Characterization of Glossy Spike Mutants and Identification of Candidate Genes Regulating Cuticular Wax Synthesis in Barley (Hordeum vulgare L.)

Abstract

Cuticular waxes comprise the hydrophobic layer that protects crops against nonstomatal water loss and biotic and abiotic stresses. Expanding on our current knowledge of the genes that are involved in cuticular wax biosynthesis and regulation plays an important role in dissecting the processes of cuticular wax metabolism. In this study, we identified the Cer-GN1 barley (Hordeum vulgare L.) mutant that is generated by ethyl methanesulfonate mutagenesis with a glossy spike phenotype that is controlled by a single recessive nuclear gene. A physiological analysis showed that the total cuticular wax loads of Cer-GN1 were one-third that of the progenitor wild-type (WT), and its water loss rate was significantly accelerated (p < 0.05). In addition, Cer-GN1 was defective in the glume’s cuticle according to the toluidine blue dye test, and it was deficient in the tubule-shaped crystals which were observed on the glume surfaces by scanning electron microscopy. Using metabolomics and transcriptomics, we investigated the impacts of cuticular wax composition and waxy regulatory genes on the loss of the glaucous wax in the spikes of Cer-GN1. Among the differential metabolites, we found that 16-hydroxyhexadecanoic acid, which is one of the predominant C16 and C18 fatty acid-derived cutin monomers, was significantly downregulated in Cer-GN1 when it was compared to that of WT. We identified two novel genes that are located on chromosome 4H and are downregulated in Cer-GN1 (HvMSTRG.29184 and HvMSTRG.29185) that encode long-chain fatty acid omega-monooxygenase CYP704B1, which regulates the conversion of C16 palmitic acid to 16-hydroxyhexadecanoic acid. A quantitative real-time PCR revealed that the expression levels of HvMSTRG.29184 and HvMSTRG.29185 were downregulated at 1, 4, 8, 12, and 16 days after the heading stage in Cer-GN1 when it was compared to those of WT. These results suggested that HvMSTRG.29184 and HvMSTRG.29185 have CYP704B1 activity, which could regulate the conversion of C16 palmitic acid to 16-hydroxyhexadecanoic acid in barley. Their downregulation in Cer-GN1 reduced the synthesis of the cuticular wax components and ultimately caused the loss of the glaucous wax in the spikes. It is necessary to verify whether HvMSTRG.29184 and HvMSTRG.29185 truly encode a CYP704B1 that regulates the conversion of C16 palmitic acid to 16-hydroxyhexadecanoic acid in barley.

Effects and mechanisms of proanthocyanidins-derived carbon dots on alleviating salt stress in rice by muti-omics analysis

“…However, after treatment with PE-CDs, D-Raffinose was significantly down-regulated compared to S, suggesting that PE-CDs alleviated the degree of salt stress. A similar trend was observed in 16-Hydroxyhexadecanoic acid, a key substance in plant keratin synthesis ( Bian et al, 2022 ).…”

Gas Chromatography–Mass Spectrometry Metabolite Analysis Combined with Transcriptomics Reveals Genes Involved in Wax Biosynthesis in Allium fistulosum L.

“…The identification of waxy mutants has contributed to understanding the precise molecular mechanisms underlying epicuticular wax formation, offering valuable insights for agricultural breeding. Although several studies have focused on wax-deficient mutants in crops such as barley [ 15 , 34 ], wheat [ 35 ], maize [ 36 ], and rice [ 37 ]. Studies on this topic in A. fistulosum are limited.…”

“…In Agave , for instance, alkanes are the main waxy components, with their content varying among different ploidy levels [ 14 ]. A significant reduction was observed in the content of 16-hydroxyhexadecanoic acid in the barley mutant Cer-GN1 [ 15 ]. However, the Allium cepa mutant exhibited considerably reduced 16-hentriacontanone content, resulting in the absence of needle-like waxy crystals [ 16 ].…”