Shen Xiaoxia, Ping Yikun, Bao Chana, Liu Chen, Tahir Muhammad Mobeen, Li Xuewei, Song Yi, Xu Weirong, Ma Fengwang, Guan Qingmei
State Key Laboratory of Crop Stress Biology for Arid Areas/Shaanxi Key Laboratory of Apple, College of Horticulture, Northwest A&F University, Yangling, Shaanxi, 712100, China.
Ningxia Engineering and Technology Research Center of Grape and Wine, Ningxia University, Yinchuan, 750021, Ningxia, China.
Plant J. 2023 Apr;114(2):262-278. doi: 10.1111/tpj.16132. Epub 2023 Feb 20.
Apple (Malus domestica) trees are vulnerable to freezing temperatures. Cold resistance in woody perennial plants can be improved through biotechnological approaches. However, genetic engineering requires a thorough understanding of the molecular mechanisms of the tree's response to cold. In this study, we demonstrated that the Mdm-miR160-MdARF17-MdWRKY33 module is crucial for apple freezing tolerance. Mdm-miR160 plays a negative role in apple freezing tolerance, whereas MdARF17, one of the targets of Mdm-miR160, is a positive regulator of apple freezing tolerance. RNA sequencing analysis revealed that in apple, MdARF17 mediates the cold response by influencing the expression of cold-responsive genes. EMSA and ChIP-qPCR assays demonstrated that MdARF17 can bind to the promoter of MdWRKY33 and promotes its expression. Overexpression of MdWRKY33 enhanced the cold tolerance of the apple calli. In addition, we found that the Mdm-miR160-MdARF17-MdWRKY33 module regulates cold tolerance in apple by regulating reactive oxygen species (ROS) scavenging, as revealed by (i) increased H O levels and decreased peroxidase (POD) and catalase (CAT) activities in Mdm-miR160e OE plants and MdARF17 RNAi plants and (ii) decreased H O levels and increased POD and CAT activities in MdmARF17 OE plants and MdWRKY33 OE calli. Taken together, our study uncovered the molecular roles of the Mdm-miR160-MdARF17-MdWRKY33 module in freezing tolerance in apple, thus providing support for breeding of cold-tolerant apple cultivars.
苹果（Malus domestica）树易受低温影响。木本多年生植物的抗寒性可通过生物技术方法提高。然而，基因工程需要深入了解树木对寒冷反应的分子机制。在本研究中，我们证明了Mdm-miR160-MdARF17-MdWRKY33模块对苹果的抗冻性至关重要。Mdm-miR160在苹果抗冻性中起负作用，而Mdm-miR160的靶标之一MdARF17是苹果抗冻性的正调节因子。RNA测序分析表明，在苹果中，MdARF17通过影响冷响应基因的表达来介导冷反应。EMSA和ChIP-qPCR分析表明，MdARF17可与MdWRKY33的启动子结合并促进其表达。MdWRKY33的过表达增强了苹果愈伤组织的耐寒性。此外，我们发现Mdm-miR160-MdARF17-MdWRKY33模块通过调节活性氧（ROS）清除来调节苹果的耐寒性，这表现为：（i）Mdm-miR160e OE植物和MdARF17 RNAi植物中H₂O水平升高，过氧化物酶（POD）和过氧化氢酶（CAT）活性降低；（ii）MdmARF17 OE植物和MdWRKY33 OE愈伤组织中H₂O水平降低，POD和CAT活性升高。综上所述，我们的研究揭示了Mdm-miR160-MdARF17-MdWRKY33模块在苹果抗冻性中的分子作用，从而为耐寒苹果品种的育种提供了支持。
