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Journal of Integrative Agriculture  2025, Vol. 24 Issue (6): 2229-2239    DOI: 10.1016/j.jia.2024.11.033
Horticulture Advanced Online Publication | Current Issue | Archive | Adv Search |
MdERF2 regulates cuticle wax formation by directly activating MdLACS2, MdCER1 and MdCER6 of apple fruit during postharvest

Xinyue Zhang*, Xinhua Zhang*, Wenwen Sun, Meng Lv, Yefei Gu, Sarfaraz Hussain, Xiaoan Li, Maratab Ali, Fujun Li#

College of Agricultural Engineering and Food Science, Shandong University of Technology, Zibo 255000, China
 Highlights 
MdERF2 positively regulated MdLACS2, MdCER1, MdCER4 and MdCER6 expression levels.  
MdERF2 directly binds to the promoters of the MdLACS2, MdCER1 and MdCER6 genes.
MdERF2 altered apple fruit cuticular wax components, contents and morphology.
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摘要  

乙烯转录因子2 (ERF2)是植物生长,果实成熟、代谢和抵御胁迫所必需的。为了深入了解MdERF2在控制苹果表皮蜡质合成中的功能,本文利用MdERF2超表达(ERF2-OE)和沉默(ERF2-AN)载体,通过侵染苹果果实和/或愈伤组织,研究了蜡质合成有关基因表达,蜡质组成、含量和超微结构的变化,并利用凝胶电泳迁移实验(EMSAs)和双荧光素酶报告基因分析(DLRs)技术,鉴定了MdERF2直接调控的蜡质合成相关的基因。结果表明,EFR2-OE上调了MdLACS2MdCER1MdCER4MdCER6等蜡质合成相关基因表达,而MdERF2沉默则抑制了上述基因的表达。同时,不同的MdERF2表达水平,还影响了蜡质的结构和积累。ERF2-OE处理显著增加了蜡质中烷烃和酮的比例,减少了脂肪酸和酯的比例。此外,EMSAsDLRs实验还证明,MdERF可以与MdLACS2MdCER1MdCER6启动子部位的GCC-box元件直接结合,激活他们的转录水平。上述结果证明,MdERF2可以靶向上调MdLACS2MdCER1MdCER6基因的表达,从而改变了苹果表皮蜡质的组成、含量和微观结构。



Abstract  

Ethylene response factors 2 (ERF2) are essential for plant growth, fruit ripening, metabolism, and resistance to stress.  In this study, the expression levels of the genes for MdERF2 implicated in the biosynthesis, composition and ultrastructure of fruit cuticular wax in apple (Malus domestica) were studied by the transfection of apple fruit and/or calli with MdERF2-overexpression (ERF2-OE) and MdERF2-interference (ERF2-AN) vectors.  In addition, the direct target genes of MdERF2 related to wax biosynthesis were identified using electrophoretic mobility shift assays (EMSAs) and dual-luciferase reporter (DLR) assays.  The findings indicated that the expression levels of four wax biosynthetic genes, long-chain acyl-CoA synthetase 2 (MdLACS2), eceriferum 1 (MdCER1), eceriferum 4 (MdCER4), and eceriferum 6 (MdCER6), were upregulated by ERF2-OE.  In contrast, the expression levels of these genes were inhibited when MdERF2 was silenced.  Furthermore, the overall structure and accumulation of fruit cuticular wax were influenced by the expression level of MdERF2.  Treatment with ERF2-OE significantly increased the proportions of alkanes and ketones and reduced the proportions of fatty acids and esters.  In addition, the EMSAs and DLR assays demonstrated that MdERF2 could bind directly to GCC-box elements in the promoters of MdLACS2, MdCER1, and MdCER6 to activate their transcription.  These results confirmed that MdERF2 targets the up-regulation of expression of the MdLACS2, MdCER1, and MdCER6 genes, thereby altering the composition, content, and microstructure of apple epidermal wax.


Keywords:  apple fruit       wax        ethylene response factor 2        target genes  
Received: 23 January 2024   Online: 26 November 2024   Accepted: 11 June 2024
Fund: 
The study was supported by the National Natural Science Foundation of China (32272384) and the Natural Science Foundation of Shandong Province, China (ZR2020MC149).  
About author:  Xinyue Zhang, E-mail: zhangxinyueyy0807@163.com; Xinhua Zhang, E-mail: zxh@sdut.edu.cn; #Correspondence Fujun Li, E-mail: lifujun@sdut.edu.cn *These authors contributed equally to this study.

Cite this article: 

Xinyue Zhang, Xinhua Zhang, Wenwen Sun, Meng Lv, Yefei Gu, Sarfaraz Hussain, Xiaoan Li, Maratab Ali, Fujun Li. 2025. MdERF2 regulates cuticle wax formation by directly activating MdLACS2, MdCER1 and MdCER6 of apple fruit during postharvest. Journal of Integrative Agriculture, 24(6): 2229-2239.

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