Citation: Busont, O.; Durambur, G.; Bernard, S.; Plasson, C.; Joudiou, C.; Baude, L.; Chefdor, F.; Depierreux, C.; Héricourt, F.; Larcher, M.; et al. Black Poplar (Populus nigra L.) Root Extracellular Trap, Structural and Molecular Remodeling in Response to Osmotic Stress. Cells 2023, 12, 858. https://doi.org/10.3390/cells12060858 Academic Editor: Andrei Smertenko Received: 16 February 2023 Revised: 4 March 2023 Accepted: 6 March 2023 Published: 9 March 2023 Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). cells Article Black Poplar (Populus nigra L.) Root Extracellular Trap, Structural and Molecular Remodeling in Response to Osmotic Stress Océane Busont 1 , Gaëlle Durambur 2 , Sophie Bernard 2,3 , Carole Plasson 2 , Camille Joudiou 1 , Laura Baude 1,4 , Françoise Chefdor 1 , Christiane Depierreux 1 , François Héricourt 1 ,Mélanie Larcher 1 , Sonia Malik 1 , Isabelle Boulogne 2 , Azeddine Driouich 2 , Sabine Carpin 1 and Frédéric Lamblin 1, * 1 Laboratoire de Biologie des Ligneux et des Grandes Cultures, Université d’Orléans, INRAE, USC 1328, CEDEX 2, F-45067 Orléans, France 2 GLYCOMEV UR 4358, SFR Normandie Végétal FED 4277, Innovation Chimie Carnot, University of Rouen Normandie, IRIB, F-76000 Rouen, France 3 INSERM, CNRS, HeRacLeS US 51 UAR 2026, PRIMACEN, University of Rouen Normandie, F-76000 Rouen, France 4 Department of Biology, University of Fribourg, CH-1700 Fribourg, Switzerland * Correspondence: frederic.lamblin@univ-orleans.fr; Tel.: +33-(0)2-3841-7127 Abstract: The root extracellular trap (RET) consists of root-associated, cap-derived cells (root AC-DCs) and their mucilaginous secretions, and forms a structure around the root tip that protects against biotic and abiotic stresses. However, there is little information concerning the changes undergone by the RET during droughts, especially for tree species. Morphological and immunocytochem- ical approaches were used to study the RET of black poplar (Populus nigra L.) seedlings grown in vitro under optimal conditions (on agar-gelled medium) or when polyethylene glycol-mediated (PEG 6000 —infused agar-gelled medium) was used to mimic drought conditions through osmotic stress. Under optimal conditions, the root cap released three populations of individual AC-DC morphotypes, with a very low proportion of spherical morphotypes, and equivalent proportions of intermediate and elongated morphotypes. Immunolabeling experiments using anti-glycan antibodies specific to cell wall polysaccharide and arabinogalactan protein (AGP) epitopes revealed the presence of homogalacturonan (HG), galactan chains of rhamnogalacturonan-I (RG-I), and AGPs in root AC- DC cell walls. The data also showed the presence of xylogalacturonan (XGA), xylan, AGPs, and low levels of arabinans in the mucilage. The findings also showed that under osmotic stress conditions, both the number of AC-DCs (spherical and intermediate morphotypes) and the total quantity of mucilage per root tip increased, whereas the mucilage was devoid of the epitopes associated with the polysaccharides RG-I, XGA, xylan, and AGPs. Osmotic stress also led to reduced root growth and increased root expression of the P5CS2 gene, which is involved in proline biosynthesis and cellular osmolarity maintenance (or preservation) in aerial parts. Together, our findings show that the RET is a dynamic structure that undergoes pronounced structural and molecular remodeling, which might contribute to the survival of the root tip under osmotic conditions. Keywords: AC-DCs; mucilage; drought stress; root cap; xylogalacturonan; xylan; arabinogalactan protein; rhamnogalacturonan-I 1. Introduction Drought is one of the most severe environmental stresses affecting plant growth and productivity [1]. The black poplar (Populus nigra L.) is an emblematic alluvial forest species that grows along large rivers with considerable seasonal flow variation. The tree is regularly exposed to periods of flooding, followed by periods of increasingly low water levels and thereby drought conditions. In the context of global warming, wild and cultivated poplars are being subjected to increasingly prolonged and intense periods of drought. The restoration and protection of riparian forests is one of the key priorities in Cells 2023, 12, 858. https://doi.org/10.3390/cells12060858 https://www.mdpi.com/journal/cells