The Functional Analysis Of Receptor-like Cytoplasmic Kinase Lrrk1 In Regulation Of Leaf Rolling In Rice

Leaf rolling is an important agronomic trait in rice(Oryza sativa)breeding.Moderate leaf rolling has proved to be useful in improving photosynthetic efficiency and grain yields.Receptor-like cytoplasmic kinases(RLCKs)in plants are the super family of receptor-like kinases(RLKs)and have important roles in development and stress responses.However,little is known about their functions in rice.Here,we report the isolation and characterization of LRRK1(Leaf rolling receptor-like cytoplasmic kinase 1),a RLCK gene involved in the regulation of leaf rolling.In this study,we have done a series of research to investigate LRRK1 how to regulate leaf rolling,the specific research results are as follows:(1)The expressed plasmid pCAMBIA1301GW-LRRK1 was transformed into kitaake,and nineteen LRRK1 transcription-inhibited type positive transgenic lines were successfully screened.Two Ubi::LRRK1-22 and Ubi::LRRK1-27 transgenic lines with high quantity of mRNA level were picked out for futher analysis,The results obviously showed that overexpression of LRRK1 results in a leaf rolling phenotype from seedling stage to mature stage,However,no obvious phenotypic change was observed in leaves between wild-type Dongjing(DJ)and lrrk1mutant.(2)LRRK1 could be a member of RLCKs family,LRRK1 with a gene size of 1104 nucleotides and deduced protein length of 367 amino acids,showed only an intracellular kinase domain(amino acids 69-340)and no transmembrane or extracellular domains.Homologous alignment analysis revealed one closely related gene in rice and two closely related genes in Arabidopsis to LRRK1.OsRLCKl(Os02g09359),AtRLCK1(Atlg52540)and AtRLCK2(At3g15890)share 54.4%,54%,50.7%protein sequence identity with rice LRRK1 respectively.(3)By paraffin section and toluidine blue staining research,Overexpression of LRRK1 in rice showed adaxially rolled leaves due to the decreased the size of bulliform cells at the adaxial cell layers.And also reduce the water content of the bulliform cells,However,loss-of-function mutant of lrrkl did not have significant difference compared with wild type.(4)Under rational close planting conditions,LRRK1-overexpressing plants have higher the panicle numbers but lower the seed setting rate than that in wild-type plants.However,there existed no significant difference in the plant height,grain yields and 1,000-grain weight between wild-type and LRRK1-overexpressing plants under rational close planting conditions.(5)To elucidate the molecular mechanism of leaf rolling via LRRK1 in rice,transcript levels of several genes known to regulate bulliform cell development were assayed by qRT-PCR analysis.The expression levels of in the leaves of the LRRK1-overexpressing plants the several negative regulators were higher than in those of the wild-type plants,whereas the expression levels of the several positive regulators were lower in the leaves of the LRRK1-overexpressing plants than in those of the wild-type plants.In addition,the expression levels of these genes were also determined in the leaves of the lrrk1.No obvious difference in the expression levels of these genes between DJ and lrrk1,These results indicated that the overexpression of LRRK1 affects expression of many bulliform cell-related genes,leading to adaxially rolled leaves.(6)The subcellular localization analysis showed that LRRK1 was predominantly localized in the plasma membrane.GUS staining of LRRK1pro::GUS transgenic plants showed that the LRRK1 is predominantly expressed in 2-d-old seedling,leaf,young root,leaf sheath and stem,suggesting LRRK1 may exercise its function in this tissues and organs.Western blot results indicate that LRRK1 is phosphorylated in vivo.Taken together,LRRK1 may through affects the expression of many bulliform cell-related genes,negative regulate the formation of bulliform cells,leading to LRRK1-ovevexpressing plants adaxially rolled leaves.Moderate leaf rolling contribute to the improvement of photosynthetic efficiency and grain yields by maintaining relatively erect plant architecture and Improving the light transmittance between leaves,rational close planting results showed that,compared with wild-type plants,the panicle number was increased in LRRK1-overexpressing plants,suggesting LRRK1 may through regulate leaf rolling enhanced light transmittance rate of plant population and improved the panicle number.Therefore,these findings suggest that LRRK1 plays an important role in rice leaf morphogenesis,especially in the regulating of leaf rolling,LRRK1 as a regulating leaf rolling candidate genes can be used for breeding rice.