Natural variation and CRISPR/Cas9 gene editing demonstrate the role of a group VII ethylene response factor, HvERF62, in regulation of barley waterlogging tolerance

Waterlogging stress is a serious problem for plants throughout the world, inhibiting plant growth and development and even leading in severe circumstances to plant death. Ethylene-responsive VII (ERFVII) is a key transcription factor regulating plant waterlogging tolerance. In this study, a major quantitative trait locus influencing chlorophyll content under waterlogging stress was detected on chromosome 4H through genome-wide association study using a natural population of barley (Hordeum vulgare). Using RNA-Seq analysis, the group VII ethylene response factor HvERF62 was identified as the candidate gene. CRISPR/Cas9-guided knockout HvERF62 mutants showed sensitivity to waterlogging, with lower chlorophyll content, fewer adventitious roots, and lower root activity. Physiological and transcriptomic analyses showed that HvERF62 plays an important role in aerenchyma formation, reactive oxygen species homeostasis, and carbohydrate accumulation under waterlogging stress, and regulates waterlogging tolerance through starch and sucrose metabolism, the mitogen-activated protein kinase signaling pathway, plant hormone signal transduction, and glycolysis/gluconeogenesis signaling. Haplotype analysis showed that the amino acid sequence of the waterlogging-intolerant haplotype Hap3 was terminated prematurely. This study provides a new genetic resource and a relevant marker for identification of waterlogging-sensitive material and deepens our understanding of the molecular mechanisms of waterlogging response in barley.