BrpNAC895 and BrpABI449 coregulate the transcription of the afflux-type cadmium transporter BrpHMA2 in Brassica parachinensis

Brassica parachinensis is a popular leafy vegetable. It is able to accumulate high concentrations of cadmium (Cd), but the molecular mechanism of Cd accumulation is unknown. This study investigated the function and regulatory mechanism of the Cd-responsive metal ion transporter gene BrpHMA2. BrpHMA2 was induced by Cd stress and specifically expressed in vascular tissues, and the protein was localized in the plasma membrane. Heterologous expression of BrpHMA2 enhanced Cd accumulation and Cd sensitivity in transgenic Arabidopsis and yeast. After Cd stress, the transcription factors BrpNAC895 and BrpABI449, which may recognize the abscisic acid-responsive elements in the BrpHMA2 promoter, were also differentially expressed. The transcriptional regulation of BrpHMA2 was further investigated using the chromatin immunoprecipitation–quantitative PCR (ChIP–qPCR) assay, the electrophoretic mobility shift assay (EMSA), and luciferase (LUC) reporter activity analysis employing the transient expression system of B. parachinensis protoplasts and tobacco leaves and the Escherichia coli expression system. By binding to the promoter, BrpNAC895 induced the transcription of BrpHMA2. BrpABI449 might bind to the BrpHMA2 promoter or interact with BrpNAC895 to interfere with the action of BrpNAC895. The findings suggest that BrpHMA2 is a membrane-based afflux-type Cd transporter involved in Cd(2+) uptake and long-distance transport in plants. BrpNAC895 and BrpABI449, which function as the transcription activator and repressor, respectively, coregulate BrpHMA2 expression.