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The enzymatic hydrolysis of cellulose into glucose, referred to as saccharification, is severely hampered by lignins. Here, we analyzed transgenic poplars (Populus tremula × Populus alba) expressing the Brachypodium (Brachypodium distachyon) p-coumaroyl-Coenzyme A monolignol transferase 1 (BdPMT1) gene driven by the Arabidopsis (Arabidopsis thaliana) Cinnamate 4-Hydroxylase (AtC4H) promoter in the wild-type (WT) line and in a line overexpressing the Arabidopsis Ferulate 5-Hydroxylase (AtF5H). BdPMT1 encodes a transferase which catalyzes the acylation of monolignols by p-coumaric acid (pCA). Several BdPMT1-OE/WT and BdPMT1-OE/AtF5H-OE lines were grown in the greenhouse, and BdPMT1 expression in xylem was confirmed by RT-PCR. Analyses of poplar stem cell walls (CWs) and of the corresponding purified dioxan lignins (DLs) revealed that BdPMT1-OE lignins were as p-coumaroylated as lignins from C3 grass straws. For some transformants, pCA levels reached 11 mg·g−1 CW and 66 mg·g−1 DL, exceeding levels in Brachypodium or wheat (Triticum aestivum) samples. This unprecedentedly high lignin p-coumaroylation affected neither poplar growth nor stem lignin content. Interestingly, p-coumaroylation of poplar lignins was not favored in BdPMT1-OE/AtF5H-OE transgenic lines despite their high frequency of syringyl units. However, lignins of all BdPMT1-OE lines were structurally modified, with an increase of terminal unit with free phenolic groups. Relative to controls, this increase argues for a reduced polymerization degree of BdPMT1-OE lignins and makes them more soluble in cold NaOH solution. The p-coumaroylation of poplar samples improved the saccharification yield of alkali-pretreated CW, demonstrating that the genetically driven p-coumaroylation of lignins is a promising strategy to make wood lignins more susceptible to alkaline treatments used during the industrial processing of lignocellulosics., The expression of a grass p-coumaroyl-CoA:monolignol transferase induces high p-coumaroylation of poplar lignins and better saccharification of alkali-pretreated poplar wood without growth penalty.

Trees can generate large mechanical stresses at the stem periphery to control the orientation of their axes. This key factor in the biomechanical design of trees, named “maturation stress”, occurs in wood fibres during cellular maturation when their secondary cell wall thickens. In this study, the spatial and temporal stiffening kinetics of the different cell wall layers were recorded during fibre maturation on a sample of poplar tension wood using atomic force microscopy. The thickening of the different layers was also recorded. The stiffening of the CML, S 1 and S 2 -layers was initially synchronous with the thickening of the S 2 layer and continued a little after the S 2 -layer reached its final thickness as the G-layer begins to develop. In contrast, the global stiffness of the G-layer, which initially increased with its thickening, was almost stable long before it reached its final maximum thickness. A limited radial gradient of stiffness was observed in the G-layer, but it decreased sharply on the lumen side, where the new sub-layers are deposited during cell wall thickening. Although very similar at the ultrastructural and biochemical levels, the stiffening kinetics of the poplar G-layer appears to be very different from that described in maturing bast fibres. Highlight New insights into the changes in mechanical properties within the cell wall of poplar tension wood fibres during maturation have been obtained using atomic force microscopy.

