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

Abstract

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.

Acknowledgements

This work was supported financially by the Natural Science Foundation of Zhejiang Province (LZ22C150001) and the China Postdoctoral Science Foundation (2019 M652064).

Author contributions

H.Z. and G.H. performed all the experiments. J.W. and J.X. participated in the bioinformatics analyses and element measurements. Y.H., L.H., and S.Z. were involved in manuscript editing. H.Z., J.Y., and W.C. conceived the research, analyzed the data, and wrote the article. All authors read and approved the final manuscript.

Data availability

Tomato seeds of AC and MET1-RNAi lines were available in our laboratory. WGBS and RNA-seq datasets have been deposited in NCBI under accession numbers PRJNA813707 (WGBS), PRJNA813389 (12-hour RNA-seq), and PRJNA681103 (72-hour RNA-seq).

Conflict of interest

The authors declare that they have no conflicts of interest.

References

[1] Briat JF, Dubos C, Gaymard F . Iron nutrition, biomass production, and plant product quality. Trends Plant Sci. 2015; 20: 33-40.

[2] Connorton JM, Balk J, Rodriguez-Celma J . Iron homeostasis in plants - a brief overview. Metallomics. 2019; 9: 813-23.

[3] Robinson NJ, Procter CM, Connolly EL et al. A ferric-chelate reductase for iron uptake from soils. Nature. 1999; 397: 694-7.

[4] Vert G, Grotz N, Dedaldechamp F et al. IRT1, an Arabidopsis transporter essential for iron uptake from the soil and for plant growth . Plant Cell. 2002; 14: 1223-33.

[5] Kobayashi T, Nozoye T, Nishizawa NK . Iron transport and its regulation in plants. Free Radic Biol Med. 2019; 133: 11-20.

[6] Riaz N, Guerinot ML . All together now: regulation of the iron deficiency response. J Exp Bot. 2021; 72: 2045-55.

[7] Yuan YX, Wu HL, Wang N et al. FIT interacts with AtbHLH38 and AtbHLH39 in regulating iron uptake gene expression for iron homeostasis in Arabidopsis. Cell Res. 2008; 18: 385-97.

[8] Wang N, Cui Y, Liu Y et al. Requirement and functional redundancy of Ib subgroup bHLH proteins for iron deficiency responses and uptake in Arabidopsis thaliana. Mol Plant. 2013; 6: 503-13.

[9] Schwarz B, Bauer P . FIT, a regulatory hub for iron deficiency and stress signaling in roots, and FIT-dependent and -independent gene signatures. J Exp Bot. 2020; 71: 1694-705.

[10] Long TA, Tsukagoshi H, Busch W et al. The bHLH transcription factor POPEYE regulates response to iron deficiency in Arabidopsis roots . Plant Cell. 2010; 22: 2219-36.

[11] Zhang J, Liu B, Li MS et al. The bHLH transcription factor bHLH104 interacts with IAA-LEUCINE RESISTANT3 and modulates iron homeostasis in Arabidopsis. Plant Cell. 2015; 27: 787-805.

[12] Liang G, Zhang H, Li X et al. bHLH transcription factor bHLH115 regulates iron homeostasis in Arabidopsis thaliana. J Exp Bot. 2017; 68: 1743-55.

[13] Kim SA, LaCroix IS, Gerber SA et al. The iron deficiency response in Arabidopsis thaliana requires the phosphorylated transcription factor URI . Proc Natl Acad Sci USA. 2019; 116: 24933-42.

[14] Gao F, Robe K, Bettembourg M et al. The transcription factor bHLH121 interacts with bHLH105 (ILR3) and its closest homologs to regulate iron homeostasis in Arabidopsis. Plant Cell. 2020; 32: 508-24.

[15] Lei RH, Li Y, Cai YR et al. bHLH121 functions as a direct link that facilitates the activation of FIT by bHLH IVc transcription factors for maintaining Fe homeostasis in Arabidopsis. Mol Plant. 2020; 13: 634-49.

