Functional characterization of BoVML1 uncovers a RER3-related network controlling chloroplast development and leaf morphogenesis in cabbage

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Cabbage (Brassica oleracea var. capitata) is a leafy Brassica vegetable crop whose leaf color and morphology critically influence yield, photosynthetic performance, and market quality. In this study, we identified a natural cabbage mutant, namely, 1180mu, that exhibits virescent-malformed leaves, reduced thylakoid content, decreased fertility, and a relatively low seed-setting rate. Physiological analyses further revealed reduced chlorophyll accumulation, impaired photosynthetic capacity, and stunted growth in 1180mu compared with the wild type (WT). Genetic analysis and map-based cloning demonstrated that BoVML1, a dominant gene homologous to Arabidopsis RER3, is causal; a 44-bp deletion in BoVML1 disrupts its function. CRISPR/Cas9 knockout of BoVML1 in the WT produced a phenotype similar to that of 1180mu, whereas BoVML1 complementation restored normal leaf color, chloroplast ultrastructure, and plant morphology in 1180mu plants. Subcellular localization assays revealed that BoVML1 is targeted to chloroplasts. Transcriptomic profiling uncovered extensive gene downregulation in 1180mu and enrichment of differentially expressed genes (DEGs) in secondary metabolic, phenylpropanoid, and hormone signaling pathways, indicating broad transcriptional reprogramming associated with the virescent phenotype. Moreover, yeast two-hybrid (Y2H) and coimmunoprecipitation assays (Co-IP) demonstrated that BoVML1 interacts with BoPMD1 and BoNAC62, both of which are associated with stress responses. Together, these findings identify BoVML1 as a key positive regulator of chloroplast development, leaf color and leaf morphology in cabbage and provide a mechanistic framework and a practical genetic target for improving leaf color and plant morphogenesis in Brassica breeding programs.