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Xylan-based nanocompartments orchestrate plant vessel wall patterning

Nature Plants volume 8, pages 295–306 (2022)Cite this article

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Abstract

Nanoclustering of biomacromolecules allows cells to efficiently orchestrate biological processes. The plant cell wall is a highly organized polysaccharide network but is heterogeneous in chemistry and structure. However, polysaccharide-based nanocompartments remain ill-defined. Here, we identify a xylan-rich nanodomain at pit borders of xylem vessels. We show that these nanocompartments maintain distinct wall patterns by anchoring cellulosic nanofibrils at the pit borders, critically supporting vessel robustness, water transport and leaf transpiration. The nanocompartments are produced by the activity of IRREGULAR XYLEM (IRX)10 and its homologues, which we show are de novo xylan synthases. Our study hence outlines a mechanism of how xylans are synthesized, how they assemble into nanocompartments and how the nanocompartments sustain cell wall pit patterning to support efficient water transport throughout the plant body.

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Fig. 1: Metaxylem vessels possess xylan nanocompartments.
Fig. 2: Xylan nanocompartments are assembled by IRX10 and its homologues.
Fig. 3: Rice IRX10 is a xylan synthase.
Fig. 4: Arabidopsis IRX10 and IRX10L are required for pit morphology.
Fig. 5: Xylan nanocompartments are essential to maintain coherent pits.
Fig. 6: Pit patterning affects xylem functions.

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Data availability

Accession numbers and gene names are available from the phylogenetic tree in Extended Data Fig. 3. The gene sequences were obtained from PLAZA 3.0 database (https://bioinformatics.psb.ugent.be/plaza/) and rice genome database (https://rice.plantbiology.msu.edu). Source data are provided with this paper.

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Acknowledgements

We thank S. Zhang and L. Wang for the kind help with AFM, A. Wu for providing seeds of Arabidopsis irx mutants, X. Fu for help with examining transpiration potential of rice plants, X. He for help with VISUAL, Y. Wu for help with MST assay, Q. Qian and D. Zeng for providing the core rice accessions, Q. Liu and S. Tang for support with field trials and C. Zheng for help with the model drawing. The super-resolution microscopy analysis was performed at the Bio-imaging Facility, Institute of Genetics and Developmental Biology, Chinese Academy of Science (CAS). This work was supported by the National Nature Science Foundation of China (grant nos. 32030077 and 31922006) to Y.Z. and B.Z., CAS grants no. XDA24010102 to Y.Z. and Youth Innovation Promotion Association CAS (Y202030) to B.Z., as well as the State Key Laboratory of Plant Genomics to Y.Z. S.P. acknowledges grants from the Australian Research Council (DP190101941), Velux (Villum Investigator grant no. 25915), Novo Nordisk (laureate grant no. NNF19OC0056076) and the Danish National Research Foundation (chair grant no. DNRF155).

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Author notes

  1. These authors contributed equally: Hang Wang, Hanlei Yang.

Authors and Affiliations

  1. State Key Laboratory of Plant Genomics, Institute of Genetics and Developmental Biology, The Innovative Academy of Seed Design, Chinese Academy of Sciences, Beijing, China

    Hang Wang, Hanlei Yang, Zhao Wen, Chengxu Gao, Yihong Gao, Yanbao Tian, Zuopeng Xu, Xiangling Liu, Baocai Zhang & Yihua Zhou

  2. University of Chinese Academy of Sciences, Beijing, China

    Hanlei Yang, Zhao Wen, Chengxu Gao, Yihong Gao & Yihua Zhou

  3. Jiangsu Key Laboratory of Crop Genetics and Physiology/Key Laboratory of the Ministry of Education for Plant Functional Genomics, College of Agriculture, Yangzhou University, Yangzhou, China

    Zuopeng Xu

  4. Department for Plant and Environmental Sciences, University of Copenhagen, Frederiksberg, Denmark

    Staffan Persson

  5. Copenhagen Plant Science Center, University of Copenhagen, Frederiksberg, Denmark

    Staffan Persson

  6. Joint International Research Laboratory of Metabolic & Developmental Sciences, State Key Laboratory of Hybrid Rice, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, China

    Staffan Persson

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Contributions

Y.Z. and B.Z. conceived the study. H.W. performed all the genetic and biochemical analyses. H.Y. performed all the super-resolution microscopy studies. Z.W. performed VISUAL treatments and compositional analyses. C.G. performed the AFM assay. Y.G. performed field trails and correlation analysis. Y.T. performed FESEM analyses. Z.X. performed the map-based cloning and conducted field trials. X.L. performed gene transformation. Y.Z., B.Z. and S.P. analysed the data and interpreted the results. Y.Z., S.P. and B.Z. wrote the article.

