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Local brassinosteroid biosynthesis enables optimal root growth

Abstract

Brassinosteroid (BR) hormones are indispensable for root growth and control both cell division and cell elongation through the establishment of an increasing signalling gradient along the longitudinal root axis. Because of their limited mobility, the importance of BR distribution in achieving a signalling maximum is largely overlooked. Expression pattern analysis of all known BR biosynthetic enzymes revealed that not all cells in the Arabidopsis thaliana root possess full biosynthetic machinery, and that completion of biosynthesis relies on cell-to-cell movement of hormone precursors. We demonstrate that BR biosynthesis is largely restricted to the root elongation zone, where it overlaps with BR signalling maxima. Moreover, optimal root growth requires hormone concentrations to be low in the meristem and high in the root elongation zone, attributable to increased biosynthesis. Our finding that spatiotemporal regulation of hormone synthesis results in local hormone accumulation provides a paradigm for hormone-driven organ growth in the absence of long-distance hormone transport in plants.

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Fig. 1: BR biosynthetic mutants are dwarfs with reduced root growth.
Fig. 2: Expression patterns of BR biosynthetic enzymes in the Arabidopsis root.
Fig. 3: BRs can move over short distances in the Arabidopsis root.
Fig. 4: BR signalling and synthesis maxima overlap in the Arabidopsis root.
Fig. 5: Root meristem and elongation zones have different demands for BRs.
Fig. 6: Local DWF4 enzyme expression determines BR levels.
Fig. 7: Localized BR biosynthesis coordinates root growth.

Data availability

The data supporting the findings in this study are available from the corresponding author upon reasonable request. Source data are provided with this paper.

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Acknowledgements

We thank Y. Yin for providing the anti-BES1 antibody, A. Caño-Delgado, Z. Ding, M. Seo and C. Koncz for providing published materials, T. Beeckman, J. Friml, G. Beemster, J. Wendrich, R. Pleskot and K. Yperman for useful discussions and M. De Cock for help in preparing the manuscript. This work was supported by Research Foundation-Flanders (project no. G022516N to E.R. and a postdoctoral fellowship no. 12R7819N to N.V.); the Chinese Scholarship Council (predoctoral fellowships to Y.W. and B.G.); the European Research Council (grant no. 803048 to M.F.); and ERDF project ‘Plants as a tool for sustainable global development’ (no. CZ.02.1.01/0.0/0.0/16_019/0000827 to M.K., P.J., J.O. and M.S.).

Author information

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Authors

Contributions

N.V., Y.W. and E.R. initiated the project and designed experiments. N.V. and Y.W. performed most of the experiments. I.V., M.F. and B.G. performed experiments and analysed data. M.K., P.J. and J.O. contributed materials and hormone measurements. N.V. and E.R. wrote the manuscript. All authors revised the manuscript.

Corresponding authors

Correspondence to Nemanja Vukašinović or Eugenia Russinova.

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The authors declare no competing interests.

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Peer review information Nature Plants thanks the anonymous reviewers for their contribution to the peer review of this work.

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

Extended data

Extended Data Fig. 1 The GFP–tagged BR biosynthetic enzymes are functional and show similar expression patterns in multiple independent transgenic lines.

a, Rescue of the BR biosynthetic mutants by expression of the corresponding GFP–tagged enzymes under the control of their endogenous promoters. Two independent transgenic lines for each gene are compared with the wild type (Col-0) and the corresponding mutant. Six-day-old seedlings are shown. Scale bars, 1 cm. b, Consistent expression patterns of GFP–tagged BR biosynthetic enzymes under the control of their endogenous promoters in multiple independent transgenic lines. Root meristems of the 6-day-old seedlings from two independent transgenic lines for each GFP–tagged enzyme are shown. For seedlings expressing pCYP90D1:CYP90D1-GFP only the root elongation zone is shown, because the signal in the root apical meristem is extremely weak. Scale bars, 50 µm. c, Six-day-old pDWF4:DWF4-GFPdwf4 roots treated with mock (DMSO), brassinazole and brassinolide for 24 h. Scale bars, 50 µm. d, Expression patterns of the PROMOTER-NLS-GFP reporters for all BR biosynthetic genes. For each reporter line, 6-day-old root meristems and cross section are shown. Scale bars, 50 µm. e, Co-expression of NLS-GFP and NLS-mCHERRY reporters under the control of CPD and ROT3 promoters, respectively. Scale bar, 50 µm. Roots were stained with propidium iodide.

