Abstract
Superoxide anion is thought to be a natural by-product with strong oxidizing ability in all living organisms and was recently found to accumulate in plant meristems to maintain stem cells in the shoot and undifferentiated meristematic cells in the root. Here we show that the DNA demethylase repressor of silencing 1 (ROS1) is one of the direct targets of superoxide in stem cells. The Fe–S clusters in ROS1 are oxidized by superoxide to activate its DNA glycosylase/lyase activity. We demonstrate that superoxide extensively participates in the establishment of active DNA demethylation in the Arabidopsis genome and that ARABIDOPSIS RESPONSE REGULATOR 12 acts downstream of ROS1-mediated superoxide signaling to maintain stem cell fate. Our results provide a mechanistic framework for superoxide control of the stem cell niche and demonstrate how redox and DNA demethylation interact to define stem cell fate in plants.
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Data availability
The bisulfite sequencing data have been deposited in the NCBI SRA and are accessible through BioProject accession number PRJNA856724. Source data are provided with this paper.
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Acknowledgements
This work was supported by grants to Z.Z. from the National Natural Science Foundation of China (32130009 and 32321001) and the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB27030105). We thank C. Huang and F. Xiang for sharing plant materials.
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Z.Z. conceived the study. Z.Z. and S.W. designed the experiments, analyzed the data and wrote the paper with input from all authors. M.L. and Z.D. analyzed the bisulfite sequencing data. D.H. performed in situ hybridization. S.W. performed all other experiments.
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Extended data
Extended Data Fig. 1 ROS1 and its homologous genes mediate superoxide signaling in the SAM.
a, Expression levels of DME in 7-day-old rdd-DME RNAi seedlings measured by qRT‒PCR with or without β-estradiol induction with 3 biological replicates. b, Ratio of the first pair of true leaves in 7-day-old wild-type Col-0 and rdd-DME RNAi seedlings with or without PG treatment under β-estradiol induction. Three biological replicates were performed, with each replicate having more than 100 plants. The data are shown as the mean ± s.d. with two-tailed Student’s t tests, and P values are shown at the top of each bar.
Extended Data Fig. 2 The ros1 mutant partially blocks superoxide signaling in regulating the WUS expression.
Expression levels of WUS in Col-0 and ros1-4 seedlings with or without MV treatment were measured by qRT‒PCR, and 5 biological replicates were performed for each sample. The data are shown as the mean ± s.d. with two-tailed Student’s t tests, and P values are shown at the top of each bar. The error bars indicate the highest and lowest values, the box indicates the middle 50%, and the centre line indicates the median, the whiskers indicate the data range within 1.5× the interquartile range, and outliers are not shown.
Extended Data Fig. 3 AtRBOHF regulates superoxide signaling in the stem cell niche.
a, b, Expression levels of NDUFS4 (a) and NDUFVI (b) in 7-day-old atrbohF seedlings with 9 biological replicates. c, Ratio of the first pair of true leaves in 7-day-old wild-type Col-0, atrbohF, ros1-4 and atrbohF ros1-4 seedlings. Three biological replicates were performed, with each replicate having more than 100 plants. d, WUS and CLV3 expression patterns in (c). e, SAM sizes of (d). (Col-0, n = 10; atrbohF, n = 13; ros1-4, n = 17; atrbohF ros1-4, n = 11). f, g, WUS (f) and CLV3 (g) expression levels in (c) were measured by qRT‒PCR with at least 6 biological replicates. Scale bar, 50 μm (d). The data are shown as the mean ± s.d. with two-tailed Student’s t tests, and P values are shown at the top of each bar. The error bars indicate the highest and lowest values, the boxes (a, b, e-g) indicate the middle 50%, and the centre line indicates the median, the whiskers indicate the data range within 1.5× the interquartile range, and outliers are not shown.
Extended Data Fig. 4 Expression of WUS activator genes in the ros1-4 and atrbohF mutants.
a, The expression levels of PHB, PHV, REV, HB8, HB15, SYD, ARR1, ARR2, ARR10 and ARR12 in 7-day-old wild-type and ros1-4 seedlings with at least 4 biological replicates. b, The expression levels of HB8, ARR10 and ARR12 in the 7-day-old wild-type and atrbohF seedlings with 4 biological replicates. The data are shown as the mean ± s.d. with two-tailed Student’s t tests, and P values are shown on the top of each bar. The error bars indicate the highest and lowest values, the box indicates the middle 50%, and the centre line indicates the median, the whiskers indicate the data range within 1.5× the interquartile range, and outliers are not shown.
Extended Data Fig. 5 ROS1 mediates superoxide signaling in regulating the DNA methylation at the ARR12 promoter.
a, DNA methylation levels of the ARR12 promoter detected by chop-PCR, including the wild-type, atrbohF, ros1-4 and atrbohF ros1-4 mutants with at least 4 biological replicates. Primers used for chop-PCR were located in the ARR12 promoter from −1659 to −1899. b, ARR12 expression levels in the atrbohF, ros1-4 and atrbohF ros1-4 mutants with at least 6 biological replicates. c, Chop-PCR detection of ARR12 promoter methylation in the wild type and ros1-4 with or without 0.25 mM PG treatment, and in the atrbohF, ndufs4, ros1-4, atrbohF ros1-4 and ndufs4 ros1-4 mutants. Primers used for chop-PCR were located in the ARR12 promoter from −852 to −2946. The experiments were repeated independently two times with similar results. d, Expression levels of ARR12 in Col-0 and ros1-4 seedlings with or without MV treatment measured by qRT‒PCR, at least 4 biological replicates were performed for each sample. The error bars (a, b and d) indicate the highest and lowest values, the box indicates the middle 50%, and the centre line indicates the median, the whiskers indicate the data range within 1.5× the interquartile range, and outliers are not shown. Two-tailed Student’s t tests and P values are shown at the top of each bar.
