In fruit of the white-fleshed peach cultivar 'Zhonghuashoutao', postharvest storage at 16°C induces anthocyanin accumulation in the flesh, while storage at 12°C or colder (or 20°C) does not. Zhu et al. (2020) showed that the permissive temperature coincides with reduced promoter methylation of anthocyanin biosynthetic genes, with transcript levels of DNA methyltransferase and demethylase genes correlating with bulk methylation, and that the methylation inhibitor 5-azacytidine can induce flesh pigmentation. What remains unresolved is the mechanistic chain: how storage temperature is sensed and transduced to the DNA methylation machinery and the anthocyanin program, why 16°C is permissive while colder temperatures are not, and whether locus-specific promoter demethylation is a cause, a consequence, or part of a feedback loop during the onset of pigmentation. A 2024 follow-up from the same group identified the transcription factors PpBBX32 and PpZAT5 (with jasmonates) as upstream activators of PpMYB10.1, resolving the transcriptional layer but leaving the epigenetic sensing and causality questions open.

Background

Anthocyanins are red-to-purple flavonoid pigments that accumulate in many plant tissues; in fruit they are major determinants of appearance and nutritional quality. Their biosynthesis proceeds through the phenylpropanoid/flavonoid pathway (genes such as PAL, C4H, 4CL, F3H, F3'H, DFR, ANS, UFGT), is transcriptionally coordinated by MYB–bHLH–WD40 ('MBW') transcription factor complexes, and requires a glutathione S-transferase transporter (in peach, PpGST1) to move the pigment into the vacuole. In peach flesh, the R2R3 MYB transcription factor PpMYB10.1 is the key activator of this program. In many plants, low temperature induces anthocyanin biosynthesis, so pigmentation is generally expected at colder, not milder, temperatures.

DNA methylation is the addition of a methyl group to the 5-position of cytosine (5-methylcytosine), a heritable epigenetic mark that in plants occurs in three sequence contexts (CG, CHG, and CHH, where H is any base other than G) and generally represses transcription when concentrated in gene promoters. Methylation is established and maintained by methyltransferases (in peach, homologs of the DRM (domains rearranged methyltransferase) and CMT (chromomethylase) family enzymes) and actively removed by demethylases of the ROS1/DML family (repressor-of-silencing 1 and demeter-like 5-methylcytosine glycosylases). Whole-genome bisulfite sequencing (WGBS) is the standard method for measuring methylation at single-cytosine resolution; 5-azacytidine is a chemical inhibitor of DNA methylation commonly used to test whether demethylation is sufficient for a response.

Zhu et al. (2020, The Plant Journal 102:965–976) studied 'Zhonghuashoutao', a normally white-fleshed peach whose flesh turns red only when fruit are stored at 16°C after harvest — not at 0, 5, 8, or 12°C, and not at 20°C. At 16°C, transcripts of anthocyanin biosynthetic genes, PpGST1, and regulators including PpMYB10.1 were elevated, and the promoters of the biosynthetic genes (PpPAL1/2, PpC4H, Pp4CL4/5/8, PpF3H, PpF3'H, PpDFR1/2/3, PpANS) and of the transcription factor gene PpbHLH3 showed lower methylation, mainly in the CHH context. Transcript levels of the methyltransferase genes PpDRM1, PpDRM2, and PpCMT2 and of the demethylase gene PpDML1 correlated strongly with bulk methylation levels. Injecting 5-azacytidine into the flesh of a different white-fleshed cultivar ('Xinyu') at 25°C induced local anthocyanin accumulation, suggesting hypomethylation can be sufficient. However, the evidence was correlative: methylation and expression were measured at a single 45-day endpoint, tissue was bulked across heterogeneous flesh zones, and the inhibitor test was performed in a different cultivar at a different temperature on a short time scale. Notably, global cytosine methylation was lowest at 0°C — yet no anthocyanin formed there — so general hypomethylation does not explain the phenotype; the permissive-temperature logic must operate at the level of specific loci and upstream signaling. The authors explicitly hypothesized that mechanisms other than promoter demethylation must account for the temperature-dependent induction of PpMYB10.1 and PpGST1, whose expression does not track promoter methylation the way the structural genes do.

Subsequent work has clarified parts of the picture without closing the mechanistic gap. Huang et al. (2024, Horticulture Research 11:uhae212), from the same laboratory, identified the B-box transcription factor PpBBX32 and the C2H2 zinc-finger transcription factor PpZAT5 as direct upstream activators of PpMYB10.1 in 'Zhonghuashoutao' flesh, forming a PpZAT5–PpBBX32–PpMYB10.1 complex, with jasmonate hormones favoring anthocyanin accumulation and correlating with PpBBX32/PpZAT5 expression; light-signaling regulators (PpHY5, PpBBX4, PpBBX10) were excluded because their expression was highest at colder temperatures where no anthocyanin forms. In apple, a related Rosaceae fruit, Yu et al. (2022, Horticulture Research 9:uhac007) showed that low temperature induces the demethylase MdROS1, which binds and demethylates promoters of anthocyanin-associated genes and is required for low-temperature anthocyanin accumulation — establishing demethylation-based causality in apple but leaving the upstream sensing mechanism unexplained and not addressing the peach system with its narrow permissive window. In peach ripening (a different process), Cao et al. (2024, Plant Physiology 194:2049–2068) demonstrated a positive feedback loop between the transcription factor PpNAC1 and the demethylase PpDML1, showing that transcription-factor–demethylase feedback architectures exist in peach. Reviews through 2025–2026 on peach anthocyanin regulation and on epigenetic regulation of fruit anthocyanin biosynthesis continue to cite the 2020 result as the state of the art, without a resolution of the temperature-sensing or causality questions.

