Identification and characterization of a new allele for ZEBRA LEAF 2, a gene encoding carotenoid isomerase in rice

Abstract The Japonica rice cultivar ‘Nipponbare’ was mutagenized by ethyl methane sulfonate (EMS) to obtain a stable and heritable “zebra leaf” mutant zb2 . Under natural conditions, yellow and green striped leaves appeared at the seedling stage of this mutant and the leaves gradually turned pale green towards the end of the tillering stage. Both temperature and illumination affected the traits of zb2 mutants. Compared with the wild type, concentrations of chlorophyll and carotenoid were significantly lower in zb2 mutants. Electron microscopic examination of the leaves revealed aberrant chloroplast structure in zb2 mutants. The zb2 mutant phenotype was found to be caused by recessive mutations in a single nuclear gene. By using map-based cloning, the target gene was mapped to a 111.9 kb region on chromosome 11. Sequencing analysis revealed a G to T mutation in exon 9 of the ZB2 gene in the mutant. This mutation transformed the 413th Glu residue in the mRNA to a stop codon, resulting in early termination of translation. The ZB2 gene encodes carotenoid isomerase, a key enzyme in carotenoid synthesis, was designated a new allele of OsCRTISO . These results indicated that zb2 is a new allele of Oscrtiso / phs3 . Real-time fluorescence quantitative PCR revealed that expression of ZB2 was highest in the leaves compared to the other organs of wild type plants. Furthermore, ZB2 expression was found to be significantly lower in the mutants compared to the wild type. Our findings further elucidate the function of the OsCRTISO gene and contribute to growing understanding of the metabolic pathway of carotenoids in rice.