Identification And Characterization Of ZEBRA LEAF2, A Gene Encoding Carotenoid Isomerase In Rice

Leaf color mutants are important materials to study chloroplastdevelopment and the metabolism of photosynthetic pigments. In recent years, morethan70genes related to leaf color have been identified by map-based cloning.Carotenoids are not only necessary for the process of photosynthesis pigments, butalso capable of absorbing free radicals and protecting chloroplasts from damage byreactive oxygen species. Carotenoid isomerase (CRTISO) encoded by OsCRTISO isa key enzyme for carotenoid synthesis. A mutation in OsCRTISO may cause blockedcarotenoid synthesis and destroyed chloroplast structure, which result in zebra leaf.Here, the Japonica rice cultivar ’Nipponbare’ was mutagenized by ethyl methanesulfonate (EMS) to obtain a stable and heritable "zebra leaf" mutant zb2. Undernatural conditions, yellow and green striped leaves appeared at the seedling stage ofthis mutant, and the leaves gradually turned pale green towards the end of thetillering stage. Both temperature and illumination affected the traits of zb2mutants.The “zebra leaf” phenotype appeared under low temperature conditions (24-30°C)but disappeared at32°C. The phenotype became more apparent at lowertemperatures. Intense light increased the yellow area of the "zebra leaf" pattern.Compared with the wild type, concentrations of chlorophyll and carotenoid weresignificantly lower in zb2mutants.In addition, mutant plants had lower height andthousand-grain weight, but higher effective tiller number. Electron microscopicexamination of the leaves revealed aberrant chloroplast structure in zb2mutants. Thezb2mutant phenotype was found to be caused by recessive mutations in a singlenuclear gene. By using map-based cloning; the target gene was mapped to a111.9kbregion on chromosome11. Sequencing analysis revealed a G to T mutation in exon9 of the ZB2gene in the mutant. This result showed that zb2is a new allele ofOscrtiso/phs3. The mutation transformed the413th Glu residue in the mRNA to astop codon, resulting in early termination of translation. The gene encodescarotenoid isomerase, a key enzyme in caretenoid synthesis, and was designated anew allele of OsCRTISO. Real-time fluorescence quantitative PCR revealed thatexpression of ZB2was the highest in the leaves compared to other organs of wildtype plants. Furthermore, ZB2expression was found to be significantly lower in themutants compared to wild type. Our findings contribute to growing understanding ofthe metabolic pathway of carotenoids in rice.