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Nuclear-localized pyruvate kinases control phosphorylation of histone H3 on threonine 11

Nature Plants volume 10, pages 1682–1697 (2024)Cite this article

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Abstract

Phosphorylation of histone H3 at threonine 11 (H3T11ph) affects transcription and chromosome stability. However, the enzymes responsible for depositing H3T11ph and the functions of H3T11ph in plants remain unknown. Here we report that in Arabidopsis thaliana, PYRUVATE KINASE 6 (PK6), PK7 and PK8 enter the nucleus under conditions of sufficient glucose and light exposure, where they interact with SWI2/SNF2-RELATED 1 COMPLEX 4 (SWC4) and phosphorylate H3 at threonine 11. Mutations in these kinases or knockdown of SWC4 resulted in FLC-dependent early flowering, short hypocotyls and short pedicels. Genome-wide, H3T11ph is highly enriched at transcription start sites and transcription termination sites, and positively correlated with gene transcript levels. PK6 and SWC4 targeted FLC, MYB73, PRE1, TCP4 and TCP10, depositing H3T11ph at these loci and promoting their transcription, and PK6 occupancy at these loci requires SWC4. Together, our results reveal that nuclear-localized PK6, PK7 and PK8 modulate H3T11ph and plant growth.

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Fig. 1: The phosphorylation of H3T11 is induced by glucose.
Fig. 2: Phosphorylation activity of nuclear-located PK6 and PK6 localization under glucose depletion and stimulation.
Fig. 3: PK6 localization under light exposure and darkness.
Fig. 4: Mutations in PK6/7/8 resulted in early flowering.
Fig. 5: PK6/7/8 are responsible for H3T11ph deposition.
Fig. 6: PK6/7/8 interact with SWC4 in the nucleus.
Fig. 7: PK6/7/8 couple with SWC4 to regulate H3T11ph and HTA9, and gene expression in the Arabidopsis genome.
Fig. 8: The co-occupancy of PK6/7/8 and SWC4 at FLC, MYB73 and PRE1.

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Data availability

The Arabidopsis mutants used in the current study are available from the corresponding author on request. The RNA-seq data and ChIP-seq data were deposited to the NCBI Sequence Read Archive with accession code PRJNA1122224. The source data underlying Figs. 1a,b, 2e,h,i, 4a and 5b,e–g; Extended Data Figs. 4a, 6d–f and 8j,k; and Supplementary Figs. 1, 3d and 8a–c are provided as a Source Data file. Other source data are provided with this paper.

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Acknowledgements

This work was supported by the National Natural Science Foundation of China (32230017, U19A2021 and 32321001), the Global Select Project (DJK-LX-2022007) of the Institute of Health and Medicine, the Hefei Comprehensive National Science Center, the Key Research and Development Projects in Anhui Province (2023n06020035), and the Fundamental Research Funds for the Central Universities (WK9100000047). We thank Y. Xiong from the FuJian Agriculture and Forestry University for kindly providing the tor-es and HA-S6K-overexpressing line seeds, and all members of the Ding group for helpful discussions.

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Authors and Affiliations

  1. Ministry of Education Key Laboratory for Membraneless Organelles and Cellular Dynamics; School of Life Sciences, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, China

    Pengcheng Hu, Yanmei Xu, Yanhua Su, Yuxin Wang & Yong Ding

  2. Haixia Institute of Science and Technology, Synthetic Biology Center, Fujian Agriculture and Forestry University, Fuzhou, China

    Yan Xiong

  3. Institute of Health and Medicine, Hefei Comprehensive National Science Center, Hefei, China

    Yong Ding

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  1. Pengcheng Hu
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Contributions

Y.D. and P.H. conceived the study and designed the experiments. P.H. performed most of the experiments. Y.D. wrote the manuscript. Y. Xu, Y.S. and Y.W. contributed to mutant generation, and Y. Xiong contributed to glucose starvation design.

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Correspondence to Yong Ding.

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Extended data

Extended Data Fig. 1 Reproducibility analysis of the biological replicates for the H3T11ph ChIP-seq.

(a) Correlation analysis of the biological replicates of the H3T11ph ChIP-seq analysis of Col-0 under glucose depletion and stimulation. The x and y axes indicate the read coverage normalized to the total mapped reads, and values are in the log2 scale. (b) Scatterplot of H3T11ph-occupied loci downregulated in Col-0 under glucose depletion vs. glucose stimulation. The x-axis indicates log2-normalized ChIP read counts, and the y-axis indicates the log2 fold change of H3T11ph levels. The dashed line indicates a fold change of 1.5. Significant (FDR < 0.05) up- and downregulated peaks are marked in red and blue, respectively. The up- and downregulated loci are indicated. (c) Metagene plots of H3T11ph enrichment in Col-0 under glucose depletion and stimulation. The log2 ratio of normalized enrichment (H3T11ph/Input) for each gene region, including the 2-kbp upstream and downstream regions from the gene, was used for the average gene analysis. The transcription start site (TSS) and transcription termination site (TTS) are indicated. (d) ChIP–seq for H3T11ph in Col-0 under glucose depletion and stimulation. The genome-browser track views are shown (chr3: 9,391,000–9,638,000 and chr4: 14,036,000–14,302,000).

