Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Redox modification of m6A demethylase SlALKBH2 in tomato regulates fruit ripening

Nature Plants volume 11, pages 218–233 (2025) Cite this article

Subjects

Abstract

Hydrogen peroxide (H2O2) functions as a critical signalling molecule in controlling multiple biological processes. How H2O2 signalling integrates with other regulatory pathways such as epigenetic modification to coordinately regulate plant development remains elusive. Here we report that SlALKBH2, an m6A demethylase required for normal ripening of tomato fruit, is sensitive to oxidative modification by H2O2, which leads to the formation of homodimers mediated by intermolecular disulfide bonds, and Cys39 serves as a key site in this process. The oxidation of SlALKBH2 promotes protein stability and facilitates its function towards the target transcripts including the pivotal ripening gene SlDML2 encoding a DNA demethylase. Furthermore, we demonstrate that the thioredoxin reductase SlNTRC interacts with SlALKBH2 and catalyses its reduction, thereby modulating m6A levels and fruit ripening. Our study establishes a molecular link between H2O2 and m6A methylation and highlights the importance of redox regulation of m6A modifiers in controlling fruit ripening.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on SpringerLink
  • Instant access to the full article PDF.

USD 39.95

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: SlALKBH2 forms homodimers through intermolecular disulfide bonds.
The alternative text for this image may have been generated using AI.
Fig. 2: SlALKBH2 is sensitive to oxidation by H2O2 in tomato fruits.
The alternative text for this image may have been generated using AI.
Fig. 3: Cys39 is a critical site for the formation of SlALKBH2 homodimers.
The alternative text for this image may have been generated using AI.
Fig. 4: SlALKBH2 oxidation affects its function.
The alternative text for this image may have been generated using AI.
Fig. 5: SlNTRC regulates the redox state of SlALKBH2.
The alternative text for this image may have been generated using AI.
Fig. 6: SlNTRC-mediated redox regulation of SlALKBH2 is responsible for fruit ripening.
The alternative text for this image may have been generated using AI.

Similar content being viewed by others

Data availability

Gene sequences and amino acid sequences used in this paper were collected from the Sol Genomics Network (https://solgenomics.net/), the Arabidopsis Information Resource (https://www.arabidopsis.org/) or Phytozome (https://phytozome-next.jgi.doe.gov/). Gene expression images and values were produced by RNA-seq and RT–qPCR analysis or directly downloaded from the Tomato Expression Atlas (http://tea.solgenomics.net/). Gene Ontology enrichment was analysed using the Gene Ontology Consortium database (http://www.geneontology.org/). High-throughput sequencing data from the m6A-seq and RNA-seq assays have been deposited in the Gene Expression Omnibus database under the accession number GSE268893 and GSE271468, respectively. All other supporting data are included in the article or its Supplementary Information. Source data are provided with this paper.

References

  1. Waszczak, C., Carmody, M. & Kangasjärvi, J. Reactive oxygen species in plant signaling. Annu. Rev. Plant Biol. 69, 209–236 (2018).

    CAS  PubMed  Google Scholar 

  2. Huang, X. et al. ROS regulated reversible protein phase separation synchronizes plant flowering. Nat. Chem. Biol. 17, 549–557 (2021).

    CAS  PubMed  Google Scholar 

  3. Kaya, H. et al. Ca2+-activated reactive oxygen species production by Arabidopsis RbohH and RbohJ is essential for proper pollen tube tip growth. Plant Cell 26, 1069–1080 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Takeda, S. et al. Local positive feedback regulation determines cell shape in root hair cells. Science 319, 1241–1244 (2008).

    CAS  PubMed  Google Scholar 

  5. Shi, W. et al. Spatially patterned hydrogen peroxide orchestrates stomatal development in Arabidopsis. Nat. Commun. 13, 5040 (2022).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Steelheart, C. et al. The effect of low ascorbic acid content on tomato fruit ripening. Planta 252, 36 (2020).

