Gene Cloning And Functional Analysis Of Arabidopsis Ch42-4 And V1 Mutants With Altered Leaf Coloration

The largest class of chloroplast-related mutations affects leaf coloration (bleached, pale green, etc.) and /or seedling viability,which influences chlorophyll biosynthesis and degradation directly or indirectly. Chlorophyll biosynthesis is catalyzed by a series of enzyme complexes. Disruption of any one step of these reactions may result in leaf color mutation. An Arabidopsis pale-green mutant ch42-4 has been isolated using a T-DNA mutagenesis strategy. Genetic analysis indicated that the pale-green phenotype was controlled by a single recessive mutation, but was not linked with T-DNA insertion. Ch42-4 gene was mapped to a region of 53kb between the molecular markers F28J12(1) and F28J12(2) on chromosome IV using a map-based cloning strategy. In the region there are 15 genes, of which At4g18480 encodes Magnesium chelatase subunit CHLI. At4g18480 was cloned and sequenced from wild type Col-0 and ch42-4. Finally ch42-4 was identified as an allelic mutant of ChlI1 gene by allelism test and sequence analysis of the ChlI1. By electron microscope we observed that no grana were formation in the chloroplast of ch42-4 mutant. RT-PCR analysis showed that expression of CHLI1, CHLD and CHLH transcripts were normal in the mutant background, but CHLI2, a homology of CHLI1 was down-regulated in the mutant. These results suggest that CHLI1 mutation changed CHLI2 expression, resulting in a serious phenotype of ch42-4 mutant. Another variegated mutant v1 was isolated by T-DNA mutagenesis, the corresponding T-DNA insertion was identified by TAIL-PCR technology.