Extended Data Fig. 2 Ectopic expression of CPD and BAS1 in the Arabidopsis root.

a, Quantification of the root meristem cell numbers of cpd seedlings expressing the pSCR:CPD-mCHERRY construct. All individual data points are plotted. Red horizontal bars represent the means and error bars represent s.d. n = number of roots analysed. The significant differences between transgenic lines and the Col-0 control were determined by one-way analysis of variance (ANOVA) and Dunnett’s multiple comparison tests. *** P < 0.001, ** P < 0.01 and * P < 0.05. The P value versus the Col-0 control for cpd is = 0.7077, line # 1 = 0.1849 and line # 9 < 0.0001. The experiment was repeated independently three times with similar results. b, Reverted root meristem diameter of the cpd mutant to that of the wild type upon ectopic expression of CPD in the endodermis. Six-day-old seedlings of the two independent transgenic lines were measured. All individual data points are plotted. Red horizontal bars represent the means and error bars represent s.d. n = number of roots analysed. The significant differences between transgenic lines and the Col-0 control were determined by one-way analysis of variance (ANOVA) and Dunnett’s multiple comparison tests. *** P < 0.001, ** P < 0.01 and * P < 0.05. The P value versus the Col-0 control for cpd is < 0.0001, line # 1 = 0.3417 and line # 9 = 0.1597. The experiment was repeated independently three times with similar results. c, Active BR depletion by the BAS1–GFP overexpression causing a typical BR-deficient phenotype. Scale bar, 1 cm. d, Thickening of the meristem and shortening of the meristem cells caused by the BAS1–GFP overexpression, typical for BR-related mutants. More than three independent experiments were performed. Representative images are shown. Scale bars, 50 µm. e, Decreased root growth by the inducible, tissue-specific expression of BAS1–GFP in the endodermis. Two independent transgenic lines were compared with the wild type (Col-0). Four-day-old seedlings were transferred to fresh medium containing 10 µM β-estradiol (EST) and imaged after 3 days. Scale bar, 1 cm. f, Non-induced pSCR-XVE:BAS1-GFP seedlings without impaired root growth. n = number of roots analysed. g, h, Quantification of meristem cell number (g) and root diameter (h) of EST-induced pSCR-XVE:BAS1-GFP seedlings. All individual data points are plotted. Red horizontal bars represent the means and error bars represent s.d. n = number of roots analysed. The significant differences between transgenic lines and the Col-0 control were determined by one-way analysis of variance (ANOVA) and Dunnett’s multiple comparison tests. *** P < 0.001, ** P < 0.01 and * P < 0.05. The P values versus the Col-0 control for transgenic lines are as follows: for (g) line # 1 = 0.9250 and line # 4 = 0.0294; for (h) line # 1 < 0.0001 and line # 4 < 0.0001. More than three independent experiments were performed. Representative images are shown. i, BES1 phosphorylation status tested by western blot with the α-BES1 antibody in roots of 7-day-old Col-0 and pSCR-XVE:BAS1-GFP seedlings. Tubulin was used as a loading control. Seedlings were germinated and grown on medium containing 10 µM β-oestradiol. The experiment was performed twice with similar outcome. j, Confocal image of a 6-day-old root meristem expressing pSCR:BAS1-mCHERRY. More than three independent experiments were performed. Representative images are shown. Scale bar, 50 µm. k-n, Quantification of root length (k) meristem cell number (l), meristem cortical cell length – first 20 cells of individual roots (m) and root diameter (n) of Col-0 and pSCR:BAS1-mCHERRY 6-day-old seedlings. All individual data points are plotted. Red horizontal bars represent the means and error bars represent s.d. n = number of roots analysed for (k), (l) and (n) and n = number of cells analysed for (m). For (m) number of individual roots used in experiment are 18, 14 and 13 for Col-0, line # 3 and line # 10, respectively. The significant differences between transgenic lines and the Col-0 control were determined by one-way analysis of variance (ANOVA) and Dunnett’s multiple comparison tests. *** P < 0.001, ** P < 0.01 and * P < 0.05. The P values versus the Col-0 control for transgenic lines are as follows: for (k) line # 3 < 0.0001 and line # 10 < 0.0001; for (l) line # 3 = 0.8157 and line # 10 = 0.1336; for (m) line # 3 < 0.0001 and line # 10 < 0.0001; for (n) line # 3 < 0.0001 and line # 10 < 0.0001. All experiments were repeated independently more than three times with similar results.

