Mechanisms Of How OsIAAGLU Modulates Plant Architecture In Rice

Plant architecture in rice,including leaf angle,plant height,tiller angle and tiller number,is an important agronomic trait that is closely related to yields.The distribution and levels of auxin or indole-3-acetic acid(IAA)within plant tissues are well known to be associated with plant architecture determination.Recently we identified an OsOxO2 T-DNA mutant(oxo2),characterized by an increased number of tillers,enlarged leaf angles and dwarfism compared to the wild-type plants(WT).Os IAAGLUoverexpressed lines showed the similar phenotypes.Os IAAGLU in the rice genome database was found to have 67% sequence similarity with IAA-glycosyltransferase of maize(ZmIAAGLU),which is known to generate IAA-glucose by conjugating free IAA to glucose.This perturbed auxin homeostasis was associated with architectural changes.The mechanisms of how OsIAAGLU modulates plant architecture in rice were analysed in this study.Main results were described as follow:(1)Overexpression of OsIAAGLU results in enlarged leaf angles and decreased heightResults of isolation and phenotypic characterization of OsIAAGLU-overexpressed transgenic line(IAAGLUOE3)showed that seven of the eleven plants carrying the HPT transgene exhibited enlarged leaf angles and decreased height,whereas plants which had lost the transgene by segregation were similar in appearance to WT.Recently,two homozygous OsIAAGLU-overexpressed lines(IAAGLUOE3 and IAAGLUOE5)were obtained.They exhibited similar phenotypes with oxo2 at the 5-leaf-stage.(2)Restoration of normal phenotypes in oxo2 by mutation of OsIAAGLUHomozygous OsIAAGLU mutants constructed by CRISPR/Cas9 system in WT and oxo2 mutant were obtained,respectively.OsIAAGLU protein couldn't be detected in OsIAAGLU mutant plants with the specific abtibody against OsIAAGLU-His by western blotting.OsIAAGLU mutants of WT exhibited increased height and a smaller leaf angle compared to WT.Moreover,the phenotype of OsIAAGLU mutants of oxo2 was consistent with WT.These results demonstrate that the aberrant phenotypes are caused by the increased expression of OsIAAGLU in oxo2.(3)Down-regulated expression of OxO in oxo2 does not alter its phenotypesEight hybrid plants of down-regulated expression of OsOxOs were obtained by crossing experiment between oxo2 as female parent and RNAi lines of OsOxOs(OsOxOsRi6)as male parent.In eight hybrid plants,protein expression level of OsOx O2 and OxO activity was significantly lower than that of female parent(oxo2),but the level of OsIAAGLU transcript had no obvious difference.Eight hybrid plants revealed similar phenotypes with oxo2,characterized by enlarged leaf angles and dwarfism at the heading stage.Therefore,the multiple morphological alterations in oxo2 mutants were caused by the enhanced expression of OsIAAGLU.(4)OsIAAGLU alters plant architecture in rice through regulating auxin homeostasisUsing transient expression of OsIAAGLU fused to a GFP in rice protoplast,the subcellular localization of Os IAAGLU was demonstrated to be in the cytoplasm.OsIAAGLU expression in the leaves was induced by IAA/NAA treatments.Overexpression of OsIAAGLU resulted in reduced sensitivity to NAA,while OsIAAGLU mutants were more sensitive to NAA than WT.Seedlings overexpressing OsIAAGLU exhibited reduced root gravitropic responses.Moreover,free IAA contents in the leaves of OsIAAGLU-overexpressing transgenic lines were lower than that of WT by HPLC,and were reduced at the lamina joint of OsIAAGLU-overexpressing transgenic lines detected by pDR5:GUS staining.The transcript abundances of OsIAA14 and OsGH3-2 involved in auxin response factors were also reduced in OsIAAGLU-overexpressing transgenic lines compared with WT,while they were induced in OsIAAGLU mutants.These results suggested that OsIAAGLU played a regulatory role in IAA homeostasis and rice plant architecture.