Yunfeng Xu, Lijun Liu, Ling Shen, Boyan Wan, Haoran Sun, Zengjie Hu, Guoping Zhang, Qiufang Shen
SUMMARY Soil salinization is one of the major abiotic stresses affecting global crop productivity and agricultural sustainability. Barley ( Hordeum vulgare L.) is one of the most salt‐tolerant cereal crops, so it is possible and imperative to explore the elite genes or genetic factors contributing to salt tolerance. Here, we identified HvCBP60‐8, a member of the CBP60 transcription factors, acting as a positive regulator of salt tolerance in barley. CRISPR/Cas9‐induced loss‐of‐function mutants of HvCBP60‐8 showed more sensitivity to salt stress, accompanied by elevated Na + accumulation in shoots due to enhanced root‐to‐shoot Na + translocation. Transcriptome analysis of wild‐type and cbp60‐8 mutants revealed three receptor‐like kinase genes ( HvRLK3/4/5 ) as downstream targets of HvCBP60‐8. Dual‐LUC assay confirmed that HvCBP60‐8 could directly activate the expression of HvRLK3/4/5 by binding to their promoters. RT‐qPCR analysis showed that HvCBP60‐8 up‐regulated the expression of three key ion transporter genes ( HvHAK1 , HvNHX1 , and HvHKT1;1 ), resulting in improved K + /Na + homeostasis under salt stress. The results also revealed the distinct variation of HvCBP60‐8 haplotypes in a natural population, with cultivated barley showing the dominant variation, probably caused by natural and artificial selection. In conclusion, HvCBP60‐8 is a pivotal transcriptional regulator of salt tolerance in barley through responding to receptor‐like kinase signaling and modulating the expression of genes encoding key ion transporters. The current results provide novel genetic targets for crop breeding to develop varieties with high salt stress tolerance.