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Cuticular waxes cover the aerial surfaces of land plants and protect them from various environmental stresses. Alkanes are major wax components and contribute to plant drought tolerance, but the biosynthesis and regulation of alkanes remain largely unknown in wheat (Triticum aestivum L.). Here, we identified and functionally characterized a key alkane biosynthesis gene ECERIFERUM1-6A (TaCER1-6A) from wheat. The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated knockout mutation in TaCER1-6A greatly reduced the contents of C27, C29, C31, and C33 alkanes in wheat leaves, while TaCER1-6A overexpression significantly increased the contents of these alkanes in wheat leaves, suggesting that TaCER1-6A is specifically involved in the biosynthesis of C27, C29, C31, and C33 alkanes on wheat leaf surfaces. TaCER1-6A knockout lines exhibited increased cuticle permeability and reduced drought tolerance, whereas TaCER1-6A overexpression lines displayed reduced cuticle permeability and enhanced drought tolerance. TaCER1-6A was highly expressed in flag leaf blades and seedling leaf blades and could respond to abiotic stresses and abscisic acid. TaCER1-6A was located in the endoplasmic reticulum, which is the subcellular compartment responsible for wax biosynthesis. A total of three haplotypes (HapI/II/III) of TaCER1-6A were identified in 43 wheat accessions, and HapI was the dominant haplotype (95%) in these wheat varieties. Additionally, we identified two R2R3-MYB transcription factors TaMYB96-2D and TaMYB96-5D that bound directly to the conserved motif CAACCA in promoters of the cuticular wax biosynthesis genes TaCER1-6A, TaCER1-1A, and fatty acyl-CoA reductase4. Collectively, these results suggest that TaCER1-6A is required for C27, C29, C31, and C33 alkanes biosynthesis and improves drought tolerance in wheat. [ABSTRACT FROM AUTHOR]