Extended Data Fig. 2 Phylogenetic analysis of pyruvate kinase (PK) family proteins in Arabidopsis.

The 14 PKs, including 10 cytosolic PKs and 4 plastidic PKs in Arabidopsis were aligned with ClustalW. The relationships of the sequences were examined with MEGA5. The evolutional scale bar is indicated at the bottom.

Extended Data Fig. 3 Characterization of the pk1, pk2, pk3, pk4, pk5, pk6, pk7, pk8, pk9, and pk10 mutants.

(a) Gene structures of PK1, PK2, PK3, PK4, PK5, PK6, PK7, PK8, PK9, and PK10 with boxes indicating exons, lines indicating introns, and triangles indicating T-DNA insertions. The primers used for genotyping are marked with arrows. (b) Genotypic analysis of the pk1, pk2, pk3, pk4, pk5, pk6, pk7, pk8, pk9, and pk10 mutants. Genotyping was performed with the left genomic primer (LP), right genomic primer (RP), and the vector primer (LBb1.3). The positions of the LPs and RPs are indicated in (A). (c) Expression levels of the full-length transcripts of PK1, PK2, PK3, PK4, PK5, PK6, PK7, PK8, PK9, and PK10 in the pk1, pk2, pk3, pk4, pk5, pk6, pk7, pk8, pk9, and pk10 mutants, respectively.

Extended Data Fig. 4 Phosphorylation activity of PK6.

(a) Global H3T11ph levels in pk1-1 pk2-1, pk2-1 pk3-1 pk4-1, pk3-1 pk4-1 pk5-1, pk9-1 pk10-1, and pk6-1 pk7-1 pk8-1 plants. H3 was used as a loading control. Replicate 1 and replicate 2 are shown. All experiments were repeated at least three times, and representative experiments are shown. Molecular mass markers in kilodaltons (kDa) are indicated on the left, and the bands are consistent with their expected sizes. The source data underlying immunoblots are provided in Source Data File. (b) Phosphorylation activity of PK6 purified from Escherichia coli on H3. SnrK2.6 was used as the positive control. The positions of MBP-PK6, GST-SnrK2.6, and H3 are marked with arrows. The star indicates the no-specific band. (c) Kinase activity of immunoprecipitated PK6 on the histones. The positions of H2A, H2B, H3, and H4 are marked with arrows. (d) The conserved arginine 87 and lysine 172 of PK6 were converted to glutamic acid and arginine, respectively (PK6R87E/K172R), to investigate the requirement of these conserved residues for the phosphorylation of Arabidopsis H3 in vitro. The conserved ATP binding sites are shown in Supplemental Fig. 7a. GFP levels were used as a loading control. (e) Activity and specificity of the immunoprecipitated PK6 kinase, assessed using different substrates. The substrates were H3 fused with His and either wild-type H3 or H3 with serine-to-alanine substitutions. GFP levels were used as a loading control.

Source data

Extended Data Fig. 5 The GUS activity of PK6 under glucose depletion and stimulation.

(a) The PK6-driven GUS activity was assessed under glucose depletion and stimulation in four-day-old seedlings. Bar, 1 mm. (b) Relative transcript levels of PK6, PK7, and PK8 under glucose depletion and stimulation. Experiments were repeated at least three times, and the results of representative experiments are shown as means ± SE, n = 3. No difference is indicated as ns. Tubulin 8 was used as a negative control. The source data underlying the statistical analysis are provided in the Source Data file.

Source data

Extended Data Fig. 6 Phosphorylation activity of H3 at threonine 11 by PK7 and PK8.

