Knockout of , a gene encoding proline-rich protein, confers enhanced cold sensitivity in rice ( L.) at the seedling stage.

Proline-rich proteins (PRPs) play multiple physiological and biochemical roles in plant growth and stress response. In this study, we reported that the knockout of induced cold sensitivity in rice. Mutant plants were generated by CRISPR/Cas9 technology to investigate the role of in cold stress and 26 mutant plants were obtained in T generation with the mutation rate of 85% including 15% bi-allelic, 53.3% homozygous, and 16.7% heterozygous and 16 T-DNA-free lines in T generation. The conserved amino acid sequence was changed and the expression level of was reduced in mutant plants. The mutant plants displayed more sensitivity to cold stress and showed low survival rate with decreased root biomass than wild-type (WT) and homozygous mutant line with large fragment deletion was more sensitive to low temperature. Mutant lines accumulated less antioxidant enzyme activity and lower levels of proline, chlorophyll, abscisic acid (ABA), and ascorbic acid (AsA) content relative to WT under low-temperature stress. The changes of antioxidant enzymes were examined in the leaves and roots with exogenous salicylic acid (SA) treatment which resulted in increased activity of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) under cold stress, while enzyme antioxidant activity was lower in untreated seedlings which showed that exogenous SA pretreatment could alleviate the low-temperature stress in rice. Furthermore, the expression of three genes encoding antioxidant enzyme activities (, , and ) was significantly down-regulated in the mutant lines as compared to WT. These results suggested that enhances cold tolerance by modulating antioxidants and maintaining cross talk through signaling pathways. Therefore, gene could be exploited for improving cold tolerance in rice and CRISPR/Cas9 technology is helpful to study the function of a gene by analyzing the phenotypes of knockout mutants generated.