[16] Rodríguez-Celma J, Connorton JM, Kruse I et al. Arabidopsis BRUTUS-LIKE E3 ligases negatively regulate iron uptake by targeting transcription factor FIT for recycling . Proc Natl Acad Sci USA. 2019; 116: 17584-91.

[17] Chang YN, Zhu C, Jiang J et al. Epigenetic regulation in plant abiotic stress responses. J Integr Plant Biol. 2020; 62: 563-80.

[18] Chen X, Schoenberger B, Menz J et al. Plasticity of DNA methylation and gene expression under zinc deficiency in Arabidopsis roots . Plant Cell Physiol. 2018; 59: 1790-802.

[19] Yong-Villalobos L, González-Morales SI, Wrobel K et al. Methylome analysis reveals an important role for epigenetic changes in the regulation of the Arabidopsis response to phosphate starvation . Proc Natl Acad Sci USA. 2015; 112: E7293-302.

[20] Secco D, Wang C, Shou HX et al. Stress induced gene expression drives transient DNA methylation changes at adjacent repetitive elements. eLife. 2015; 4: e09343.

[21] Xu JM, Wang ZQ, Wang JY et al. Low phosphate represses histone deacetylase complex1 to regulate root system architecture remodeling in Arabidopsis. New Phytol. 2020; 225: 1732-45.

[22] Widiez T, Kafafi ESE, Girin T et al. High nitrogen insensitive 9 (HNI9)-mediated systemic repression of root NO3-uptake is associated with changes in histone methylation. Proc Natl Acad Sci USA. 2011; 108: 13329-34.

[23] Li Y, Mukherjee I, Thum KE et al. The histone methyltransferase SDG8 mediates the epigenetic modification of light and carbon responsive genes in plants. Genome Biol. 2015; 16: 79.

[24] Wu K, Wang S, Song W et al. Enhanced sustainable green revolution yield via nitrogen-responsive chromatin modulation in rice. Science. 2020; 367: eaaz2046.

[25] Mager S, Schonberger B, Ludewig U et al. The transcriptome of zinc deficient maize roots and its relationship to DNA methylation loss. BMC Plant Biol. 2018; 18: 372.

[26] Chandrika NNP, Sundaravelpandian K, Yu SM et al. ALFIN-LIKE 6 is involved in root hair elongation during phosphate deficiency in Arabidopsis. New Phytol. 2013; 198: 709-20.

[27] Chen CY, Wu K, Schmidt W et al. The histone deacetylase HDA19 controls root cell elongation and modulates a subset of phosphate starvation responses in Arabidopsis. Sci Rep. 2015; 5: 15708.

[28] Fan HJ, Zhang ZL, Wang N et al. SKB1/PRMT5-mediated histone H4R3 dimethylation of Ib subgroup bHLH genes negatively regulates iron homeostasis in Arabidopsis thaliana. Plant J. 2014; 77: 209-21.

[29] Xing JW, Wang TY, Liu ZS et al. GENERAL CONTROL NON-REPRESSED PROTEIN5-mediated histone acetylation of FERRIC REDUCTASE DEFECTIVE3 contributes to iron homeostasis in Arabidopsis. Plant Physiol. 2015; 168: 1309-20.

[30] Park EY, Tsuyuki KM, Hu FL et al. PRC2-mediated H3K27me3 contributes to transcriptional regulation of FIT-dependent iron deficiency response. Front Plant Sci. 2019; 10: 627.

[31] Singh S, Kailasam S, Lo JC et al. Histone H3 lysine4 trimethylation-regulated GRF11 expression is essential for the iron-deficiency response in Arabidopsis thaliana. New Phytol. 2021; 230: 244-58.

[32] Bocchini M, Bartucca ML, Ciancaleoni S et al. Iron deficiency in barley plants: phytosiderophore release, iron translocation, and DNA methylation. Front Plant Sci. 2015; 6: 514.