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Correspondence to Baocai Zhang or Yihua Zhou.

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Extended data

Extended Data Fig. 1 Depositing xylan in the developing rice internodes.

a, Diagram of a developing wild type (Nipponbare, NP) internode (left), a large vein (centre), and a macerated metaxylem vessel (right). S1‒S9 indicate the sequential segments of internode from the base to the tip. SCW, Secondary cell wall. b, Xylan abundance in each segment, analysing the monosaccharide content. Error bars represent the mean ± s.d. n = 5 biological replicates. c, Representative electrophoretogram of the released xylo-oligosaccharides. d, Xylo-oligosaccharides released by xylanase M6 digestion. Error bars represent the mean ± s.d. n = 3 biological replicates. e, Morphology of vessel pits in the NP developing internodes. The vessel walls were stained with S4B. f, Boxplot of pit size of vessel walls shown in (e). Box boundaries represent the 25th and 75th percentile, centre line represents the median, × indicates the mean, and whiskers represent the 25th percentile − 1.5 * the interquartile range and the 75th percentile + 1.5 * the interquartile range. P < 0.0001 by one-way ANOVA. n = 200 vessel pits from three individual plants. Letters a–d indicate statistical significance according to Tukey’s multiple range test. P < 0.05. Bars = 1 cm in (a) and 2 μm in (e).

Source data

Extended Data Fig. 2 Genetic characterization of the bc18 mutant.

a, Map-based cloning of bc18. The letters on the chromosome indicate molecular markers for mapping. Number inside the parentheses indicate recombinant individuals. Arrows represent the genes within the mapping region. b, The mature plants of wild type (WT) and irx10-1. Bar = 12 cm. c, Western blotting of total membrane proteins extracted from the indicated plants using the anti-IRX10 antibodies. The reduced IRX10 signal in bc18 (a missense mutant) and absence of IRX10 signal in irx10-1 (a null mutant) demonstrated specificity of anti-IRX10 antibodies. Anti-BiP antibody was used for load control. d, Diagram of the IRX10 construct for complementation assay. e, Measurement of breaking force of the internodes in wild type, bc18, irx10-1, and the complementary line (Com). Error bars represent the mean ± s.d. n = 15 internodes from individual plants. P < 0.0001 by one-way ANOVA. Letters a–c indicate statistical significance according to Tukey’s multiple range test. P < 0.05. The experiment in (c) was repeated independently for three times.

Source data

Extended Data Fig. 3 Comparison of IRX10 and its homologues in rice.

a, Phylogenetic tree of GT47 family members from rice and Arabidopsis. Neighbour-join method was used with 1000 replicates. b, Phylogenetic tree and domain structure of rice IRX10 and the homologues. The genotypes of triple mutants (Tm1−Tm3) and quadruple mutants (Qm) generated in this study are indicated by different colour lines, respectively. c, qPCR analysis of IRX10 and the homologues in the indicated tissues, showing the relative expression levels to rice TP1. Error bars represent the mean ± s.d. n = 3 biological replicates. d, qPCR analysis of genes expressed in vessels (V), fibre cells (FC), and parenchyma cells (PC) collected by laser microdissection in wild type. Error bars represent the mean ± s.d. n = 3 biological replicates. Rice TP1 was used as an internal control.

Source data

Extended Data Fig. 4 Generation of the rice mutants of IRX10 homologues.

a, Diagram of IRX10 and its homologous genes. The mutant alleles are indicated by short bars. b, The mature plants of the indicated genotypes. c, Microscopy images of the large vein in wild type and the indicated mutants. The boxed parts were magnified in Fig. 2b. d, Transmission electronic microscopy images of vessel walls in the indicated genotypes. Asterisks indicate the vessel walls. e-g, Boxplot of diameter (e), length (f), and wall thickness (g) of vessels in the indicated genotypes. Box boundaries represent the 25th and 75th percentile, centre line represents the median, × indicates the mean, and whiskers represent the 25th percentile − 1.5 * the interquartile range and the 75th percentile + 1.5 * the interquartile range. P < 0.0001 by one-way ANOVA. n = 50 in (e) and 21~47 in (f) of vessels from three individual indicated plants. In (g), n = 100 measurements in at least four vessels from three individual indicated plants. Letters a–e indicate statistical significance according to Tukey’s multiple range test. P < 0.05. Tm1, Tm2, Tm3, and Qm indicate triple and quadruple mutants defined in Extended Data Fig. 3b. Bars = 12 cm in (b), 20 μm in (c) and 1 μm in (d).