Extended Data Fig. 6 ARR12 mediates superoxide signaling in the SAM.
a, Morphology of SAM in Col-0 and arr12 mutants with or without PG treatment determined using toluidine blue staining. The experiments were repeated independently at least two times on pools of apices of at least 21 plants, with similar results. The white dotted line represents the SAM. Scale bars, 100 μm. b, SAM sizes of Col-0 and arr12 seedlings with or without PG treatment. (Col-0 mock, n = 24; Col-0 PG, n = 25; arr12 mock, n = 23; arr12 PG, n = 21). The error bars indicate the highest and lowest values, the box indicates the middle 50%, and the centre line indicates the median, the whiskers indicate the data range within 1.5× the interquartile range, and outliers are not shown. c, Ratio of the first pair of true leaves in 7-day-old wild-type Col-0 and arr12 seedlings with or without PG treatment. Three biological replicates were performed, with each replicate having more than 100 plants. The data are shown as the mean ± s.d. with two-tailed Student’s t tests, and P values are shown at the top of each bar.
Extended Data Fig. 7 Overexpression of ARR12 partially rescued the defects of the atrbohF and ros1-4 mutants.
a, Phenotypes and WUS and CLV3 expression patterns in wild-type Col-0, 35S::ARR12, ros1-4, 35S::ARR12 ros1-4, atrbohF, and 35S::ARR12 atrbohF seedlings. The experiments were repeated independently two times on pools of 100 seedlings, with similar results. Arrowheads: true leaves. b, The true leaf initiation defects of 7-day-old ros1-4 and atrbohF were partially rescued by the overexpression of ARR12. Three biological replicates were performed, with each replicate having 100 plants. c, The reduced SAM sizes in ros1-4 and atrbohF seedlings were partially restored by 35S::ARR12. (Col-0, n = 15; 35S::ARR12, n = 17; ros1-4, n = 17; 35S::ARR12 ros1-4, n = 11; atrbohF, n = 11; 35S::ARR12 atrbohF, n = 16). Scale bars, 1.5 mm (a, seedlings); 50 μm (a, in situ hybridization). The data are shown as the mean ± s.d. (b) with two-tailed Student’s t tests, and P values are shown at the top of each bar. The error bars (c) indicate the highest and lowest values, boxes indicate the middle 50%, and the centre line indicates the median, the whiskers indicate the data range within 1.5× the interquartile range, and outliers are not shown.
Extended Data Fig. 8 Iron-sulfur clusters in ROS1 mediate superoxide regulation of ARR12.
ARR12 expression levels in the wild-type Col-0, pROS1::mROS1, ros1-4 and pROS1::ROS1, ros1-4 with or without PG treatment measured by qRT‒PCR with 5 biological replicates. The error bars indicate the highest and lowest values, the box indicates the middle 50%, and the centre line indicates the median, the whiskers indicate the data range within 1.5× the interquartile range, and outliers are not shown. P values are shown at the top of each bar with two-tailed Student’s t tests.
Extended Data Fig. 9 Superoxide oxidation of iron-sulfur clusters activates ROS1 glycosylases/lyases.
a, Enzyme activity of His-MBP, His-MBP-mROS1 and His-MBP-ROS1 with or without superoxide oxidation in vitro. b, Enzyme activity of His-MBP, His-MBP-mROS1 and His-MBP-ROS1 with or without 10 μM hydrogen peroxide treatment in vitro. All experiments were repeated independently two times, with similar results.
Extended Data Fig. 10 The superoxide anion mediates AT4G14400 expression in response to cold through DNA demethylation.
a, Expression levels of AT4G14400 in Col-0, atrbohF, ndufs4 and ros1-4 seedlings with or without cold treatment (4 °C for 24 h) measured by qRT‒PCR with 4 biological replicates. b, Chop-PCR detection of AT4G14400 promoter methylation in the wild type, atrbohF, ndufs4 and ros1-4 with or without cold treatment. Primers used for chop-PCR were located in the AT4G14400 promoter from −1812 to −2516. The error bars (a) indicate the highest and lowest values, the box indicates the middle 50%, and the centre line indicates the median. Two-tailed Student’s t tests, and P values are shown at the top of each bar.
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Unprocessed western blots and/or gels and statistical source data for Figs. 1–3 and 5 and Extended Data Figs. 1–10.
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Wang, S., Liu, M., Hu, D. et al. Control of DNA demethylation by superoxide anion in plant stem cells. Nat Chem Biol (2024). https://doi.org/10.1038/s41589-024-01737-8
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DOI: https://doi.org/10.1038/s41589-024-01737-8
