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Plasmodesmata mediate cell-to-cell transport of brassinosteroid hormones

Nature Chemical Biology volume 19, pages 1331–1341 (2023)Cite this article

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Abstract

Brassinosteroids (BRs) are steroidal phytohormones that are essential for plant growth, development and adaptation to environmental stresses. BRs act in a dose-dependent manner and do not travel over long distances; hence, BR homeostasis maintenance is critical for their function. Biosynthesis of bioactive BRs relies on the cell-to-cell movement of hormone precursors. However, the mechanism of the short-distance BR transport is unknown, and its contribution to the control of endogenous BR levels remains unexplored. Here we demonstrate that plasmodesmata (PD) mediate the passage of BRs between neighboring cells. Intracellular BR content, in turn, is capable of modulating PD permeability to optimize its own mobility, thereby manipulating BR biosynthesis and signaling. Our work uncovers a thus far unknown mode of steroid transport in eukaryotes and exposes an additional layer of BR homeostasis regulation in plants.

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Fig. 1: PD permeability modulates BR signaling.
Fig. 2: BL but not its precursor 22-OHCR rescues dwf4 mutants after PD closure.
Fig. 3: Localization of CSA in Arabidopsis root tips.
Fig. 4: BRs regulate PD permeability.
Fig. 5: PD-mediated transport and homeostasis of BR biosynthesis and signaling.

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Data availability

Numerical source data files and uncropped scans of blots are provided for figures and extended data figures. Primer lists, Fiji macro for callose deposition quantification and notes on synthesis of chemical compound used in this study can be found in Supplementary Information. Source data are provided with this paper.

Code availability

Single-cell RNA-sequencing data analysis code used in this study is deposited at GitHub at https://github.com/tmnolan/Plasmodesmata-mediate-cell-to-cell-transport-of-brassinosteroid-hormones.

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Acknowledgements

We thank Y. Benitez-Alfonso (University of Leeds, UK), Y. Helariutta (University of Helsinki, Finland) and L. De Veylder (VIB-Ghent University) for providing published materials; J. Oklestkova (Palacký University, Czech Republic) for the kind gift of castasterone; Q. Yu (VIB-Ghent University) for pUBQ10:PDCB1–mCherry construct; and Y. Yin (Iowa State University, Ames, USA) for providing the anti-BES1 antibody. We thank S. Vanneste (Ghent University, Belgium), M. Strnad and J. Oklestkova (Palacký University, Czech Republic) for useful discussions and M. De Cock for help in preparing the manuscript. This work was supported by the Research Foundation-Flanders (project G002121N to E.R. and a postdoctoral fellowships 12R7822N and 12R7819N to N.V.), Chinese Scholarship Council (predoctoral fellowships to Y.W.), European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (project 772103-BRIDGING to E.M.B.), Human Frontier long-term postdoctoral fellowship (LT000340/2019 to M.P.P.), ERDF project ‘Plants as a tool for sustainable global development’ (CZ.02.1.01/0.0/0.0/16_019/0000827 to M.K.), National Institute of General Medical Sciences of the National Institutes of Health (R01GM127759 to W.B. and MIRA 1R35GM131725 to P.N.B.), US National Science Foundation (Postdoctoral Research Fellowships in Biology Program IOS-2010686 to T.M.N.), Howard Hughes Medical Institute to P.N.B. as an Investigator, Grants-in-Aid for Scientific Research from the JSPS (JP21H05644 to T.S.) and JSPS Research Fellowship for Young Scientists (to Y.L.). Two-photon cell diffusion assay and root whole-mount immunostaining was done on the Bordeaux Imaging Center, member of the national infrastructure France-BioImaging supported by the French National Research Agency (ANR-10-INBS-04).

