Plant surface properties are crucial determinants of resilience to abiotic and biotic stresses. The outer layer of the plant cuticle consists of chemically diverse epicuticular waxes. The WAX INDUCER1/SHINE subfamily of APETALA2/ETHYLENE RESPONSIVE FACTORS regulates cuticle properties in plants. In this study, four barley genes homologous to the Arabidopsis thaliana AtWIN1 gene were mutated using RNA-guided Cas9 endonuclease. Mutations in one of them, the HvWIN1 gene, caused a recessive glossy sheath phenotype associated with β-diketone deficiency. A complementation test for win1 knockout (KO) and cer-x mutants showed that Cer-X and WIN1 are allelic variants of the same genomic locus. A comparison of the transcriptome from leaf sheaths of win1 KO and wild-type plants revealed a specific and strong downregulation of a large gene cluster residing at the previously known Cer-cqu locus. Our findings allowed us to postulate that the WIN1 transcription factor in barley is a master mediator of the β-diketone biosynthesis pathway acting through developmental stage- and organ-specific transactivation of the Cer-cqu gene cluster.

In barley having adherent hulls, an irreversible connection between the pericarp with both palea and lemma is formed during grain maturation. A mutation in the NUDUM 1 (NUD1) gene prevents this connection and leads to the formation of barley with non-adherent hulls. A genetic model of two isogenic lines was used to elucidate the genetic mechanisms of hull adhesion: a doubled haploid line having adherent hulls and its derivative with non-adherent hulls obtained by targeted mutagenesis of the NUD1 gene. Comparative transcriptomics analysis of the grain coats was performed at two stages of development: the milk stage, when the hulls can still be easily detached from the pericarp, and the dough stage when the hull adhesion process occurs. It was shown that the main differences in the transcriptomes lie in the genes related to DNA replication and chromatin assembly, cell wall organization, and cuticle formation. Meanwhile, genes involved in lipid biosynthesis mostly show minor differences in expression between stages and genotypes and represent a limited set of active genes. Among the 3-ketoacyl-CoA synthase (KCS) genes active during grain development, candidates for key enzymes responsible for very long-chain fatty acid elongation were identified.