Transgenic poplars (Populus tremula x Populus alba, clone INRA 717-1B4) were produced by introducing the Brachypodium distachyon Bradi2g36910 (BdPMT1) gene driven by the Arabidopsis (Arabidopsis thaliana) Cinnamate 4-Hydroxylase (AtC4H) promoter in the wild-type (WT) line and in a line overexpressing the Arabidopsis Ferulate 5-Hydroxylase (AtF5H). BdPMT1 encodes a transferase which catalyzes the acylation of monolignols by p-coumaric acid (CA). Several BdPMT1- OE/WT and BdPMT1-OE/AtF5H-OE transgenic lines were grown in the greenhouse and BdPMT1 expression in xylem was confirmed by RT-PCR. The analysis of the cell walls (CW) of poplar stems and of corresponding purified dioxan lignins (DL) revealed that the BdPMT1-OE lignins were as p-coumaroylated as the lignins of C3 grass straws. For some transformants, CA levels even reached about 11 mg/g CW and 66 mg/g DL, which by far exceeds those of Brachypodium or wheat samples. This unprecedentedly high p-coumaroylation of poplar lignins affected neither the poplar growth, nor the stem lignin content. By contrast, the transgenic lignins were structurally modified, with an increase of terminal units with free phenolic groups. Relative to controls, this increase argues for a reduced polymerization degree of BdPMT1-OE lignins and makes them more soluble in cold NaOH solution. The p-coumaroylation of poplar samples, up to the levels of C3 grasses, improved the saccharification yield of alkali-pretreated poplar CW. These results establish that the genetically-driven p-coumaroylation of lignins is a promising strategy to make wood lignins more susceptible to the alkaline treatments that can be used during the industrial processing of lignocellulosics.One-sentence summaryThe expression of a grass p-coumaroyl-CoA:monolignol transferase induces a high p-coumaroylation of poplar lignins and a better saccharification of alkali-pretreated poplar wood without growth penalty

International audience; Les arbres atteignent des hauteurs et des durées de vie considérables grâce aux propriétés remarquables de leur bois. En effet, le bois remplit trois fonctions principales : (1) la conduction de l’eau de la racine au houppier, (2) le soutien mécanique de la masse toujours en augmentation de l’arbre en croissance et (3) le stockage de réserves temporaires, capitales pour la pérennité de l’arbre. Chez les angiospermes, les vaisseaux, les fibres et les rayons parenchymateux sont, respectivement, affiliés à ces fonctions [1]. La composition et la structure des parois de ces cellules varient considérablement en fonction des stades de développement et des conditions environnementales. Par exemple, en réponse à des contraintes mécaniques, les angiospermes produisent un bois dit de tension qui se caractérise par la présence d’une couche surnuméraire dans les parois des fibres, appelée couche G, et composée exclusivement de cellulose et de polysaccharides non-cellulosiques [2-3]. Le bois est donc un assemblage complexe des parois secondaires des fibres et vaisseaux, interconnectés aux cellules vivantes de rayons. Afin de s’affranchir de cette grande complexité, les mécanismes moléculaires de la formation du bois doivent être préférentiellement étudiés au niveau cellulaire.Ici, nous présentons le développement d’une méthode de microphénotypage non destructrice basée sur la microspectroscopie ATR-FTIR. Nous avons appliqué cette technique sur du bois de peupliers inclinés pour induire la formation de bois de tension. Grâce aux analyses d’images hyperspectrales obtenues, nous avons montré que(i) les couches G sont principalement composées de cellulose et de polysaccharides non cellulosiques,(ii) les lignines des parois cellulaires des vaisseaux sont plus riches en unités G alors que celles des fibres sont plus riches en unités S.Ces résultats sont conformes aux études antérieures [3-6], avec une résolution spatiale cinq fois plus élevée. Nous avons enfin pu étudier avec précision la composition des parois de fibres de bois de tension hors couche G et de rayons, jusqu’alors peu étudiées.En résumé, la microspectroscopie ATR-FTIR offre de nouvelles possibilités pour l’étude de la composition de la paroi cellulaire à l’échelle de la cellule.

In insects, the gall-inducing life-style has evolved independently many times. Several evolutionary pathways leading to this lifestyle have been proposed. While there is compelling evidence supporting surface-feeders and stem-borers as ancestral states of insect gall-inducers, an evolutionary pathway from leaf-miners remains hypothetical. Here we explored this question by comparing the developmental processes of two micromoths, a gall-inducer Caloptilia cecidophora (Lep., Gracillariidae), and its non-gall-inducing relative C. ryukyuensis. Like other Caloptilia, the first and second instars of C. cecidophora are leaf-miners and the gall is initiated inside the leaf mine by the third instar, thus suggesting leaf-mining as an ancestral, plesiomorphic state in this case. This is the first example of an insect species switching from leaf-mining to gall-inducing during larval development. The first two leaf-mining instars of C. cecidophora exhibit an absence of growth and a reduced time duration compared to C. ryukyuensis. The shortening of the duration of leaf-mining stages is apparently compensated in C. cecidophora by a larger egg size than C. ryukyuensis, and an additional larval instar during the gall phase.

