild type,the accumula tion of ROS in the LRRK1 transgenic plants was significantly higher than WT,while the knockout mutant LRRK1 was significantly decreased.At the same time,ascorbic acid(ASA),one of the important antioxidants,was significantly increased in mutant LRRK1-1,while it was significantly decreased in overexpression strain LRRK1.(7)LRRK1 may be involved in salt stress response regulation through CSN5 regulating the stability of VTC1,a key enzyme in ASA synthesis.Previous studies have shown that CSN5 negatively regulates salt tolerance in rice and is involved in regulating the degradation process of VTC1 via ubiquitination pathway.VTC1 accumulation of LRRK1 overexpressed lines and mutant LRRK1 after salt stress was detected by polyclonal antibody of VTC1.It was found that the accumulation of LRRK1 overexpressed lines was the least,the wild-type was in the middle,and the accumulation of VTC1 in mutant LRRK1 was the highest.Combined with the above changes in ROS and ASA contents,we speculated that LRRK1 mi ght regulate the stability of VTC1 through CSN5,thus regulating ASA biosynthesis and ROS dynamic balance.In conclusion,these results reveal that there is a signal transduction pathway of STRK2 ?LRRK1?CSN5?VTC1 in rice,which is involved in the regulation of salt stress response by regulating ASA biosynthesis and ROS dynamic balance.Although the signal transduction patterns in this pathway still need to be further investigated,but the resolve of the salt stress signal transduction pathway can further lay the foundation of the molecular mechanism of plant salt stress response,meanwhile,it will provide the gene resources and theoretical guidance of cultivation of salt-tolerant rice varieties.