Knockout Mutants of Generated Using CRISPR/Cas9 Revealed Abiotic Stress Tolerance in Rice

Plants produce and accumulate stress-resistant substances when exposed to abiotic stress, which involves a protein conversion mechanism that breaks down stress-damaged proteins and supplies usable amino acids. Eukaryotic protein turnover is mostly driven by the ubiquitination pathway. Among the three enzymes required for protein degradation, E3 ubiquitin ligase plays a pivotal role in most cells, as it determines the specificity of ubiquitination and selects target proteins for degradation. In this study, to investigate the function of (Plant U-box gene in ), we constructed a CRISPR/Cas9 vector, generated gene-edited individuals, and evaluated resistance to abiotic stress using gene-edited lines. A stress-tolerant phenotype was observed as a result of drought and salinity stress treatment in the T gene-edited null lines (PUB7-GE) lacking the T-DNA. In addition, although PUB7-GE did not show any significant change in mRNA expression analysis, it showed lower ion leakage and higher proline content than the wild type (WT). Protein-protein interaction analysis revealed that the expression of the genes (, , , and ) known to be involved in stress increased in PUB7-GE and this, by forming a 1-node network with and , acted as a negative regulator of drought and salinity stress. This result provides evidence that will be a useful target for both breeding and future research on drought tolerance/abiotic stress in rice.