National audience; Trees are able to grow high and to live old thanks to the remarkable properties of their wood. As a matter of a fact, wood delivers three major functions: (1) water conduction from roots to crown, (2) support of the ever-increasing mass of the growing tree and (3) storage of temporary reserves, important for tree growth over the years. In angiosperm trees, different wood cell types are affected to each of these functions. Fibers are involved in tree mechanical support, vessels in water conduction and parenchyma rays in starch and/or lipid storage during the resting period. In addition, these cell types have distinct developmental programs. While fibers and vessels are early-dying cells, parenchyma rays stay alive longer. Therefore wood is a complex patchwork of cells and its structure results from the three-dimensional assembly of the cell walls of dead fibers and vessels, interconnected with still living parenchyma rays. This great complexity stands as an obstacle when studying wood formation and the construction of wood properties. However, this can be circumvented thanks to the development of cell-specific approaches. We developed a non-destructive method based on ATR-FTIR imaging on poplar wood sections. This technology enables to collect IR-absorbance spectra from small areas of cross-sections, which makes possible to differentiate between wood cell-types or even between the different cell wall layers from a single fiber. We first demonstrated that spectra taken from fiber cell walls on cross-sections differed from spectra obtained from wood powder. We also showed that ATR-FTIR imaging is able to discriminate the cell walls of fibers, vessels and rays. These findings are in accordance with other studies [1], but with an improved spatial resolution. ATR-FTIR microspectroscopy is thus a promising tool to finely characterize the cell wall of different wood cell types. This work has been partly supported by the OPeNSPeNU project (funded by the Centre Val de Loire Region)

International audience; Successful plant manipulation by herbivores requires a reconfiguration of the primary and secondary metabolisms of the host-plant. Water deficit is generally predicted to negatively affect the development of gall-inducing insects, by impairing their ability to remodel the primary metabolism of their host. We assessed whether host genotype could modulate water deficit effects on a gall-inducing aphid, Phloeomyzus passerinii, developing on poplars, which are known to be among the most susceptible trees to water deficit. Stem-cuttings of two poplar genotypes, with a different resistance level to P. passerinii, were grown under three irrigation treatments, and subsequently infested with the same aphid clone. Plant growth parameters and aphid colonies development were recorded, and the development and organization of the pseudogall induced by-the aphid was observed using histological investigations. The three irrigation treatments resulted in three levels of water deficit. Mean predawn leaf water potential reached -0.10, -0.49 and -0.75 MPa in the optimal, intermediate and low irrigation treatments respectively. In both genotypes, growth, architecture and photosynthesis were similarly affected. The severe water deficit reduced the development of aphids on both genotypes, which is in agreement with the general prediction for gall-inducing insects. Nonetheless, while the intermediate water deficit did not affect the development of aphids on the susceptible genotype, it enhanced their development on the resistant one. This latter observation contradicts the prediction for gall-inducing insects, but is in agreement with the plant stress hypothesis for sucking insects. The size of the pseudogall induced by the aphids in the bark of their host progressively diminished when water deficit increased. Lignification, believed to be an important component of poplar resistance to P. passerinii, also progressively decreased with water deficit. The involvement of physiological and architecture modifications due to water deficit on the development of aphids is discussed. The results demonstrate that host genotype can modulate water deficit effects on the development of a galling aphid, thereby compromising predictions on the outcome of droughts on stand resistance to plant-manipulating herbivores.

REALLY INTERESTING NEW GENE (RING) proteins play important roles in the regulation of many processes by recognizing target proteins for ubiquitination. Previously, we have shown that the expression of PtaRHE1, encoding a Populus tremula × Populus alba RING-H2 protein with E3 ubiquitin ligase activity, is associated with tissues undergoing secondary growth. To further elucidate the role of PtaRHE1 in vascular tissues, we have undertaken a reverse genetic analysis in poplar. Within stem secondary vascular tissues, PtaRHE1 and its corresponding protein are expressed predominantly in the phloem. The downregulation of PtaRHE1 in poplar by artificial miRNA triggers alterations in phloem fibre patterning, characterized by an increased portion of secondary phloem fibres that have a reduced cell wall thickness and a change in lignin composition, with lower levels of syringyl units as compared with wild-type plants. Following an RNA-seq analysis, a biological network involving hormone stress signalling, as well as developmental processes, could be delineated. Several candidate genes possibly associated with the altered phloem fibre phenotype observed in amiRPtaRHE1 poplar were identified. Altogether, our data suggest a regulatory role for PtaRHE1 in secondary phloem fibre development.