Source data

Extended Data Fig. 3 Expression patterns of BR biosynthetic enzymes in the root elongation zone.

a, The expression domains of several genes expanding in the root elongation zone. DWF4–GFP expression was very broad and the GFP signal could be observed in all cell types of the elongation zone. The CPD–GFP expression was restricted to the stele of the elongation zone. The DET2–GFP expression was limited to the stele, similarly to the expression of CPD–GFP. The ROT3–GFP expression had maxima in the stele, but a signal also occured in all other cell types. The expression of the ROT3 homolog, CYP90D1, was limited to the stele. The BR6OX1 and BR6OX2 enzymes both had expression maxima in endodermis and pericycle, but the expression pattern of the BR6OX1 enzyme was expanded in all cell types. Roots were stained with propidium iodide. Scale bars, 50 µm. b, Increase in the expression levels of most of the BR biosynthetic enzymes in the transition and elongation zones as visualized by colour-based fluorescence intensity coding. All reporter lines showed an increase in expression higher in the root except for DET2–GFP and GFP–BR6OX2. The signal intensity of the BR receptor, BRI1-mCITRINE in pBRI1:BRI1-mCITRINE/bri1 line did not exhibit signal increase in the elongation zone. Maximum Z projections of the same roots from Fig. 2a are shown for BR biosynthetic enzyme lines. Scale bars, 100 µm. c, Roots expressing pBR6OX2:BR6OX2-GFP (left panel). Increased GFP signal could be observed in the elongation zone as visualized by colour-based fluorescence intensity coding (right panel). Scale bars, 100 µm. All transgenic lines in (a), (b) and (c) were imaged in more than three independent experiments. Representative images are shown. d, Expression levels of BR biosynthetic genes and BRI1 in the Arabidopsis root. All BR biosynthetic genes had an increased expression in the elongation zone, while BRI1-mCITRINE exhibited the highest expression in the meristem. Extracted from Huang and Schiefelbein (2015). MZ, meristematic zone; EZ, elongation zone; DZ, differentiation zone. Symbols depict individual values. Bars represent s.d.

Extended Data Fig. 4 The DWF4 expression maximum precedes the BR signalling peak monitored by the nuclear fluorescence intensity of BES1–GFP.

a, Six-day-old Arabidopsis roots expressing DWF4–GFP/dwf4 and BES1–GFP/Col-0 under the control of their native promoters. Scale bars, 100 µm. b, Fluorescence intensity of DWF4–GFP and BES1–GFP in single cells within single cell files, along the longitudinal root axis shown in a. Note that the DWF4–GFP expression peak precedes the BES1–GFP nuclear accumulation peak in the elongation zone. The DWF4–GFP fluorescence intensity was measured for the whole cells along the trichoblast file whereas the BES1–GFP florescence intensity was measured in the nuclei along cortical file.

Extended Data Fig. 5 Effects of exogenous brassinolide (BL) on root growth.

