Functional Analysis Of Oryza Sativa Brassinosteroid Biosynthesis Enzyme Gene CYP90A3 And CYP90A4

Brassinosteroids(BRs),are a class of important plant sterols hormones,and play an essential role in seed germination,cell division,cell elongation,vascular differentiation,root and stem development and biotic and abiotic stress responses in plant.For food crop rice,a series of agriculturally valuable traits,like leaf angles,plant height and grain shape,are influenced by Brassinosteroid.CPD(Constitude Photomorphogenesisand Dwarfism)belonging to cytochrome P450 oxidase family members,is a key enzyme of BR synthesis.Compared with a single At CPD/CYP90A1 in Arabidopsis,two CPD genes(CYP90A3/Os CPD1 and CYP90A4/Os CPD2)are found in rice.Due to the lack of knockout mutant materials,so far,whether CYP90A3/Os CPD1 and CYP90A4/Os CPD2 participate in rice BR synthesis is still unclear.To address this issue,we investigated the function of CYP90A3 and CYP90A4 in this study,and the main findings are as follows:1.CYP90A3 and CYP90A4 are highly homologus.The amino acid identity between CYP90A3 and CYP90A4 is as high as 98%.Both of them consist of seven exons and six introns,and contain five conserved CYP450 domains,rich in proline domain,A(molecular oxygen binding),B(steroid binding),C and heme binding domain.2.Construction of cyp90a3 and cyp90a4 knockout mutant with CRISPR/Cas9 gene editing tool.Mutant plants including cyp90a3,cyp90a4 and cyp90a3 cyp90a4 double mutant are obtained.Compared with the wild-type,cyp90a3 and cyp90a4 single mutants have not significant alteration in growth and development,while cyp90a3 cyp90a4 double mutants exhibit a series of typical BR deficent mutant phenotypes such as severe plant dwarf,dark green and curly leaves.Further physiological experiments have shown that the dwarf phenotype of cyp90a3 cyp90a4 double mutant can be rescued by application of24-epi Brassinolide(24-e BL).3.CYP90A3 and CYP90A4 affect the cell morphology.The leaves of cyp90a3,cyp90a4 and cyp90a3 cyp90a4 double mutant were dissected using the scanning electron microscope.Consistent with the dwarf phenotype of double mutant plant,the cell length of cyp90a3 cyp90a4 double mutant are significantly reduced,wherase cyp90a3 and cyp90a4 single mutants are similar to that of the wild-type.4.Overexpression of CYP90A3 and CYP90A4 promote the leaf angle and grain length.CYP90A3 and CYP90A4 overexpressing rice plants driven by the Ca MV-35 S promoter were constructed.Compared with the wild-type,CYP90A3 and CYP90A4 overexpression plants showed a significant increase of leaf-angle and grain length.5.CYP90A3 and CYP90A4 are expressed in a variety of tissues,including roots,stems and leaves.In addition,the subcellular localization analysis showed that CYP90A3 and CYP90A4 distribution in endoplasmic reticulum,which is consistent with the endoplasmic reticulum mediated BR synthesis.All tegther,these results revealed an essential role of CYP90A3 and CYP90A4 in rice BR synthesis.Moreover,overexpression of CYP90A3 and CYP90A4 increased the length of grain,providing a potential application in rice breeding.