Received: 15 May 2019 | Revised: 18 June 2019 | Accepted: 21 June 2019 DOI: 10.1002/pld3.151 ORIGINAL RESEARCH Cell wall composition and transcriptomics in stem tissues of stinging nettle (Urtica dioica L.): Spotlight on a neglected fibre crop Xuan Xu1 | Aurélie Backes1 | Sylvain Legay1 | Roberto Berni2,3 | Claudia Faleri2 | Edoardo Gatti4 | Jean‐Francois Hausman1 | Giampiero Cai2 | Gea Guerriero1 1Environmental Research and Innovation (ERIN) Department, Luxembourg Institute of Abstract Science and Technology (LIST), Esch/Alzette, Stinging nettle (Urtica dioica L.) produces silky cellulosic fibres, as well as bioactive Luxembourg molecules. To improve the knowledge on nettle and enhance its opportunities of 2Department of Life Sciences, University of Siena, Siena, Italy exploitation, a draft transcriptome of the “clone 13” (a fibre clone) is here presented. 3Trees and Timber Institute-National The transcriptome of whole internodes sampled at the top and middle of the stem is Research Council of Italy (CNR-IVALSA), Follonica, Italy then compared with the core and cortical tissues sampled at the bottom. Young inter- 4Institute of Biometeorology nodes show an enrichment in genes involved in the biosynthesis of phytohormones (IBIMET), National Research Council, (auxins and jasmonic acid) and secondary metabolites (flavonoids). The core of inter- Bologna, Italy nodes collected at the bottom of the stem is enriched in genes partaking in different Correspondence aspects of secondary cell wall formation (cellulose, hemicellulose, lignin biosynthe- Gea Guerriero, Luxembourg Institute of Science and Technology (LIST), sis), while the cortical tissues reveal the presence of a C starvation signal probably Environmental Research and Innovation due to the UDP-glucose demand necessary for the thickening phase of bast fibres. (ERIN) Department, Esch/Alzette L-4362, Luxembourg. Cell wall analysis indicates a difference in rhamnogalacturonan structure/composi- Email: [email protected] tion of mature bast fibres, as evidenced by the higher levels of galactose measured, Present address as well as the occurrence of more water-soluble pectins in elongating internodes. The Aurélie Backes, Unité de Recherche targeted quantification of phenolics shows that the middle internode and the cortical Résistance Induite et BioProtection des Plantes, UFR Sciences Exactes et tissues at the bottom have higher contents than top internodes. Ultrastructural anal- Naturelles, SFR Condorcet FR CNRS yses reveal the presence of a gelatinous layer in bast fibres with a lamellar structure. 3417, Université de Reims-Champagne- Ardenne, Reims Cedex 2, France The data presented will be an important resource and reference for future molecular studies on a neglected fibre crop. KEYWORDS bast fibres, electron microscopy, immunohistochemistry, RNA-Seq cell wall composition, Urtica dioica L. Xu and Backes equally contributed as first authors. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes. © 2019 The Authors. Plant Direct published by American Society of Plant Biologists, Society for Experimental Biology and John Wiley & Sons Ltd. Plant Direct. 2019;3:1–17. wileyonlinelibrary.com/journal/pld3 | 1 2 | XU ET AL. 1 | INTRODUCTION produces around 4%–5% bast fibres; Hartl & Vogl, 2002; Harwood & Edom, 2012). Harnessing plant biomass as energy feedstock and source of (macro) The present study also provides the transcriptomic signature molecules for industrial applications is an undeniable need to foster of nettle internodes sampled at different stem heights (top, mid- a sustainable, low C bio-economy. Herbaceous crops are important dle and bottom which reflect progressive maturation stages of renewable resources, because they produce biomass in a shorter extraxylary fibres and vascular tissues) and gives a special em- time as compared to woody species. phasis to cell wall-related processes. The results are discussed in Among herbaceous crops, plants producing bast fibres (belong- relation to the current knowledge concerning bast fibre devel- ing to the group of fibre crops) are very attractive, because they pro- opment in other fibre crops, namely flax, hemp and ramie. The vide long and strong fibres containing high amounts of crystalline data are complemented by the chemical analysis of the cell walls cellulose. These fibres are not only used in the textile sector, but extracted from the different internodes, the quantification of fla- are also appreciated by the biocomposite industry as eco-friendly vonoids and by the immunolocalization of specific polysaccharide alternatives to man-made fibres. components. Flax, hemp, ramie and nettle produce bast fibres with