a, Root growth rates of Col-0 plants grown on increasing concentrations of BL. Six-day-old seedlings were transferred to fresh media containing BL or mock and root growth was followed for 4 days. Values represent the means of the root growth rates ± s.d. n = number of roots analysed. b, c, Quantification of the root meristem diameter (b) and the root meristem area size measured from the quiescent center to the first elongated cell (c) of Col-0 and dwf4 treated with increasing concentrations of BL. Roots were treated with BL for 24 h. All individual data points are plotted. Red horizontal bars represent the means and error bars represent s.d. n = number of roots analysed. The significant differences between Col-0 and the dwf4 seedlings treated with BL and Col-0 grown on mock (0) were determined by two-way analysis of variance (ANOVA) and Tukey’s multiple comparisons tests. *** P < 0.001, ** P < 0.01 and * P < 0.05. The P values versus the Col-0 + DMSO control for different treatments are as follows: for (b) Col-0 + 10 pM BL < 0.0001, Col-0 + 50 pM BL < 0.0001, Col-0 + 100 pM BL < 0.0001, Col-0 + 250 pM BL < 0.0001, dwf4 + DMSO < 0.0001, dwf4 + 10 pM BL = 0.0142, dwf4 + 50 pM BL < 0.0001, dwf4 + 100 pM BL < 0.0001, dwf4 + 250 pM BL < 0.0001; for (c) Col-0 + 10 pM BL = 0.9019, Col-0 + 50 pM BL = 0.3871, Col-0 + 100 pM BL > 0.9999, Col-0 + 250 pM BL = 0.9972, dwf4 + DMSO = 0.0638, dwf4 + 10 pM BL > 0.9999, dwf4 + 50 pM BL = 0.5746, dwf4 + 100 pM BL = 0.3938, dwf4 + 250 pM BL = 0.0037. All experiments were repeated independently more than three times with similar results. d, Experimental setup for measurements of the root meristem cell production rate (left panel). Six-day-old Col-0 and dwf4 seedlings were transferred to agar plates supplemented with BL and mock, and after 3 days of growth, elongation of individual roots was recorded for the period of 24 h (E). Subsequently, roots were imaged with confocal microscope, and the average mature cell length was calculated (Lavg) for each root and used to calculate the meristem production rate (P). e, Quantification of the root meristem production rate (P) for cortical cells. All individual data points are plotted. Red horizontal bars represent the means and error bars represent s.d. n = number of roots analysed. The significant differences between Col-0 and the dwf4 seedlings treated with BL and Col-0 grown on mock (0) were determined by two-way analysis of variance (ANOVA) and Tukey’s multiple comparison tests. *** P < 0.001, ** P < 0.01 and * P < 0.05. The P value versus the Col-0 + DMSO control for Col-0 + 10 pM BL is = 0.0874, Col-0 + 100 pM BL = 0.7849, dwf4 + DMSO < 0.0001, dwf4 + 10 pM BL = 0.9623 and dwf4 + 100 pM BL < 0.0001. The experiment was repeated independently three times with similar results. f, Quantification of mature cell length of individual roots used to calculate the meristem production rate in (e). All individual data points are plotted. Red horizontal bars represent the means and error bars represent s.d.

Source data

Extended Data Fig. 6 Ectopic expression of DWF4 enzyme.

a, Confocal images of meristems of long and short p35S:DWF4-mCHERRY (DWF4-OE) roots. Six-day-old seedlings were used for imaging. Scale bars, 50 µm. Confocal imaging was performed in three independent experiments. Representative images are shown. b, Two biological replicates of the western blot shown in Fig. 6c. The experiment was repeated independently three times with similar results. c, Eight-day-old seedlings expressing DWF4–GFP in dwf4 mutant background under the control of native or WEREWOLF (WER) promoters. Two independent transgenic lines for each construct were compared with wild type (Col-0) and dwf4. Plants were grown for 6 days until the point where homozygous dwf4 plants could be recognized and transferred to fresh agar plates. Root tips were marked (yellow bar), and grown for two additional days. d, Confocal images of root meristems of Col-0, dwf4 and two independent dwf4 mutant lines expressing DWF4–GFP under the control of the DWF4 and WER promoters. Six-day-old plants were imaged. Roots were stained with propidium iodide. All transgenic lines were imaged in more than three independent experiments. Representative images are shown. Scale bars, 50 µm. e-g, Quantification of cortical meristem cell number (e), root meristem diameter (f) and cortical meristem cell length of the first 20 cells of individual roots (g). All individual data points are plotted. Red horizontal bars represent the means and error bars represent s.d. n = number of roots (e, f) and cells (g). For (g) number of individual roots used in the experiment are 19, 18, 13, 19, 21 and 20 for Col-0, dwf4, line # 18, line # 20, line # 1 and line # 5, respectively. The significant differences between transgenic lines and the Col-0 control were determined by one-way analysis of variance (ANOVA) and Dunnett’s multiple comparison tests. *** P < 0.001, ** P < 0.01 and * P < 0.05. The P value versus the Col-0 control for different transgenic lines were as follows: for (e) dwf4 = 0.5724, line # 18 = 0.8377, line # 20 = 0.4041, line # 1 = 0.0308 and line # 5 = 0.0763; for (f) dwf4 < 0.0001, line # 18 = 0.7739, line # 20 = 0.1982, line # 1 < 0.0001 and line # 5 < 0.0001; for (g) dwf4 < 0.0001, line # 18 = 0.0938, line # 20 = 0.0022, line # 1 < 0.0001 and line # 5 < 0.0001. The experiment was repeated independently three times with similar results.