    CAS  PubMed  Google Scholar 

  7. Bi, G. et al. The cytosolic thiol peroxidase PRXIIB is an intracellular sensor for H2O2 that regulates plant immunity through a redox relay. Nat. Plants 8, 1160–1175 (2022).

    CAS  PubMed  Google Scholar 

  8. Silva, A. M. N., Vitorino, R., Domingues, M. R. M., Spickett, C. M. & Domingues, P. Post-translational modifications and mass spectrometry detection. Free Radic. Biol. Med. 65, 925–941 (2013).

    CAS  PubMed  Google Scholar 

  9. Sevilla, F., Martí, M. C., De Brasi-Velasco, S. & Jiménez, A. Redox regulation, thioredoxins, and glutaredoxins in retrograde signalling and gene transcription. J. Exp. Bot. 74, 5955–5969 (2023).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Liu, J. et al. A METTL3-METTL14 complex mediates mammalian nuclear RNA N6-adenosine methylation. Nat. Chem. Biol. 10, 93–95 (2014).

    CAS  PubMed  Google Scholar 

  11. Shi, H., Wei, J. & He, C. Where, when, and how: context-dependent functions of RNA methylation writers, readers, and erasers. Mol. Cell 74, 640–650 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Jia, G. et al. N6-methyladenosine in nuclear RNA is a major substrate of the obesity-associated FTO. Nat. Chem. Biol. 7, 885–887 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Zheng, G. et al. ALKBH5 is a mammalian RNA demethylase that impacts RNA metabolism and mouse fertility. Mol. Cell 49, 18–29 (2013).

    CAS  PubMed  Google Scholar 

  14. Sikorski, V., Selberg, S., Lalowski, M., Karelson, M. & Kankuri, E. The structure and function of YTHDF epitranscriptomic m6A readers. Trends Pharmacol. Sci. 44, 335–353 (2023).

    CAS  PubMed  Google Scholar 

  15. Shen, L., Ma, J., Li, P., Wu, Y. & Yu, H. Recent advances in the plant epitranscriptome. Genome Biol. 24, 43 (2023).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Zhou, L. et al. m6A-mediated regulation of crop development and stress responses. Plant Biotechnol. J. 20, 1447–1455 (2022).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Tang, J., Chen, S. & Jia, G. Detection, regulation, and functions of RNA N6-methyladenosine modification in plants. Plant Commun. 4, 100546 (2023).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Kong, Y., Yu, J., Ge, S. & Fan, X. Novel insight into RNA modifications in tumor immunity: promising targets to prevent tumor immune escape. Innovation 4, 100452 (2023).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Vu, L. D., Gevaert, K. & De Smet, I. Protein language: post-translational modifications talking to each other. Trends Plant Sci. 23, 1068–1080 (2018).

    CAS  PubMed  Google Scholar 

  20. Sun, H. L. et al. Stabilization of ERK-phosphorylated METTL3 by USP5 increases m6A methylation. Mol. Cell 80, 633–647.e7 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Wang, X. et al. Structural basis of N6-adenosine methylation by the METTL3-METTL14 complex. Nature 534, 575–578 (2016).

    CAS  PubMed  Google Scholar 

  22. Du, Y. et al. SUMOylation of the m6A-RNA methyltransferase METTL3 modulates its function. Nucleic Acids Res. 46, 5195–5208 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Yu, F. et al. Post-translational modification of RNA m6A demethylase ALKBH5 regulates ROS-induced DNA damage response. Nucleic Acids Res. 49, 5779–5797 (2021).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. De Jesus, D. F. et al. Redox regulation of m6A methyltransferase METTL3 in β-cells controls the innate immune response in type 1 diabetes. Nat. Cell Biol. 26, 421–437 (2024).

    PubMed  PubMed Central  Google Scholar 

  25. Zhu, T., Yong, X. L. H., Xia, D., Widagdo, J. & Anggono, V. Ubiquitination regulates the proteasomal degradation and nuclear translocation of the fat mass and obesity-associated (FTO) protein. J. Mol. Biol. 430, 363–371 (2018).

