Wheat TaMYB96-2D positively regulates glaucousness and drought tolerance by promoting cuticular wax accumulation

Cuticular wax protects the aerial organs of land plants from environmental stresses, especially drought. Glaucousness is an important wax phenotype that is closely related to drought tolerance in wheat (Triticum aestivum). However, how wheat glaucousness is regulated remains largely unknown. In this study, 3 homologous R2R3-type MYB transcription factors, TaMYB96-2A, TaMYB96-2B, and TaMYB96-2D, were identified from wheat sub-genomes 2A, 2B, and 2D, respectively. Compared with wild-type, TaMYB96-2D-overexpressing lines exhibited a more severe glaucous phenotype with significantly enhanced total wax loads and amounts of individual cuticular wax components in flag leaves, flag leaf sheaths, peduncles, and glumes. In addition, overexpression of TaMYB96-2D led to lower water loss rates, less chlorophyll leaching, and increased drought tolerance. By contrast, the 3 TaMYB96-2A/2B/2D gene knockout lines displayed a green phenotype with reduced total wax loads and amounts of individual cuticular wax components, particularly diketones (β-diketone and hydroxy-β-diketone). The knockout lines also showed higher water loss rates, more chlorophyll leaching, and reduced drought tolerance. TaMYB96-2D directly bound to the consensus CAACCA motif present in the promoter regions of 3 diketone biosynthetic genes, TaDMH, TaDMP, and TaDMC, and positively regulated their expression. Taken together, these results demonstrate that TaMYB96-2D enhances the glaucous phenotype and drought tolerance by promoting cuticular wax accumulation on the aerial organs of wheat. TaMYB96-2D can serve as a valuable tool to improve wax production and enhance drought tolerance in wheat breeding programs.