[33] Sun S, Zhu JM, Guo RZ et al. DNA methylation is involved in acclimation to iron-deficiency in rice (Oryza sativa). Plant J. 2021; 107: 727-39.

[34] Jin CW, Du ST, Chen WW et al. Elevated carbon dioxide improves plant iron nutrition through enhancing the iron-deficiency-induced responses under iron-limited conditions in tomato. Plant Physiol. 2009; 150: 272-80.

[35] Zhu HH, Wang JY, Jiang D et al. The miR157-SPL-CNR module acts upstream of bHLH101 to negatively regulate iron deficiency responses in tomato. J Integr Plant Biol. 2022; 64: 1059-75.

[36] Yao MQ, Chen WW, Kong JH et al. METHYLTRANSFERASE1 and ripening modulate vivipary during tomato fruit development . Plant Physiol. 2020; 183: 1883-97.

[37] Law JA, Jacobsen SE . Establishing, maintaining and modifying DNA methylation patterns in plants and animals. Nat Rev Genet. 2010; 11: 204-20.

[38] Sudan J, Raina M, Singh R . Plant epigenetic mechanisms: role in abiotic stress and their generational heritability. 3 Biotech. 2018; 8: 172.

[39] Chen WW, Zhu HH, Wang JY et al. Comparative physiological and transcriptomic analyses reveal altered Fe-deficiency responses in tomato epimutant Colorless non-ripening. Front Plant Sci. 2022; 12: 796893.

[40] Kou HP, Li Y, Song XX et al. Heritable alteration in DNA methylation induced by nitrogen-deficiency stress accompanies enhanced tolerance by progenies to the stress in rice (Oryza sativa L.). J Plant Physiol. 2011; 168: 1685-93.

[41] Fan SK, Ye JY, Zhang LL et al. Inhibition of DNA demethylation enhances plant tolerance to cadmium toxicity by improving iron nutrition. Plant Cell Environ. 2020; 43: 275-91.

[42] Tian P, Lin ZT, Lin DB et al. The pattern of DNA methylation alteration, and its association with the changes of gene expression and alternative splicing during phosphate starvation in tomato. Plant J. 2021; 108: 841-58.

[43] He L, Huang H, Bradai M et al. DNA methylation-free Arabidopsis reveals crucial roles of DNA methylation in regulating gene expression and development . Nat Commun. 2022; 13: 1335.

[44] Dowen RH, Pelizzola M, Schmitz RJ et al. Widespread dynamic DNA methylation in response to biotic stress. Proc Natl Acad Sci USA. 2012; 109: E2183-91.

[45] Meng DZ, Dubin M, Zhang P et al. Limited contribution of DNA methylation variation to expression regulation in Arabidopsis thaliana. PLoS Genet. 2016; 12: e1006141.

[46] Yen MR, Suen DF, Hsu FM et al. Deubiquitinating enzyme OTU5 contributes to DNA methylation patterns and is critical for phosphate nutrition signals. Plant Physiol. 2017; 175: 1826-38.

[47] Zhang XY, Yazaki J, Sundaresan A et al. Genome-wide high-resolution mapping and functional analysis of DNA methylation in Arabidopsis. Cell. 2006; 126: 1189-201.

[48] Wang Y, Zuo L, Wei T et al. CHH methylation of genes associated with fatty acid and jasmonate biosynthesis contributes to cold tolerance in autotetraploids of Poncirus trifoliata. J Integr Plant Biol. 2022; 64: 2327-43.

[49] Zhang HM, Lang ZB, Zhu JK . Dynamics and function of DNA methylation in plants. Nat Rev Mol Cell Biol. 2018; 19: 489-506.

[50] Srikant T, Yuan W, Berendzen KW et al. Canalization of genome-wide transcriptional activity in Arabidopsis thaliana accessions by MET1-dependent CG methylation . Genome Biol. 2022; 23: 263.