    CAS  PubMed  Google Scholar 

  26. Chen, Y. et al. O-GlcNAcylation determines the translational regulation and phase separation of YTHDF proteins. Nat. Cell Biol. 25, 1676–1690 (2023).

    CAS  PubMed  Google Scholar 

  27. Yang, Y. et al. O-GlcNAcylation of YTHDF2 promotes HBV-related hepatocellular carcinoma progression in an N6-methyladenosine-dependent manner. Signal Transduct. Target. Ther. 8, 63 (2023).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Zhou, L., Tian, S. & Qin, G. RNA methylomes reveal the m6A-mediated regulation of DNA demethylase gene SlDML2 in tomato fruit ripening. Genome Biol. 20, 156 (2019).-methyladenosine recognition

    PubMed  PubMed Central  Google Scholar 

  29. Jeong, H. S., Hayashi, G. & Okamoto, A. Diazirine photocrosslinking recruits activated FTO demethylase complexes for specific N6-methyladenosine recognition. ACS Chem. Biol. 10, 1450–1455 (2015).

    CAS  PubMed  Google Scholar 

  30. Lang, Z. et al. Critical roles of DNA demethylation in the activation of ripening-induced genes and inhibition of ripening-repressed genes in tomato fruit. Proc. Natl Acad. Sci. USA 114, E4511–E4519 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Michalska, J., Zauber, H., Buchanan, B. B., Cejudo, F. J. & Geigenberger, P. NTRC links built-in thioredoxin to light and sucrose in regulating starch synthesis in chloroplasts and amyloplasts. Proc. Natl Acad. Sci. USA 106, 9908–9913 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. Hou, L. Y. et al. NTRC plays a crucial role in starch metabolism, redox balance, and tomato fruit growth. Plant Physiol. 181, 976–992 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Martínez-Pérez, M. et al. Arabidopsis m6A demethylase activity modulates viral infection of a plant virus and the m6A abundance in its genomic RNAs. Proc. Natl Acad. Sci. USA 114, 10755–10760 (2017).

    PubMed  PubMed Central  Google Scholar 

  34. Tang, J. et al. OsALKBH9-mediated m6A demethylation regulates tapetal PCD and pollen exine accumulation in rice. Plant Biotechnol. J. 22, 2410–2423 (2024).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Castro, B. et al. Stress-induced reactive oxygen species compartmentalization, perception and signalling. Nat. Plants 7, 403–412 (2021).

    CAS  PubMed  PubMed Central  Google Scholar 

  36. Zhou, H., Huang, J., Willems, P., Van Breusegem, F. & Xie, Y. Cysteine thiol-based post-translational modification: what do we know about transcription factors? Trends Plant Sci. 28, 415–428 (2023).

    CAS  PubMed  Google Scholar 

  37. Trinh, V. H. et al. Redox regulation of phosphatase and tensin homolog by bicarbonate and hydrogen peroxide: implication of peroxymonocarbonate in cell signaling. Antioxidants 13, 473 (2024).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. Singh, V. P. et al. Evolution of reactive oxygen species cellular targets for plant development. Trends Plant Sci. 29, 865–877 (2024).

    CAS  PubMed  Google Scholar 

  39. Midorikawa, T., Endow, J. K., Dufour, J., Zhu, J. & Inoue, K. Plastidic type I signal peptidase 1 is a redox-dependent thylakoidal processing peptidase. Plant J. 80, 592–603 (2014).

    CAS  PubMed  Google Scholar 

  40. Jiang, G. et al. Redox regulation of the NOR transcription factor is involved in the regulation of fruit ripening in tomato. Plant Physiol. 183, 671–685 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Kirchsteiger, K., Ferrandez, J., Pascual, M. B., Gonzalez, M. & Cejudo, F. J. NADPH thioredoxin reductase C is localized in plastids of photosynthetic and nonphotosynthetic tissues and is involved in lateral root formation in Arabidopsis. Plant Cell 24, 1534–1548 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. Pérez-Ruiz, J. M., Naranjo, B., Ojeda, V., Guinea, M. & Cejudo, F. J. NTRC-dependent redox balance of 2-Cys peroxiredoxins is needed for optimal function of the photosynthetic apparatus. Proc. Natl Acad. Sci. USA 114, 12069–12074 (2017).