Problem Statement

Identify the mechanism by which postharvest storage temperature is sensed and transduced to the DNA methylation machinery and the anthocyanin biosynthetic program in 'Zhonghuashoutao' peach flesh, and determine the causal role of temperature-dependent promoter demethylation in the onset of anthocyanin accumulation. Concretely: (i) what upstream temperature-sensing or signaling mechanism distinguishes the permissive 16°C condition from non-permissive temperatures (12°C and below, and 20°C), given that colder storage produces even lower global cytosine methylation yet no pigmentation; (ii) how is the temperature signal conveyed to the methyltransferase genes (PpDRM1, PpDRM2, PpCMT2) and the demethylase gene PpDML1 whose transcript levels correlate with the observed methylation changes, and to the promoter demethylation of the anthocyanin biosynthetic genes; (iii) is the locus-specific promoter demethylation a cause of transcriptional activation, a consequence of it, or part of a feedback loop; and (iv) which additional mechanisms, beyond the now-identified PpBBX32/PpZAT5/jasmonate transcriptional layer, account for the temperature-dependent induction of PpMYB10.1 and PpGST1, whose expression does not track their promoter methylation. The question concerns the peach postharvest system in which the phenomenon was discovered, and its generality across peach cultivars and storage regimes is part of the question.

Sub-questions (i)–(iv) are distinct, jointly posed aspects of this mechanism. A sub-question is resolved once a candidate component or mechanism is named and shown to discriminate the permissive 16°C condition from the non-permissive conditions at the loci it concerns.

References

  1. Zhu Y-C, Zhang B, Allan AC, Lin-Wang K, Zhao Y, Wang K, Chen K-S, Xu C-J (2020) DNA demethylation is involved in the regulation of temperature-dependent anthocyanin accumulation in peach. The Plant Journal 102(5):965–976. doi:10.1111/tpj.14680, https://doi.org/10.1111/tpj.14680
  2. Huang D, Xue L, Lu Y, Liu M, Lin-Wang K, Allan AC, Zhang B, Chen K, Xu C (2024) PpBBX32 and PpZAT5 modulate temperature-dependent and tissue-specific anthocyanin accumulation in peach fruit. Horticulture Research 11(10):uhae212. doi:10.1093/hr/uhae212, https://doi.org/10.1093/hr/uhae212
  3. Yu L, Sun Y, Zhang X, Chen M, Wu T, Zhang J, Xing Y, Tian J, Yao Y (2022) ROS1 promotes low temperature-induced anthocyanin accumulation in apple by demethylating the promoter of anthocyanin-associated genes. Horticulture Research 9:uhac007. doi:10.1093/hr/uhac007, https://doi.org/10.1093/hr/uhac007
  4. Cao X, Li X, Su Y, Zhang C, Wei C, Chen K, Grierson D, Zhang B (2024) Transcription factor PpNAC1 and DNA demethylase PpDML1 synergistically regulate peach fruit ripening. Plant Physiology 194(4):2049–2068. doi:10.1093/plphys/kiad627, https://doi.org/10.1093/plphys/kiad627
  5. Zhong S, Zhu H, Li W, Wu D, Miao Y, Dong B, Wang Y, Xiao Z, Fang Q, Deng J, Zhao H (2024) DNA methylome analysis reveals novel insights into active hypomethylated regulatory mechanisms of temperature-dependent flower opening in Osmanthus fragrans. Horticulture Research 11(3):uhae010. doi:10.1093/hr/uhae010, https://doi.org/10.1093/hr/uhae010
  6. Wang Y, Zhang F, Ren D-S, Zeng K, Sun Y, Wu N, Zhang Z (2026) Epigenetic regulation of fruit anthocyanin biosynthesis in horticultural crops. Horticultural Plant Journal 12(3):529–538. doi:10.1016/j.hpj.2025.11.004, https://doi.org/10.1016/j.hpj.2025.11.004
  7. Lu Y, Yang W, Huang D, Liang L, Xu C (2025) Advances in understanding of the mechanisms regulating anthocyanin accumulation in peach. Scientia Horticulturae 342:114022. doi:10.1016/j.scienta.2025.114022, https://doi.org/10.1016/j.scienta.2025.114022
  8. Zhang Y, Sun L, Chi M, Cheng X, Hu H, Zhou H, Yao M, Guo Z, Ling J, Li P (2026) Suppression of ethylene metabolism serves as a key factor underlying high temperature-induced promotion of anthocyanin synthesis of peach skin. Postharvest Biology and Technology 232:113998. doi:10.1016/j.postharvbio.2025.113998, https://doi.org/10.1016/j.postharvbio.2025.113998
  9. Hou H, Kong X, Xiao J, Jiang G, Zhu H, Jiang Y, Qu H, Li T (2026) An increase in DNA methylation contributes to anthocyanin biosynthesis in the pericarp as litchi (Litchi chinensis Sonn.) fruit ripens. Journal of Advanced Research (2026). doi:10.1016/j.jare.2026.02.004, https://doi.org/10.1016/j.jare.2026.02.004