Functional Analysis Of Lignin Biosynthesis Regulation Gene OsIDD2 And Grain Size Related Gene OsPPKL2 In Rice(Oryza Sativa L.)

Rice,as an important food crop in the world,is also a model plant for the study of molecular biology of monocotyledons.About half of the population in China cosumes rice as the staple food,and rice plant breeding has always been a hot spot in the research field of rice crops,which has played an important role in the improvement of high-yielding rice varieties in China.The traits of rice ideal plant breeding are mainly concerned with important aspects such as plant height,tiller,leaf angle,spike type and grain weight.Thus this paper focuses on the different molecular mechanisms of OsIDD2 regulating the formation of secondary cell wall and OsPPKL2 in the regulation of rice plant type and grain size.The main findings are as follows:1.OsIDD2,a zinc finger and INDETERMINATE DOMAIN protein,regulates secondary cell wall formationDifferent types and stages of plant cells vary in terms of the composition and structure of their cell walls that mainly function to provide mechanical support to cells,tissues,and the whole plant body.Generally,in secondary cell walls(SCW)of mature plant cells,the most prominent component is lignin,along with cellulose and hemicellulose.Previously,we found 123 transcription factors(TFs)as candidate regulators of secondary cell wall(SCW)formation in rice by using phylogenetic and co-expression network analyses.From the results of co-expression network analysis,in addition to AtC3H14,a plant-specific tandem CCCH zinc finger protein,other zinc finger transcription factors have not been found to be involved in the study of SCW formation.Therefore,this study is of great significance for the regulation of secondary cell wall formation by transcription factors.Among them,we examined in this work the role of OsIDD2,a zinc finger and indeterminate domain(IDD)family TF.Its overexpressors showeddwarfism,fragile leaves,and decreased lignin content,which are typical phenotypes of plants defective in SCW formation,whereas its knockout plants showedslightly increased lignin content.We also studied the expression pattern of OsIDD2,9,and 11 in various organs of rice using a public database.As predicted,the expression patterns of OsIDD2 and 11 were similar to each other,while that of OsIDD9 differed from the two.This suggests that OsIDD2 and 11 might have redundant functions as discussed below.The RNA-seq and quantitative reverse transcription polymerase chain reaction analyses confirmed that some lignin biosynthetic genes were downregulated in the OsIDD2-overexpressing plants,and revealed thesame case for other genes involved in cellulose synthesis and sucrose metabolism.The transient expression assay revealed that the expression of CAD2/3 and SUSS was diminished in the presence of OsIDD2,while that of CesA4,7,and 9 remained the same.This indicates that OsIDD2 directly interacts with the target sequences of the CADs and SUS5 to negatively regulate their transcription,whereas the downregulation of CesA genes in OsIDD2-ox might have been indirectly caused by OsIDD2 through the associated transcriptional regulatory network(s).Whereas an AlphaScreen assay,which can detect the interaction between TFs and their target DNA sequences,directly confirmed the interaction between OsIDD2 and the target sequences located in the promoter regions of CAD2 and CAD3.Based on these observations,we conclude that OsIDD2 is negatively involved in SCW formation and other biological events by downregulating its target genes.Interestingly,OsIDD2 also negatively regulates the expression of SUS5 in a direct manner.Sucrose synthases are key enzymes for sucrose metabolism in plant cells,and considered to be responsible for supplying UDP-glucose for cellulose synthesis in the cell wall.Further,it has been demonstrated that regulation of sucrose synthesis can alter the composition of cell wall components.And since SUS5 is predominantly associated with the plasma membrane,it is reasonable to think that the regulation of SUSS expression by OsIDD2 might play an important part in its role to regulate SCW formation.2.OsPPKL2 regulates rice plant architecture and grain size through BR signaling pathwayRice plant architecture and grain size are important factors that determine rice yield and quality.Therefore,it is of great significance to study the plant type and grain development of rice.Based on the OsPPKLl gene,which has been reported in our laboratory regulates the BR signaling pathway in rice.We found two homologous genes,OsPPKL2 and OsPPKL3 in rice.Bioinformatics analysis revealed that OsPPKL2 is homologous to BSU1(Bril suppressor protein 1)in Arabidopsis thaliana.We hypothesize that OsPPKL2 may regulate the grain development by participating in Brassinosteroid(BR)signal transduction or synthetic pathways.Therefore,it is of great significance to study the function and mechanism of OsPPKL2 in BR signaling pathway.The phenotypic identification revealed that mutant osppkl2-1/2 showed dwarfing plant type,decreased plant height,smaller grain weight,smaller thousand grains,but osppkl2-3 showed longer grain length.We found that in the BL treatment the wild-type leaf angle was significantly larger than osppkl2-1/2,and osppkl2-3 was significantly larger than wild-type.Western-blotting experiments demonstrated that the key genes expression in brassinosteroids path way OsBZR1 and DLT protein expression were inhibited in the mutant osppkl2-1/2.Application of exogenous BL can cause changes in transcriptional expression level of OsPPKL2.Therefore,it was shown that OsPPKL2 affects the signaling or synthetic pathway of brassinosteroid hormones.OsPPKL2 is homologous to BSU1.BSU1 dephosphorylates the negative regulator BIN2 in the brassinosteroid(BR)signaling pathway.Subcellular localization shows localization in the cytoplasm.In vitro phosphatase activity assay revealed that OsPPKL2 had dephosphorylation activity and had self-phosphorylation activity.The yeast two-hybrid and BiFC experiments were used to verify the interaction of OsPPKL2 four interacting proteins,STK(Serine/Threonine kinase 1),HEAT(Protein transporter),YTH(YT521-B-like protein family protein)and OsPPKL2.Therefore,OsPPKL2 is able to form homodimers and has its self-dephosphorylation activity.Mutant phenotype analysis revealed that stk showed smaller grain.For exogenous BL treatment,stk is more sensitive than wild-type DJ.Western-blotting experiments showed that the OsBZR1 and DLT protein expression were inhibited in the mutant stk.It probly also has the function of regulating grain size in brassinosteroid pathway.