gelati- nous cell walls characterized by a high content of crystalline cel- 2 | METHODS lulose (>70%) and hypolignified (lignin content between 2%–7%) (Guerriero, Behr, Backes, et al., 2017; Guerriero, Sergeant, & 2.1 | Plant material, growth conditions and confocal Hausman, 2013). Several papers have been published on the molec- microscopy ular study of bast fibre formation in flax, ramie and hemp, however no study is yet available on stinging nettle (Urtica dioica L.), which Urtica dioica was propagated via stem cuttings and grown in incu- is one of the most undervalued plants among the economically in- bators following a cycle of 16 hr light 25°C/8 hr dark 20°C. After teresting ones. 5 weeks, stem internodes were collected along three regions of the Nettle is a perennial herbaceous plant growing in temperate stem localized at different heights (i.e., top, middle and bottom, as regions and producing long bast fibres between 43–58 mm (Bacci, described in Guerriero, Behr, Legay, et al., 2017). Three biological Baronti, Predieri, & di Virgilio, 2009) with high tensile strength. It replicates were used, each consisting of seven nettle plants se- has a long history as a fibre crop in Austria and Germany and was lected among all incubators. The “TOP” segment is located at the widely cultivated during the Second World War in Central Europe to top (below the apex). The “MID” (middle) is the segment possibly in- manufacture the uniforms of the German army. cluding the snap point and the “BOT” (bottom) segment corresponds Urtica dioica grows well on over-fertilized soils, it is naturally re- to the second internode below the “MID”. A segment of 2.5 cm was sistant to pests and diseases and it is a robust weed relying on an collected from the middle of each internode to minimise variations underground system of roots and rhizomes. This fibre crop has a in gene expression, caused by the differences in developmental multitude of applications in the medicinal, textile and biocomposite stages of the tissues (Guerriero, Behr, Legay, et al., 2017). The TOP sectors and is therefore a multi-purpose crop: its fibres are silkier and MID internodes were taken as a whole, following a sampling in texture than flax, it can be used to treat skin conditions and has strategy previously published for flax (Gorshkov, Behr, Legay, et al., expectorant, hemostatic, anti-inflammatory and diuretic properties 2017). The BOT sample was peeled to separate the cortex from (Kregiel, Pawlikowska, & Antolak, 2018). the core. The total sample numbers was therefore 12 (three bio- Similarly to hemp, the nettle stem is composed of a cortex con- logical replicates for the TOP, three for the MID, three for the BOT taining bast fibres and a woody core. It has been shown that the stem core and three for the BOT peels). In order to generate the de novo of nettle displays a lignification gradient from the top to the bottom transcriptome, the reads obtained for the internodes of the three (necessary for mechanical strength at the stem base), accompanied by above-described stem regions were pooled with those obtained different stages of bast fibre development (from elongation at the top, for other nettle tissues (leaves, roots, whole stem core tissues and to cell wall thickening at the bottom) (Backes et al., 2018). The differ- peels, calli). The calli were sampled from in vitro-micropropagated ential cell wall composition of nettle stem tissues and the sequential nettle stems (according to Gatti, Di Virgilio, & Bacci, 2008). Young developmental stages of bast fibres make this plant ideal to address leaves were taken from the stem apex, while roots (fine roots) were bast fibre‐ and cell wall-related studies, as previously shown in textile quickly sampled, washed under tap water to remove soil particles, hemp. blotted dry and cut to smaller pieces with a sterile scalpel. All col- The availability of an annotated transcriptome is important for lected samples were frozen in liquid nitrogen and stored at −80°C plant molecular studies and for addressing fundamental questions until analysis. The libraries used to enrich the de novo transcriptome related to the function of specific genes. (i.e., those obtained using leaves, whole stems, roots and calli) were We here provide for the first time a draft transcriptome of the run as single replicates. nettle “clone 13” selected by Bredemann for fibre yield between Sample preparation for confocal microscopy analysis with the 1927–1950 (Harwood & Edom, 2012). This clone is characterized different antibodies (PlantProbes) was carried out as previously de- by a higher bast fibre yield (16% as compared to wild nettle, which scribed