Cytochrome P450 family member CYP96B5 hydroxylates alkanes to primary alcohols and is involved in rice leaf cuticular wax synthesis

Odd-numbered primary alcohols are components of plant cuticular wax, but their biosynthesis remains unknown. We isolated a rice wax crystal-sparse leaf 5 (WSL5) gene using a map-based cloning strategy. The function of WSL5 was illustrated by overexpression and knockout in rice, heterologous expression in Arabidopsis and transient expression in tobacco leaves. WSL5 is predicted to encode a cytochrome P450 family member CYP96B5. The wsl5 mutant lacked crystalloid platelets on the surface of cuticle membrane, and its cuticle membrane was thicker than that of the wild-type. The wsl5 mutant is more tolerant to drought stress. The load of C-23-C-33 alkanes increased, whereas the C-29 primary alcohol reduced significantly in wsl5 mutant and WSL5 knockout transgenic plants. Overexpression of WSL5 increased the C-29 primary alcohol and decreased alkanes in rice leaves. Heterologous expression of WSL5 increased the C-29 primary alcohol and decreased alkanes, secondary alcohol, and ketone in Arabidopsis stem wax. Transient expression of WSL5 in tobacco leaves also increased the production C-29 primary alcohol. WSL5 catalyzes the terminal hydroxylation of alkanes, yielding odd-numbered primary alcohols, and is involved in the formation of epidermal wax crystals on rice leaf, affecting drought sensitivity.