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Article|12 May 2023|OPEN
CG hypermethylation of the bHLH39 promoter regulates its expression and Fe deficiency responses in tomato roots
Huihui Zhu1 ,† , Guanghao Han2 ,† , Jiayi Wang1 , Jiming Xu1 , Yiguo Hong2 , Li Huang3 , Shaojian Zheng1 , Jianli Yang1 , and Weiwei Chen,1,2 ,
1State Key Laboratory of Plant Physiology and Biochemistry, College of Life Sciences, Zhejiang University, Hangzhou 310058, China
2Research Centre for Plant RNA Signaling, College of Life and Environmental Sciences, Hangzhou Normal University, Hangzhou 311121, China
3Laboratory of Cell & Molecular Biology, Institute of Vegetable Science, Zhejiang University, Hangzhou 310058, China
*Corresponding author. E-mail:,
Both authors contributed equally to the study.

Horticulture Research 10,
Article number: uhad104 (2023)
Views: 122

Received: 09 Nov 2022
Accepted: 08 May 2023
Published online: 12 May 2023


Iron (Fe) is an essential micronutrient for all organisms, including plants, whose limited bioavailability restricts plant growth, yield, and nutritional quality. While the transcriptional regulation of plant responses to Fe deficiency have been extensively studied, the contribution of epigenetic modulations, such as DNA methylation, remains poorly understood. Here, we report that treatment with a DNA methylase inhibitor repressed Fe deficiency-induced responses in tomato (Solanum lycopersicum) roots, suggesting the importance of DNA methylation in regulating Fe deficiency responses. Dynamic changes in the DNA methylome in tomato roots responding to short-term (12 hours) and long-term (72 hours) Fe deficiency identified many differentially methylated regions (DMRs) and DMR-associated genes. Most DMRs occurred at CHH sites under short-term Fe deficiency, whereas they were predominant at CG sites following long-term Fe deficiency. Furthermore, no correlation was detected between the changes in DNA methylation levels and the changes in transcript levels of the affected genes under either short-term or long-term treatments. Notably, one exception was CG hypermethylation at the bHLH39 promoter, which was positively correlated with its transcriptional induction. In agreement, we detected lower CG methylation at the bHLH39 promoter and lower bHLH39 expression in MET1-RNA interference lines compared with wild-type seedlings. Virus-induced gene silencing of bHLH39 and luciferase reporter assays revealed that bHLH39 is positively involved in the modulation of Fe homeostasis. Altogether, we propose that dynamic epigenetic DNA methylation in the CG context at the bHLH39 promoter is involved in its transcriptional regulation, thus contributing to the Fe deficiency response of tomato.