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TOP1α fine-tunes TOR-PLT2 to maintain root tip homeostasis in response to sugars

Nature Plants volume 8, pages 792–801 (2022)Cite this article

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A Publisher Correction to this article was published on 08 September 2022

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Abstract

Plant development is highly dependent on energy levels. TARGET OF RAPAMYCIN (TOR) activates the proximal root meristem to promote root development in response to photosynthesis-derived sugars during photomorphogenesis in Arabidopsis thaliana. However, the mechanisms of how root tip homeostasis is maintained to ensure proper root cap structure and gravitropism are unknown. PLETHORA (PLT) transcription factors are pivotal for the root apical meristem (RAM) identity by forming gradients, but how PLT gradients are established and maintained, and their roles in COL development are not well known. We demonstrate that endogenous sucrose induces TOPOISOMERASE1α (TOP1α) expression during the skotomorphogenesis-to-photomorphogenesis transition. TOP1α fine-tunes TOR expression in the root tip columella. TOR maintains columella stem cell identity correlating with reduced quiescent centre cell division in a WUSCHEL RELATED HOMEOBOX5-independent manner. Meanwhile, TOR promotes PLT2 expression and phosphorylates and stabilizes PLT2 to maintain its gradient consistent with TOR expression pattern. PLT2 controls cell division and amyloplast formation to regulate columella development and gravitropism. This elaborate mechanism helps maintain root tip homeostasis and gravitropism in response to energy changes during root development.

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Fig. 1: TOP1α represses TOR expression.
Fig. 2: TOR controls cell division and PLT2 accumulation.
Fig. 3: TOR phosphorylates and stabilizes PLT2.

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Data availability

The RNA-seq raw data have been deposited in the NCBI SRA (https://www.ncbi.nlm.nih.gov/sra/) under the accession number: PRJNA700788. Sequence data for the genes in this article can be found in the Arabidopsis Genome Initiative or GenBank/EMBL databases under the following accession numbers: CDC6, AT2G29680; CDF4, AT2G34140; CDKB1, AT3G54180; CLE40, AT5G12990; CYCB1;1, AT4G37490; CYCD3;3, AT3G50070; CYCD5;1, AT4G37630; E2Fa, AT2G36010; ETG1, AT2G40550; GIN2, AT4G29130; KIN10, AT3G01090; KIN11, AT3G29160; MCM5, AT2G07690; MCM7, AT4G02060; PLT1, AT3G20840; PLT2, AT1G51190; SCR, AT3G54220; SHR, AT4G37650; SS1, AT5G24300; SS2, AT3G01180; SS3, AT1G11720; SS4, AT4G18240; TOP1α, AT5G55300; TOR, AT1G50030; UBQ5, AT3G62250; WOX5, AT3G11260. Source data are provided with this paper.

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Acknowledgements

We thank X. Chen and Y. Xiong for helpful discussion, Y. Xiong for sharing the e2fa and tor-es mutants and the TOR constructs for Y2H, L. Pi for the CSC marker J2341, B. Menand for the AtTOR::GUS line, C. Li for the pPLT2::CFP line and X. Huang and Y. Wang for the technical support on IP–MS. This research was supported by the project 'Full-time introduction of high-end talent research project' (2020HBQZYC004; A202105008 to X.L.) from Hebei province, funding from Hebei Natural Science Foundation (grant nos C2021205013 to X.L. and C2021205043 to L.G.) and grants from NSFC (grant nos 31970824 to X.L. and 31401039 to L.G.).

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  1. These authors contributed equally: Hao Zhang, Lin Guo, Yongpeng Li, Dan Zhao.