(a) Complementation of the pk7-1 and pk8-1 mutants with full-length PK7 or PK8 sequences, respectively, fused with HA driven by the PK7 (ProPK7:HA-PK7) or PK8 (ProPK8:HA-PK8) promoters, respectively. Over 40 independent transgenic lines were generated for each transformant, and two representative transgenic plants (T7 and T10 for the pk7-1 complementation, and T2 and T8 for the pk8-1 complementation) are shown. (b) Total leaf number of Col-0, pk7-1, pk8-1, and the complemented lines under a long-day photoperiod. Flowering time was assessed by counting the number of rosette leaves (white boxes) and cauline leaves (grey boxes) at bolting under a long-day photoperiod. Values shown are means ± SD of total leaves; over 25 plants were scored for each line. The source data underlying the statistical analysis are provided in the Source Data file. (c) Activity of the immunoprecipitated PK7 (left panel) and PK8 (right panel) kinases on H3 under glucose depletion and stimulation. Experiments were repeated at least three times and the results of representative experiments are shown. (d) Activity of the immunoprecipitated PK7 and PK8 kinases on threonine 11 of H3 under glucose stimulation. Experiments were repeated at least three times and the results of representative experiments are shown. The source data underlying immunoblots are provided in Source Data File. (e) The total PK7 levels and PK7 in the nucleus and cytoplasm of ProPK7:HA-PK7 plants (T7) under glucose depletion and stimulation, determined using a western blot. A total of 4% nuclear proteins and 20% cytoplasmic proteins were used for western blotting. The nuclear and cytoplasmic proteins were validated by anti-H3 and anti-β-Tublin, respectively. (f) The total PK8 levels and PK8 levels in the nucleus and cytoplasm of ProPK8:HA-PK8 plants (T2) under glucose depletion and stimulation, determined using a western blot. The nuclear and cytoplasmic proteins were validated by anti-H3 and anti-β-Tubulin, respectively. The source data underlying immunoblots in (E) and (F) are provided in Source Data File.

Source data

Extended Data Fig. 7 The PK activity of PK6, PK7, and PK8.

(a) Sequence alignment of PK6 with PKM2 and Pyk1. The conserved ATP binding sites are shown in red boxes, and PEP binding sites are shown in green boxes. (b) PK6, PK7, PK8, and PK6 with conserved arginine 46 and lysine 236 converted to glutamic acid and arginine, respectively (PK6R46E/K236R), as shown in a Coomassie blue-stained gel. (c) The PK activity of PK6, PK7, PK8, and PK6R46E/K236R. Values are means ± SD (n = 3 independent biological replicates) (d) PK activity in Col-0 and pk6/7/8 seedlings under glucose depletion and glucose stimulation. Values are means ± SD (n = 3 independent biological replicates). (e) Glucose levels in Col-0 under dark treatment and LD treatment. Values are means ± SD (n = 3 independent biological replicates). The source data underlying the statistical analysis in (c), (d) and (e) are provided in the Source Data file.

Source data

Extended Data Fig. 8 The translocation of PK6, PK7, and PK8 under glucose stimulation and depletion.

(a), (c), (e), and (h) Cellular sublocations of PK6-GFP (a), PK7-GFP (c), PK8-GFP (e), and GFP alone (h) in Arabidopsis protoplasts under glucose stimulation and glucose plus AZD8055 or Torin2. H3-RFP was used as a nuclear marker. GFP, H3-RFP, bright-field microscopy (differential interference contrast; DIC), and merged images are shown. Scale bars, 10 µm. GFP alone (h) was used as the negative control. GFP, green fluorescent protein. RFP, red fluorescent protein. (b), (d), (f), and (i) Quantitative confocal imaging of PK6-GFP (b), PK7-GFP (d), PK8-GFP (f), and GFP alone (i) in Arabidopsis protoplasts, determined by calculating the cytoplasm/nucleus (C/N) signal intensity ratio. Values shown are means ± SD; over 10 cells were scored for each treatment. Different letters mark statistically significant differences, as revealed using a one-way ANOVA (p < 0.01). (j) and (k) H3T11ph in Col-0 and PK6 levels in 35S:GFP-PK6 plants were measured during a darkness plus glucose and re-light treatment (j), and constituted long day plus glucose treatment (k) using a western blot. Molecular mass markers in kilodaltons (kDa) are indicated on the left in, and the bands are consistent with their expected sizes. H3 was used as a loading control. Experiments were repeated at least three times, and the results of representative experiments are shown. The source data underlying immunoblots are provided in Source Data File. (l) Relative transcription levels of PK6, PK7, and PK8 under long day photoperiod, darkness, and re-light conditions. Experiments were repeated at least three times. and the results of representative experiments are shown as means ± SE, n = 3. Ubiquitin 10 was used as internal control. The source data underlying the statistical analysis in (b), (d), (f), (i) and (l) are provided in the Source Data file.

Source data

Extended Data Fig. 9 Characterization of the pk6 mutants.

