Search

Changements biochimiques pendant la maturation des parois. Workshop final du projet ANR Blanc StressInTrees "Approches couplées physiologique et micro-mécanique de la génération des contraintes de maturation dans le bois de tension"

International audience; Angiosperm trees are able to reorient their axes thanks to their capacities to differentiate tension wood on the upper side of stems and branches. In poplar, tension wood fibres develop an extra cell wall layer, named G-layer, responsible for the peculiar mechanical properties of tension wood. G-layer is composed of highly crystalline cellulose microfibrils embedded in a polysaccharide/glycoprotein matrix devoid of lignin. During the G-layer deposition, cellulose microfibrils get oriented parallel to the fibre axis and placed under the state of high tensile stress. Chitinase-like proteins (CTL) have been proposed to play different roles in plant growth and development. Genes encoding CTL have been associated to cellulose synthesis and their mutation causes ectopic deposition of lignin. Transcripts of CTL2 gene were shown to be highly abundant in differentiating tension wood (Déjardin et al., 2004). Its homologue in Arabidopsis is specifically expressed in stems and co-expressed with the CesA genes involved in cellulose synthesis in secondary cell walls (Persson et al., 2005). In our study, we investigated potential function of CTL2 in the formation and mechanical properties of tension wood. Expression of CTL2 was quantified in different tissues and the effects of CTL2 down-regulation have been characterized in GM poplar trees. CTL2 gene appeared highly expressed in xylem with no difference in expression between tension and opposite wood. Downregulation of CTL2 did not seem to impact growth and development of trees in greenhouse conditions. However, stems appeared to be highly breakable in the transverse direction. Anatomical observations of xylem showed altered formation of Glayers in vessel-surrounding fibres in transgenic poplars. Further analyses indicated a substantial increase in wood lignin content. Cellulose crystallinity, microfibril angle and stem mechanical properties are currently assessed. Results will be presented and discussed in the light of present hypothesis regarding CTL2 function in plant development.

Lakhal W., Boizot N., Lesage-Descauses M-C., Rogier O., Lainé-Prade V., Laurans F., Ader K., Charpentier J-P., Decoville M., Bénédetti H., Leplé J-C., Pilate G., Déjardin A, 2015. Both ChIP-SEQ and in planta gene modification demonstrate the involvement of MYB090 and MYB156 in secondary cell wall formation in poplar. INUPRAG Meeting, 6-8 October 2015, Nancy, France, communication orale.; Both ChIP-SEQ and in planta gene modification demonstrate the involvement of MYB090 and MYB156 in secondary cell wall formation in poplar. INUPRAG-Meeting 2015

National audience

International audience; In hardwood trees, tension wood formation is a remarkable adaptive mechanism that makes possible for the tree to reorient its axis. Tension wood differs from normal wood by anatomical, chemical and physical characteristics: it is less lignified, and contains more cellulose of higher crystallinity, with longitudinally oriented microfibrils. In poplar, tension wood fibers harbor an extra cell wall layer, named G-layer, responsible for the peculiar mechanical properties of tension wood (1). We propose that differences between tension and normal wood mechanical properties are linked to the polysaccharide composition of the G-layer. In order to investigate the nature of these links, we realized immunolocalisation studies with light microscope observation. 158 monoclonal antibodies (from CCRC, LM, JIM and MAC series) raised against various epitopes specific to different families of plant cell wall polysaccharides were tested. For each antibody tested, we analyzed where labeling was present, not only between tension and opposite wood (that is completely devoid of G-fibres), but also according to cell type, cell wall layer, and this along the gradient of xylem cell differentiation. Most antibodies directed against xyloglucans, fucosylated or not, specifically labeled the primary wall / middle lamella in both tension and opposite wood fibers. This was also the case for several antibodies specific to rhamnogalacturonans I. Some other antibodies labeled only the S2 layer, such as most antibodies directed against xylans, whereas, unexpectedly, a few other xylan-specific antibodies exhibited a wider range of labeling. None of the antibody tested was able to specifically label opposite wood cell walls. On the contrary, among all the antibodies directed against rhamnogalacturonan I or arabinogalactan epitopes that were tested, eighteen of them specifically labeled the G-layer in tension wood fibers and, for some of them, the labeling was restricted to newly formed G-layers. Overall, from the signals obtained with each of the antibody tested, differences between tension and opposite wood fibers when observed were only detected at the level of the G-layer whose composition appears to evolve throughout its maturation. Furthermore, our study strongly suggests the involvement of specific arabinogalactan-proteins and pectins during G-layer construction. Finally, we optimized a procedure for G-layer isolation in order to analyze the polysaccharide composition of purified G-layers by mass spectrometry. In the presentation, we will discuss more in details all the results obtained using these two complementary approaches.

