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Distribution of cell-wall polysaccharides and proteins during growth of the hemp hypocotyl

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Abstract

Main conclusion

The immuno-ultrastructural investigation localized cell-wall polysaccharides of bast fibers during hemp hypocotyl growth. Moreover, for the first time, the localization of a peroxidase and laccase is provided in textile hemp.

Abstract

In the hypocotyl of textile hemp, elongation and girth increase are separated in time. This organ is therefore ideal for time-course analyses. Here, we follow the ultrastructural rearrangement of cell-wall components during the development of the hemp hypocotyl. An expression analysis of genes involved in the biosynthesis of cellulose, the chief polysaccharide of bast fiber cell walls and xylan, the main hemicellulose of secondary cell walls, is also provided. The analysis shows a higher expression of cellulose and xylan-related genes at 15 and 20 days after sowing, as compared to 9 days. In the young hypocotyl, the cell walls of bast fibers show cellulose microfibrils that are not yet compacted to form a mature G-layer. Crystalline cellulose is detected abundantly in the S1-layer, together with unsubstituted/low-substituted xylan and, to a lesser extent, in the G-layer. The LM5 galactan epitope is confined to the walls of parenchymatic cells. LM6-specific arabinans are detected at the interface between the cytoplasm and the gelatinous cell wall of bast fibers. The class III peroxidase antibody shows localization in the G-layer only at older developmental stages. The laccase antibody shows a distinctive labelling of the G-layer region closest to the S1-layer; the signal becomes more homogeneous as the hypocotyl matures. The data provide important insights on the cell wall distribution of polysaccharide and protein components in bast fibers during the hypocotyl growth of textile hemp.

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Acknowledgements

The authors wish to thank Aude Corvisy and Laurent Solinhac for technical support.

Funding

The authors acknowledge the Fonds National de la Recherche, Luxembourg (Project CANCAN C13/SR/5774202) for financial support.

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Authors and Affiliations

  1. Research and Innovation Department, Luxembourg Institute of Science and Technology, 5 Avenue des Hauts-Fourneaux, 4362, Esch/Alzette, Luxembourg

    Marc Behr, Jean-Francois Hausman, Sébastien Planchon, Jenny Renaut & Gea Guerriero

  2. Department of Life Sciences, University of Siena, via P.A. Mattioli 4, 53100, Siena, Italy

    Claudia Faleri & Giampiero Cai

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  1. Marc Behr
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  2. Claudia Faleri
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  3. Jean-Francois Hausman
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  7. Gea Guerriero
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Correspondence to Giampiero Cai or Gea Guerriero.

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425_2019_3245_MOESM1_ESM.docx

Supplementary material 1 Supplementary Document S1 Nucleotide sequences used for the design of qPCR primers targeting CELLULOSE SYNTHASE genes (DOCX 20 kb)

425_2019_3245_MOESM2_ESM.tif

Supplementary material 2 Supplementary Fig. S2 Expression of genes involved in xylan biosynthesis as determined by RNA-Seq (RPKM values for expression). For each gene, different letters correspond to significant differences (Tukey post hoc test). ESK ESKIMO, FRA FRAGILE FIBER, GAUT GALACTURONOSYLTRANSFERASE, GUT Β-1,4-XYLOSYLTRANSFERASE, GUX GLUCURONIC ACID SUBSTITION OF XYLAN, GXM GLUCURONOXYLAN METHYLTRANSFERASE, IRX IRREGULAR XYLEM, RWA REDUCED WALL ACETYLATION (TIFF 703 kb)

425_2019_3245_MOESM3_ESM.tif

Supplementary material 3 Supplementary Fig. S3 (a) Expression of genes involved in cellulose deposition as determined by RNA-Seq (RPKM values for expression). For each gene, different letters correspond to significant differences (Tukey post hoc test). CTL CHITINASE LIKE, COB COBRA, IRX IRREGULAR XYLEM, CESA CELLULOSE SYNTHASE, FLA FASCICLIN-LIKE ARABINOGALACTAN PROTEIN. (b) Expression of genes involved in cellulose deposition as determined by RT-qPCR. For each gene, different letters correspond to significant differences (ANOVA one-way with Tukey’s post hoc test) (TIFF 1872 kb)

425_2019_3245_MOESM4_ESM.tif

Supplementary material 4 Supplementary Fig. S4 Densitometric analysis of immunogold labelling signals. (a) Analysis of the distribution of xylans as shown by the LM10 antibody. (b) Analysis of the crystalline cellulose distribution as shown by the CBM3a probe. (c) Analysis of laccase distribution as shown by the Lac17 antibody. Measurements were performed at the three developmental stages H9, H15, and H20. The signal intensity (ordinate axis) is expressed as a percentage of gray values. The x-axis shows the distance where point 0 indicates the boundary of the primary cell wall (PCW). The thickness of the secondary cell wall layers is indicated. (TIFF 1309 kb)

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Behr, M., Faleri, C., Hausman, JF. et al. Distribution of cell-wall polysaccharides and proteins during growth of the hemp hypocotyl. Planta 250, 1539–1556 (2019). https://doi.org/10.1007/s00425-019-03245-9

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