Source data

Extended Data Fig. 7 Confined expression of CPD enzyme can only locally rescue the root phenotype of cpd mutant.

a, Six-day-old cpd mutant roots expressing CPD–GFP and CPD–mCHERRY under the control of WEREWOLF (WER) and COBRA-LIKE9 (COBL9) promoters, respectively. All transgenic lines were imaged in more than three independent experiments. Representative images are shown. Scale bars, 100 µm. b, c, Quantification of the root lengths (b) and mature cortical cell lengths (c) of the Col-0, cpd, pWER:CPD-GFP/cpd and pCOBL9:CPD-mCHERRY/cpd lines. All individual data points are plotted. Red horizontal bars represent the means and error bars represent s.d. n = number of roots (b) and cells (c) analysed. For (c) the number of individual roots used in the experiment was 12 for all lines. The significant differences between transgenic lines and the Col-0 control were determined by one-way analysis of variance (ANOVA) and Dunnett’s multiple comparison tests. *** P < 0.001, ** P < 0.01 and * P < 0.05. The P value versus the Col-0 control for different transgenic lines are as follows: for (b) cpd < 0.0001, pWER:CPD-GFP < 0.0001 and pCOBL9:CPD-mCHERRY < 0.0001; for (c) cpd < 0.0001, pWER:CPD-GFP < 0.0001 and pCOBL9:CPD-mCHERRY = 0.049. The experiment was repeated independently three times with similar results. d, Confocal images of root meristems of Col-0, cpd, pWER:CPD-GFP/cpd and pCOBL9:CPD-mCHERRY/cpd lines. All transgenic lines were imaged in more than three independent experiments. Representative images are shown. Scale bars, 50 µm. e-g, Quantification of cortical meristem cell number (e), cortical meristem cell length of the first 20 cells of individual roots (f) and root meristem diameter (g). All individual data points are plotted. Red horizontal bars represent the means and error bars represent s.d. n = number of roots (e, g) and cells (f). For (f) the number of individual roots used in the experiment was 12 for all lines. The significant differences between transgenic lines and the Col-0 control were determined by one-way analysis of variance (ANOVA) and Dunnett’s multiple comparison tests. *** P < 0.001, ** P < 0.01 and * P < 0.05. The P value versus the Col-0 control for different transgenic lines are as follows: for (e) cpd = 0.0808, pWER:CPD-GFP = 0.0370 and pCOBL9:CPD-mCHERRY = 0.0323; for (f) cpd < 0.0001, pWER:CPD-GFP = 0.011 and pCOBL9:CPD-mCHERRY = < 0.0001; for (g) cpd < 0.0001, pWER:CPD-GFP = 0.2712 and pCOBL9:CPD-mCHERRY = < 0.0001. The experiment was repeated independently three times with similar results.

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Supplementary information

Supplementary Information

Supplementary Table 1.

Reporting Summary

Supplementary Video 1

Long-term confocal imaging of 6-day-old pDWF4WF4-GFP/dwf4 roots by vertical-stage microscopy.

Supplementary Video 2

Long-term confocal imaging of 6-day-old pROT3:ROT3-GFP/rot3 roots by vertical-stage microscopy.

Supplementary Video 3

Long-term confocal imaging of 6-day-old pCPD:CPD-GFP/cpd roots by vertical-stage microscopy.

Supplementary Video 4

Long-term confocal imaging of 6-day-old pBES1:BES1-GFP roots by vertical-stage microscopy.

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Unprocessed immunolots.

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Unprocessed immunoblots.

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Vukašinović, N., Wang, Y., Vanhoutte, I. et al. Local brassinosteroid biosynthesis enables optimal root growth. Nat. Plants 7, 619–632 (2021). https://doi.org/10.1038/s41477-021-00917-x

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