Demande Internationale PCT n° PCT/IB2010/052590; The invention relates to the production of plants having a reduced lignin content and in which the cellulose hydrolysis of the walls is increased, via the total or partial inhibition of the expression and/or the activity of two laccases in said plant.

International audience

International audience; Lignin is a major component of the secondary cell wall. This complex phenolic polymer is derived mainly from three hydroxycinnamyl alcohol monomers named monolignols. After transport of the monolignols to the cell wall, lignin is formed through an enzymatically-initiated oxidative polymerization of these precursors. Whereas many studies have established that this polymerization involves peroxidases, the involvement of laccases is more speculative. The identification of the peroxidases and laccases specifically involved in lignification is complicated by the fact that these enzymes belong to multigene families. For instance, in Arabidopsis, there are 73 peroxidase and 17 laccase genes. In order to determine which peroxidases and/or laccases could be involved in the polymerisation of monolignols, several strategies have been considered: • Expression profiling in the highly lignified infloresecence stem by RT-PCR and promoter/GUS fusions • Presence in the cell wall proteome of stems • Characterization of homozygous knock-out Arabidopsis TDNA mutants and determination of the impact of the mutation on lignin content and composition. The results of this study will be presented and new information on the importance of several genes in lignification will be provided. Orthologs of poplar have been identified and are currently under study.

International audience; Wood formation is a complex process under both genetic and environmental controls and it is known to be strongly affected by water availability. This study aims to finely describe both wood anatomy and gene expression modifications induced by the sequential application of a water stress followed by a rewatering step on greenhouse-grown poplar plants induced to form tension wood by leaning of their stem. Water stress followed by a rewatering step induced striking modifications in wood anatomy: water stress lead to both a reduction in fibre dimensions and an increase in cell wall thickness, when rewatering induces a transient and strong increase in the vessel/fibre ratio, particularly in tension wood area. This anatomical study has been complemented by a global gene expression analysis using whole poplar genome Affymetrix GeneChip. Transcriptome analysis has been performed on very young xylem tissues collected shortly after rewatering, that is when initial fusiform cells were preferentially oriented toward vessel differentiation. We will present and discuss the results obtained in this experiment.

International audience; In response to environmental stimuli such as wind, snow, slope or asymmetric crown shape, leaning woody plants form a special tissue, named reaction wood, which allows tree axis reorientation. In angiosperm trees, this reaction wood is associated with tensile strains and consequently has been named tension wood (TW). It occurs at the upper face of leaning stems and branches, and can be obtained in a controlled way on young trees by simply tilting the stem. Inpoplar, TW is mainly characterized by xylem fibres which are poorly lignified and have an extra thick gelatinous secondary layer, named G layer, in the secondary cell wall. This G layer contains almost completely pure crystalline cellulose with microfibrils oriented nearly parallel to the axis of the fibre. Different models have been proposed in order to link these biochemical and ultrastructural features to the mechanical properties of TW. To assess the validity of these models we need to find out the molecular mechanisms involved in the formation of the G layer. With the aim to identify such mechanisms we looked for genes differentially expressed between TW and OW. We have previously shown that l0 fasciclin-like arabinogalactan (FLAs) poplar genes were highly expressed in the xylem of tension wood [1, 2]. Five other popFLAs (l I to l5) were shown to be expressed in xylem but not differentially between TW and ow t2l. These 15 popFLAs belong to the class of arabinogalactan-proteins (AGPs) containing one fasciclin-like domain surounded by two AGP- like domains. While the fasciclin-like domain appears well conserved among the different proteins, we find more variability in the AGPlike domains which carry the arabinogalaotosylation sites. Thanks to the recent publication of the poplar genome sequence we identified 41 additional poplar genes encoding putative fasciclin-like protein. Two-thirds of the predicted genes contain one FASI (fasciclin-like) domain as defined by the reference "IPR000782" in InterPro database, the other 17 genes carrying 2 FAS1 domains. In addition, 70% of the sequences are predicted to be anchored to a glycosyl-phosphatidylinositol (GpI). It has been postulated that this dual presence of fasciclin-like domain and GPI-anchor could support a putative function of these proteins in cell adhesion and signaling. In regard to the phylogenic analysis of this large gene family we investigated the expression profiling of these 56 poplar FLAs. This has been done through electronic northern and from genome-wide expression data collected on Affymetrix poplar array experiments. Electronic northern were done from 200.199 expressed sequence tags (ESTs) available in public databases representing 37 different non normalized cDNA libraries. Genome-wide expression patterns were investigated in tension wood in response l) to nyctemeral variation and 2) to re-watering after water shortage. The aim of thefirst experiment was to see if nyctemer variation of FLAs gene could be correlated with expression pattern of other cell-wall related genes. In the second experiment, re-watering induces xylem re-growth together with a significant increase of differentiation of vessel elements. Expression analyses of these FLAs in these experiments will be presented and discussed.