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Authors and Affiliations

  1. Department of Plant Biotechnology and Bioinformatics, Ghent University, Ghent, Belgium

    Yaowei Wang, Yongming Luo, Nemanja Vukašinović & Eugenia Russinova

  2. Center for Plant Systems Biology, VIB, Ghent, Belgium

    Yaowei Wang, Yongming Luo, Nemanja Vukašinović & Eugenia Russinova

  3. Laboratoire de Biogenèse Membranaire, Unité Mixte de Recherche 5200, Université de Bordeaux, Centre National de la Recherche Scientifique, Villenave d’Ornon, France

    Jessica Perez-Sancho, Marija Smokvarska & Emmanuelle M. Bayer

  4. Plant Molecular and Cellular Biology Laboratory and Integrative Biology Laboratory, Salk Institute for Biological Studies, La Jolla, CA, USA

    Matthieu Pierre Platre & Wolfgang Busch

  5. Department of Organic and Macromolecular Chemistry, Ghent University, Ghent, Belgium

    Brenda Callebaut & Johan M. Winne

  6. Laboratory of Growth Regulators, Institute of Experimental Botany, The Czech Academy of Sciences and Palacký University, Olomouc, Czech Republic

    Karoll Ferrer & Miroslav Kvasnica

  7. Faculty of Science, Hokkaido University, Sapporo, Japan

    Yongming Luo & Takeo Sato

  8. Department of Biology, Duke University, Durham, NC, USA

    Trevor M. Nolan & Philip N. Benfey

  9. Howard Hughes Medical Institute, Duke University, Durham, NC, USA

    Philip N. Benfey

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Contributions

Y.W., N.V., E.M.B and E.R. initiated the project and designed experiments. Y.W. performed most of the experiments. N.V. prepared constructs and performed imaging. Y.L. performed imaging and calculated the PD index. J.P.S. and M.S. performed callose immunostaining and DRONPA-s imaging and analyzed the data. T.M.N. analyzed scRNA-seq data. M.P.P. generated transgenic lines and contributed materials. B.C. and J.M.W. synthetized CSA, and M.K. and K.F. synthetized 22-OHCR. Y.W., N.V., T.S., E.M.B., W.B., P.N.B. and E.R. analyzed the data and wrote the article. All authors revised the manuscript.

Corresponding authors

Correspondence to Nemanja Vukašinović or Eugenia Russinova.

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Competing interests

P.N.B. is the cofounder and Chair of the Scientific Advisory Board of Hi Fidelity Technologies, a company that works on crop root growth. The remaining authors declare no competing interests.

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Nature Chemical Biology thanks Y. Benitez-Alfonso 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 Subcellular localization of BR biosynthetic enzymes.

a,b,c,d, The BR biosynthetic enzyme ROT3-GFP and DWF4-GFP co-localize with the ER marker C4H-mCherry when transiently co-expressed in tobacco leaves (a,c) and in Arabidopsis root epidermal cells expressing ROT3-GFP and DWF4-GFP under the control of their native promoters (b,d). The fluorescence intensity profiles along the white arrows are shown on the right. A.U., arbitrary units. e, Co-localization of HDEL-BFP and DWF4-GFP with plasmodesmata (PD) marker PDLP1-mCherry and MCTP3-GFP with PD marker PDCB1-mCherry when transiently co-expressed in tobacco leaves. These co-expression combinations were used to calculate PD indexes in (f). f, The PD index of DWF4-GFP biosynthetic enzyme compared to the indexes of ER marker HDEL-BFP and PD-resident protein MCTP3-GFP. DWF4-GFP index above 1 indicates partial enrichment at PD. All individual data points are plotted. Horizontal and error bars represent the means and s.d., respectively. n, number of ROIs used to calculate average PD index. The significant differences were determined with one-way analysis of variance (ANOVA) and Tukey’s multiple comparison tests. *** P < 0.001 g, Co-localization of DWF4-GFP and ROT3-GFP biosynthetic enzymes expressed under their native promoters with the PD marker PDLP1-mCherry in Arabidopsis roots. Epidermal and cortical cells of the root transition zone were imaged for DWF4-GFP and ROT3-GFP, respectively. White arrowheads mark PD, labeled by PDLP1-mCherry. No clear co-localization of BR biosynthetic enzymes and PDLP1-mCherry were observed. Scale bars, 10 μm (a,c,g), 20 μm (e) and 25 μm (b,d). For a,b,c,d,e,f, the experiment was repeated three times and for g,h, twice with similar results.

