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SUMMARY: Cold stress has always been a major abiotic factor affecting the yield and quality of temperate fruit crops. Ethylene plays a critical regulatory role in the cold stress response, but the underlying molecular mechanisms remain elusive. Here, we revealed that ethylene positively modulates apple responses to cold stress. Treatment with 1‐aminocyclopropane‐1‐carboxylate (an ethylene precursor) and aminoethoxyvinylglycine (an ethylene biosynthesis inhibitor) respectively increased and decreased the cold tolerance of apple seedlings. Consistent with the positive effects of ethylene on cold stress responses, a low‐temperature treatment rapidly induced ethylene release and the expression of MdERF1B, which encodes an ethylene signaling activator, in apple seedlings. Overexpression of MdERF1B significantly increased the cold tolerance of apple plant materials (seedlings and calli) and Arabidopsis thaliana seedlings. A quantitative real‐time PCR analysis indicated that MdERF1B upregulates the expression of the cold‐responsive gene MdCBF1 in apple seedlings. Moreover, MdCIbHLH1, which functions upstream of CBF‐dependent pathways, enhanced the binding of MdERF1B to target gene promoters as well as the consequent transcriptional activation. The stability of MdERF1B–MdCIbHLH1 was affected by cold stress and ethylene. Furthermore, MdERF1B interacted with the promoters of two genes critical for ethylene biosynthesis, MdACO1 and MdERF3. The resulting upregulated expression of these genes promoted ethylene production. However, the downregulated MdCIbHLH1 expression in MdERF1B‐overexpressing apple calli significantly inhibited ethylene production. These findings imply that MdERF1B–MdCIbHLH1 is a potential regulatory module that integrates the cold and ethylene signaling pathways in apple. Significance Statement: Here, we show that the ethylene response factor MdERF1B acting in the CBF/DREB1 signaling pathway was upregulated by cold treatment. Further investigation revealed that MdCIbHLH1, which functions upstream of CBF‐regulated pathways, is essential for MdERF1B‐mediated cold tolerance and ethylene biosynthesis. [ABSTRACT FROM AUTHOR]

Summary: In plants, flavonoids play critical roles in resistance to biotic and abiotic stresses, and contribute substantially to the quality, flavor, and nutritional quality of many fruit crops. In apple (Malus × domestica), inbreeding has resulted in severe decreases in the genetic diversity and flavonoid content. Over the last decade, we have focused on the genetic improvement of apple using wild red‐fleshed apple resources (Malus sieversii f. niedzwetzkyana). Here, we found that the MYB transcription factors (TFs) involved in the synthesis of proanthocyanidins can be classified into TT2 and PA1 types. We characterized a PA1‐type MYB transcription factor, MdMYBPA1, from red‐fleshed apple and identified its role in flavonoid biosynthesis using overexpression and knockdown‐expression transgenes in apple calli. We explored the relationship between TT2‐ and PA1‐type MYB TFs, and found that MdMYB9/11/12 bind the MdMYBPA1 promoter. In addition, MdMYBPA1 responded to low temperature by redirecting the flavonoid biosynthetic pathway from proanthocyanidin to anthocyanin production. In binding analyses, MdbHLH33 directly bound to the low‐temperature‐responsive (LTR) cis‐element of the MdMYBPA1 promoter and promotes its activity. In addition, the calli expressing both MdMYBPA1 and MdbHLH33, which together form a complex, produced more anthocyanin under low temperature. Our findings shed light on the essential roles of PA1‐type TFs in the metabolic network of proanthocyanidin and anthocyanin synthesis in plants. Studies on red‐fleshed wild apple are beneficial for breeding new apple varieties with high flavonoid contents. [ABSTRACT FROM AUTHOR]