International audience

National audience; Le bois de tension chez le peuplier est produit lorsque les tiges se trouvent, suite à une contrainte environnementale, dans une position inclinée par rapport à la verticale. Ce bois, permet le redressement des tiges et se caractérise par la présence de fibres particulières à parois très épaisses appelées fibres G. L'objectif de ce travail consiste en l'identification de facteurs de transcription impliqués dans la différenciation des fibres G du bois de tension. Parmi les 10 000 clones des banques ESTs de bois de peuplier (Populus tremula x alba) de l'INRA d'Orléans (Déjardin et al 2004), la famille LIM (37 EST) est la famille de facteurs de transcription la plus représentée. Six gènes PtaLlM (PtaLlMla, PtaLlMlb, PtaWLlMla, PtaWLlMlb, PtaWLlM2a et PtaPLIM2a) ont été identifiés et les ADNc pleine longueur correspondant à ces gènes ont été obtenus. L'analyse de la répartition de ces EST LIM dans nos banques ainsi que dans les banques populusDB (Populus tremula x tremuloides), nous montre qu'une forte proportion d'EST est issue de jeune xylème de bois tendu. PtaLlMl a est le consensus ayant le plus d'EST provenant en majorité de bois tendu et de xylème mature. Dans le génome du peuplier (Populus trichocarpa), il existe au moins 12 modèles de gène de cette famille: PtLIMla et b, PtWLIM2a et b, PtPLlM2a et b, PtWLIMla et b, PtLIM2a et b, et PtLla et b. La forte homologie de séquence entre ces 6 couples de gènes indique que les gènes PtLIM sont probablement des gènes dupliqués. Des analyses phylogénétiques nous montrent que les protéines PtPLIM2a et b appartiennent au groupe PLIM2 spécifique du pollen chez les plantes et que les protéines PtaWLIM1a et b et PtWLIM2a et b appartiennent respectivement aux groupes WLIM1 et WLIM2 exprimé de manière ubiquitaire (Eliasson et al 2000). Les protéines PtaLIM1a et b, PtL1a et b et PtLIM2a et b formeraient des groupes phylogénétiques distincts. Les résultats d'analyse d'expression par RT-PCR semi-quantitative entre les différents organes de peuplier nous montrent que les gènes PtaLIMont des profils d'expression différents. Tout d'abord, les gènes PtaLIM sont tous plus fortement exprimés dans le jeune xylème du tronc. PtaLIMl a, PtaLIMl b et Pta WLIMI b sont spécifiques des tissus vasculaires (feuilles, racines, tronc). PtaWLlM2a et PtaWLIMla sont exprimés constitutivement dans tous les organes. Et PtaPLlM2a, aussi exprimé dans le xylème, est surtout exprimé dans les étamines. Les analyses RT-PCR menées sur des échantillons de jeune xylème tendu et opposé d'un arbre incliné pendant 15 jours indiquent que l'expression de tous les gènes PtaLlM est induite dans le bois tendu. Enfin, l'induction de l'expression de ces gènes dans le bois tendu varie en fonction du niveau de différenciation du xylème. L'ensemble de ces données sera complété par western blot et immunohistolocalisation. Pour cela, nous avons à notre disposition un anticorps pouvant reconnaître les protéines PtaLIM1a, PtaLIMlb, PtaWLIM1a, et PtaWLIM1b. Et d'autre part nous utiliserons un anticorps dirigé contre la protéine recombinante 6His- PtaLIM 1 a.