Authors and Affiliations

  1. Ministry of Education Key Laboratory of Molecular and Cellular Biology, Hebei Key Laboratory of Molecular and Cellular Biology, College of Life Sciences, Hebei Normal University, Shijiazhuang, China

    Hao Zhang, Lin Guo, Dan Zhao, Yichao Zheng, Baowen Zhang, Yuru Ma, Yanxiao Niu, Sujuan Cui, Shuzhi Zheng, Wenqiang Tang & Xigang Liu

  2. State Key Laboratory of North China Crop Improvement and Regulation, Key Laboratory of Hebei Province for Plant Physiology and Molecular Pathology, Hebei Agricultural University, Baoding, China

    Hao Zhang, Ke Zhang, Ying Zhang, Kang Zhang, Jihong Xing, Fengru Wang & Jingao Dong

  3. State Key Laboratory of Plant Cell and Chromosome Engineering, Center for Agricultural Resources Research, Institute of Genetics and Developmental Biology, The Innovative Academy of Seed Design, Chinese Academy of Sciences, Shijiazhuang, China

    Hao Zhang, Lin Guo, Yongpeng Li, Luping Liu, Wenwen Chang & Xigang Liu

  4. Hebei Collaboration Innovation Center for Cell Signaling, Shijiazhuang, China

    Hao Zhang, Lin Guo, Dan Zhao, Yichao Zheng, Baowen Zhang, Yuru Ma, Yanxiao Niu, Sujuan Cui, Shuzhi Zheng, Wenqiang Tang & Xigang Liu

  5. College of Life Sciences, Hengshui University, Hengshui, China

    Dan Zhao

  6. Key Laboratory of Animal Physiology, Biochemistry and Molecular Biology of Hebei Province, College of Life Science, Hebei Normal University, Shijiazhuang, China

    Jiajie Hou & Ke Tan

  7. Food Science College, Shenyang Agricultural University, ShenYang, China

    Chenghao Fu

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  1. Hao Zhang
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Contributions

J.D., L.G. and X.L. designed the experiments. H.Z., Y.L., D.Z., L.L., W.C., Y.N., Y.M., Y. Zheng, Y. Zhang and Ke Z. carried out most of the experiments with technical support from F.W., J.X., Kang Z. and S.C. H.Z., B.Z., S.Z. and W.T. performed the IP–MS experiment. J.H., K.T. and C.F. performed the CPT treatment NSCLC cell assay. X.L., S.Z. and W.T. analysed data. H.Z., Y.L., L.G. and X.L. wrote the manuscript with input from J.D.

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Correspondence to Lin Guo, Jingao Dong or Xigang Liu.

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Nature Plants thanks José Manuel Pérez-Pérez, Benoit Menand and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Maintenance of root tip homeostasis.

a, Cartoon depicting a longitudinal section of a wild type (WT) Arabidopsis root tip. QC: quiescent center; CSC: columella stem cell; PM: proximal root meristem; DM: distal root meristem; COL: columella; S1-S4: columella layers 1–4. b, Morphological observation of seedlings grown under the indicated treatments. Seedlings were grown on sugar-free medium for 5 days in the dark, transferred to different conditions, and grown for 3 more days. Scale bar: 5 mm. c, Examination of cell division in the RAMs of pCYCB1;1::GUS plants under the indicated treatments. Scale bar: 100 µm. d, f, Quantification of PM and COL size in WT plants grown under the indicated conditions. Data were analyzed from 15 seedlings for each treatment from 3 experiments. Statistical significance was determined by one-way ANOVA and Tukey test (P < 0.05) (see Supplementary Data 1 for specific P values) (d) or by Student’s t-test (two-tailed) (f) and are expressed as mean ± s.d. e, DM and CC homeostasis is maintained during the skotomorphogenesis-to-photomorphogenesis transition. The seedlings described in (b) harboring the QC marker pWOX5::GFP were selected for structural observation of the root tip. Arrows mark the CSC layer; lines mark the columella story (S). Scale bar: 50 µm.