Source data

Extended Data Fig. 2 Reduced cell-to-cell connectivity negatively affects BR signaling.

a,b, Quantification of root meristem cell length in (a) and root diameter in (b) of 5-d-old Col-0 and pEN7:icals3m seedlings which were grown for 12 h on agar medium containing either estradiol (EST) (5 μM) or DMSO (mock). For (a,b) the significant differences were determined with two-way analysis of variance (ANOVA) and Šídák’s multiple comparisons test. *** P < 0.001 and * P < 0.05. c, Aniline blue staining of callose deposition in the root tips of pEN7:icals3m plants. Five-day-old seedlings were transferred to agar medium containing estradiol (EST) (5 μM) for the indicated times and DMSO (mock), followed by aniline blue staining. Scale bars, 100 µm. d, Confocal images of pCPD:PDLP5-BFP/pCPD:CPD-GFP/cpd and pCPD:PDLP5-BFP/pSCR:CPD-mCherry/cpd lines. Scale bars, 50 µm. e, The relative gene expression of PDLP5 in pCPD:PDLP5-BFP/pCPD:CPD-GFP/cpd (line #15) and pCPD:PDLP5-BFP/pSCR:CPD-mCherry/cpd (line #14) compared to their segregating siblings that do not express pCPD:PDLP5-BFP. Error bars represent s.d. f, Phenotypes of 6-d-old pCPD:CPD-GFP/cpd, pCPD:PDLP5-BFP/pCPD:CPD-GFP/cpd, pSCR:CPD-mCherry/cpd, and pCPD:PDLP5-BFP/pSCR:CPD- mCherry/cpd seedlings from two independent transgenic lines. For each line, segregating siblings that do not express pCPD:PDLP5-BFP are shown. Scale bar, 1 cm. g, The quantification of primary root length of transgenic lines shown in (f). h,i, Quantification of root meristem cell length in (h) and root diameter in (i) of seedlings shown in (f). j, Phosphorylation status of BES1 detected by immunoblotting (IB) with α-BES1 antibody in roots. Tubulin detected with α-tubulin antibody was used as loading control. pBES1, phosphorylated BES1, dBES1, dephosphorylated BES1. Two panels from each row are from the same blots and were cropped and arranged for clarity. For a,b,g,h,i, all individual data points are plotted. Horizontal and error bars represent the means and s.d., respectively. n, number of roots used in b,g,i, and cells used in a,h. The significant differences for g,h,i, were determined with one-way ANOVA and Tukey’s multiple comparison tests. *** P < 0.001, ** P < 0.01, and * P < 0.05. For a,b,h,i,j, the experiment was repeated twice and for f,g, three times with similar results.

Source data

Extended Data Fig. 3 Increased cell-to-cell connectivity positively affects BR signaling.

a, A confocal image of 6-d-old p35S:PdBG1-mCitrine (PdBG1-OE) root meristem. Cell walls were stained with propidium iodide. b, Aniline blue staining of callose deposition in the root tips of PdBG1-OE plants. Scale bars, 100 µm (a,b). c, Phenotype of 6-d-old PdBG1-OE seedlings. Scale bar, 1 cm. d,e,f, Quantification of the primary root length (d), root meristem cell length (e) and root meristem diameter (f) shown in (c). All individual data points are plotted. Horizontal and error bars represent the means and s.d., respectively. n, number of roots (d,f) and cells (e). The significant difference was determined with two-tailed Student’s unpaired t-test analysis. *** P < 0.001, ** P < 0.01, and * P < 0.05. g, Phosphorylation status of BES1 detected by immunoblotting with α-BES1 antibody in whole seedlings. Tubulin detected with α- tubulin antibody was used as loading control. pBES1, phosphorylated BES1, dBES1, dephosphorylated BES1. h, Quantification of BES1 dephosphorylation in (g) represented as a ratio of dephosphorylated BES1 (dBES1) relative to the total BES1. pBES1, phosphorylated BES1. i, The relative gene expression of DWF4 in Col-0 and PdBG1-OE line. Error bars represent the s.d. For h,i, the significant difference was determined with two-tailed Student’s unpaired t-test analysis. *** P < 0.001, ** P < 0.01, and * P < 0.05. For c,d,e,f, the experiment was done in two and for g,h,i, in three independent biological repeats with similar results.

