AcNPR1 enhances freezing tolerance by targeting AcPIF3 for degradation in <i>Actinidia chinensis</i>

Abstract Due to the high sensitivity of kiwifruit to cold stress, improving cold resistance has become a major bottleneck for the kiwifruit industry. Our previous studies demonstrated that AcCBF1 and AcCBF2 positively regulate cold tolerance in Actinidia chinensis. Nevertheless, the regulatory mechanisms underlying the CBF-COR signaling pathway in kiwifruit remain largely unknown. Here, we found that AcNPR1 could positively regulate freezing tolerance of A. chinensis. Importantly, our evidence suggests a model in which AcNPR1 acts as a substrate adaptor for the CRL3 E3 ligase, interacting with AcPIF3 and promoting its ubiquitination and degradation. We identified that AcPIF3 could negatively regulate freezing tolerance of A. chinensis at least in part through directly inhibiting AcCBF1 and AcCBF2. Interestingly, we found that AcNPR1 is primarily localized to the cytoplasm under normal conditions. However, cold stress not only increases the abundance of AcNPR1 protein, but also induces its shuttling from the cytoplasm to and accumulation in the nucleus, potentially accelerating the ubiquitination and degradation of AcPIF3. Therefore, we concluded that the cold induced increase in AcNPR1 protein level and its cytoplasmic to nuclear translocation contributes to the ubiquitination and degradation of AcPIF3, thereby alleviating the inhibitory effect of AcPIF3 on AcCBF1 and AcCBF2 and enhancing the freezing resistance of A. chinensis.