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GDSL-domain proteins have key roles in suberin polymerization and degradation

Nature Plants volume 7, pages 353–364 (2021)Cite this article

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Abstract

Plant roots acquire nutrients and water while managing interactions with the soil microbiota. The root endodermis provides an extracellular diffusion barrier through a network of lignified cell walls called Casparian strips, supported by subsequent formation of suberin lamellae. Whereas lignification is thought to be irreversible, suberin lamellae display plasticity, which is crucial for root adaptative responses. Although suberin is a major plant polymer, fundamental aspects of its biosynthesis and turnover have remained obscure. Plants shape their root system via lateral root formation, an auxin-induced process requiring local breaking and re-sealing of endodermal lignin and suberin barriers. Here, we show that differentiated endodermal cells have a specific, auxin-mediated transcriptional response dominated by cell wall remodelling genes. We identified two sets of auxin-regulated GDSL lipases. One is required for suberin synthesis, while the other can drive suberin degradation. These enzymes have key roles in suberization, driving root suberin plasticity.

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Fig. 1: Using genetics to map auxin responses in the differentiated endodermis.
Fig. 2: Suberin is degraded while the lateral root cap cuticle is established during lateral root formation.
Fig. 3: A cluster of five auxin-repressed GELPs is expressed in the differentiated endodermis.
Fig. 4: Suberin deposition requires a cluster of auxin-repressed GELPs.
Fig. 5: A cluster of auxin-induced GELPs is required for suberin degradation.

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Data availability

All data to support the conclusions of this manuscript are included in the main text and the supplementary materials. The full RNA-seq dataset was deposited in the NCBI Gene Expression Omnibus under accession GSE153478. Source data are provided with this paper.

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Acknowledgements

We thank C. Grefen and M. Barberon for insightful discussions about GDSL-domain-containing proteins, experimental approaches and stimulating discussions; and the Electron Microscopy Facility and Imaging Facility of the University of Lausanne and the Center of Microcopy and Image Analysis of the University of Zurich for excellent service and support. Work in the Geldner laboratory was supported by an ERC Consolidator Grant (GA-N: 616228-ENDOFUN) and two consecutive SNSF grants (CRSII3_136278 and 31003A-156261). R.U. was supported by an EMBO Long-Term Fellowship (EMBO ALTF 1046‐2015). Work in the Nawrath laboratory was supported by a Swiss National Science Foundation grant (no. 310030_188672/1). Work in the Vermeer laboratory was supported by grants from the Swiss National Science Foundation (Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung; PP00P3_157524 and 316030_164086), the Netherlands Organization for Scientific Research (Nederlandse Organisatie voor Wetenschappelijk Onderzoek; NWO 864.13.008) and support from the University of Neuchâtel.

Author information

Author notes

  1. Emanuel Schmid-Siegert

    Present address: NGSAI, Epalinges, Switzerland

  2. Tonni Grube Andersen

    Present address: Max Planck Institute for Plant Breeding Research, Cologne, Germany

Authors and Affiliations

  1. Department of Plant Molecular Biology, University of Lausanne, Lausanne, Switzerland

    Robertas Ursache, Valérie Dénervaud Tendon, Kay Gully, Damien De Bellis, Tonni Grube Andersen, Christiane Nawrath & Niko Geldner

  2. Laboratory of Cell and Molecular Biology, Institute of Biology, University of Neuchâtel, Neuchâtel, Switzerland

    Cristovāo De Jesus Vieira Teixeira, Vinay Shekhar & Joop E. M. Vermeer

  3. Electron Microscopy Facility, University of Lausanne, Lausanne, Switzerland

    Damien De Bellis

  4. Vital-IT Competence Center, Swiss Institute of Bioinformatics, Lausanne, Switzerland

    Emanuel Schmid-Siegert, Sandra Calderon & Sylvain Pradervand

  5. Department of Plant and Microbial Biology & Zurich–Basel Plant Science Centre, University of Zurich, Zurich, Switzerland

    Vinay Shekhar & Joop E. M. Vermeer

  6. Genomic Technologies Facility, University of Lausanne, Lausanne, Switzerland

    Sandra Calderon & Sylvain Pradervand

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Contributions

R.U., N.G. and J.E.M.V. conceived, designed and coordinated the project. R.U., C.D.J.V.T., V.D.T., D.D.B., K.G., V.S., T.G.A. and J.E.M.V. performed all experimental work. E.S.-S., S.C. and S.P. analysed RNA-seq data. J.E.M.V. wrote the first draft of the manuscript. R.U., T.G.A., C.N., N.G. and J.E.M.V. revised the manuscript and all authors were involved in the discussion of the work.

Corresponding authors

Correspondence to Robertas Ursache, Niko Geldner or Joop E. M. Vermeer.

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The authors declare no competing interests.

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Peer review information Nature Plants thanks Owen Rowland 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 SHY2pro::NLS-3xmVENUS dynamics in Col-0 (a), slr-1 (b) and CASP1pro::shy2-2/slr-1 treated with NAA.

a. Maximum image projections of roots expressing SHY2pro::NLS-3xmVENUS in Col-0 (a), slr-1 (b) and CASP1pro::shy2-2/slr-1 (c) after 2, 4, 8, 16 and 24 hours of NAA treatment. Yellow dots indicate SHY2pro::NLS-3xmVENUS signal in the endodermis. The images in are representatives of each experiment repeated 3 times. Scale bar in (a) = 50 µm.

Extended Data Fig. 2 Differentiated endodermal cells have a distinct transcriptional response to auxin.

a, Confocal images of a root expressing SLRpro::CITRINE:SYP122 confirming that SLR is expressed in the epidermis, cortex, pericycle and weakly in the stele, but not in the endodermis (indicated by asterisks). The images are representatives of the experiment repeated 3 times. b and c, Comparison of the current dataset (Ursache) with the data set of Lewis et al.14. b, Heatmap showing that both datasets cluster separately and do not have significant overlap, which is confirmed by the analysis of correlation between the two data sets (c). d and e, Comparison of the current dataset (Ursache) with the data set of Voß et al.15. d, Heatmap showing that both datasets cluster separately and do not have significant overlap, which is confirmed by the analysis of correlation between the two data sets (e). p values in c and e are derived from a Pearson correlation test, are two-sided t-test values and not corrected for multiple testing. The corresponding cut-points (*/**/***) represent here p < 0.05, p < 0.01 and p < 0.001. The r value measures the correlation between 2 sets of data. The p-value states whether there are enough observations to believe that an observed correlation is not appearing by chance. Scale bar = 25 µm.

Extended Data Fig. 3 A high number of differentially regulated genes are expressed in the endodermis.

a, Confocal images of GELP12pro::NLS-3xmVENUS expression in different genetic backgrounds, showing repression in slr-1 under control conditions. b, Auxin treatment (10 µM NAA, 16hrs) results in induction of GELP12pro::NLS-3xmVENUS expression in the endodermis of slr-1 roots. NLS-3xmVENUS signal is shown in green and CFW staining of cell walls is shown in grey. c, Confocal images of roots expressing transcriptional markers of candidate genes differentially expressed between slr-1 and CASP1pro::shy2-2/slr-1 roots and showing specific expression in the endodermis. d, Confocal images showing xylem-specific expression of LAC2pro::NLS-3xmVENUS. e, Heatmap showing the expression dynamics of suberin-related genes significant differentially expressed. NLS-3xmVENUS signal is shown in green, CFW staining of cell walls in gray and cell wall staining by PI in red. The images in (a-d) are representatives of each experiment repeated at least 3 times. Scale bars = 20 µm.

Extended Data Fig. 4 Expression of suberin biosynthesis-related GELPs is induced by ABA and CIF2 treatment.

a, Confocal images showing effect of ABA treatment (1 µM, 24hr) on the expression domain of GPAT5pro::NLS-3xmVENUS and GELP38pro::NLS-3xmVENUS in Arabidopsis roots. The images are representatives of the experiment repeated 3 times. b, Quantification of the effect of ABA (1 µM, 24hr) and CIF2 peptide (500 nM, 24h) treatment on the expression of suberin biosynthesis-related GELPs identified as being repressed by auxin treatment. c, Schematic representation of the different single mutants of the suberin biosynthesis-related GELPs used in this study. d, qPCR results showing the relative gene expression in Col-0 control (100%) and T-DNA insertion lines of the suberin synthesis-related GELPs used in this study. The results are based on three biological replicates. The p value versus the Col-0 control for gelp49-1 is <0.000001 and for gelp51-1 is < 0.000001. ND, not detected. e, Quantification of suberin occupancy in the endodermis of the single mutants of the suberin biosynthesis-related GELPs using FY staining (n = 10 biologically independent samples). The p value versus the Col-0 control for gelp38-c1 is 0.1269, for gelp38-c2 is 0.2616, for gelp51-1 is > 0.9999, for gelp96-1 is 0.9385, for gelp22-1 is > 0.9999 and for gelp49-1 is > 0.9999. Different letters in (d) and (e) (p < 0.05) indicate statistically significant differences between means by ANOVA and two-sided t-test analysis. f, Schematic representation of the mutations in the gelpquint-1 and gelpquint-2 mutants. The mutations are indicated in red and the PAM sites in blue. Error bars in (b), (d) and (e) are SD. Scale bar in (a) = 25 µm.

Extended Data Fig. 5 Extended characterization of the phenotype of gelpquint mutants.

a, Nile Red staining of wild-type and gelpquint-1 and gelpquint-2 roots confirms the absence of suberin in the mutants. b, TEM micrographs of high-pressure frozen roots of wild-type and gelpquint-1 showing the absence of suberin lamella in the mutant. A region without lateral root primordia has been chosen to highlight the absence of suberin lamella. c, FY staining of CIF2 peptide treated Col-0 and gelpquint-1 seedlings showing absence of CIF2-mediated suberin deposition in gelpquint-1. d, TEM micrograph of high-pressure frozen roots of wild-type and gelpquint-1 showing that the lateral root cap cuticle is not affected in the gelpquint-1 mutant. e, Fluorescein di-acetate (FDA) uptake assay in wild-type roots showing a suberin mediated block of uptake at the level of the endodermis. f, Confocal image of a root expressing GELP38-XVEpro::GELP38- mCITRINE (green) in gelpquint-1 after β-Estradiol treatment (5 µM) stained with FM4-64 dye. g, TEM micrographs of roots of gelpquint-1 and the complementation by GELP38-XVEpro::GELP38- mCITRINE showing the complete recovery of endodermis suberin lamella. The images in (a-g) are representatives of each experiment repeated at least 3 times. h, Counting of PI-stained cells as a proxy for Casparian strip barrier in the roots of wild-type and gelpquint-1 and gelpquint-2 seedlings. All individual data points are plotted. No statistically significant difference was detected in using ANOVA and Bonferroni-adjusted paired two-sided t-test. The p value versus the Col-0 control for gelpquint-1 is 0.1680 and for gelpquint-2 is > 0.9999. i-j, Basic Fuchsin staining of the Casparian strip in early and differentiated endodermal cells of wild-type and gelpquint-1 and gelpquint-2 roots. k, Salt stress assay showing that gelpquint-1 and gelpquint-2 mutant seedlings are more sensitive to mild salt stress (85 mM NaCl) compared to wild-type. The images in (i-k) are representatives of each experiment repeated at least 3 times. l, Quantification of the effect of prolonged salt stress on the fresh weight of wild-type and gelpquint-1 and gelpquint-2 seedlings. All individual data points are plotted. The p value versus the Col-0 control for Col-0 transferred to salt is 0.7857, for gelpquint1 versus gelpquint1 transferred to salt is < 0.000001 and for gelpquint-2 versus gelpquint-2 transferred to salt is < 0.000001. m, Quantification of emerged lateral roots in wild-type and gelpquint-1 and gelpquint-2 mutants after 8 days of exposure to salt. All individual data points are plotted (n = 10). n, Quantification of the expression of known suberin biosynthesis-related genes in gelpquint-1 and gelpquint-2 mutants. Results are presented as fold-change compared to their expression levels in Col-0 (n = 9). No statistically significant difference was detected in using ANOVA and Bonferroni-adjusted paired two-sided t-test. For GPAT5, the p value versus Col-0 for gelpquint-1 is > 0.9999 and for gelpquint-2 is 0.4546, for ASFT the p values are > 0.9999 and > 0.9999; for HORST the p values are 0.1122 and > 0.9999; for FAR1 the p values are > 0.9999 and > 0.9999; for FAR4 the p values are 0.1467 and > 0.9999; for KSC2 the p values are 0.1136 and > 0.9999 correspondingly. Different letters in (m) (p < 0.001) and asterisks in (l) (p < 0.001) indicate statistically significant differences between means by ANOVA and two-sided t-test analysis. ns, not significant. For the boxplots in (l) and (m) the center depicts the median while the lower and upper box limits depict the 25th and 75th percentile respectively. Whiskers represent minima and maxima. Closed dots depict individual samples. Data in (h) and (n) are presented as mean +/- SD. Scale bars for (a), (d), (e), (f), (g), (h-j) = 25 µm. Scale bars for (b) and (c) = 1 µm, for (k) = 5 mm.

Extended Data Fig. 6 Auxin-upregulated GELPs show three distinct expression patterns.

a, FY staining of a Col-0 root at the site of lateral root emergence highlighting the presence of cuticle (indicated by arrow). b-c, FY staining of bodyguard mutant root at the site lateral root emergence. The absence of a proper cuticle is highlighted by the gap in FY staining. d-g, Confocal images showing the expression patterns of GELP12 (d), GELP72 (e), GELP73 (f) and GELP81 (g) during lateral root emergence. h-j, Confocal images showing the expression of GELP12 (h), GELP55 (i) and GELP72 (j) after 10 µM NAA treatment. NLS-3xmVENUS signal is in green and Calcofluor White staining of cell walls is in gray. The images are representatives of each experiment repeated at least 3 times. Scale bars = 25 µm.

Extended Data Fig. 7 Overexpression of three auxin-induced GELPs leads to suberin degradation.

a, FY staining on roots of Col-0 treated with b-Estradiol results in normal suberin pattern, whereas inducible endodermis-specific overexpression of GELP12, GELP55 or GELP72 results in degradation of suberin highlighted by absence of FY signal. The overexpression of GELP73 and GELP81 results in a normal suberin pattern similar to wild-type. b, FY staining of ELTPproXVE>>GELP12 lines germinated on normal 1/2MS medium for 4 days and transferred to the plates supplemented with 5 µM β-estradiol for 36 hours to observe the suberin degradation. c, TEM micrographs of ELTPproXVE>>GELP12 grown on plates with and without β-estradiol to highlight the absence of suberin lamella upon induction of GELP12 expression in endodermis. d, Schematic representation of the mutations in the auxin-upregulated single GELP mutants. The mutations are indicated in red and the PAM sites in blue. The images in (a-c) are representatives of each experiment repeated at least 3 times. Scale bars in (a) and (b) = 500 µm. Scale bars in (c) = 1 µm.

Extended Data Fig. 8 Some auxin-inducible GELPs facilitate lateral root emergence.

a-k, Gravistimulation-mediated induction of lateral root formation to functionally characterize the role of auxin-induced GELPs during lateral root formation. Staging of lateral root development was performed at 18hr and 42hr after gravistimulation. a, Col-0. b and c, gelp12-c1 and gelp12-c2. d and e, gelp55-c1 and gelp55-c2. f and g, gelp72-1 and gelp72-c1. h and i, gelp73-c1 and gelp73-c2. j and k, p values are indicated. A p value below 0.05 indicates a statistically significant difference as determined using a Pearson’s χ2 test. Experiments were repeated three times with a minimal of 15 seedlings per genotype and time point.

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Ursache, R., De Jesus Vieira Teixeira, C., Dénervaud Tendon, V. et al. GDSL-domain proteins have key roles in suberin polymerization and degradation. Nat. Plants 7, 353–364 (2021). https://doi.org/10.1038/s41477-021-00862-9

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