Genetic complementation reveals distinct glabrous classes and a BrBPC2-BrGL1/BrGL3/BrTTG1 regulatory module controlling trichome development in Chinese cabbage

Abstract Trichome development affects both stress tolerance and commercial quality in Chinese cabbage. Although trichome formation has been extensively studied, the genetic heterogeneity underlying glabrous phenotypes in Chinese cabbage and the mechanism underlying trichome restoration in glabrous × glabrous hybrids remain incompletely understood. In this study, we combined genetic complementation analysis, map-based cloning, transgenic validation, yeast and in planta interaction assays, and DAP-seq-based downstream screening to dissect the regulatory mechanism controlling trichome development. Pairwise crosses among nine glabrous inbred lines identified two complementation groups, indicating that phenotypically similar glabrous lines can carry defects in distinct genetic components. Fine mapping and sequence analysis identified defective alleles of BrGL1 in HY and Jin75, whereas ShiF carried a 94-bp deletion in BrTTG1 that was predicted to cause premature translation termination. Overexpression of BrGL1 restored trichome formation, whereas CRISPR/Cas9-mediated disruption of BrGL1 abolished trichome formation and disruption of BrTTG1 severely impaired normal trichome development, confirming their essential roles. Mechanistically, BrGL1 and BrTTG1 showed only a weak or unstable direct association, whereas BrGL3 interacted with both proteins and acted as a central interaction hub within a dynamic BrGL1/BrGL3/BrTTG1 regulatory module. Upstream, BrBPC2 bound to the BrGL1 promoter and activated its expression. Downstream, BrETC1, BrETC3, and BrGL2 were identified as candidate transcriptional outputs responsive to BrGL1- and BrGL3-containing effector combinations. Together, these results uncover a complementation-based genetic framework for glabrous classification and establish a BrBPC2–BrGL1/BrGL3/BrTTG1 regulatory pathway that coordinates trichome development in Chinese cabbage.