Brassinosteroid promotes leaf wrinkling in <i>Brassica campestris</i> by regulating a key transcription factor and its downstream genes

Chinese cabbage (Brassica campestris syn. B. rapa) is an important leafy vegetable featuring a variety of cultivars with diverse leaf morphologies. Among these, leaf wrinkling is a distinctive trait that not only defines cultivar types but also influences key agronomic traits such as light interception, photosynthetic capacity, and consumer preferences related to taste and texture. Despite its importance, the genetic regulation underlying this trait remains unclear. In this study, we compared the transcriptomes of 2 Chinese cabbage cultivars, 'Huangya14' ('HY14', with wrinkled leaf) and 'Suzhouqing' ('SZQ', with flat leaf), at 3 developmental stages. Clustering analysis suggested that the difference between wrinkled and flat leaves was already apparent 7 d after sowing. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analysis revealed that a multifaceted network involving adaxial-abaxial axis polarity, epidermal cell division, and brassinosteroid (BR) signaling mediates leaf wrinkling. Notably, the determination of endogenous BR content and exogenous treatment revealed the differentially expressed genes BRASSINAZOLE RESISTANT 1 (BcBZR1), PHABULOSA (BcPHB), and CYCLIN D3;1 (BcCYCD3;1), which were further characterized to be sensitive to either endogenous or exogenous BR levels. Overexpression of BcBZR1 and BcPHB contributed to wrinkled leaves with irregular tissue arrangement, increased epidermal cell number, and the elevated expression level of BcCYCD3;1. Meanwhile, silencing the 2 genes reduced the degree of leaf wrinkling. Interestingly, yeast 1-hybrid, dual-LUC, and chromatin immuno-precipitation quantitative PCR assays revealed that BcBZR1 specifically binds to the promoter of BcPHB and BcCYCD3;1 to activate their expression, and BcPHB also promoted the expression of BcCYCD3;1. This study unveils a mechanism whereby BR contributes to leaf wrinkling in Chinese cabbage by regulating the key component BcBZR1 and its downstream genes BcPHB and BcCYCD3;1.