Brchli1 mutation induces bright yellow leaves by disrupting magnesium chelatase I subunit function in Chinese cabbage (Brassica rapa L. ssp. pekinensis)

Magnesium chelatase (MgCh) plays a pivotal role in photosynthesis, catalyzing the insertion of magnesium into protoporphyrin IX (Proto IX), a key intermediate in chlorophyll (Chl) biosynthesis. MgCh is a heteromeric complex composed of the MgCh D subunit (CHLD), the MgCh H subunit (CHLH), and the MgCh I subunit (CHLI). The <i>bright yellow leaves</i> (<i>byl</i>) mutant was obtained through ethyl methanesulfonate (EMS) mutagenesis of the 'FT' Chinese cabbage (<i>Brassica rapa</i> L. ssp. <i>pekinensis</i>) doubled haploid line, whose Chl content, net photosynthetic rate (<i>Pn</i>), and non-photochemical quenching coefficient (NPQ) were decreased, and whose chloroplast development was incomplete. <i>byl</i> recovered to a light green phenotype under weak light conditions. Genetic analysis revealed that the bright yellow leaves phenotype of <i>byl</i> was caused by a single recessive nuclear gene. Using Mutmap sequencing and Kompetitive allele-specific PCR (KASP) identification, <i>BraA01g010040.3.5C</i>, encoding the CHLI subunit of MgCh, was identified as the candidate gene and named <i>Brchli1.</i> A nonsynonymous G-to-A mutation in the <i>Brchli1</i> exon resulted in the substitution of aspartic acid with asparagine. <i>Brchli1</i>-silenced Chinese cabbage displayed bright yellow leaves with decreased <i>Brchli1</i> expression. Transiently overexpressed <i>Brchli1</i> in the <i>byl</i> mutant restored the green leaf phenotype and significantly increased relative <i>Brchli1</i> expression levels. Both BrCHLI1 and its mutated variant were localized in chloroplasts. Yeast two-hybrid and luciferase complementation imaging assays demonstrated that BrCHLI1 interacted with both BrCHLD and itself. BrCHLI1 mutations did not affect its interaction with BrCHLD. Together, <i>Brchli1</i> mutations impaired the function of MgCh, providing insights into the molecular mechanism of leaf coloration.