CRISPR/Cas9-mediated mutation in auxin efflux carrier confers chilling tolerance by modulating reactive oxygen species homeostasis in rice.

Phytohormone auxin plays a vital role in plant development and responses to environmental stresses. The spatial and temporal distribution of auxin mainly relies on the polar distribution of the PIN-FORMED (PIN) auxin efflux carriers. In this study, we dissected the functions of , a monocot-specific auxin efflux carrier gene, in modulating chilling tolerance in rice. The results showed that expression was dramatically and rapidly suppressed by chilling stress (4°C) in rice seedlings. The homozygous mutants were generated by CRISPR/Cas9 technology and employed for further research. mutant roots and shoots were less sensitive to 1-naphthaleneacetic acid (NAA) and -1-naphthylphthalamic acid (NPA), indicating the disturbance of auxin homeostasis in the mutants. The chilling tolerance assay showed that mutants were more tolerant to chilling stress than wild-type (WT) plants, as evidenced by increased survival rate, decreased membrane permeability, and reduced lipid peroxidation. However, the expression of well-known ()/ ()-dependent transcriptional regulatory pathway and Ca signaling genes was significantly induced only under normal conditions, implying that defense responses in mutants have probably been triggered in advance under normal conditions. Histochemical staining of reactive oxygen species (ROS) by 3'3-diaminobenzidine (DAB) and nitroblue tetrazolium (NBT) showed that mutants accumulated more ROS than WT at the early stage of chilling stress, while less ROS was observed at the later stage of chilling treatment in mutants. Consistently, antioxidant enzyme activity, including catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD), improved significantly during the early chilling treatments, while was kept similar to WT at the later stage of chilling treatment, implying that the enhanced chilling tolerance of mutants is mainly attributed to the earlier induction of ROS and the improved ROS scavenging ability at the subsequent stages of chilling treatment. In summary, our results strongly suggest that the gene regulates chilling tolerance by modulating ROS homeostasis in rice.