    PubMed  PubMed Central  Google Scholar 

  43. Petreikov, M. et al. Carbohydrate balance and accumulation during development of near-isogenic tomato lines differing in the AGPase-L1 allele. J. Am. Soc. Hortic. Sci. 134, 134–140 (2009).

    Google Scholar 

  44. Ma, X. et al. A robust CRISPR/Cas9 system for convenient, high-efficiency multiplex genome editing in monocot and dicot plants. Mol. Plant 8, 1274–1284 (2015).

    CAS  PubMed  Google Scholar 

  45. Delaunay, A., Isnard, A. D. & Toledano, M. B. H2O2 sensing through oxidation of the Yap1 transcription factor. EMBO J. 19, 5157–5166 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. Li, X. et al. Deciphering the regulatory network of the NAC transcription factor FvRIF, a key regulator of strawberry (Fragaria vesca) fruit ripening. Plant Cell 35, 4020–4045 (2023).

    PubMed  PubMed Central  Google Scholar 

  47. Chen, H. et al. Firefly luciferase complementation imaging assay for protein–protein interactions in plants. Plant Physiol. 146, 368–376 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. Chen, C. et al. A pathogen-induced putative NAC transcription factor mediates leaf rust resistance in barley. Nat. Commun. 14, 5468 (2023).

    CAS  PubMed  PubMed Central  Google Scholar 

  49. Jumper, J. et al. Highly accurate protein structure prediction with AlphaFold. Nature 596, 583–589 (2021).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. Moore, S., Payton, P., Wright, M., Tanksley, S. & Giovannoni, J. Utilization of tomato microarrays for comparative gene expression analysis in the Solanaceae. J. Exp. Bot. 56, 2885–2895 (2005).

    CAS  PubMed  Google Scholar 

  51. Schmittgen, T. D. & Livak, K. J. Analyzing real-time PCR data by the comparative CT method. Nat. Protoc. 3, 1101–1108 (2008).

    CAS  PubMed  Google Scholar 

  52. Dominissini, D., Moshitch-Moshkovitz, S., Salmon-Divon, M., Amariglio, N. & Rechavi, G. Transcriptome-wide mapping of N6-methyladenosine by m6A-seq based on immunocapturing and massively parallel sequencing. Nat. Protoc. 8, 176–189 (2013).

    CAS  PubMed  Google Scholar 

  53. Duan, H. C. et al. ALKBH10B is an RNA N6-methyladenosine demethylase affecting Arabidopsis floral transition. Plant Cell 29, 2995–3011 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  54. Zhou, L. et al. N6-methyladenosine RNA modification regulates strawberry fruit ripening in an ABA-dependent manner. Genome Biol. 22, 168 (2021).

    CAS  PubMed  PubMed Central  Google Scholar 

  55. Juttner, J., Olde, D., Langridge, P. & Baumann, U. Cloning and expression of a distinct subclass of plant thioredoxins. Eur. J. Biochem. 267, 7109–7117 (2000).

    CAS  PubMed  Google Scholar 

  56. Zhou, L. et al. Increasing flavonoid contents of tomato fruits through disruption of the SlSPL-CNR, a suppressor of SlMYB12 transcription activity. Plant Biotechnol. J. 22, 290–292 (2024).

    PubMed  Google Scholar 

Download references

Acknowledgements

This work was supported by the National Natural Science Foundation of China (31925035 to G.Q. and 32202557 to L.Z.), the CAS Project for Young Scientists in Basic Research (YSRB-093 to Z.L. and G.Q.) and the China National Postdoctoral Program for Innovative Talents (BX20220340 to L.Z.). We thank H. Su (Institute of Botany, Chinese Academy of Sciences) for the LC–MS/MS assay; Z. Lu (Institute of Botany, Chinese Academy of Sciences) for the protein mass spectrometry analysis; J. Li (Institute of Botany, Chinese Academy of Sciences) for assistance with subcellular localization; and Y. Liu (South China Agriculture University) for providing the pYLCRISPR/Cas9Pubi-H binary vector.

