BnaHSFA2, a heat shock transcription factor interacting with HSP70 and MPK11, enhances freezing tolerance in transgenic rapeseed

Heat shock transcription factors (Hsfs) play important roles in plant developmental regulations and various abiotic stress responses. However, their evolutionary mechanism of freezing tolerance remains poorly understood. In our previous transcriptomics study based on DNA methylation sequencing, the BnaHsfA2 was found to be significantly accumulated in winter rapeseed (Brassica rapa L.) under freezing stress, and the expression levels of BnaHsfA2 showed a gradual increasing trend over three years. In this study, BnaHsfA2 was isolated and characterized. Its' encoding protein has a relatively high phylogenetic relationship with the AtHsfA2; Subcellular localization results indicated that BnaHsfA2 was a nuclear protein; BnaHsfA2 exhibited higher expression levels in mature seed coats and seeds, seedling leaves, flowering filaments as well as anthers. The transcription level of BnaHsfA2 in leaves of rapeseed seedling was significantly increased at -4 °C stress for 12h and 24h. BnaHsfA2 promoter has many stress-responsive cis-regulatory elements. β-glucuronidase (GUS) staining assays indicated that the BnaHsfA2 promoter was induced under freezing stress, and it's 5'-deletion fragment from 465 to 1284 was essential for the transcriptional expression in response to freezing stress. The BnaHsfA2-transgenic rapeseed lines showed greater freezing resistance in comparison with the wild type (WT); the BnaHsfA2 overexpression lines showed increased antioxidant enzyme activities, decreased level of lipid peroxidation and reactive oxygen species (ROS) accumulation compared to the WT. Finally, yeast two-hybrid assay demonstrated that BnaHsfA2 interacted with rapeseed mitogen-activated protein kinase 11 (BnaMPK11) and heat shock factor-binding protein (BnaHsp70). The study will pave the way for further understanding the regulatory networks of BnaHsfA2 in plants under abiotic stress.