Molecular Biochemical Analysis And Functional Studies Of FLR Gene Family In Rice

As one of the most important food crops,rice(Oryza Sativa L.)is the staple food for more than half of the population in China.With the increasing population in China,to improve the capacity of rice is an important foun dation for national food security.Therefore,the high yield,high quality and high resistance of rice have become the major goal of the current rice breeding.Rice growth and development are affected by a variety of external signals,and Receptor-like Kinases(RLKs)play an important role in the response process of external signals.RLKs recognizes external signaling molecules through the extracellular receptor domain,and triggers the intracellular kinase domain to recruit and/or phosphorylate specific substrates,so as to realize the signal transmission from the extracellular to the intracellular.FERONIA belongs to the CrRLK1 L subfamily,which is involved in almost all aspects of Arabidopsis growth and survival.It has been reported that the FERONIA-like receptor 1(FLR1)and FLR2,homologs of Arabidopsis FERONIA,can regulate plant height,tillering,fertility and yield with redundant functions in rice.FLR9(also name RUPO)is involved in regulating the growth of pollen tube and intracellular potassium balance in rice.However,the mechanisms that RLKs regulate rice yield,rice quality and resistance remain unclear.This study mainly focused on the functions of FLR gene family in rice yield,rice quality and resistance and their related mechanisms.The main results are as follows:(1)Using bioinformatics method,16 CrRLK1 L gene family members(named FLR1-16)were idenditified in japonica rice.Most FLR genes do not have intron and the FLR gene family is expanded mainly through tandem replication and frag ment replication.Four members of FLR gene family(FLR3,8,11,and 14)are located at the cell membrane,which is also consistent with the typical feature of RLKs.FLR gene family is expressed in various tissues at distinct stages in rice and some members may respond to stress.(2)Our previous studies have shown that FLR1 and FLR2 have functional redundancy in regulating plant height and fertility in rice,flr1 and flr2 showed plant dwarfing and poor fertility.In the present study,we obtained single mut ants of 12 FLR genes by CRISPR-Cas9 assay.The agronomic traits of transgenic plants were compared and flr15 showed semi-dwarfing phenotype and severe sterility.Therefore,FLR1,FLR2 and FLR15 are all regulating plant height and fertility of rice.(3)FLR1,FLR2,FLR8 and FLR15 participated in the regulation of grain size:FLR1,FLR2,and FLR8 negatively regulated grain width,grain length,and grain size,respectively,whereas FLR15 positively regulated grain size.Moreover,FLR1 can negatively regulate grain filling rate and modulate spikelet hull development by inhibiting cell elongation and division.(4)FLR1,FLR3 and FLR14 positively regulated rice grain quality.Percentage of grains with chalkiness of all three gene mutants is significantly increased,and flr3 and flr14 mutants showed obvious white-core floury endosperm.In flr1 mutant,the content of total starch and amylose increased while protein content decreased.Its endosperm was packed with small,loose,and irregular polyhedral starch grains.We found that FLR1 affects grain quality through regulating the expression of starch biosynthesis-related genes in the developing endosperm.(5)Four members of FLR gene family responded to the stress of rice blast disease.Among which,FLR1 and FLR13 positively regulated rice immunity,while FLR2 and FLR11 negatively did so.flr2 mutant produced more ROS during the infection of M.oryzae,and the transcripts of key resistance genes in jasmonic acid and salicylic acid pathways was elevated.flr2 mutant not only resist different physiological species of M.oryzae,but also could had better disease resistance in the blast nursery.(6)FLR1-OsRacGEF1-OsRac1 signaling pathways regulated rice growth and immunity.FLR1 can interact with and phosphorylated OsRacGEF 1.The phosphorylated OsRacGEF1 activated OsRac1.The activated OsRac1 and FLR1 formed a complex on the plasma membrane,regulating the production of ROS and the phosphorylation of downstream OsMAPK6,and ultimately affecting the growth,development and immunity of rice.