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Journal of Integrative Agriculture  2023, Vol. 22 Issue (6): 1704-1719    DOI: 10.1016/j.jia.2023.04.033
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MdWRKY40is directly promotes anthocyanin accumulation and blocks MdMYB15L, the repressor of MdCBF2, which improves cold tolerance in apple

XU Peng-yue1*, XU Li1*, XU Hai-feng1, HE Xiao-wen1, HE Ping1, CHANG Yuan-sheng1, WANG Sen1, ZHENG Wen-yan1, WANG Chuan-zeng2, CHEN Xin1, LI Lin-guang1#, WANG Hai-bo1#

1 Shandong Institute of Pomology, Shandong Academy of Agricultural Sciences, Tai’an 271000, P.R.China

2 Modern Agriculture Research Institute of Yellow River Delta, Shandong Academy of Agricultural Sciences, Dongying 257000, P.R.China

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冷胁迫是影响苹果生产的重要限制因素。在本研究中,我们以苹果砧木‘M9T337’‘60-160’的组织培养幼苗为试材进行检测,发现它们对冷胁迫分别表现为抗性和敏感性。‘M9T337’‘60-160’幼苗在冷胁迫(1℃)处理48小时后,差异表达基因(DEGs)的富集途径和生理变化明显不同,表明它们对冷胁迫的反应存在差异。两个砧木幼苗WRKY转录因子差异表达分析表明MdWRKY40isMdWRKY48为潜在冷耐性调控子。在苹果愈伤中分别过表达MdWRKY40isMdWRKY48,结果发现过表达MdWRKY48的愈伤没有明显效果,而MdWRKY40is能促进花青苷积累和提高愈伤冷耐性,并促进花青苷合成结构基因MdDFR和冷信号核心基因MdCBF2的表达。酵母单杂和凝胶阻滞( EMSA )分析表明MdWRKY40is仅能结合MdDFR的启动子。酵母双杂和双分子荧光互补(BiFC)表明MdWRKY40is能通过其蛋白NLeu ZipperCBF2抑制子MdMYB15L互作。当敲除MdWRMY40is蛋白NLeu Zipper后,在愈伤中过表达发现其不能影响MdCBF2的表达水平和愈伤冷耐性,表明MdWRKY40is参与冷信号途径是通过与MdMYB15L互作来实现的。综上,MdWRKY40is能直接绑定MdDFR启动子促进花青苷积累,并通过与MdMYB15L互作,干扰其对MdCBF2抑制作用,间接促进MdCBF2表达,从而提高冷耐性。这些结果为苹果砧木抗冷机制的研究提供了新视角,为未来筛选抗寒砧木提供分子依据


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 MdWRKY48 in 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 two-hybrid screening and bimolecular fluorescence complementation showed that MdWRKY40is interacts with the CBF2 inhibitor 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.

Keywords:  MdWRKY40is        anthocyanin accumulation        MdMYB15L        MdCBF2        cold tolerance  
Received: 02 December 2022   Online: 28 April 2023   Accepted: 24 March 2023

This work was 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) and the earmarked fund for China Agriculture Research System (CARS-27).

About author:  XU Peng-yue, E-mail:; #Correspondence LI Lin-guang, E-mail:; WANG Hai-bo, E-mali: * These authors contributed equally to this study.

Cite this article: 

XU Peng-yue, XU Li, XU Hai-feng, HE Xiao-wen, HE Ping, CHANG Yuan-sheng, WANG Sen, ZHENG Wen-yan, WANG Chuan-zeng, CHEN Xin, LI Lin-guang, WANG Hai-bo. 2023.

MdWRKY40is directly promotes anthocyanin accumulation and blocks MdMYB15L, the repressor of MdCBF2, which improves cold tolerance in apple . Journal of Integrative Agriculture, 22(6): 1704-1719.

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