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Golgi-localized putative S-adenosyl methionine transporters required for plant cell wall polysaccharide methylation

Nature Plants volume 8, pages 656–669 (2022)Cite this article

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Abstract

Polysaccharide methylation, especially that of pectin, is a common and important feature of land plant cell walls. Polysaccharide methylation takes place in the Golgi apparatus and therefore relies on the import of S-adenosyl methionine (SAM) from the cytosol into the Golgi. However, so far, no Golgi SAM transporter has been identified in plants. Here we studied major facilitator superfamily members in Arabidopsis that we identified as putative Golgi SAM transporters (GoSAMTs). Knockout of the two most highly expressed GoSAMTs led to a strong reduction in Golgi-synthesized polysaccharide methylation. Furthermore, solid-state NMR experiments revealed that reduced methylation changed cell wall polysaccharide conformations, interactions and mobilities. Notably, NMR revealed the existence of pectin ‘egg-box’ structures in intact cell walls and showed that their formation is enhanced by reduced methyl esterification. These changes in wall architecture were linked to substantial growth and developmental phenotypes. In particular, anisotropic growth was strongly impaired in the double mutant. The identification of putative transporters involved in import of SAM into the Golgi lumen in plants provides new insights into the paramount importance of polysaccharide methylation for plant cell wall structure and function.

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Fig. 1: Identification of putative GoSAMTs.
Fig. 2: GoSAMT mutants show reduced polysaccharide methylation.
Fig. 3: Two-dimensional NMR experiments show marked reduction of pectin methyl esterification and increased egg-box structures.
Fig. 4: DIPSHIFT experiments show important changes in polysaccharide mobility.
Fig. 5: Long-mixing-time PDSD experiments suggest reduced methyl esterification strengthens cellulose–pectin interactions.
Fig. 6: gosamt1 gosamt2 mutants display strong growth and developmental phenotypes.

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Data availability

Unprocessed NMR data files of PDSD experiments are available from https://doi.org/10.17863/CAM.82899. The co-expression network was obtained from ATTED-II (http://atted.jp) version 9.2 using Ath-m.c7-1 dataset. Alphafold models of CeSAMT1 (Q9N3A9) and Arabidopsis GoSAMT1 (Q6NLR2) were obtained from Alphafold (https://alphafold.ebi.ac.uk). Protein sequences for phylogeny of Supplementary Fig. 1c were obtained from Plaza genomics (https://bioinformatics.psb.ugent.be/plaza/). Expression data of GoSAMTs from the eFP browser can be found in (http://bar.utoronto.ca/efp_arabidopsis/cgi-bin/efpWeb.cgi). Source data are provided with this paper.

References

  1. Temple, H., Saez-Aguayo, S., Reyes, F. C. & Orellana, A. The inside and outside: topological issues in plant cell wall biosynthesis and the roles of nucleotide sugar transporters. Glycobiology 26, 913–925 (2016).

    Article  CAS  PubMed  Google Scholar 

  2. Reyes, F. & Orellana, A. Golgi transporters: opening the gate to cell wall polysaccharide biosynthesis. Curr. Opin. Plant Biol. 11, 244–251 (2008).

    Article  CAS  PubMed  Google Scholar 

  3. Urbanowicz, B. R. et al. 4-O-methylation of glucuronic acid in Arabidopsis glucuronoxylan is catalyzed by a domain of unknown function family 579 protein. Proc. Natl Acad. Sci. USA 109, 14253–14258 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Temple, H. et al. Two members of the DUF579 family are responsible for arabinogalactan methylation in Arabidopsis. Plant Direct 3, e00117 (2019).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  5. O’Neill, M. A. et al. Locating methyl-etherified and methyl-esterified uronic acids in the plant cell wall pectic polysaccharide rhamnogalacturonan II. SLAS Technol. 25, 329–344 (2020).

    Article  PubMed  CAS  Google Scholar 

  6. Atmodjo, M. A., Hao, Z. & Mohnen, D. Evolving views of pectin biosynthesis. Annu Rev. Plant Biol. 64, 747–779 (2013).

    Article  CAS  PubMed  Google Scholar 

  7. Levesque-Tremblay, G., Pelloux, J., Braybrook, S. A. & Müller, K. Tuning of pectin methylesterification: consequences for cell wall biomechanics and development. Planta 242, 791–811 (2015).

