Identification of cyp703a3-3 and analysis of regulatory role of CYP703A3 in rice anther cuticle and pollen exine development

Anther cuticle and pollen exine are two elaborated lipid-soluble barriers protecting pollen grains from environmental and biological stresses. However, less is known about the mechanisms underlying the synthesis of these lipidic polymers. Here, we identified a no-pollen male-sterility mutant cyp703a3-3 from the indica restorer line Zhonghui 8015 (Zh8015) mutant library treated with 60Coγ-ray radiation. Histological analysis indicated that cyp703a3-3 underwent abnormal tapetal cells development, produced few orbicules and secreted less sporopollenin precursors to anther locule, as well as cutin monomers on anther. Genetic analysis revealed that cyp703a3-3 was controlled by a single recessive gene. Map-based cloning was performed to narrow down the mutant gene to a 47.78-kb interval on the chromosome 8 between two markers S15-29 and S15-30. Sequence analysis detected three bases (GAA) deletion in the first exon of LOC_Os08g03682, annotated as CYP703A3 with homologous sequences related to male sterility in Arabidopsis, causing the Asparagine deletion in the mutant site. Moreover, we transformed genomic fragment of CYP703A3 into cyp703a3-3, which male-sterility phenotype was recovered. Both the wild-type and cyp703a3-3 mutant 3D structure of CYP703A3 protein were modeled. Results of qPCR suggested CYP703A3 mainly expressed in anthers with greatest abundance at microspore stage, and genes involved in sporopollenin precursors formation and transportation, such as GAMYB, TDR, CYP704B2, DPW2, OsABCG26 and OsABCG15, were significantly reduced in cyp703a3-3. Collectively, our results further elaborated CYP703A3 plays vital role in anther cuticle and pollen exine development in rice (Oryza sativa L.).