Mutation of a magnesium chelatase BrCHLD affects the chlorophyll content and magnesium chelatase activity in Chinese...

Magnesium chelatase (MgCh) catalyzes the insertion of Mg²⁺ into protoporphyrin IX, which is essential for chlorophyll (Chl) biosynthesis. MgCh consists of three subunits, CHLI, CHLD, and CHLH subunits. However, previous studies have cloned only BrCHLI and BrCHLH genes in Chinese cabbage, and the mechanism by which BrCHLD regulates chlorophyll synthesis in Chinese cabbage has not been identified. In this study, we characterized a Chl defective mutant M1685 in Chinese cabbage EMS-mutagenized population. The M1685 mutant exhibits yellow leaves and low Chl level with impaired photosynthesis and chlorophyll fluorescence parameters. MutMap and kompetitive allele specific PCR (KASP) results indicated that BraA06g005760.3.5C, a homolog of AtCHLD that encodes the CHLD subunit of the Mg-chelatase enzyme, was the candidate gene, designated BrCHLD. The G to A mutation at 1763 bp led to the arginine to lysine variation at 588 aa of BrCHLD protein. Silencing of BrCHLD by virus-induced gene silencing (VIGS) resulted in the mutant phenotype in Chinese cabbage. Quantitative real-time-PCR (qRT-PCR) assays and promoter activity analysis showed that BrCHLD was expressed in all tissues and stages in 'FT'. BrCHLD was localized to the chloroplast, and the mutation did not affect the subcellular localization. The mutation did not affect the self-interaction of BrCHLD and the interaction between BrCHLD and BrCHLI. However, the Mg-protoporphyrin IX content and MgCh activity were significantly reduced in M1685. Our study demonstrated that BrCHLD was required for the Chl biosynthesis, laying a theoretical foundation for revealing Chl synthesis in Chinese cabbage.