Cold stress is an important factor that limits apple production. In this study, we examined the tissue-cultured plantlets of apple rootstocks ‘M9T337' and ‘60-160', which are resistant and sensitive to cold ...Cold stress is an important factor that limits apple production. In this study, we examined the tissue-cultured plantlets of apple rootstocks ‘M9T337' and ‘60-160', which are resistant and sensitive to cold stress, respectively. The enriched pathways of differentially expressed genes(DEGs) and physiological changes in ‘M9T337' and ‘60-160' plantlets were clearly different after cold stress(1°C) treatment for 48 h, suggesting that they have differential responses to cold stress. The differential expression of WRKY transcription factors in the two plantlets showed that MdWRKY40is and MdWRKY48 are potential regulators of cold tolerance. When we overexpressed MdWRKY40is and MdWRKY48in apple calli, the overexpression of MdWRKY48 had no significant effect on the callus, while MdWRKY40is overexpression promoted anthocyanin accumulation, increased callus cold tolerance, and promoted the expression of anthocyanin structural gene MdDFR and cold-signaling core gene MdCBF2. Yeast one-hybrid screening and electrophoretic mobility shift assays showed that MdWRKY40is could only bind to the MdDFR promoter. Yeast twohybrid screening and bimolecular fluorescence complementation showed that MdWRKY40is interacts with the CBF2inhibitor MdMYB15L through the leucine zipper(LZ). When the LZ of MdWRMY40is was knocked out, MdWRKY40is overexpression in the callus did not affect MdCBF2 expression or callus cold tolerance, indicating that MdWRKY40is acts in the cold signaling pathway by interacting with MdMYB15L. In summary, MdWRKY40is can directly bind to the MdDFR promoter in order to promote anthocyanin accumulation, and it can also interact with MdMYB15L to interfere with its inhibitory effect on MdCBF2, indirectly promoting MdCBF2 expression, and thereby improving cold tolerance.These results provide a new perspective for the cold-resistance mechanism of apple rootstocks and a molecular basis for the screening of cold-resistant rootstocks.展开更多
基金supported by the Natural Science Foundation of Shandong Province, China (ZR2021MC045)the Key Research & Development Plan (Major Scientific and Technological Innovation Project) of Shandong Province, China (2021LZGC024)the earmarked fund for China Agriculture Research System(CARS-27)。
文摘Cold stress is an important factor that limits apple production. In this study, we examined the tissue-cultured plantlets of apple rootstocks ‘M9T337' and ‘60-160', which are resistant and sensitive to cold stress, respectively. The enriched pathways of differentially expressed genes(DEGs) and physiological changes in ‘M9T337' and ‘60-160' plantlets were clearly different after cold stress(1°C) treatment for 48 h, suggesting that they have differential responses to cold stress. The differential expression of WRKY transcription factors in the two plantlets showed that MdWRKY40is and MdWRKY48 are potential regulators of cold tolerance. When we overexpressed MdWRKY40is and MdWRKY48in apple calli, the overexpression of MdWRKY48 had no significant effect on the callus, while MdWRKY40is overexpression promoted anthocyanin accumulation, increased callus cold tolerance, and promoted the expression of anthocyanin structural gene MdDFR and cold-signaling core gene MdCBF2. Yeast one-hybrid screening and electrophoretic mobility shift assays showed that MdWRKY40is could only bind to the MdDFR promoter. Yeast twohybrid screening and bimolecular fluorescence complementation showed that MdWRKY40is interacts with the CBF2inhibitor MdMYB15L through the leucine zipper(LZ). When the LZ of MdWRMY40is was knocked out, MdWRKY40is overexpression in the callus did not affect MdCBF2 expression or callus cold tolerance, indicating that MdWRKY40is acts in the cold signaling pathway by interacting with MdMYB15L. In summary, MdWRKY40is can directly bind to the MdDFR promoter in order to promote anthocyanin accumulation, and it can also interact with MdMYB15L to interfere with its inhibitory effect on MdCBF2, indirectly promoting MdCBF2 expression, and thereby improving cold tolerance.These results provide a new perspective for the cold-resistance mechanism of apple rootstocks and a molecular basis for the screening of cold-resistant rootstocks.
