Mapping and Functional Analysis of LE Gene in a Lethal Etiolated Rice Mutant at Seedling Stage

Abstract

An EMS-induced mutant le (lethal etiolated) obtained from a rice variety Zhongjian 100, was characterized by lethal etiolated phenotypes with significantly lower contents of chlorophyll a, chlorophyll b, total chlorophyll and carotenoids, as well as significantly decreased the number of chloroplast grana and irregular and less-stacked grana lamella. The root length, root surface area and root volume of le plants were markedly reduced relative to the wild type. The mutant le showed significantly lower catalase activity and total protein content, significantly higher peroxidase activity,. Using the map-based cloning method, LE gene was mapped to an interval of 48 kb between markers RM16107 and RM16110 on rice chromosome 3. A mutation (from T to C) at the nucleotide position 692 bp of LOC_Os03g59640 (ChlD) occurred, resulting in a change from leucine to proline. By crossing HM133 (a pale green mutant with a single-base substitution of A for G on the exon 10 of ChlD subunit) with a heterozygous line of le (LEle), two plant lines heterozygous at the LE locus and HM133 locus were obtained. Among 15 transgenic plants, 3 complementation lines displayed normal leaf color with significant higher total chlorophyll and chlorophyll a and b contents. The ChlD subunit of the mutant was unable to interact with the ChlI subunit, as demonstrated by a yeast two-hybrid assay. The mutation in le led to lethal etiolated phenotype, but no such phenomenon was observed in the mutants. The interaction between the ChlD and ChlI subunits, chlorophyll synthesis, and chloroplast development may all be impacted by the mutation, which could ultimately result in plant death. The mutation in the AAA+ domain of ChlD blocked the interaction of ChlDle with ChlI, leading to the loss of function of ChlD and hindering chlorophyll synthesis and chloroplast development, and ultimately causing lethal etiolated phenotype of the mutant.