Extended Data Fig. 2 TOP1α controls root gravitropism in response to sucrose.

a, b, Root gravitropism (a) and statistical analysis of vertical growth index (VGI) (b) in roots of the indicated genotypes under ±Suc treatment. Data were analyzed from 15 seedlings for each treatment from 3 experiments. Statistical significance was determined by Student’s t-test (two-tailed) (b) and are expressed as mean ± s.d. Scale bar: 1 cm. c, Graphical representation of VGI to define root gravitropism. d, Time course of the gravity response and amyloplast formation in WT and top1α ± Suc treatment. Seedlings were turned 90° from the vertical axis and fixed at the indicated time points. Amyloplast formation and levels in layers S1 and S2 were examined. Yellow lines represent amyloplast levels. Green arrows mark amyloplasts in premature CSCs. (m/n) indicates m in n of biological repeats showing the displayed features. Scale bar: 25 µm. e, f, Root gravity response (e) and statistical analysis (f) of WT and top1α in response to Suc. Data represent mean ± s.d. of 3 biological repeats. Statistical significance was determined by Student’s t-test (two-tailed) (f) and are expressed as mean ± s.d. Scale bar: 0.5 mm. (m/n) in (d and e) indicates m in n of biological repeats showing the displayed features.

Extended Data Fig. 3 TOP1α controls root tip homeostasis in response to sucrose.

a-c, Quantification of QC cell number (a), CSC layer (b), and CC number (c) of WT and top1α ± Suc treatment. Data represent mean ± s.d. of 3 biological repeats (a, b). Data were analyzed from 15 seedlings for each treatment from 3 experiments (c). Statistical significance was determined by one-way ANOVA with Tukey’s post hoc test (P < 0.05). d, RT-qPCR of CLE40 and CDF4 expression in WT and top1α under the indicated treatments. Gene expression in WT –Suc was set to 1. Data represent mean ± s.d. of 3 biological repeats. Student’s t test (two-tailed). e, Glucose contents in Ler and top1a. Statistical significance was determined by one-way ANOVA and Tukey test (P < 0.05). Data represent mean ±s.d. of 3 biological repeats (n = 3). Each replicate (n) is from 500–550 root tips. f. TOP1α expression patterns in pTOP1a::GFP root tips. Scale bar: 50 µm. g. TOP1α expression in pTOP1a::GFP root under indicated treatments. (m/n) indicates m in n of biological repeats showing the displayed features. Scale bar: 100 μm.

Extended Data Fig. 4 The role of TOP1α is independent of Glc and energy sensors.

a, Genetic analysis of the roles of GIN2 and TOP1α in root gravitropism. Scale bars: 1 cm. b, RT-qPCR of GIN2 expression in WT and top1α under the indicated treatments. Gene expression in WT –Suc was set to 1. Data represent mean ± s.d. of 3 biological repeats. Student’s t test (two-tailed).

Extended Data Fig. 5 Transcriptome analysis of TOP1α and TOR regulated genes.

a, Venn diagram showing the number of overlapping TOR-activated and TOR-repressed as well as Glc-TOR-activated and Glc-TOR repressed DEGs identified by Xiong et al., (2013). b, Venn diagram showing the number of overlapping DEGs in top1α vs. Ler and AZD-8055-treated Ler vs. Ler (left); and overlapping DEGs in top1α vs. Ler and AZD-8055-treated top1α vs. top1α (right). c, GO analysis of TOR-activated genes (upper panel) and TOR-repressed genes (lower panel). d, GO analysis of TOP1α-activated genes (left) and TOP1α-repressed genes (right). e, f, Hierarchical clustering analysis of TOR-activated (e) and TOR-repressed genes (f). TOR-activated and TOP1α-repressed DEGs (e) and TOR-repressed and TOP1α-activated DEGs (f) were grouped and subjected to GO analysis, as shown in the graphs. g, h, Hierarchical clustering analysis of TOP1α-activated (g) genes and TOP1α-repressed genes (h). TOP1α-activated and TOR-repressed DEGs (g) and TOP1α-repressed and TOR-activated DEGs (h) were grouped and subjected to GO analysis, as shown in the graphs. The GO enrichment analysis was performed by R package ClusterProfiler3.0, in which the pvalue was calculated by Fisher’ exact test, and was adjusted by Benjamini-Hochbery method in c-h.

