TaMYB96-5D regulates diketone biosynthesis and glaucousness by activating <i>WAX1</i> 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 mainly associated with the glaucousness appearance in wheat (Triticum aestivum). WAX1 (W1) locus consists of three genes, TaDMH, TaDMP and TaDMC, which are reported to directly participate in wheat wax diketones biosynthesis. However, the regulatory mechanism underlying diketones biosynthesis and glaucousness formation is still largely unclear in wheat. Here, we tested the involvement of the R2R3-type MYB transcription factor TaMYB96-5D in the regulation of wheat wax diketones biosynthesis and glaucousness 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 components, 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 diketones biosynthesis and glaucousness formation in wheat.