Author information

Authors and Affiliations

  1. State Key Laboratory of Plant Diversity and Specialty Crops, Institute of Botany, Chinese Academy of Sciences, Beijing, China

    Leilei Zhou, Guangtong Gao, Renkun Tang, Jinying Liu, Yuying Wang, Zhenchang Liang, Shiping Tian & Guozheng Qin

  2. China National Botanical Garden, Beijing, China

    Leilei Zhou, Guangtong Gao, Renkun Tang, Jinying Liu, Yuying Wang, Zhenchang Liang, Shiping Tian & Guozheng Qin

  3. University of Chinese Academy of Sciences, Beijing, China

    Guangtong Gao, Renkun Tang, Jinying Liu, Zhenchang Liang, Shiping Tian & Guozheng Qin

Authors
  1. Leilei Zhou
    View author publications

    Search author on:PubMed Google Scholar

  2. Guangtong Gao
    View author publications

    Search author on:PubMed Google Scholar

  3. Renkun Tang
    View author publications

    Search author on:PubMed Google Scholar

  4. Jinying Liu
    View author publications

    Search author on:PubMed Google Scholar

  5. Yuying Wang
    View author publications

    Search author on:PubMed Google Scholar

  6. Zhenchang Liang
    View author publications

    Search author on:PubMed Google Scholar

  7. Shiping Tian
    View author publications

    Search author on:PubMed Google Scholar

  8. Guozheng Qin
    View author publications

    Search author on:PubMed Google Scholar

Contributions

G.Q. and L.Z. conceived and designed the experiments. S.T., Z.L. and Y.W. provided critical discussions. L.Z., G.G., R.T. and J.L. performed the experiments and analysed the data. G.Q. and L.Z. wrote the manuscript. All authors commented on the final draft of the paper.

Corresponding author

Correspondence to Guozheng Qin.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Nature Plants thanks Mingchun Liu, Paula Muñoz and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Exogenous H2O2 treatment promotes tomato fruit ripening.

a, Examination of specificity of the anti-SlALKBH2 antibody. Total proteins extracted from fruits of the WT and slalkbh2 mutants (slalkbh2-25/28) at 42 days post-anthesis were subjected to SDS-PAGE with (+) or without (-) DTT (50 mM) in the sample buffer and analyzed by immunoblot using a polyclonal anti-SlALKBH2 antibody. Actin was used as a loading control. The experiment was repeated independently three times with similar results. b, Expression images and values of the H2O2 biosynthetic gene SOD1 in the cultivar M82 fruits at different developmental stages. The expression images and values were downloaded from the Tomato Expression Atlas (http://tea.solgenomics.net/). SOD1, superoxide dismutase 1. RPM, reads of exon model per million mapped reads. c, H2O2 treatment accelerated fruit ripening in tomato. Five representative fruits are presented. Scale bar, 1 cm. d,e, Alterations of lycopene content (d) and transcript levels of ripening-related genes SlPSY1, SlACS2 and SlPG2a (e) in H2O2-treated fruits. For c-e, fruits harvested at the mature green 3 (MG-3) stage were treated with 10 mM H2O2 for 1 h, and then incubated for 3 days. Data are means ± s.d. (n = 3 biological replicates). Asterisks indicate significant differences (**P < 0.01, ***P < 0.001; two-tailed Student’s t-test).