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"

Plant-manipulating herbivores can modify their host nutrients and defensive compounds to their own advantage. This generally results in enhanced performance of herbivores on modified tissues, but the anatomical and biochemical modifications undergone by host tissues could also modify the herbivores' behaviour. Phloeomyzus passerinii induces a reaction tissue in the bark of its susceptible poplar hosts, suggesting a host-plant manipulation by the aphid. We investigated whether the formation of this reaction tissue, confirmed by histological sections, affected the performance and feeding behaviour of the aphid. In addition we studied how host resistance affected these parameters by comparing two host genotypes of different susceptibility. Aphids developed faster on the preinfested susceptible host genotype, whereas no effect was detected on the resistant one, confirming an improvement in the host's nutritional value due to the preinfestation, in agreement with the nutrition hypothesis. The DC-EPG technique indicated that in both host genotypes, preinfestation shortened the delay until the first probe and increased both the rate and duration of sustained ingestion, and consequently host acceptance. This suggests that host manipulation can adjust not only the nutritional value of plant tissues at an optimal level for development but also host acceptance by the herbivores. In the resistant host genotype though, the number of probes and the duration of parenchyma exploration increased in the galled tissue, which was congruent with extensive cell wall modifications indicated by more extended induced lignification. The adaptive consequences of this modified feeding behaviour are discussed.

RG-I and AGP, but not XG, are associated to the building of the peculiar mechanical properties of tension wood. Hardwood trees produce tension wood (TW) with specific mechanical properties to cope with environmental cues. Poplar TW fibers have an additional cell wall layer, the G-layer responsible for TW mechanical properties. We investigated, in two poplar hybrid species, the molecules potentially involved in the building of TW mechanical properties. First, we evaluated the distribution of the different classes of non-cellulosic polysaccharides during xylem fiber differentiation, using immunolocalization. In parallel, G-layers were isolated and their polysaccharide composition determined. These complementary approaches provided information on the occurrence of non-cellulosic polysaccharides during G-fiber differentiation. We found no evidence of the presence of xyloglucan (XG) in poplar G-layers, whereas arabinogalactan proteins (AGP) and rhamnogalacturonan type I pectins (RG-I) were abundant, with an apparent progressive loss of RG-I side chains during G-layer maturation. Similarly, the intensity of immunolabeling signals specific for glucomannans and glucuronoxylans varies during G-layer maturation. RG-I and AGP are best candidate matrix components to be responsible for TW mechanical properties.

article en open access; International audience; In the search for renewable energy sources, genetic engineering is a promising strategy to improve plant cell wall composition for biofuel and bioproducts generation. Lignin is a major factor determining saccharification efficiency and, therefore, is a prime target to engineer. Here, lignin content and composition were modified in poplar (Populus tremula 3 Populus alba) by specifically down-regulating CINNAMYL ALCOHOL DEHYDROGENASE1 (CAD1) by a hairpin-RNA-mediated silencing approach, which resulted in only 5% residual CAD1 transcript abundance. These transgenic lines showed no biomass penalty despite a 10% reduction in Klason lignin content and severe shifts in lignin composition. Nuclear magnetic resonance spectroscopy and thioacidolysis revealed a strong increase (up to 20-fold) in sinapaldehyde incorporation into lignin, whereas coniferaldehyde was not increased markedly. Accordingly, ultra-high-performance liquid chromatography-mass spectrometry-based phenolic profiling revealed a more than 24,000-fold accumulation of a newly identified compound made from 8-8 coupling of two sinapaldehyde radicals. However, no additional cinnamaldehyde coupling products could be detected in the CAD1-deficient poplars. Instead, the transgenic lines accumulated a range of hydroxycinnamate-derived metabolites, of which the most prominent accumulation (over 8,500-fold) was observed for a compound that was identified by purification and nuclear magnetic resonance as syringyl lactic acid hexoside. Our data suggest that, upon down-regulation of CAD1, coniferaldehyde is converted into ferulic acid and derivatives, whereas sinapaldehyde is either oxidatively coupled into S'(8-8) S' and lignin or converted to sinapic acid and derivatives. The most prominent sink of the increased flux to hydroxycinnamates is syringyl lactic acid hexoside. Furthermore, low-extent saccharification assays, under different pretreatment conditions, showed strongly increased glucose (up to +81%) and xylose (up to +153%) release, suggesting that down-regulating CAD1 is a promising strategy for improving lignocellulosic biomass for the sugar platform industry.

