Biosynthesis and drought resistance of cuticular wax alkanes in wheat

[Objective]The aim of the work was to identify and functionally characterize a key alkane biosynthesis gene TaCER1-6A from wheat.[Method]Overexpression and CRISPR/Cas9-mediated gene editing of TaCER1-6A were carried out in wheat,and the wax content,cuticle permeability and drought tolerance of TaCER1-6A transgenic lines and wild-type(WT)were further analyzed by gas chromatography-mass spectrometry(GC-MS),gas chromatography-flame ionization detection(GC-FID),chlorophyll leaching,and water loss assays.The biological function of TaCER1-6A was characterized in detail.And dual-luciferase(LUC),yeast one-hybrid(Y1H),and β-glucuronidase(GUS)activation assays were used to confirm the interactions of R2R3-type MYB transcription factors TaMYB96-2D/5D and the promoter of TaCER1-6A.[Result]The CRISPR/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 C27,C29,C31,and C33 alkanes in wheat leaves,suggesting that TaCER1-6A is specifically involved in the biosynthesis of C27-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(ABA).TaCER1-6A was located in the endoplasmic reticulum(ER),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 TaCER1-6A.[Conclusion]TaCER1-6A is required for C27-C33 alkanes biosynthesis and improves drought resistance in wheat,suggesting that TaCER1-6A is a promising tool aiming at generating wheat cultivars with more alkanes contents and improved drought tolerance via molecular breeding and transgenic strategies.