Source data

Extended Data Fig. 2 Cys39 site is essential for oxidation and abundance of SlALKBH2 proteins.

a, An enhanced CaMV 35S promoter increased the abundance of SlALKBH2-HA and SlALKBH2C39S-HA fusion proteins expressed in N. benthamiana leaves. b, Quantification of protein levels shown in a. c, Effects of multiple Cys mutations on the formation of SlALKBH2 homodimers. d, Effects of Cys279 mutation on the formation of SlALKBH2 homodimers. For c and d, the proteins were expressed in N. benthamiana leaves treated with 100 mM H2O2 for 30 min. e, Quantification of homodimer versus monomer shown in d (-DTT). f, The Cys279 mutation did not affect SlALKBH2 protein levels. g, The Cys279 mutation did not affect SlALKBH2 stability. For f and g, the proteins were expressed in N. benthamiana leaves treated with or without translation inhibitor cycloheximide (CHX). h, Effects of Cys39 mutation and deletion on SlALKBH2 protein levels. i, Quantification of protein levels shown in h. j, Co-IP assay revealing the self-interaction of SlALKBH2 upon Cys36 mutation. k, Mutation of Cys36 enhanced the self-interaction of SlALKBH2. l, Quantification of protein levels shown in k (IB:anti-HA). m, Effects of Cys36 mutation on SlALKBH2 protein levels. Protein levels for f, g and m are quantified as shown. For a, c, d, f-h, j, k and m, total proteins were extracted from N. benthamiana leaves and subjected to immunoblot using an anti-HA or anti-Flag antibody. Actin was used as a loading control. For b, e and i, data are means ± s.d. (n = 3 biological replicates). Statistical significance was assessed through one-way ANOVA analysis, with lowercase letters indicating significant differences (P < 0.05, Tukey’s test). For f, g, l and m, data are means ± s.d. (n = 3 biological replicates). Asterisks indicate significant differences (*P < 0.05, ***P < 0.001; two-tailed Student’s t-test). NS, no significance.

Source data

Extended Data Fig. 3 Changes in transcriptome-wide m6A methylomes in fruits of the slalkbh2 mutant.

a, Representative photographs of the fruits used for m6A-seq analysis. Fruits of the WT and slalkbh2 mutants (slalkbh2-25 and slalkbh2-28) were harvested at 39 day post-anthesis (dpa), corresponding to the mature green 3 (MG-3) stage. Scale bar, 1 cm. b, Pearson correlation coefficients between m6A-seq samples. The reads within m6A peak regions were employed for the Pearson correlation analysis. Rep, replicate. c, Venn diagrams showing the overlap of m6A peaks from three independent m6A-seq experiments. d, The percentage of m6A peaks in the five non-overlapping transcript segments. TSS, transcription start site; UTR, untranslated region; CDS, coding sequence. e, The relative enrichment of m6A peaks in the five non-overlapping transcript segments. f, m6A motif identified within m6A peak regions by HOMER software. g,h, Volcano plot showing the hypermethylated (green) and hypomethylated (orange) m6A peaks in fruits of the slalkbh2-25 (g) and slalkbh2-28 (h) mutants compared to the WT. i, Changes in transcript levels of ripening-related genes in fruits of slalkbh2-25 and slalkbh2-28 mutants at 39 dpa, as determined by RT-qPCR analysis. Data are means ± s.d. (n = 3 biological replicates). Statistical significance was assessed through one-way ANOVA analysis, with lowercase letters indicating significant differences (P < 0.05, Tukey’s test).

Source data

Extended Data Fig. 4 Sequence alignment of SlALKBH2 homologs across various plant species.

Tomato (Solanum lycopersicum) and other 15 plant species were analyzed, including tobacco (Nicotiana tabacum), eggplant (Solanum melongena), potato (Solanum tuberosum), Arabidopsis (Arabidopsis thaliana), watermelon (Citrullus lanatus), cucumber (Cucumis sativus), strawberry (Fragaria vesca), apple (Malus domestica), peach (Prunus persica), grape (Vitis vinifera), rice (Oryza sativa), maize (Zea mays), peanut (Arachis hypogaea), soybean (Glycine max) and sorghum (Sorghum bicolor). A conserved peptide sequence at the N terminus of SlALKBH2 and its homologues was identified and highlighted with a blue line, and the two critical Cys sites (Cys36 and Cys39) in SlALKBH2 are denoted by asterisks. The Fe (II) and α-ketoglutaramate (α-KG) binding sites are indicated by rectangles and square, respectively. The sequences corresponding to the conserved AlkB domains are indicated by a red line.