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.

The lignification process in poplar tension wood lignified cell wall layers, specifically the S(1) and S(2) layers and the compound middle lamella (CML), was analysed using ultraviolet (UV) and transmission electron microscopy (TEM). Variations in the thickness of the gelatinous layer (G-layer) were also measured to clarify whether the lignified cell wall layers had completed their lignification before the deposition of G-layers, or, on the contrary, if lignification of these layers was still active during G-layer formation. Observations using UV microscopy and TEM indicated that both UV absorbance and the degree of potassium permanganate staining increased in the CML and S(1) and S(2) layers during G-layer formation, suggesting that the lignification of these lignified layers is still in progress during G-layer formation. In the context of the cell-autonomous monolignol synthesis hypothesis, our observations suggest that monolignols must go through the developing G-layer during the lignification of CML and the S(1) and S(2) layers. The alternative hypothesis of external synthesis (in the rays) does not require that monolignols go through the G-layer before being deposited in the CML, or the S(1) and S(2) layers. Interestingly, the previous observation of lignin in the poplar G-layer was not confirmed with the microscopy techniques used in the present study.

Wood formation is a complex biological process, involving five major developmental steps, including (1) cell division from a secondary meristem called the vascular cambium, (2) cell expansion (cell elongation and radial enlargement), (3) secondary cell wall deposition, (4) programmed cell death, and (5) heartwood formation. Thanks to the development of genomic studies in woody species, as well as genetic engineering, recent progress has been made in the understanding of the molecular mechanisms underlying wood formation. In this review, we will focus on two different aspects, the lignification process and the control ofmicrofibril angle in the cell wall of wood fibres, as they are both key features of wood material properties., La formation du bois est un processus de développement complexe, impliquant cinq étapes majeures : (1) la division des cellules à partir d’un méristème secondaire, appelé le cambium; (2) l’élongation des cellules ; (3) le dépôt d’une paroi secondaire épaisse ; (4) la mort programmée des cellules ; et (5) leur imprégnation par des composés phénoliques conduisant à la formation du bois de coeur. Grâce au développement des techniques de génomique et aux travaux menés sur la caractérisation d’arbres transgéniques, certains mécanismes moléculaires impliqués dans la formation du bois commencent à être mieux connus. Dans cette revue, deux aspects importants pour la qualité du bois seront approfondis : la lignification et le contrôle de l’angle des microfibrilles de cellulose dans la paroi des fibres de bois.

International audience; Six Populus deltoides Bartr. ex Marsh. × P. nigra L. genotypes were selected to investigate whether stem xylem anatomy correlated with gas exchange rates, water-use efficiency (WUE) and growth performance. Clonal copies of the genotypes were grown in a two-plot common garden test under contrasting water regimes, with one plot maintained irrigated and the other one subjected to moderate summer water deficit. The six genotypes displayed a large range of xylem anatomy, mean vessel and fibre diameter varying from about 40 to 60 μm and from 7.5 to 10.5 μm, respectively. Decreased water availability resulted in a reduced cell size and an important rise in vessel density, but the extent of xylem plasticity was both genotype and trait dependent. Vessel diameter and theoretical xylem-specific hydraulic conductivity correlated positively with stomatal conductance, carbon isotope discrimination and growth performance-related traits and negatively with intrinsic WUE, especially under water deficit conditions. Vessel diameter and vessel density measured under water deficit conditions correlated with the relative losses in biomass production in response to water deprivation; this resulted from the fact that a more plastic xylem structure was generally accompanied by a larger loss in biomass production.

