Genome-wide identification of C2H2-ZFPs and functional analysis of BcZAT12 and BcZAT10 under cold stress in non-heading Chinese cabbage

Low temperature affects the quality and yield of non-heading Chinese cabbage.C2H2-type zinc finger proteins (C2H2-ZFPs) play an important role in various environmental stresses. To date, the functions of C2H2-ZFPs in non-heading Chinese cabbage are still uncharacterized. Here, 163 C2H2-ZFPs in non-heading Chinese cabbage were identified using the sequenced NHCC001 genome and classified into nine subfamilies. Then characterization of the C2H2-ZFPs was comprehensively investigated. Synteny analysis suggested that these genes these genes were highly conserved during evolution, which may be related to the triploid (WGT) event of NHCC001. Through a combination of genome-wide and transcriptome analysis, we identified two zinc finger protein factors BcZAT12 and BcZAT10 that are closely related to cold. Furthermore, it was found that both BcZAT12 and BcZAT10 were localized in the nucleus and highly expressed in leaves. VIGS and transient overexpression experiments indicated that BcZAT12 and BcZAT10 acted as transcriptional repressors to negatively regulate cold tolerance in non-heading Chinese cabbage during cold resistance. Surprisingly, we demonstrated that BcZAT12 can directly bind to the ACT core sequence of A (G/C) T-X3-4-A (G/C) T on the BcCBF1 promoter and inhibit its activity. Meanwhile, we discovered that BcNTL4 and BcNAC055 can bind to the G-BOX element on the promoter of BcZAT12 and negatively regulate the expression of BcZAT12. Interestingly, we found that BcABF2/4 can interact with BcZAT12, speculating that BcZAT12 may interact with BcABF2/4 to regulate the expression of cold-related genes. This study laid a foundation to further understand the functional role and regulatory mechanism of C2H2-ZFPs of non-heading Chinese cabbage under cold stress.