Source data

Extended Data Fig. 4 22-Hydroxycampesterol (22-OHCR) is an inactive BR precursor.

a, BR biosynthetic pathway with all known enzymes and their presumed position within the pathway. The position of 22-OHCR is highlighted. b, 22-OHCR rescued the root phenotype of dwf4, but not of cpd. Five-day-old Arabidopsis wild type (Col-0), dwf4, and cpd seedlings were transferred to agar medium containing BL (500 pM), 22-OHCR (500 nM), and DMSO (mock), and imaged after 24 h. Root tips were marked immediately after the transfer (white bars). Scale bars, 5 mm. c, Quantification of the primary root length of Col-0, dwf4 and cpd in (b). Horizontal and error bars represent the means and the s.d., respectively. n, number of roots analyzed. The significant differences between the wild type (Col-0) and the mutants were determined with one-way analysis of variance (ANOVA) and Tukey’s multiple comparison tests. *** P < 0.001 d, Phosphorylation status of BES1 detected by immunoblotting (IB) with the α-BES1 antibody in seedlings in (b). Tubulin detected with the α-tubulin antibody was used as loading control. pBES1, phosphorylated BES1, dBES1, dephosphorylated BES1. e, Confocal images of 6-day-old root tips of pBES1:gBES1-GFP/pEN7:icals3m/dwf4 Arabidopsis seedlings stained with PI. Scale bars, 100 µm. f,g, Quantification of the meristem cell length (f) and root meristem diameter (g) of roots shown in (e). Horizontal and error bars represent the means and s.d., respectively. n, number cells. The significant differences were determined with one-way analysis of variance (ANOVA) and Tukey’s multiple comparison tests. *** P < 0.001. For b,c,d,e,f,g, the experiment was repeated twice with similar results.

Source data

Extended Data Fig. 5 Biological activity and uptake of castasterone-alkyne (CSA).

a, CSA retains the biological properties of castasterone (CS). Five-day-old seedlings were transferred to agar media containing different concentrations of CS or CSA as indicated and DMSO (mock) for 24 h. Root tips were marked immediately after the transfer (white bars). Scale bar, 5 mm. b, Quantification of primary root length in (a). Horizontal and error bars represent the means and the s.d., respectively. n, number of roots analyzed. The significant differences were determined with one-way analysis of variance (ANOVA) and Tukey’s multiple comparison tests. *** P < 0.001 c, Aniline blue staining of callose deposition in 6-day-old pWER:icals3m plants. Wild type (Col-0) was used as control (left panel). Scale bars, 100 µm. d, Phenotypes of 6-d-old wild type (Col-0) and pWER:icals3m seedlings. Scale bar, 5 mm. e, Quantification of primary root length in (c). Horizontal and error bars represent the means and the s.d., respectively. n, number of roots analyzed. The significant differences were determined with two-tailed Student’s unpaired t-test analysis. *** P < 0.001. f, Accumulation of CS-BDP-FL signal in in the epidermal cells of Col-0 and pWER:icals3m 6-d-old seedlings. Scale bars, 25 µm. For a,b,d,e, the experiment was repeated twice with similar results.

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Extended Data Fig. 6 BRs positively regulate callose deposition in roots.

Callose immunostaining of Arabidopsis wide type (Col- 0), dwf4 and cpd roots. Four-day-old seedlings were transferred to agar medium containing brassinolide (BL) (200 nM), brassinazole (BRZ) (1 µM) and DMSO (mock) for 24 h. Cell nuclei were stained by 4′,6-diamidino-2-phenylindole (DAPI). Epidermal cell layers are shown. Scale bars, 50 μm. The same experimental material was used to quantify callose deposition at PD in Fig. 4b.

Extended Data Fig. 7 Expression levels of callose deposition-related genes are positively regulated by BRs.

a, Heatmap showing relative expression of PD-related genes across all cells from publicly available brassinolide (BL) 2-hour scRNA-seq compared to brassinazole (BRZ) scRNA-seq. Color represent log2 fold-change of BL 2-hour versus BRZ. b, Two-dimensional uniform manifold approximation and projection (UMAP) embedding of BRZ and BL 2-hour treated cells from scRNA-seq dataset36. Colors indicate cell type (top) or developmental stage (bottom). c, PDLP3 and CalS8/CSL4 expression in BRZ and BL 2-hour scRNA-seq. The color scale on the UMAP projection represents log-normalized, corrected UMI counts.

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Supplementary Tables 1 and 2 and Supplementary Notes 1–3.

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Wang, Y., Perez-Sancho, J., Platre, M.P. et al. Plasmodesmata mediate cell-to-cell transport of brassinosteroid hormones. Nat Chem Biol 19, 1331–1341 (2023). https://doi.org/10.1038/s41589-023-01346-x

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