Article en open access; International audience; Xylem resistance to drought-induced cavitation is a key trait of plant water relations. This study assesses the genetic variation expressed for stem cavitation resistance within a population of a riparian species, the European black poplar (Populus nigra L.), and explores its relationships with xylem anatomy, water-use efficiency (WUE), and growth. Sixteen structural and physiological traits related to cavitation resistance, xylem anatomy, growth, bud phenology, and WUE were measured on 33 P. nigra genotypes grown under optimal irrigation in a 2-year-old clonal experiment in a nursery. Significant genetic variation was expressed for the xylem tension inducing 50% loss of hydraulic conductivity (Ψ50) within the studied population, as attested by the high value of broad-sense heritability estimated for this trait (H 2 ind = 0.72). Stem cavitation resistance was associated with xylem structure: the more cavitation-resistant genotypes exhibited lower hydraulic efficiency and higher mechanical reinforcement as assessed from stem xylem cross sections. By contrast, Ψ50 was not significantly related to shoot height increment, total above-ground dry mass, or bulk leaf carbon isotope discrimination, a proxy for intrinsic WUE. These findings indicate that the trade-offs between xylem resistance to cavitation, hydraulic efficiency, and mechanical reinforcement can occur at the within-population level. Given that the studied genotypes were exposed to the same environmental conditions and evolutionary drivers in situ, the trade-offs detected at this scale are expected to reflect true functional relationships.

International audience; Wood is a complex and highly variable tissue, the formation of which is developmentally and environmentally regulated. In reaction to gravitropic stimuli, angiosperm trees differentiate tension wood, a wood with specific anatomical, chemical and mechanical features. In poplar the most significant of these features is an additional layer that forms in the secondary wall of tension wood fibres. This layer is mainly constituted of cellulose microfibrils oriented nearly parallel to the fibre axis. Tension wood formation can be induced easily and strongly by bending the stem of a tree. Located at the upper side of the bent stem, tension wood can be compared with the wood located on its lower side. Therefore tension wood represents an excellent model for studying the formation of xylem cell walls. This review summarizes results recently obtained in the field of genomics on tension wood. In addition, we present an example of how the application of functional genomics to tension wood can help decipher the molecular mechanisms responsible for cell wall characteristics such as the orientation of cellulose microfibrils.

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

Phloeomyzus passerinii can induce a pseudogall within the bark of susceptible poplar genotypes, while in resistant genotypes the induction seems to be impeded by lignification processes. Phloeomyzus passerinii is a major pest of poplar stands in Europe, North Africa and the Near East. This aphid feeds in the cortical parenchyma of different poplar species and hybrids, and can affect their growth and survival through unknown mechanisms. In some genotypes, however, resistance prevents either the settlement or the development of aphid colonies. For a better understanding of tree reactions to aphid probing, we compared the anatomical and biochemical modifications undergone within the bark of stem cuttings, after different delays of either aphid colonization or mechanical wounding. To assess how resistance may modulate tree reactions, the comparison was performed using three poplar genotypes exhibiting different resistance levels. In these three genotypes, mechanical wounding induced a similar, localized, wound periderm. In contrast, aphid colonization triggered more extended reactions, which differed among genotypes. In the susceptible genotype, aphids induced a reaction tissue, characterized after a month by thin-walled hypertrophied cells and a depletion of soluble phenolic compounds and starch. Anatomical features of this reaction tissue suggest that the aphid initiates a pseudogall in the cortical tissues of its susceptible host. In the resistant genotypes, however, the differentiation of the reaction tissue was totally or partially inhibited, probably because of extended lignification processes. The implications of a pseudogall induction on susceptible hosts’ physiology, and the impact of lignification on aphid development and behavior, are discussed.

