Wheat drought-responsive WXPL transcription factors regulate cuticle biosynthesis genes

The cuticle forms a hydrophobic waxy layer that covers plant organs and provides protection from biotic and abiotic stresses. Transcription of genes responsible for cuticle formation is regulated by several types of transcription factors (TFs). Five orthologous to WAX PRODUCTION (WXP1 and WXP2) genes from Medicago truncatula were isolated from a cDNA library prepared from flag leaves and spikes of drought tolerant wheat (Triticum aestivum, breeding line RAC875) and designated TaWXP-like (TaWXPL) genes. Tissue-specific and drought-responsive expression of TaWXPL1D and TaWXPL2B was investigated by quantitative RT-PCR in two Australian wheat genotypes, RAC875 and Kukri, with contrasting glaucousness and drought tolerance. Rapid dehydration and/or slowly developing cyclic drought induced specific expression patterns of WXPL genes in flag leaves of the two cultivars RAC875 and Kukri. TaWXPL1D and TaWXPL2B proteins acted as transcriptional activators in yeast and in wheat cell cultures, and conserved sequences in their activation domains were localised at their C-termini. The involvement of wheat WXPL TFs in regulation of cuticle biosynthesis was confirmed by transient expression in wheat cells, using the promoters of wheat genes encoding two cuticle biosynthetic enzymes, the 3-ketoacyl-CoA-synthetase and the cytochrome P450 monooxygenase. Using the yeast 1-hybrid (Y1H) assay we also demonstrated the differential binding preferences of TaWXPL1D and TaWXPL2B towards three stress-related DNA cis-elements. Protein structural determinants underlying binding selectivity were revealed using comparative 3D molecular modelling of AP2 domains in complex with cis-elements. A scheme is proposed, which links the roles of WXPL and cuticle-related MYB TFs in regulation of genes responsible for the synthesis of cuticle components.