A BolMYB34-l-BolTRY-l Regulatory Module Negatively Regulates Trichome Initiation in Brassica oleracea

Brassica oleracea exhibits remarkable morphological diversity and is cultivated worldwide. We previously showed that dense trichomes in a wild accession (B. incana, C01) confer resistance to insect pests, yet the underlying regulatory mechanisms remain unclear. Here, we developed a cleaved amplified polymorphic site (CAPS) marker to distinguish sequence variations in BolTRY-l between trichome-rich C01 and glabrous C41. Functional analyses revealed that overexpression of BolTRY-l in the Arabidopsis try mutant and in C01 markedly suppressed trichome initiation, identifying BolTRY-l as a negative regulator. Promoter sequence comparison and activity assays further indicated that divergence in the BolTRY-l promoter underlies the contrasting trichome phenotypes between C01 and C41. Using the HDOCK server, yeast one-hybrid (Y1H), and dual-luciferase reporter (Dual-LUC) assays, we showed that BolMYB34-l directly binds to MYB-binding site (MBS) elements within the BolTRY-l promoter in C01. In addition, co-immunoprecipitation (Co-IP), bimolecular fluorescence complementation (BiFC), and luciferase complementation (LCA) assays confirmed a physical interaction between BolMYB34-l and BolTRY-l. Overexpression and virus-induced gene silencing (VIGS) analyses further supported that BolMYB34-l functions as an upstream negative regulator of BolTRY-l. Collectively, our findings reveal a novel regulatory module in which the BolMYB34-l-BolTRY-l complex negatively regulates trichome formation in B. oleracea.