BrANL2 negatively regulates the anthocyanin biosynthesis by depressing BrPAL, BrC4H, and Br4CL in purple flowering Chinese cabbage (Brassica rapa var. parachinensis).

A new deep‑purple flowering Chinese cabbage germplasm (22M‑699) was developed, and BrANL2 was identified as a negative regulator of anthocyanin biosynthesis that directly represses BrPAL, BrC4H, and Br4CL, providing a target for purple-color breeding in Brassica crops. Purple flowering Chinese cabbage is highly valued in the market for its vibrant color and potential health benefits, especially the rich anthocyanin contents. Previously, we introduced the purple trait from mustard (Brassica juncea) into heading Chinese cabbage (Brassica rapa var. pekinensis) via distant hybridization, which resulted in a novel germplasm designated 18M-245. Subsequently, this purple trait was introgressed into flowering Chinese cabbage (Brassica rapa var. parachinensis) through hybridization and successive backcrossing, yielding another new germplasm named 22M-699, which exhibits uniformly deep-purple leaves similar to those of 18M-245. To investigate the genetic basis of the purple trait in purple flowering Chinese cabbage, we sequenced several genes involved in anthocyanin biosynthesis regulation. The results revealed insertions and large-fragment structural variations in the promoter region of BrANL2, which impair its transcriptional activity, leading to reduced expression in the purple germplasm 22M‑699. BrANL2 expression is negatively correlated with anthocyanin accumulation under low‑temperature conditions. Using DAP-seq and dual-luciferase reporter assays, we demonstrated that BrANL2 directly binds to the promoters of BrPAL, BrC4H, and Br4CL and represses their expression, thereby inhibiting anthocyanin biosynthesis. Furthermore, overexpression of BrANL2 reverses the purple phenotype in 22M-699, while its knockout induces purple pigmentation in a green line. Collectively, our findings reveal a suppressive role of BrANL2 targeting BrPAL, BrC4H, and Br4CL in anthocyanin biosynthesis in contrast to the previously reported positive functions of ANL2-like transcription factors and provide a potential strategy for generating purple materials in cruciferous vegetables.