National audience; La formation du bois ou xylogenèse résulte d'un processus de différenciation complexe, initié par des divisions cellulaires au niveau du cambium, une couche cellulaire méristématique présente sur toute la hauteur de l'arbre, sous l'écorce. Les nouvelles cellules formées passent ensuite par une phase d'élongation, avant de subir une maturation, caractérisée par la lignification des parois. Ce processus de différenciation se produit selon un gradient allant du cambium jusqu'au centre de la tige. Le bois est donc un tissu complexe, avec une organisation spatiale bien particulière, qui lui confère ses propriétés. Les caractéristiques anatomiques et chimiques du bois peuvent être également fortement modifiées sous l'influence de l'environnement. Par exemple, quand un arbre penche, du bois de réaction se forme en réponse à des stimuli gravitationnels et mécaniques. La variabilité et l'hétérogénéité du bois, et en conséquence ses propriétés, résultent au moins en partie de l'expression concertée d'un grand nombre de gènes. Pour accroître nos connaissances sur la formation du bois, nous avons développé un programme de séquençage d'EST sur peuplier (Expressed Sequence Tag ou Etiquette de Séquence Transcrite). Profitant du haut degré d'organisation de la tige, nous avons prélevé différentes zones du bois (zone cambiale, jeune xylème en différenciation et xylème mature) à la fois sur du bois de tension et du bois opposé pour générer quatre banques d'ADNc différentes. 10 062 EST, reflétant les gènes exprimés dans chaque zone du bois, ont été séquencées et réparties en 1337 clusters et 3595 singletons. Pour les plus gros clusters, la distribution en EST a été comparée entre les différentes banques pour identifier des gènes spécifiques ou très fortement exprimés pendant une des phases du processus de xylogenèse. Par exemple, nous avons ainsi clairement mis en évidence que les gènes exprimés entre zone cambiale et jeune xylème sont très différents, traduisant un changement profond dans le processus de développement. Neuf clusters spécifiques ou très fortement exprimés dans le bois de tension ont été détectés, correspondant à des protéines à arabinogalactanes, une saccharose synthase, une fructokinase, un facteur de transcription et une protéine de fonction Inconnue. Cette première étape d'identification se poursuit par des études globales d'expression qui permettront à terme d'identifier des clusters de gènes présentant des profils d'expression similaires, au cours d'étapes de la xylogenèse déterminantes dans la définition de propriétés du bois. Une première puce ADN a été réalisée à partir de 1500 EST sélectionnées et les premières hybridations réalisées ont montré une expression différentielle d'un groupe de protéines particulières, les AGP , entre bois tendu et bois opposé.

International audience; Lignification, a major biological event in the plant kingdom, is still poorly understood due to the structural complexity and to the high variability of native lignins. Poplars underexpressing two enzymes of the monolignol biosynthetic pathway (caffeic acid/5 hydroxyferulic O-methyltransferase or cinnamyl alcohol dehydrogenase) or Arabidopsis thaliana knock-out T-DNA insertion mutants in the genes encoding these enzymes have been obtained and analysed for the quantity and quality of their lignins. COMT or CAD down-regulation induced in poplar, a woody plant, and in Arabidopsis, the annual crucifer model plant, show similar specific alterations in lignin structure due to the incorporation of unusual phenolics in the polymer. The specific structural traits of lignins, which account for higher (CAD deficiency) or lower (COMT deficiency) susceptibility to alkaline delignification processes have been delineated. Lignins of COMT depressed plants essentialy comprise guaiacyl (G) units together with substantial amounts of 5-hydroxyguaiacyl (5-OH-G) units. They display a markedly higher frequency of resistant interunit bonds including new structures (benzodioxanes) and a lower frequency of free phenolic lignin units. These changes make the lignin polymer less amenable ta alkaline delignification. Lignins of CAD deficient plants display a higher content of free phenolic groups, and incorporation of sinapaldehyde units, both associated with a greater solubility in dilute alkali at room temperature. The lignin characteristics of the CAD antisense poplars allow a reduction in the amount of alkali required for paper pulp making using the Kraft procedure.

National audience

International audience

International audience; The current development of biotechnologies opens up major potential in the area of genetic improvement of forest tree species. In particular, they should enable new improved varieties that are needed to maintain competitiveness of the timber industry to be disseminated. This article reviews the possibilities and limitations of using biotechnologies for forest trees; in vitro vegetative propagation by cell embryogenesis and microcutting, genetic transformation and marker-assisted selection.; Le développement actuel des biotechnologies ouvre des potentialités importantes en matière d'amélioration génétique des arbres forestiers. Notamment, celles-ci devraient permettre de diffuser rapidement les nouvelles variétés aux performances accrues nécessaires au maintien de la compétitivité de la filière-bois. Cette revue fait le point sur les possibilités et les limitations de l'utilisation des biotechnologies chez les arbres forestiers : multiplication végétative in vitro par embryogénèse somatique et par microbouturage, transformation génétique et sélection assistée par marqueurs.

National audience

International audience

International audience

International audience

International audience

International audience

International audience

International audience

National audience

International audience

National audience

National audience

National audience

National audience

National audience

International audience

International audience

International audience

International audience

302 ref. *INRA, URD, Centre d'Orleans Diffusion du document : INRA, URD, Centre d'Orleans Diplôme : Dr. d'Université

International audience; Despite the importance of forest trees all over the world, molecular studies on these species are rare. Many problems encountered in transformation and regeneration in combination with long generation time and a large genomic size, make trees unattractive for molecular research. ln this paper, we report on research developments in genetic engineering of forest trees with emphasis on transformation and gene expression. We discuss the possibility of adding new phenotypic traits to trees and show that some tree species (such as poplar) can be easily manipulated genetically.

40 ref.; International audience

International audience

44 ref.; International audience

International audience