CG hypermethylation of the bHLH39 promoter regulates its expression and Fe deficiency responses in tomato roots

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.

Automated summary

Iron (Fe) is an essential micronutrient for all organisms. However, its limited bioavailability restricts plant growth, yield, and nutritional quality. The transcriptional regulation of plant responses to Fe deficiency has been extensively studied, but the contribution of DNA methylation remains poorly understood. In this study, it was found that DNA methylation plays a role in regulating Fe deficiency responses in tomato roots. Dynamic changes in the DNA methylome in response to Fe deficiency were identified, with differentially methylated regions (DMRs) occurring at different sites depending on the duration of Fe deficiency. Furthermore, the changes in DNA methylation levels did not correlate with the changes in gene expression levels, except for the CG hypermethylation at the bHLH39 promoter, which was positively correlated with its transcriptional induction. The involvement of bHLH39 in the modulation of Fe homeostasis was confirmed through gene silencing and luciferase reporter assays. Overall, these findings suggest that dynamic epigenetic DNA methylation at the CG context at the bHLH39 promoter contributes to the Fe deficiency response in tomato.