HvbZIP33 and HvbZIP76 have overlapping roles in foliar transpiration in drought-stressed barley

Basic leucine zippers (bZIP) constitute one of the biggest protein families and evolutionarily conserved transcription factors (TFs) in plants. We obtained mutant lines for two bZIP TFs, HvbZIP33 and HvbZIP76, in the genetic background of the barley cultivar Golden Promise (GP) via targeted gene-specific mutagenesis using an RNA-guided Cas9 endonuclease. A comprehensive morphological, physiological and transcriptomic analysis was performed in wild-type GP compared with hvbzip33 and hvbzip76 mutants under drought stress. The morphological and physiological changes were similar in both mutants and in the wild-type GP. Most strikingly, the mutants exhibited accelerated wilting and increased water loss. This effect was primarily caused by higher stomatal conductance (gs) and transpiration rate (E) in mutants compared to wild-type GP under both control and drought conditions, which in turn had a detrimental effect on the mutants intrinsic water use efficiency (iWUE). Likewise, the transcriptome profiles of hvbzip33 and hvbzip76 were more similar to each other than those of wild-type GP. We found that the number of differentially regulated genes under control versus drought-stress conditions was higher in the mutants than in wild-type GP, suggesting that the mutants try to compensate for accelerated foliar water loss. The study highlights the essential roles of HvbZIP33 and HvbZIP76 in balancing water loss in barley. These findings provide a foundation for engineering enhanced drought tolerance in barley through targeted manipulation of these genes to optimize transpiration rates.