Characterization Of FERONIA Receptor-like Kinase MdMRLK2 In Response To Drought,Cold And Valsa Mali Infection In Malus

China is the largest apple cultivation country in the world.However,drought and cold stress seriously restrict the development of apple industry,and Valsa canker caused by Valsa mali also poses a serious threat to apple production.MRLK(malectin like domain containing receptor like kinases)family is a larger number of receptor like kinases containing malectin domain.FERONIA(FER),as the most well-studied member of this family,plays an important role in plant response to abiotic and biotic stresses.Recently,the research on the function of FER mainly focuses on Arabidopsis,and its function in apple is unclear.Therefore,we used ’gala-3’ and Md MRLK2 overexpression apple plants as experimental materials to explore the function of Md MRLK2 under drought,cold and Valsa canker inoculation conditions.The main results are as follows:Firstly,we found Md MRLK2 responded to drought,cold,ABA and chitosan(simulated inoculation)treatments.Through identification of MRLKs in apple,it was found that there were 10 Md MRLKs,phylogenetic tree analysis showed that Md MRLK2 was close to FER of Arabidopsis among this menbers.Md MRLK2 was up-regulated under drought,cold and Valsa canker inoculation conditions,and it was highly expressed in apple flowers and leaf buds.In addition,Md MRLK2 was localized to the plasma membrane and that kinase domain was highly conserved.Secondly,we found Md MRLK2 improved apple drought tolerance by regulating free amino acids and energy metabolism.Overexpression of Md MRLK2 enhanced the activity of antioxidant enzymes,reduced the accumulation of ROS,and alleviated the photosystem damage,therefore improving the photosynthetic capacity of apple plants under natural drought stress.Analizing of ABA content and stomatal characteristics showed that the significant increase of ABA level led to the decrease in stomatal aperture and the reduction of plant transpiration rate.The results showed that the interaction between Md MRLK2 and Md GAPB by Y2 H and Bi FC,increasing Md GAPB protein level and GAPDH enzyme activity,so as to maintain a high energy level of transgenic plants.The interaction between Md MRLK2 and Md CYS4 promoted the accumulation of a large number of free amino acids and eventually enhanced the drought resistance of apple.Thirdly,we found Md MRLK2 enhances apple adaptability to long-term moderate drought stress by promoting the development of roots.Overexpression of Md MRLK2 had maintained high photosynthetic capacity and improved the accumulation of plant biomass of apple plants under long-term moderate drought stress.We confirmed the interaction between Md MRLK2 and Md CCR1,a key lignin synthase,by Y2 H,Bi FC,Spit-Luc and Co-IP,which probably promoted the lignin accumulation and vessel density in transgenic apple plants under long-term and moderate drought stress,resulting in higher root activity,water potential and water use efficiency.In order to further verify the effect of Md CCR1 on plant root growth,Md MRLK2 RNAi and Md CCR1 RNAi transgenic roots were obtained by Agrobacterium rhizogenes.Their roots was inhabited under drought alone,but exogenous lignin treatment could partially recover the growth of Md MRLK2 RNAi and Md CCR1 RNAi roots under long-term moderate drought stress.These results showed that overexpression of Md MRLK2 could promote plant photosynthesis,improve water use efficiency,lignin content and promote biomass accumulation,increasing apple adaptability to long-term moderate drought stress.Fourthly,we found Md MRLK2 enhances apple cold tolerance by maintaining cell wall components,inducing osmotic adjustments and activating anthocyanin biosynthesis.We found that overexpression of Md MRLK2 had reduced polygalacturonase,pectinate lyase pectinesterase,and cellulase activities and maintained the contents of water insoluble pectin(WIP),lignin,cellulose,and hemicellulose in cell walls under cold conditions.Md MRLK2 overexpression also promoted soluble sugar and free amino acid accumulation and reduced photosystem damage upon cold stress.The anthocyanin content accumulated in transgenic plants is also significantly higher than that in WT plants,which may be caused by the interaction between Md MRLK2 and MYB transcription factor Md MYBPA1.We do further analysis and found that Md MRLK2 promote the combination of Md MYBPA1 with the promoter of anthocyanin key synthase pro Md ANS and pro Md UFGT upon cold stress,thus increasing the anthocyanin content and improving the cold tolerance of apple.At last,we found Md MRLK2 negatively regulates Valsa canker resistance by suppressing defense responses and hypersensitive reaction(HR).After inoculation with V.mali,we found that Md MRLK2 overexpression apple plants had enhanced ABA and reduced SA levels,Md MRLK2 overexpression also suppressed polyphenol accumulation and inhibited the activities of phenylalanine ammonia-lyase(PAL),β-1,3-glucanase(GLU),and chitinase(CHT)during V.mali infection.Moreover,Md MRLK2 interacted with Md HIR1,a hypersensitive-induced response protein,and blocking Md HIR1-mediated HR by disrupting Md HIR1 self-interaction.