ZmDREB1A regulates RAFFINOSE SYNTHASE controlling raffinose accumulation and plant chilling stress tolerance in maize.

Raffinose accumulation is positively correlated with plant chilling stress tolerance; however, the understanding of the function and regulation of raffinose metabolism under chilling stress remains in its infancy. RAFFINOSE SYNTHASE (RAFS) is the key enzyme for raffinose biosynthesis. We report here that two independent maize (Zea mays) zmrafs mutant lines, in which raffinose was completely abolished, were more sensitive to chilling stress and their net photosynthetic product (total soluble sugars and starch) accumulation were significantly decreased compared with controls after chilling stress. A similar characterization of the maize Dehydration Responsive Element (DRE) Binding Protein 1A mutant (zmdreb1a) showed that ZmRAFS expression and raffinose content were significantly decreased compared with its control under chilling stress. Overexpression of maize ZmDREB1A in maize leaf protoplasts increased ZmDREB1A amounts which consequently up-regulated the expression of maize ZmRAFS and the Renilla LUCIFERASE (Rluc) which was controlled by the ZmRAFS promoter. Deletion of the single drought responsive element (DRE) in the ZmRAFS promoter abolished ZmDREB1A's influence on Rluc expression while addition of three copies of the DRE in the ZmRAFS promoter dramatically increased Rluc expression when ZmDREB1A was simultaneously overexpressed. Electrophoretic mobility shift assays and CHIP-qPCR demonstrated that ZmDREB1A directly binds to the DRE motif in the promoter of ZmRAFS both in vitro and in vivo. These data demonstrate that ZmRAFS, which was directly regulated by ZmDREB1A, enhances both raffinose biosynthesis and plant chilling stress tolerance.