Source data

Extended Data Fig. 5 Immunolabelling analysis of xylan in vessel walls.

a,b, The vessel walls of wild type, bc18 and triple mutant (Tm1, irx10 10l1 10l3) stained using antibodies of LM10 (a) and LM11 (b) and FITC-conjugated secondary antibodies. Cell walls were stained using propidium iodide (PI). Bars = 5 µm. The experiments were repeated independently for three times.

Extended Data Fig. 6 Subcellular localization of rice IRX10.

a, The confocal images of Nicotiana benthamiana leaf epidermal cells that transiently expressed IRX10-GFP and Golgi-marker Man49-mCherry. b, Representative confocal image of Nicotiana benthamiana leaf epidermal cells that transiently expressed IRX10-mCherry and Arabidopsis VND6. The wall bands were visualized using UV illumination (405 nm). Bars = 10 μm. The experiments were repeated independently for at least three times.

Extended Data Fig. 7 Enzyme activity assay of rice IRX10.

a, The mutating amino acids examined in this study were indicated in the predicted 3D structure of rice IRX10. b, Recombinant proteins of rice IRX10 and the variants (Upper panel). Western blotting of IRX10 and the variants using anti-His antibodies (Low panel). 10D represents D311N/D312N. 10R indicates R295K. CBB, Coomassie brilliant blue staining. c,d, Electrophoretograms of the reaction products generated by IRX10 and its variants using UDP-xylose (c) and xylopentaose plus UDP-xylose (d) as the substrates, respectively. Xyl1‒Xyl8 indicate standard markers of xylo-oligosaccharides. Asterisks indicate unspecific bands. e, Activity analysis of recombinant IRX10 and the variants. ++ indicates more activated than +; − indicates undetectable activity. The experiments in (b-d) were repeated independently for at least three times.

Source data

Extended Data Fig. 8 Immunolabelling analysis of xylan in Arabidopsis vessels induced by VISUAL.

a,b, Tracheary elements induced in the Arabidopsis cotyledons of wild type (Col) and atirx10 at different inducing time points (hai). Xylan was probed by LM10 antibody and FITC-conjugated secondary antibodies. The cell walls were stained using cellulose specific dye S4B. Bars = 10 μm. The experiments were repeated independently for three times.

Extended Data Fig. 9 FESEM and AFM analysis of pitted vessel walls.

a, The representative FESEM images of pitted vessel walls of the indicated genotypes. b, Magnification of the boxed regions of pit borders in (a). The arrows indicate the clear boundary of pits. c, The representative AFM Images of pitted vessel walls of the indicated genotypes. The arrows indicate the fused boundary of adjacent pores. Bars = 400 nm in (a), 100 nm in (b), and 1 μm in (c). The experiments were repeated independently for three times.

Extended Data Fig. 10 Transport and phenotype analyses.

a, Two-week-old seedlings of the indicated genotypes. b,c, Leaf cross section of wild-type seedlings treated in rhodamine B (RB, b) and rhodamine B-tagged CEP6.1 peptide (RB-CEP, c), showing fluorescent signals mainly in vessels cells. d, Length of the normal (green) and wilting (yellow) parts of leaves from the indicated genotypes shown in Fig. 6d. Error bars represent the mean ± s.d. n = 20 leaves from individual plants. e-g, Correlation analysis of pit area with the 2nd internode length (e), internode weight (f), and panicle length (g) in 42 core rice accessions. Tm1, Tm2 and Tm3 indicate triple mutants defined in Extended Data Fig. 3b. Bars = 5 cm in (a) and 20 μm in (b,c). Letters a–d in (d) indicate statistical significance according to Tukey’s multiple range test. P < 0.05.

Source data

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Supplementary Information

Supplementary Figs. 1–4, Tables 1–5 and unprocessed gels for supplementary Fig. 3b.

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Supplementary Video

The predicted 3D structure of rice IRX10 to show the amino acids for mutation analysis.

Supplementary Data

Statistical source data for Supplementary Information.

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Unprocessed western blots and gels.

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Wang, H., Yang, H., Wen, Z. et al. Xylan-based nanocompartments orchestrate plant vessel wall patterning. Nat. Plants 8, 295–306 (2022). https://doi.org/10.1038/s41477-022-01113-1

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