TaMYB96-5D regulates diketone biosynthesis and glaucousness by activating WAX1 expression in wheat

Cuticular wax covers the aerial surfaces of land plants and protects them from environmental stresses. Diketones (β-diketone and hydroxy-β-diketone) are the most important components of cuticular wax, and are mainly associated with the glaucous appearance in wheat (Triticum aestivum). The WAX1 (W1) locus consists of three genes, TaDMH, TaDMP, and TaDMC, which are reported to directly participate in biosynthesis of wheat wax diketones. However, the regulatory mechanism underlying diketone biosynthesis and formation of glaucousness is still largely unclear in wheat. Here, we tested the involvement of the R2R3-type MYB transcription factor TaMYB96-5D in the regulation of biosynthesis of wheat wax diketones and glaucous appearance. The overexpression of TaMYB96-5D caused a significant increase in the total wax load and the amounts of individual wax components, resulting in increased glaucousness and improved drought tolerance. Conversely, three genes of TaMYB96-5A/5B/5D knockout lines exhibited large decreases in the total wax load and the amounts of each wax component, especially diketones, along with a glossy green phenotype and reduced drought tolerance. TaMYB96-5D bound directly to the CAACCA motif in promoters of the TaDMH, TaDMP, and TaDMC genes, and activated their transcription. Overall, these findings provide new insights into the regulation of diketone biosynthesis and formation of glaucousness in wheat.