Extended Data Fig. 6 TOP1a regulates cell cycle gene expression.

a, Heatmap showing the expression of cell-cycle-related genes in WT and top1α ± AZD-8055 treatment. b, RT-qPCR of CYCD3;3 and E2Fa as well as E2Fa target gene expression in WT and top1α under the indicated treatments. Gene expression in WT –Suc was set to 1. Data represent mean ± s.d. of 3 biological repeats. Statistical significance was determined by one-way ANOVA and Tukey test (P < 0.05) (see Supplementary Data 1 for specific P values). c, Diagram of the CYCD3;3 locus. Gray and black rectangles and black lines represent untranslated regions, coding regions, and introns, respectively. Two nucleotides (GA) were deleted, resulting in a premature stop codon (red), in the cycd3;3 mutant. d, e, Quantification of CSC layer (d) and CC number (e) of the indicated genotypes. Data represent mean ± s.d. of 3 biological repeats (d). Data were analyzed from 15 seedlings for each genotype from 3 experiments (e). Statistical significance was determined by one-way ANOVA and Tukey test (P < 0.05) (see Supplementary Data 1 for specific P values). f, Root gravity responses and amyloplast formation in the indicated genotypes. Seedlings were turned 90° from the vertical axis and fixed at the indicated time points. Amyloplast formation and levels in layers S1 and S2 were examined. Yellow lines represent amyloplast levels, white stars mark the QC, green arrows mark the CSC layer, white arrows mark amyloplast in CSC. (m/n) indicates m in n of biological repeats showing the displayed features. Scale bar: 25 µm.

Extended Data Fig. 7 TOP1 regulates mTOR and cell cycle gene expression in response to Glc.

a, The cell viability of human NSCLC cell line HCC827 under the indicated treatments. Data represent mean ± s.d. of 3 biological repeats. Student’s t test (two-tailed). b, RT-qPCR of mTOR and cell cycle gene expression in HCC827 under the indicated treatments. Gene expression in untreated cells in 5 mM Glc was set to 1. Data represent mean ± s.d. of 3 biological repeats. Student’s t test (two-tailed). GAPDH served as negative control.

Extended Data Fig. 8 TOP1α and TOR regulate PLT2 expression.

a, b, Gene expression (a) and protein distribution of SHR, SCR, and PLT1 in plants (b) of the indicated genotypes. Gene expression in WT –Suc was set to 1. Data represent mean ± s.d. of 3 biological repeats. Student’s t test (two-tailed). (m/n) indicates m in n of biological repeats showing the displayed features. Scale bars: 50 µm. c, Diagram of the PLT2 locus. Gray and black rectangles and black lines represent untranslated regions, coding regions, and introns, respectively. A DNA fragment (shown in red) was deleted in the plt2 mutant. d, Quantification of CC number in plants of the indicated genotypes. Data were analyzed from 15 seedlings for each genotype from 3 experiments. Statistical significance was determined by one-way ANOVA and Tukey test (P < 0.05) (see Supplementary Data 1 for specific P values). e, Root gravity responses and amyloplast formation in top1α and top1α plt2 plants of the indicated genotypes. Seedlings were turned 90° from the vertical axis and fixed at the indicated time points. Amyloplast formation and levels in layers S1 and S2 were examined. Yellow lines represent amyloplast levels, white arrow marks amyloplasts and green arrows mark the premature CSC indicating that plt2 mutation failed to rescue the QC quiescence. (m/n) indicates m in n of biological repeats showing the displayed features. Scale bars: 25 µm. f, Diagram of the SS1/2/3/4 and CYCD5;1 loci. Gray and black rectangles and black lines represent untranslated regions, coding regions, and introns, respectively. The regions tested by ChIP are marked by black lines. g, RT-qPCR of SS1-4 in Ler and top1a plants under the indicated treatment. Gene expression in WT was set to 1. Data represent mean ± s.d. of 3 biological repeats. Statistical significance was determined by one-way ANOVA and Tukey test (P < 0.05) (see Supplementary Data 1 for specific P values).