Extended Data Fig. 5 Oxidative regulation of SlALKBH2 appears to be conserved.

a, Protein similarity assay revealing a conserved peptide sequence at the N terminus of SlALKBH2 homologues across 15 plant species. The Cys36 and Cys39 sites of SlALKBH2 are highlighted in the conserved peptide sequence, and the type of amino acid (aa) in the homologues are shown. b, AtALKBH9B and OsALKBH9 form homodimers under oxidation by H2O2. c, The mutation of Cys37 in AtALKBH9B and Cys49 in OsALKBH9 impaired the formation of homodimers. d, Quantification of homodimer versus monomer shown in c (-DTT). For b and c, the SlALKBH2 homologues or their mutated variants were expressed in N. benthamiana leaves treated with 100 mM H2O2 for 30 min. e, The mutation of Cys37 in AtALKBH9B or Cys49 in OsALKBH9 resulted in an obvious decrease in their protein levels. f, Protein levels are quantified as shown. The SlALKBH2 homologues or their mutated variants were expressed in N. benthamiana leaves. For d and f, data are means ± s.d. (n = 3 biological replicates). Asterisks indicate significant differences (*P < 0.05, **P < 0.01, ***P < 0.001; two-tailed Student’s t-test). NS, no significance. g, AtALKBH10B proteins failed to form homodimers upon H2O2 exposure. AtALKBH10B-HA was expressed in N. benthamiana leaves treated with 100 mM H2O2 for indicated times. h, Mutation of the cysteine residues in AtALKBH10B did not impact its protein levels. i, Quantification of protein levels shown in h. AtALKBH10B-HA or its mutated variants were expressed in N. benthamiana leaves for 36 h. For b, c, e, g and h, total proteins were extracted and subjected to immunoblot using an anti-HA antibody. Actin was used as a loading control. For i, data are means ± s.d. (n = 3 biological replicates). Statistical significance was assessed through one-way ANOVA analysis (P < 0.05, Tukey’s test).

Source data

Extended Data Fig. 6 Identification of tomato SlNTRC as an active thioredoxin reductase.

a, Phylogenetic analysis of NADPH-dependent thioredoxin reductase (NTR) isoforms among various plant species. The phylogenetic tree was produced by MEGA 5.2 software. Bootstrap values from 1000 replications for each branch are presented. NTR proteins are composed of three isoforms, including NTRA and NTRB, which contain a conserved pyr_redox_2 domain, and NTRC containing both pyr_redox_2 and thioredoxin domains. b, Expression pattern of SlNTRC in tomato fruits at different developmental stages, as determined by RT-qPCR analysis. Actin gene was used as an internal control. IM, immature green; MG, mature green; Br, breaker; OR, orange; RR, red ripe. Data are means ± s.d. (n = 3 biological replicates). c, Expression images of SlNTRC in the cultivar M82 fruits at different developmental stage. The expression images were downloaded from the Tomato Expression Atlas (http://tea.solgenomics.net/). LR, light red; dpa, days post-anthesis. d, Subcellular localization of SlNTRC. The SlNTRC or its N-terminal signal peptide (NSP) fused with eGFP (SlNTRC-eGFP and SlNTRCNSP-eGFP) was transiently expressed in N. benthamiana leaves. Arabidopsis AtNTRC and its NSP were employed as positive controls, and eGFP alone was utilized as an internal control. Scale bars, 20 µm. e, Expression analysis of the eGFP-tagged fusion proteins used for subcellular localization. The total proteins were extracted and subjected to immunoblot analysis using an anti-GFP antibody. Actin was used as a loading control.