(a) Total leaf number of pk6-1, pk6-2, pk7-1, pk8-1, and pk6/7/8 mutants under a long-day photoperiod, a short-day photoperiod, and a short-day photoperiod with gibberellic acid (GA3) treatment. White boxes indicate rosette leaves and grey boxes indicate cauline leaves. Values shown are means ± SD of total leaves; over 25 plants were scored for each line. (b) Flowering-time phenotypes of Col-0, pk6-1, and two complemented lines (T16 and T18) under a long-day photoperiod. Over 40 independent transgenic lines were generated, and two representative transgenic plants are shown. (c) Total leaf number of Col-0, pk6-1, and two complemented lines under a long-day photoperiod. Flowering time was assessed by counting the number of rosette leaves and cauline leaves at bolting under the long-day photoperiod. White boxes indicate rosette leaves, and grey boxes indicate cauline leaves. Values shown are means ± SD of total leaves; over 25 plants were scored for each line. (d) Representative images of FRI, FRI pk6-1, FRI pk6-2, FRI pk7-1, FRI pk8-1, and FRI pk6/7/8 plants under a long-day photoperiod. (e) Total leaf number of FRI, FRI pk6-1, FRI pk6-2, FRI pk7-1, FRI pk8-1, and FRI pk6/7/8 plants under a long-day photoperiod. White boxes indicate rosette leaves, and grey boxes indicate cauline leaves. Values shown are means ± SD of total leaves; over 25 plants were scored for each line. (f) and (g) Relative transcript levels of FLC (f) and FT (g) in FRI pk6-1, FRI pk6-2, FRI pk7-1, FRI pk8-1, and FRI pk6/7/8. Experiments were repeated at least three times and the results of representative experiments are shown as means ± SE, n = 3. Asterisks indicate a significant difference using Student’s t-test (P < 0.01). No difference is indicated as ns. Ubiquitin 10 was used as internal control. The source data underlying the statistical analysis in (a), (c), (e), (f) and (g) are provided in the Source Data file.

Source data

Extended Data Fig. 10 Reproducibility analysis of the biological replicates for the RNA-seq.

(a) and (b) Correlation analysis of the biological replicates of the RNA-seq analysis of Col-0 (a) and pk6/7/8 (b) under a long-day photoperiod. The x and y axes indicate the read coverage normalized to the total mapped reads, and values are in the log2 scale. (c) Volcano plot of the RNA-seq results of Col-0 and pk6/7/8. The negative log of the p-value (base 10) was plotted on the y axis, while the log of the fold change (FC; base 2) was plotted on the x axis. The upregulated and downregulated genes in pk6/7/8 compared with Col-0 are indicated in the Venn diagram. (d) Gene ontology (GO) enrichment of functions associated with biological processes in the 2355 downregulated genes in pk6/7/8. The differentially expressed genes were analysed using TBtools (v.2.052). The p-value in Fisher exact test is computed.

Supplementary information

Supplementary Information

Supplementary Figs. 1–10 and Source Data for unprocessed western blots in supplementary figures.

Reporting Summary

Supplementary Table 1

H3T11ph peaks with or without glucose.

Supplementary Table 2

Upregulated and downregulated genes in pk6/7/8 mutants.

Supplementary Table 3

H3T11ph peaks in Col-0 and pk6/7/8 plants.

Supplemental Dataset 1

The primers and constructs.

Supplemental Dataset 2

Statistical source data for supplementary figures.

Source data

Source Data Fig. 1

Unprocessed western blots for Figs. 1a,b,d.

Source Data Fig. 2

Unprocessed western blots for Fig. 2e.

Source Data Fig. 2–4, 7 and 8, and Source Data Extended Data Figs. 5–9

Statistical source data for Fig. 2d. Statistical source data for Fig. 3b. Statistical source data for Fig. 4b,d,f,g,j. Statistical source data for Fig. 7f,g,i,j. Statistical source data for Fig. 8b–d. Statistical source data for Extended Data Fig. 5b. Statistical source data for Extended Data Fig. 6b. Statistical source data for Extended Data Fig. 7c–e. Statistical source data for Extended Data Fig. 8b,d,f,i,l. Statistical source data for Extended Data Fig. 9a,c,e–g.

Source Data Fig. 3

Unprocessed western blots for Fig. 3c,d.

Source Data Fig. 5

Unprocessed western blots for Fig. 5a.

Source Data Fig. 6

Unprocessed western blots for Fig. 6b,e–g.

Source Data Extended Data Fig. 4

Unprocessed western blots for Extended Data Fig. 4a.

Source Data Extended Data Fig. 6

Unprocessed western blots for Extended Data Fig. 6d–f.

Source Data Extended Data Fig. 8

Unprocessed western blots for Extended Data Fig. 8j,k.

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Hu, P., Xu, Y., Su, Y. et al. Nuclear-localized pyruvate kinases control phosphorylation of histone H3 on threonine 11. Nat. Plants 10, 1682–1697 (2024). https://doi.org/10.1038/s41477-024-01821-w

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