    Article  CAS  PubMed  Google Scholar 

  8. Daher, F. B. & Braybrook, S. A. How to let go: pectin and plant cell adhesion. Front. Plant Sci. 6, 523 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  9. Palin, R. & Geitmann, A. The role of pectin in plant morphogenesis. Biosystems 109, 397–402 (2012).

    Article  CAS  PubMed  Google Scholar 

  10. Goldberg, R. et al. in Pectins and Pectinases. Progress in Biotechnology Vol. 14 (eds Visser, J. & Voragen, A. G. J.) 151–172 (Elsevier, 1996).

  11. Xiao, C., Somerville, C. & Anderson, C. T. POLYGALACTURONASE INVOLVED IN EXPANSION1 functions in cell elongation and flower development in Arabidopsis. Plant Cell 26, 1018–1035 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Phyo, P., Wang, T., Xiao, C., Anderson, C. T. & Hong, M. Effects of pectin molecular weight changes on the structure, dynamics, and polysaccharide interactions of primary cell walls of Arabidopsis thaliana: insights from solid-state NMR. Biomacromolecules 18, 2937–2950 (2017).

    Article  CAS  PubMed  Google Scholar 

  13. Yang, Y., Anderson, C. T. & Cao, J. Polygalacturonase45 cleaves pectin and links cell proliferation and morphogenesis to leaf curvature in Arabidopsis thaliana. Plant J. 106, 1493–1508 (2021).

    Article  CAS  PubMed  Google Scholar 

  14. Zhang, G. F. & Staehelin, L. A. Functional compartmentation of the Golgi apparatus of plant cells: immunocytochemical analysis of high-pressure frozen- and freeze-substituted sycamore maple suspension culture cells. Plant Physiol. 99, 1070–1083 (1992).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Goubet, F. & Mohnen, D. Subcellular localization and topology of homogalacturonan methyltransferase in suspension-cultured Nicotiana tabacum cells. Planta 209, 112–117 (1999).

    Article  CAS  PubMed  Google Scholar 

  16. Du, J. et al. Mutations in the pectin methyltransferase QUASIMODO2 influence cellulose biosynthesis and wall integrity in Arabidopsis. Plant Cell 32, 3576–3597 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Mouille, G. et al. Homogalacturonan synthesis in Arabidopsis thaliana requires a Golgi-localized protein with a putative methyltransferase domain. Plant J. 50, 605–614 (2007).

    Article  CAS  PubMed  Google Scholar 

  18. Krupkova, E., Immerzeel, P., Pauly, M. & Schmulling, T. The TUMOROUS SHOOT DEVELOPMENT2 gene of Arabidopsis encoding a putative methyltransferase is required for cell adhesion and coordinated plant development. Plant J. 50, 735–750 (2007).

    Article  CAS  PubMed  Google Scholar 

  19. Miao, Y., Li, H. Y., Shen, J., Wang, J. & Jiang, L. QUASIMODO 3 (QUA3) is a putative homogalacturonan methyltransferase regulating cell wall biosynthesis in Arabidopsis suspension-cultured cells. J. Exp. Bot. 62, 5063–5078 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Held, M. A. et al. CGR3: a Golgi-localized protein influencing homogalacturonan methylesterification. Mol. Plant 4, 832–844 (2011).

    Article  CAS  PubMed  Google Scholar 

  21. Kim, S. J., Held, M. A., Zemelis, S., Wilkerson, C. & Brandizzi, F. CGR2 and CGR3 have critical overlapping roles in pectin methylesterification and plant growth in Arabidopsis thaliana. Plant J. 82, 208–220 (2015).

    Article  CAS  PubMed  Google Scholar 

  22. Ibar, C. & Orellana, A. The import of S-adenosylmethionine into the Golgi apparatus is required for the methylation of homogalacturonan. Plant Physiol. 145, 504–512 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Palmieri, L. et al. Molecular identification of an Arabidopsis S-adenosylmethionine transporter. Analysis of organ distribution, bacterial expression, reconstitution into liposomes, and functional characterization. Plant Physiol. 142, 855–865 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Bouvier, F. et al. Arabidopsis SAMT1 defines a plastid transporter regulating plastid biogenesis and plant development. Plant Cell 18, 3088–3105 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Drew, D., North, R. A., Nagarathinam, K. & Tanabe, M. Structures and general transport mechanisms by the major facilitator superfamily (MFS). Chem. Rev. 121, 5289–5335 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Yan, N. Structural biology of the major facilitator superfamily transporters. Annu. Rev. Biophys. 44, 257–283 (2015).

    Article  CAS  PubMed  Google Scholar 

  27. Nikolovski, N. et al. Putative glycosyltransferases and other plant Golgi apparatus proteins are revealed by LOPIT proteomics. Plant Physiol. 160, 1037–1051 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Nikolovski, N., Shliaha, P. V., Gatto, L., Dupree, P. & Lilley, K. S. Label-free protein quantification for plant Golgi protein localization and abundance. Plant Physiol. 166, 1033–1043 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  29. Tejada-Jimenez, M., Galvan, A. & Fernandez, E. Algae and humans share a molybdate transporter. Proc. Natl Acad. Sci. USA 108, 6420–6425 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Wohlschlager, T. et al. Methylated glycans as conserved targets of animal and fungal innate defense. Proc. Natl Acad. Sci. USA 111, E2787–E2796 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Obayashi, T., Aoki, Y., Tadaka, S., Kagaya, Y. & Kinoshita, K. ATTED-II in 2018: a plant coexpression database based on investigation of the statistical property of the mutual rank index. Plant Cell Physiol. 59, e3 (2018).

    Article  PubMed  CAS  Google Scholar 

  32. Schmid, M. et al. A gene expression map of Arabidopsis thaliana development. Nat. Genet. 37, 501–506 (2005).

    Article  CAS  PubMed  Google Scholar 

  33. Li, X. et al. Development and application of a high throughput carbohydrate profiling technique for analyzing plant cell wall polysaccharides and carbohydrate active enzymes. Biotechnol. Biofuels 6, 94 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Wang, T., Park, Y. B., Cosgrove, D. J. & Hong, M. Cellulose–pectin spatial contacts are inherent to never-dried Arabidopsis thaliana primary cell walls: evidence from solid-state NMR. Plant Physiol. 168, 871–883 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  35. Hediger, S., Emsley, L. & Fischer, M. Solid-state NMR characterization of hydration effects on polymer mobility in onion cell-wall material. Carbohydr. Res. 322, 102–112 (1999).

    Article  CAS  Google Scholar 

  36. Jarvis, M. C. & Apperley, D. C. Chain conformation in concentrated pectic gels—evidence from C-13 NMR. Carbohyd. Res. 275, 131–145 (1995).

    Article  CAS  Google Scholar 

  37. Ha, M.-A. et al. in Pectins and Pectinases. Progress in Biotechnology Vol. 14 (eds Visser, J. & Voragen, A. G. J.) 561–568 (Elsevier, 1996).

  38. Wang, T., Phyo, P. & Hong, M. Multidimensional solid-state NMR spectroscopy of plant cell walls. Solid State Nucl. Magn. Reson. 78, 56–63 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Kohorn, B. D. et al. Pectin dependent cell adhesion restored by a mutant microtubule organizing membrane protein. Plants 10, 690 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Willis, L. et al. Cell size and growth regulation in the Arabidopsis thaliana apical stem cell niche. Proc. Natl Acad. Sci. USA 113, E8238–E8246 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Gendreau, E. et al. Cellular basis of hypocotyl growth in Arabidopsis thaliana. Plant Physiol. 114, 295–305 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Peaucelle, A. et al. Arabidopsis phyllotaxis is controlled by the methyl-esterification status of cell-wall pectins. Curr. Biol. 18, 1943–1948 (2008).

    Article  CAS  PubMed  Google Scholar 

  43. Peaucelle, A. et al. Pectin-induced changes in cell wall mechanics underlie organ initiation in Arabidopsis. Curr. Biol. 21, 1720–1726 (2011).

    Article  CAS  PubMed  Google Scholar 

  44. Lee, C. et al. Three Arabidopsis DUF579 domain-containing GXM proteins are methyltransferases catalyzing 4-O-methylation of glucuronic acid on xylan. Plant Cell Physiol. 53, 1934–1949 (2012).

    Article  CAS  PubMed  Google Scholar 

  45. Bouton, S. et al. QUASIMODO1 encodes a putative membrane-bound glycosyltransferase required for normal pectin synthesis and cell adhesion in Arabidopsis. Plant Cell 14, 2577–2590 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Cao, L., Lu, W., Mata, A., Nishinari, K. & Fang, Y. Egg-box model-based gelation of alginate and pectin: a review. Carbohydr. Polym. 242, 116389 (2020).

    Article  CAS  PubMed  Google Scholar 

  47. Liners, F., Thibault, J.-F. & Van Cutsem, P. Influence of the degree of polymerization of oligogalacturonates and of esterification pattern of pectin on their recognition by monoclonal antibodies. Plant Physiol. 99, 1099–1104 (1992).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Draye, M. & Van Cutsem, P. Pectin methylesterases induce an abrupt increase of acidic pectin during strawberry fruit ripening. J. Plant Physiol. 165, 1152–1160 (2008).

    Article  CAS  PubMed  Google Scholar 

  49. Hocq, L., Pelloux, J. & Lefebvre, V. Connecting homogalacturonan-type pectin remodeling to acid growth. Trends Plant Sci. 22, 20–29 (2017).

    Article  CAS  PubMed  Google Scholar 

  50. Ralet, M.-C. et al. Xylans provide the structural driving force for mucilage adhesion to the Arabidopsis seed coat. Plant Physiol. 171, 165–178 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Broxterman, S. E. & Schols, H. A. Characterisation of pectin–xylan complexes in tomato primary plant cell walls. Carbohydr. Polym. 197, 269–276 (2018).

    Article  CAS  PubMed  Google Scholar 

  52. Biswal, A. K. et al. Working towards recalcitrance mechanisms: increased xylan and homogalacturonan production by overexpression of GAlactUronosylTransferase12 (GAUT12) causes increased recalcitrance and decreased growth in Populus. Biotechnol. Biofuels 11, 1–26 (2018).

    Article  CAS  Google Scholar 

  53. Simmons, T. J. et al. Folding of xylan onto cellulose fibrils in plant cell walls revealed by solid-state NMR. Nat. Commun. 7, 13902 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Phyo, P. et al. Gradients in wall mechanics and polysaccharides along growing inflorescence stems. Plant Physiol. 175, 1593–1607 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Daher, F. B. et al. Anisotropic growth is achieved through the additive mechanical effect of material anisotropy and elastic asymmetry. Elife 7, e38161 (2018).

    Article  Google Scholar 

  56. Zhang, Y. et al. Molecular insights into the complex mechanics of plant epidermal cell walls. Science 372, 706–711 (2021).

    Article  CAS  PubMed  Google Scholar 

  57. Fujita, M. et al. The anisotropy1 D604N mutation in the Arabidopsis cellulose synthase1 catalytic domain reduces cell wall crystallinity and the velocity of cellulose synthase complexes. Plant Physiol. 162, 74–85 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Bethke, G. et al. Pectin biosynthesis is critical for cell wall integrity and immunity in Arabidopsis thaliana. Plant Cell 28, 537–556 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Hibara, K.-I. et al. Abnormal shoot in youth, a homolog of molybdate transporter gene, regulates early shoot development in rice. Am. J. Plant Sci. 4, 1–9 (2013).

    Article  CAS  Google Scholar 

  60. Benedetti, M. et al. Plant immunity triggered by engineered in vivo release of oligogalacturonides, damage-associated molecular patterns. Proc. Natl Acad. Sci. USA 112, 5533–5538 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Pontiggia, D., Benedetti, M., Costantini, S., De Lorenzo, G. & Cervone, F. Dampening the DAMPs: how plants maintain the homeostasis of cell wall molecular patterns and avoid hyper-immunity. Front Plant Sci. 11, 613259 (2020).

    Article  PubMed  PubMed Central  Google Scholar 

  62. Cabrera, J. C., Boland, A., Messiaen, J., Cambier, P. & Van Cutsem, P. Egg box conformation of oligogalacturonides: the time-dependent stabilization of the elicitor-active conformation increases its biological activity. Glycobiology 18, 473–482 (2008).

    Article  CAS  PubMed  Google Scholar 

  63. Rocha, P. S. et al. The Arabidopsis HOMOLOGY-DEPENDENT GENE SILENCING1 gene codes for an S-adenosyl–l-homocysteine hydrolase required for DNA methylation-dependent gene silencing. Plant Cell 17, 404–417 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Moffatt, B. A. & Weretilnyk, E. A. Sustaining S‐adenosyl–l‐methionine‐dependent methyltransferase activity in plant cells. Physiol. Plant. 113, 435–442 (2001).

    Article  CAS  Google Scholar 

  65. Parra-Rojas, J. P. et al. New steps in mucilage biosynthesis revealed by analysis of the transcriptome of the UDP-rhamnose/UDP-galactose transporter 2 mutant. J. Exp. Bot. 70, 5071–5088 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  66. Patron, N. J. et al. Standards for plant synthetic biology: a common syntax for exchange of DNA parts. N. Phytol. 208, 13–19 (2015).

    Article  CAS  Google Scholar 

  67. Engler, C. et al. A golden gate modular cloning toolbox for plants. ACS Synth. Biol. 3, 839–843 (2014).

    Article  CAS  PubMed  Google Scholar 

  68. Shimada, T. L., Shimada, T. & Hara‐Nishimura, I. A rapid and non‐destructive screenable marker, FAST, for identifying transformed seeds of Arabidopsis thaliana. Plant J. 61, 519–528 (2010).

    Article  CAS  PubMed  Google Scholar 

  69. Nelson, B. K., Cai, X. & Nebenführ, A. A multicolored set of in vivo organelle markers for co‐localization studies in Arabidopsis and other plants. Plant J. 51, 1126–1136 (2007).

    Article  CAS  PubMed  Google Scholar 

  70. Clough, S. J. & Bent, A. F. Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16, 735–743 (1998).

    Article  CAS  PubMed  Google Scholar 

  71. Labun, K. et al. CHOPCHOP v3: expanding the CRISPR web toolbox beyond genome editing. Nucleic Acids Res. 47, W171–W174 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  72. Lawrenson, T. et al. Induction of targeted, heritable mutations in barley and Brassica oleracea using RNA-guided Cas9 nuclease. Genome Biol. 16, 1–13 (2015).

    Article  CAS  Google Scholar 

  73. Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676–682 (2012).

    Article  CAS  PubMed  Google Scholar 

  74. Yang, W. et al. Regulation of meristem morphogenesis by cell wall synthases in Arabidopsis. Curr. Biol. 26, 1404–1415 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  75. Wightman, R., Wallis, S. & Aston, P. Hydathode pit development in the alpine plant Saxifraga cochlearis. Flora 233, 99–108 (2017).

    Article  Google Scholar 

  76. de Reuille, P. B. et al. MorphoGraphX: a platform for quantifying morphogenesis in 4D. Elife 4, e05864 (2015).

    Article  Google Scholar 

  77. Gilbert, H. J., Hazlewood, G. P., Laurie, J. I., Orpin, C. G. & Xue, G. P. Homologous catalytic domains in a rumen fungal xylanase: evidence for gene duplication and prokaryotic origin. Mol. Microbiol. 6, 2065–2072 (1992).

    Article  CAS  PubMed  Google Scholar 

  78. Anthon, G. E. & Barrett, D. M. Comparison of three colorimetric reagents in the determination of methanol with alcohol oxidase. Application to the assay of pectin methylesterase. J. Agric. Food Chem. 52, 3749–3753 (2004).

    Article  CAS  PubMed  Google Scholar 

  79. Munowitz, M. G., Griffin, R. G., Bodenhausen, G. & Huang, T. H. Two-dimensional rotational spin-echo nuclear magnetic resonance in solids: correlation of chemical-shift and dipolar interactions. J. Am. Chem. Soc. 103, 2529–2533 (1981).

    Article  CAS  Google Scholar 

  80. Hong, M. et al. Coupling amplification in 2D MAS NMR and its application to torsion angle determination in peptides. J. Magn. Reson. 129, 85–92 (1997).

    Article  CAS  PubMed  Google Scholar 

  81. Bielecki, A., Kolbert, A. C. & Levitt, M. H. Frequency-switched pulse sequences—homonuclear decoupling and dilute spin NMR in solids. Chem. Phys. Lett. 155, 341–346 (1989).

    Article  CAS  Google Scholar 

  82. Rienstra, C. M. et al. De novo determination of peptide structure with solid-state magic-angle spinning NMR spectroscopy. Proc. Natl Acad. Sci. USA 99, 10260–10265 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  83. Tsirigos, K. D., Peters, C., Shu, N., Käll, L. & Elofsson, A. The TOPCONS web server for consensus prediction of membrane protein topology and signal peptides. Nucleic Acids Res. 43, W401–W407 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  84. Derba-Maceluch, M. et al. Suppression of xylan endotransglycosylase PtxtXyn10A affects cellulose microfibril angle in secondary wall in aspen wood. New Phytol. 205, 666–681 (2015).

    Article  CAS  PubMed  Google Scholar 

  85. Kumar, S., Stecher, G., Li, M., Knyaz, C. & Tamura, K. MEGA X: molecular evolutionary genetics analysis across computing platforms. Mol. Biol. Evol. 35, 1547 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

The characterization of gosamt mutants was supported as part of The Center for Lignocellulose Structure and Formation, an Energy Frontier Research Center funded by the US Department of Energy, Office of Science, Basic Energy Sciences, under Award number DE-SC0001090. This study used NMR spectrometers at the MIT–Harvard Center for Magnetic Resonance, which is supported by NIH grant P41 GM132079. Initial gene identification, mutant isolation and preliminary pectin methylation studies were done by H.T. and P.D. under grant EPSRC/BBSRC OpenPlant (BB/L014130/1) and A.O., J.P.P.-R. and S.S.-A. supported by Fondo de Areas Prioritarias–Centro de Regulacion del Genoma-15090007, FONDECYT 1190695 and FONDECYT 1201467. Most of the microscopy experiments used The Sainsbury Laboratory Microscopy Core Facility, which is supported by the Gatsby Charitable Foundation. We thank F. López-Hernández for his advice on dark-grown hypocotyl experiments, R. Wightman for his helpful support with microscopy experiments and L. Wilson and N. Anders for their helpful suggestions during manuscript writing.

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Author notes

  1. Weibing Yang

    Present address: National Key Laboratory of Plant Molecular Genetics, CAS Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences (CAS) and CAS-JIC Center of Excellence for Plant and Microbial Sciences (CEPAMS), Shanghai, China

  2. Jan J. Lyczakowski

    Present address: Department of Plant Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland

Authors and Affiliations

  1. Department of Biochemistry, University of Cambridge, Cambridge, UK

    Henry Temple, Jan J. Lyczakowski, Alberto Echevarría-Poza, Igor Yakunin, Oliver M. Terrett & Paul Dupree

  2. Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA, USA

    Pyae Phyo & Mei Hong

  3. Sainsbury Laboratory, University of Cambridge, Cambridge, UK

    Weibing Yang

  4. Centro de Biotecnología Vegetal, FONDAP Center for Genome Regulation, Facultad de Ciencias de la Vida, Universidad Andrés Bello, Santiago, Chile

    Juan Pablo Parra-Rojas, Susana Saez-Aguayo & Ariel Orellana

  5. Department of Physics, University of Warwick, Coventry, UK

    Ray Dupree

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Contributions

H.T., A.O., M.H. and P.D. conceived and designed the study. H.T. conducted most of the molecular genetic, microscopy and biochemical experiments, assisted by W.Y., J.J.L., A.E.-P., I.Y., J.P.P.-R., O.M.T. and S.S.-A. NMR experiments were conducted mostly by P.P. with contribution from R.D. Data analysis and interpretation was conducted by H.T., P.P., A.O., M.H. and P.D. The paper was written by H.T. and P.D. with contributions from all authors.

Corresponding authors

Correspondence to Mei Hong or Paul Dupree.

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The authors declare no competing interests.

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Nature Plants thanks Chaowen Xiao 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 GoSAMTs are present throughout the plant kingdom.

(A) Sequence identity and similarity matrix generated based on MUSCLE alignments of whole length protein sequences of CeSAMT1 and Arabidopsis GoSAMTs. (B) CeSAMT1 and Arabidopsis GoSAMTs topology using TOPCONS83. (C) Alphafold models of CeSAMT1 and Arabidopsis GoSAMT1, aligned using the PDB pairwise alignment tool (https://www.rcsb.org/alignment). The two models superimposed remarkably well, with alignment scores of RMSD = 1.3 and TM-score = 0.88. Models were visualised using UCSF ChimeraX (Pettersen et al., 2021)84. (D) Phylogenetic tree of MFS_5 proteins from Arabidopsis thaliana (At), Amborella thricopoda (Atr), Caenorhabditis elegans, Chlamydomona reinhardtii, Klebsormidium nitens, Homo sapiens, Micromonas commoda, Oryza sativa (Os), Ostreococcus lucimarinus, Picea abies (PAB), Physcomitrella patens (Pp) Selaginella moellendorffii (SMO), Solanum lycopersicum (Solyc). All sequences were obtained from PLAZA (https://bioinformatics.psb.ugent.be/plaza/). Arabidopsis sequences are highlighted in red. Phylogenetic tree was generated using Molecular Evolutionary Genetics Analysis MEGA X85 and visualized by FigTree 1.4.2. (E) Expression data points of the GoSAMT family members, obtained from eFP browser (http://bar.utoronto.ca/efp_arabidopsis/cgi-bin/efpWeb.cgi)32. (F) Subcellular localisation of GoSAMT2-GFP and GoSAMT1-mCherry expressed under their endogenous promoters, observed in cotyledon epidermal cells of Arabidopsis stable lines. Similar results were observed in three different plants.

Extended Data Fig. 2 Complementation of the gosamt1 gosamt2 mutant.

(A) Pictures of adult plants of WT, gosamt single mutants and gosamt1 gosamt2 mutant molecular complemented lines. (B) Western blot, expression analysis of GoSAMT1-GFP and GoSAMT2-GFP complemented lines using Anti-GFP antibody (ab290) from Abcam at a dilution of 1:10000 in milk-TBS buffer. (C) Box and whiskers plot representing plant fresh weight of the different genotypes. n ≥15 plants measurements per genotype. Box boundaries represent the 25th and 75th percentile, and centre line represent the median, whiskers represent the minimum and maximum data point. D) Ratio of released Xyl4GlcA and Xyl4MeGlcA products after endoxylanase treatment of basal stem AIR, coupled to capillary electrophoresis experiments of secondary cell wall xylan of WT, gosamt1, gosamt2, gosamt1 gosamt2 and molecular complemented lines. Values correspond to the mean of n= 3 biological replicates. (E) Methanol release experiments of leaf AIR of WT, gosamt1, gosamt2, gosamt1 gosamt2 and molecular complemented lines. Values correspond to the mean of n= 3 biological replicates. Asterisks in C, D and E indicate significant differences between gosamt1 gosamt2 mutant and the rest of the genotypes defined by one-way ANOVA followed by Dunnett’s multiple comparison test:*,P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.

Source data

Extended Data Fig. 3 Homogalacturonan molecule schemes and their 13C chemical shift index.

Schemes illustrate 31 and 21 HG conformations and not intended to be conformationally accurate. Yellow circles highlight GA carbons from GAC1-GAC5, pink circles highlight GAC1 and GAC4 in their 21 conformation, blue circles highlight methyl-esterified GAC6, green circles highlight carboxylate GAC6, turquoise circle represent αGAC1re and purple circle represent βGAC1re. Chemical shifts in green represent HG carbons and chemical shifts in pink represent the changes in 21 HG conformation, as presented in Fig. 3.

Extended Data Fig. 4 CP-DIPSHIFT experiment.

CP-DIPSHIFT curves of WT (black) and gosamt1 gosamt2 (red) cell walls measured at 293 K. Best-fit 13C-1H dipolar coupling values (scaled by FSLG) and SCH order parameters are given in each panel. The experiment was conducted with C-H dipolar doubling, a CP contact time of 500 μs under 7.8 kHz MAS. The 101 ppm, 99.0 ppm, and 79.8 ppm peaks of pectin backbones are more rigid in the mutant than in the WT cell wall.

Extended Data Fig. 5 gosamt1 gosamt2 has smaller leaves with smaller cells.

(A) Rosette leaves of five-weeks-old WT and gosamt1 gosamt2 plants. (B) SEM pictures of the second-largest leaf of WT and gosamt1 gosamt2. Yellow arrows show cell adhesion defects in the mutant. Similar results were observed in three different plants per genotype.

Extended Data Fig. 6 gosamt1 gosamt2 mutants display strong cell elongation and adhesion phenotypes.

(A) Quantification of etiolated hypocotyls of WT, gosamt1, gosamt2 and gosamt1 gosamt2, one to five days after germination (DAG) grown etiolated hypocotyls length grown in ½MS media and ½MS supplemented with CaCl2 to a final concentration of 15mM. Error bars correspond to SD of at least n ≥73 seedling measurements per genotype per time point. (B) WT and gosamt1 gosamt2 etiolated hypocotyls length, grown in ½MS media and ½MS supplemented with CaCl2 to a final concentration of 15mM, stained with ruthenium red. Bar 5mm. (C) Scanning electron microscopy (SEM) of WT and in MS and in MS supplemented with CaCl2 to a final concentration of 15 mM. Bar 100μm.

Extended Data Fig. 7 gosamt3 mutant plants do not show obvious growth or developmental phenotypes.

(A) RT-PCR of GoSAMT1, GoSAMT2, GoSAMT3 and EF1α in WT and gosamt mutant plants. NTC, no template control. All PCR reactions were carried out using 30 cycles. Primers used in this experiment are listed in Table S1. Same results, including GoSAMT3, have been observed at least two times. (B) Representative images of 21-day-old WT, single gosamt mutant and gosamt1 gosamt2 plants. Bar, 2cm. (C) Representative images of 5-week-old WT, single gosamt3 mutant and gosamt1 gosamt2 plants. Bar, 2cm.

Source data

Extended Data Fig. 8 Severe phenotypes resulting from GoSAMT3 CRISPR-Cas9 gene editing in gosamt1 gosamt2 plants.

(A) Schematic diagram of the GoSAMT3 gene. Green arrowheads show sgRNA targets, and blue arrows show primer positions for genotyping experiments. The red arrowhead shows T-DNA position of the gosamt3 mutant described in Supplemental Fig. 7. (B) Schematic diagram of the CRISPR-Cas9 construct. Transcriptional units were assembled into L2 vectors using Golden Gate Modular Cloning. (C) Representative images of 16-day-old WT, gosamt1 gosamt2 mutant plants, and WT and gosamt1 gosamt2 plants after transformation with the GoSAMT3 CRISPR-Cas9 construct. A range of severity of phenotypes was seen, only in the transformed gosamt1 gosamt2 mutants, which were grouped into three severity classes. (D) Quantitation of individual phenotypes and ungerminated seeds from the GoSAMT3 CRISPR-Cas9 transformed gosamt1 gosamt2 seeds. (E) Representative scanning electron microscope images of seedlings in group 3. Bar = 500μm Similar results were observed in three different plants of this group. (F) GoSAMT3 genotyping of WT, gosamt1 gosamt2, gosamt3 and three GoSAMT3 CRISPR-Cas9 transformed WT (#1, #2 and #3) and gosamt1 gosamt2 mutant plants (#5, #6, #7). Deletions indicating partial gene editing were observed in all T1 individuals. The blue asterisk corresponds to WT GoSAMT3 amplicon size using the primers shown in A. Red asterisks correspond to GoSAMT3 amplification products containing deletions. (G) Sanger sequencing of the PCR products from individuals in (F), revealing deletion of regions of the GoSAMT3 gene.

Source data

Supplementary information

Supplementary Information

Supplementary Table 1 lists primers used in this study.

Reporting Summary

Source data

Source Data Fig. 1

Unprocessed gels.

Source Data Extended Data Fig. 2

Unprocessed western blot.

Source Data Extended Data Fig. 7

Unprocessed gels.

Source Data Extended Data Fig. 8

Unprocessed gels.

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Temple, H., Phyo, P., Yang, W. et al. Golgi-localized putative S-adenosyl methionine transporters required for plant cell wall polysaccharide methylation. Nat. Plants 8, 656–669 (2022). https://doi.org/10.1038/s41477-022-01156-4

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