Source data

Extended Data Fig. 7 The N terminus of SlALKBH2 interacts with the thioredoxin domain of SlNTRC.

a, Schematic illustration for the interaction assay of protein fragments involving SlALKBH2 and SlNTRC. The sequence of the SlALKBH2 protein was divided into three segments: the N-terminal sequence (SlALKBH2-N), the AlkB domain (SlALKBH2-D) and the C-terminal sequence (SlALKBH2-C). These segments were separately utilized to assess their interactions with the thioredoxin domain (SlNTRC-C) of SlNTRC. Numerical values indicate the positions of the first and last amino acid in their respective sequences. b, Y2H assay revealing a strong interaction between the N terminus of SlALKBH2 and the thioredoxin domain of SlNTRC. The SlALKBH2-N, SlALKBH2-D or SlALKBH2-C fused to the binding domain (BD) of GAL4 (BD-SlALKBH2-N, BD-SlALKBH2-D or BD-SlALKBH2-C) and the SlNTRC-C fused to the activation domain (AD) of GAL4 (AD-SlNTRC-C) were co-expressed in yeast cells. The transformants were cultured on SD/-Leu/-Trp (-LW) and then selected on SD/-Leu/-Trp/-His/-Ade (-LWHA) with or without the X-α-gal. The transformants carrying empty AD or BD vector were employed as negative controls. c, LCI assay revealing the interaction between the N terminus of SlALKBH2 and the thioredoxin domain of SlNTRC. The SlALKBH2-N, SlALKBH2-D or SlALKBH2-C fused to the C terminus of firefly luciferase (cLUC-SlALKBH2-N, cLUC-SlALKBH2-D or cLUC-SlALKBH2-C) and the SlNTRC-C fused to the N terminus of firefly luciferase (SlNTRC-C-nLUC) were co-expressed in N. benthamiana leaves.

Extended Data Fig. 8 SlNTRC is indispensable for the normal growth of tomato plants.

a, Genotyping of slntrc-21 and slntrc-29 mutants generated by CRISPR/Cas9-mediated gene editing. The single guide RNAs (sgRNAs) containing different target sequences (T1 and T2) were designed to specifically target the fourth and fifth exons of SlNTRC. The red letters indicate the protospacer adjacent motif (PAM). b, Schematic illustration for the predicted truncated SlNTRC proteins in the slntrc-21 and slntrc-29 mutants. c, Changes in the transcript levels of SlNTRC in the leaves of slntrc-21 and slntrc-29 mutants, as determined by RT-qPCR analysis. Actin gene was used as an internal control. d, The growth phenotypes of slntrc mutant lines. Scale bar, 1 cm. e, Changes in redox state of endogenous SlALKBH2 proteins in the slntrc mutant lines. Total proteins extracted from 35-day-old leaves were subjected to SDS-PAGE with (+) or without (-) DTT (50 mM) and analyzed using a polyclonal anti-SlALKBH2 antibody. Actin was used as a loading control. f, Quantification of SlALKBH2 homodimer versus monomer shown in e (-DTT). g, Quantification of SlALKBH2 protein levels shown in e (+DTT). h, Genotyping of SlNTRC heterozygous mutants generated by CRISPR/Cas9-mediated gene editing. i, Changes in the transcript level of SlNTRC in fruits of slntrc-H1 and slntrc-H3, as determined by RT-qPCR analysis. j, The OE-NTRC-2 and OE-NTRC-6 overexpression lines displayed no obvious change in vegetative growth compared to the WT. Scale bar, 1 cm. k, Changes in transcript levels of ripening-related genes in fruits of OE-NTRC-2 and OE-NTRC-6 at 42 day post-anthesis, as determined by RT-qPCR analysis. For c, f, g, i and k, data are means ± s.d. (n = 3 biological replicates). Statistical significance was assessed through one-way ANOVA analysis, with lowercase letters indicating significant differences (P < 0.05, Tukey’s test).

Source data

Supplementary information

Source data

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Zhou, L., Gao, G., Tang, R. et al. Redox modification of m6A demethylase SlALKBH2 in tomato regulates fruit ripening. Nat. Plants 11, 218–233 (2025). https://doi.org/10.1038/s41477-024-01893-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • Version of record:

  • Issue date:

  • DOI: https://doi.org/10.1038/s41477-024-01893-8

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing