Functional Analysis Of OsSCL7,a GRAS Family Protein Conferring Growth And Regulation Of Stress Response In Rice

Rice is an important grain crop,providing food for nearly half of the world’s population.GRAS transcription factors belong to an important and large family in plants,which are involved in plant growth and development,signal transduction and stress responses.OsSCL7 is an important member of SCL4 / 7 subfamily,whose function has rarely been reported,so excavating the functions of OsSCL7 is important for its application in rice production.In this study,the gene editing technology of Crisper/Cas9 was used to construct scl7 mutants in nipponbare background.The phenotypes and responses to abiotic and biotic stresses of scl7 were analyzed,as well as the target genes and interacted proteins.The main results are as follows:The scl7 mutant exhibits dwarf,decreased tillering numbers,shorter internodes,wider and shorter leaves,and reduced leaf angle and seed setting rates compared to the wild type.Meanwhile,the cell characteristics of the stem and leaf in scl7 mutant were observed through resin sections.The longitudinal sections showed that the stem cells became smaller and irregular and arranged densely in scl7 mutant compared to the wild type,leading to dwarfing in scl7 mutant.Cross-sections showed that the stem cells of scl7 mutant decreased in sizes,but increased in numbers,which lead to the thicker stems in scl7 mutants.Observation of leaf longitudinal sections revealed that the number of mesophyll cells,parenchyma cells,and sclerenchyma cells in the scl7 leaves increased compared to those of the wild type,which result in wider leaves in the mutant.Furthermore,the complementary experiment proved that the abnormal phenotypes of scl7 is due to the loss of OsSCL7 function,indicated that OsSCL7 is essential for vegetative growth and reproductive development in rice.In order to clarify the gene expression characteristics of OsSCL7,OsSCL7 promoter-driven GUS expression analysis was performed.GUS staining results revealed that OsSCL7 expressed in all tissues,but relatively higher in root,bud,and flower tissues,while lower in leaves,which was also be verified by q RT-PCR analysis.Studies have shown that SCL7 family proteins play important roles in plant stress responses.Therefore,q RT-PCR was used to detect the expression level of OsSCL7 in different time after treatment with PEG6000,Na Cl and M.oryzae.The results showed that OsSCL7 expression was down-regulated after treatment of drought and high salt,while was up-regulated after induction by rice blast.Nine days after treatment with drought,all wild-type were found dead and the scl7 mutants recovered to normal growth.However,9 days after treatment with 150 m M Na Cl,wild-type and mutants showed different degrees of curled leaf and shriveled plants,but there was no significant difference between them.After 3 days inoculated with Guy11,scl7 exhibited more disease lesions than those of the wild type,decreased expression of PR genes and increased fungul growth.Moreover,after the leaf sheath was injected with e GFP-tagged M.oryzae strain Zhong1,the growth and infection speed of the fungus in the scl7 mutant was faster than that in the wild type at different time periods.The results above indicated that OsSCL7 positively regulates plant immunity and negatively regulates abiotic stress responses.The crystal structure of OsSCL7 shows that it can form a dimer and has the ability to bind DNA,suggesting that OsSCL7 may be a potential transcription factor.And we also proved that OsSCL7 can interact with itself to form a dimer in yeast.In addition,subcellular localization observation shows OsSCL7 is mainly localized in the nucleus,with a small amount of expression in cell membrane and cytoplasm.As a transcription factor,what is its target genes? To solve this problem,we performed RNA-Seq analysis.The results showed that there were 772 differently expressed genes,519 of them were up-regulated and 253 of them were down-regulated compared with the wild type.Four genes with significantly different expression were selected,and using the luciferase reporter gene system,we found that the promoters of the four genes co-transformed with OsSCL7 were significantly up-regulated compared with only transformed the promoters,indicating that OsSCL7 has transcriptional activity.Additionlly,in order to clarify the signal pathway of OsSCL7,two interacted proteins,G13(LOC＿Os03g63020)and GF14c(LOC＿Os08g33370)were obtained by screening the yeast library,and the interactions were further verified by COIP(immunoprecipitation),Bi FC(bimolecular fluorescence complementary),LUC(luciferase),and yeast dual hybridization analysis.Subcellular localization of GF14 c and G13 showed that GF14 c was mainly localized on the membrane,while G13 was localized on the nucleus and membrane.q RT-PCR analysis showed that both of them were induced by M.oryzae.And g13 and gf14 c mutants were constructed by Crisper / Cas9 method and they showed more susceptible to blast than the wild type.In summary,OsSCL7 plays important roles in rice growth and development,as well as in biotic and abiotic stress responses.And GF14 c has been reported to participate in rice growth and development and regulation of stress responses.Therefore,OsSCL7 may regulate rice growth development and stress responses by interacting with GF14 c and G13.