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

Abstract

An EMS (ethy methanesulfonate)-induced lethal etiolated (le) mutant obtained from the rice variety Zhongjian 100 was characterized by lethal etiolated phenotypes, with significantly reduced levels of chlorophyll a, chlorophyll b, total chlorophyll, and carotenoids. Additionally, the mutant displayed a significantly decreased number of chloroplast grana, along with irregular and less-stacked grana lamellae. The le mutant showed markedly diminished root length, root surface area, and root volume compared with the wild type. It also exhibited significantly lower catalase activity and total protein content, while peroxidase activity was significantly higher. Using the map-based cloning method, we successfully mapped the LE gene to a 48-kb interval between markers RM16107 and RM16110 on rice chromosome 3. A mutation (from T to C) was identified at nucleotide position 692 bp of LOC_Os03g59640 (ChlD), resulting in a change from leucine to proline. By crossing HM133 (a pale green mutant with a single-base substitution of A for G in exon 10 of ChlD subunit) with a heterozygous line of le (LEle), we obtained two plant lines heterozygous at both the LE and HM133 loci. Among 15 transgenic plants, 3 complementation lines displayed normal leaf color with significantly higher total chlorophyll, chlorophyll a, and chlorophyll b contents. The mutation in le led to a lethal etiolated phenotype, which has not been observed in other ChlD mutants. The mutation in the AAA+ domain of ChlD disrupted the interaction of ChlDle with ChlI as demonstrated by a yeast two-hybrid assay, leading to the loss of ChlD function and hindering chlorophyll synthesis and chloroplast development. Consequently, this disruption is responsible for the lethal etiolated phenotype in the mutant.

Keywords

• Oryza sativa
• lethal etiolated mutant
• gene cloning
• functional analysis
• reactive oxygen species