Knockout Mutants of OsPUB7 Generated Using CRISPR/Cas9 Revealed Abiotic Stress Tolerance in Rice

Abstract

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 OsPUB7 (Plant U-box gene in Oryza sativa), we constructed a CRISPR/Cas9 vector, generated OsPUB7 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 T2 OsPUB7 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 (OsPUB23, OsPUB24, OsPUB66, and OsPUB67) known to be involved in stress increased in PUB7-GE and this, by forming a 1-node network with OsPUB66 and OsPUB7, acted as a negative regulator of drought and salinity stress. This result provides evidence that OsPUB7 will be a useful target for both breeding and future research on drought tolerance/abiotic stress in rice.