Low temperature affects the yield and quality of crops. Inducer of CBF expression 1 (ICE1) plays a positive role in plant cold tolerance by promoting the expression of CRT binding factor (CBF) and cold-responsive (COR) genes. Several ICE1-interacting transcription factors (TFs) that regulate plant cold tolerance have been identified. However, how these TFs affect the function of ICE1 and CBF expression under cold conditions remains unclear. Here, we identified the MYC-type TF MdbHLH4, a negative regulator of cold tolerance in Arabidopsis (Arabidopsis thaliana) and apple (Malus domestica) plants. Under cold conditions, MdbHLH4 inhibits the expression of MdCBF1 and MdCBF3 by directly binding to their promoters. It also interacts with MdICE1L, a homolog of AtICE1 in apple, and inhibits the binding of MdICE1L to the promoters of MdCBF1/3 and thus their expression. We showed that MdCAX3L-2, a CAX (Ca2+/H+ exchanger) family gene that negatively regulates plant cold tolerance, is also a direct target of MdbHLH4. MdbHLH4 reduced apple cold tolerance by promoting MdCAX3L-2 expression. Moreover, overexpression of either MdCAX3L-2 or MdbHLH4 promoted the cold-induced ubiquitination and degradation of MdICE1L. Overall, our results reveal that MdbHLH4 negatively regulates plant cold tolerance by inhibiting MdCBF1/3 expression and MdICE1L promoter-binding activity, as well as by promoting MdCAX3L-2 expression and cold-induced MdICE1L degradation. These findings provide insights into the mechanisms by which ICE1-interacting TFs regulate CBF expression and ICE1 function and thus plant cold tolerance.

Abiotic stresses are increasingly harmful to crop yield and quality. Calcium and its signaling pathway play an important role in modulating plant stress tolerance. As specific Ca2+ sensors, calcineurin B-like (CBL) proteins play vital roles in plant stress response and calcium signaling. The CBL family has been identified in many plant species; however, the characterization of the CBL family and the functional study of apple MdCBL proteins in salt response have yet to be conducted in apple. In this study, 11 MdCBL genes were identified from the apple genome. The coding sequences of these MdCBL genes were cloned, and the gene structure and conserved motifs were analyzed in detail. The phylogenetic analysis indicated that these MdCBL proteins could be divided into four groups. The functional identification in Na+-sensitive yeast mutant showed that the overexpression of seven MdCBL genes could confer enhanced salt stress resistance in transgenic yeast. The function of MdCBL10.1 in regulating salt tolerance was also verified in cisgenic apple calli and apple plants. These results provided valuable insights for future research examining the function and mechanism of CBL proteins in regulating apple salt tolerance.

SummaryLow temperature is the main environmental factor affecting the yield, quality and geographical distribution of crops, which significantly restricts development of the fruit industry. The NAC (NAM, ATAF1/2 and CUC2) transcription factor (TF) family is involved in regulating plant cold tolerance, but the mechanisms underlying these regulatory processes remain unclear. Here, the NAC TF MdNAC104 played a positive role in modulating apple cold tolerance. Under cold stress, MdNAC104‐overexpressing transgenic plants exhibited less ion leakage and lower ROS (reactive oxygen species) accumulation, but higher contents of osmoregulatory substances and activities of antioxidant enzymes. Transcriptional regulation analysis showed that MdNAC104 directly bound to the MdCBF1 and MdCBF3 promoters to promote expression. In addition, based on combined transcriptomic and metabolomic analyses, as well as promoter binding and transcriptional regulation analyses, we found that MdNAC104 stimulated the accumulation of anthocyanin under cold conditions by upregulating the expression of anthocyanin synthesis‐related genes, including MdCHS‐b, MdCHI‐a, MdF3H‐a and MdANS‐b, and increased the activities of the antioxidant enzymes by promoting the expression of the antioxidant enzyme‐encoding genes MdFSD2 and MdPRXR1.1. In conclusion, this study revealed the MdNAC104 regulatory mechanism of cold tolerance in apple via CBF‐dependent and CBF‐independent pathways.

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SUMMARYSalt stress adversely affects the yield and quality of crops and limits their geographical distribution. Studying the functions and regulatory mechanisms of key genes in the salt stress response is important for breeding crops with enhanced stress resistance. Autophagy plays an important role in modulating the tolerance of plants to various types of abiotic stressors. However, the mechanisms underlying salt‐induced autophagy are largely unknown. Cation/Ca2+ exchanger proteins enhance apple salt tolerance by inhibiting Na+ accumulation but the mechanism underlying the response to salt stress remains unclear. Here, we show that the autophagy‐related gene MdATG18a modulated apple salt tolerance. Under salt stress, the autophagic activity, proline content, and antioxidant enzyme activities were higher and Na+ accumulation was lower in MdATG18a‐overexpressing transgenic plants than in control plants. The use of an autophagy inhibitor during the salt treatment demonstrated that the regulatory function of MdATG18a depended on autophagy. The yeast‐one‐hybrid assay revealed that the homeodomain‐leucine zipper (HD‐Zip) transcription factor MdHB7‐like directly bound to the MdATG18a promoter. Transcriptional regulation and genetic analyses showed that MdHB7‐like enhanced salt‐induced autophagic activity by promoting MdATG18a expression. The analysis of Na+ efflux rate in transgenic yeast indicated that MdCCX1 expression significantly promoted Na+ efflux. Promoter binding, transcriptional regulation, and genetic analyses showed that MdHB7‐like promoted Na+ efflux and apple salt tolerance by directly promoting MdCCX1 expression, which was independent of the autophagy pathway. Overall, our findings provide insight into the mechanism underlying MdHB7‐like‐mediated salt tolerance in apple through the MdHB7‐like‐MdATG18a and MdHB7‐like‐MdCCX1 modules. These results will aid future studies on the mechanisms underlying stress‐induced autophagy and the regulation of stress tolerance in plants.