Extended Data Fig. 9 TOR stabilizes PLT2.

a, CFP florescence showing PLT2 transcript levels in pPLT2::CFP and pPLT2::CFP top1α under the indicated treatments. Scale bars: 50 µm. b, e, Y2H assays to examine TOR-PLT2 and TOR-PLT1 interactions (b) and to identify particular domains of PLT2 for the TOR-PLT2 interaction (e). The indicated construct combinations were co-transformed into yeast cells and screened on plates containing SD/–Leu,–Trp or SD/–Ade,–His,–Leu,–Trp medium. c, Amino acid sequence alignment of PLT1 and PLT2. The AP2 domains are marked by red lines. d, Diagram of the constructs used in the yeast two-hybrid (Y2H) assay. F: full length; N: N-terminus; C: C-terminus; KD: kinase domain; AP2: AP2 domain. f, g, Examination of PLT2 stability in a cell-free system. MBP-PLT2 was purified and added to cell extracts from WT plants treated with DMSO or MG132 (f) or from WT and tor-es plants (g). Anti-MBP and anti-P-S/T antibodies were used to detect MBP-PLT2 and phosphorylated MBP-PLT2, respectively. h, MBP-PLT2 signal intensity of (g) quantified by ImageJ. Values represent the mean ± s.d. (n = 3). Statistical significance was determined by Student’s t-test (two-tailed). i, Examination of MBP-PLT2S12A stability in a cell-free system. MBP-PLT2 and MBP-PLT2S12A were purified and added to cell extracts from WT plants. Anti-MBP and anti-P-S/T antibodies were used to detect MBP-PLT2 and phosphorylated MBP-PLT2, respectively. j, l, CC number (j) and meristematic zone (l) of the indicted plants. Seedling was grown on -Suc under dark condition for 5 days. The CC number (j) and PLT2-GFP level (Fig. 3j) were examined. Data were analyzed from 15 seedlings for each genotype from 3 experiments. Statistical significance was determined by Student’s t test (two-tailed). k, The root phenotypes of the indicated plants. The seedlings were grown on 1/2MS plate under light condition for 5 days after germination. The PLT2-GFP level and RAM size were examined. The white arrows indicate the first elongated cortex cell and the yellow bars indicate the RAM size. Scale bars: 50 µm. For a and k, (m/n) indicates m in n of biological repeats showing the displayed features. For f, g and i, experiments were repeated three times with similar results. For gel source data, see Unmodified_Gels_Fig2.

Source data

Extended Data Fig. 10 A model of the role of TOP1α in fine-tuning TOR-PLT2 to maintain DM and CC homeostasis in the root tip.

Schematic diagram of the TOP1α- and TOR-mediated regulatory network used by plants to maintain DM and CC homeostasis in response to photosynthesis-derived Suc. Arrows represent activation; lines with bars represent repression. Dashed lines represent indirect regulation, and solid lines represent direct regulation.

Supplementary information

Supplementary Information

Supplementary Fig. 1.

Reporting Summary

Supplementary Data 1

Exact P values of one-way ANOVA and Tukey test.

Supplementary Data 2

Gene expression in RNA-seq analysis.

Supplementary Data 3

Primers used for plasmid constructs, RT–qPCR and mutagenesis.

Supplementary Data 4

All raw data for the statistical analysis that are visualized in the article.

Source data

Source Data Fig. 3

Unprocessed western blots and/or gels.

Source Data Extended Data Fig. 9

Unprocessed western blots and/or gels.

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Zhang, H., Guo, L., Li, Y. et al. TOP1α fine-tunes TOR-PLT2 to maintain root tip homeostasis in response to sugars. Nat. Plants 8, 792–801 (2022). https://doi.org/10.1038/s41477-022-01179-x

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