Autorisation de diffusion par l'éditeur; Le puceron lanigère du peuplier. Phloeomyzus passerinii est un ravageur important des peupleraies du sud de l'Europe, du Proche-Orient et d'Afrique du Nord. Après une insertion de ses stylets, à la fois intercellulaire et intracellulaire, il se nourrit exclusivement dans le parenchyme cortical du tronc des peupliers. Durant les pullulations, ce puceron Cause des nécroses corticales étendues et un dessèchement des rameaux, pouvant conduire à la mort de l'arbre. Nous avons comparé la réaction corticale après blessure mécanique et après piqure du puceron entre deux génotypes hybrides Populus x canadensis avec des niveaux de résistance contrastés ('I214' qui est sensible et 'I45/51' qui affecte les paramètres démographiques du puceron). Chaque semaine durant un mois, nous avons réalisé des coupes histologiques autour des zones d'insertion des stylets et des blessures mécaniques pour comparer les modifications anatomiques et biochimiques des tissus de l'écorce. Les blessures mécanique ont induit des péridermes de blessures très localisés alors que l'alimentation du puceron a entraîné des réactions élendues, présentant des similarités avec un bourrelet cicatriciel, mais avec également des cellules hypertrophiées à parois minces, une localisation particulière des dépôts pariétaux de lignine et des accumulations périphériques de tanins et flavanols. Les dépôts de lignine étaient plus étendus et importants chez le génotype résistant. Notre hypothèse est que P. passerinzrii, aveC son mode destructif de pénétration des tissus de son hôte, déclenche les mécanismes de défense du peuplier, puis les manipule pour induire la formation d'une pseudogalle.

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.

International audience; Forty percent of aphids live wholly or partly on trees, most species being associated with leaves or petioles. Species able to exploit woody parts have either specific adaptations, such as extra long stylets that allow them to reach the phloem, or the ability to induce galls. The woolly poplar aphid, Phloeomyzus passerinii (Signoret) (Hemiptera: Aphididae), colonizes the trunks and base of the lower branches of mature poplars and causes cortical necrosis leading to the death of trees where infestation is heavy. Very little is known about the mode of feeding of P. passerinii. This study looked at the feeding behavior of P. passerinii on stem-cuttings of Populus x canadensis Moench using: (i) histological analyses of the feeding site and stylet pathway and (ii) electrical penetration graphs (EPG, DC) based on parthenogenetic apterous females on woody tissues. The histological and EPG results showed that stylets of P. passerinii penetrated into the plant tissues following a straight unbranched extracellular and intracellular pathway to reach the cortical parenchyma. Compared to EPGs for phloem sap feeding aphids, there were differences in the waveforms A and C whereas a new waveform Icp was described. Based on histological analyses and previous descriptions of EPG waveforms, correlations with the stylet tip position and aphid activities within bark tissues are discussed. A pathway and a sustained intracellular phase were distinguished, both occurring in the cortical parenchyma cells. The bark aphid feeding mode is discussed in relation to the damage caused and in terms of changes in the aphid's diet.

We present results and insights gained from the characterization of the hydraulic architecture of eight field-grown Populus deltoides × Populus nigra genotypes, already known to differ in terms of growth performance, water-use and xylem anatomy (Marron et al. 2005, Monclus et al. 2005, 2006; Fichot et al. 2009). Under well-watered conditions, we provide evidence that (1) there are wide genotypic variations for hydraulic efficiency inferred from the leaf specific hydraulic conductance (kSL) and for resistance to drought-induced cavitation inferred from the water potential inducing 50% loss in hydraulic conductance, (2) a strong trade-off between water transport efficiency and xylem safety occurs at the whole plant level, indicating that genotypes with lower kSL apparently compensate the greater risks of embolism by building a safer xylem, (3) higher relative growth rate is tightly associated with lower whole-plant hydraulic efficiency and higher resistance to cavitation, (4) estimates of leaf water-use efficiency (intrinsic water use efficiency and carbon isotope discrimination against 13C) are weakly associated with whole-plant hydraulics. Under limited water supply, the characterization of xylem resistance to cavitation provides additional evidence that (5) xylem safety acclimates to moderate drought in a genotype-dependent manner, (6) there is no clear relationship between xylem safety and either xylem water transport efficiency or xylem biomechanics, contrary to what is observed at inter-specific scales. Our results provide interesting insights for the possible role of whole plant hydraulic architecture and xylem hydraulics in mediating key aspects of whole-plant physiology in poplar, such as growth and water-use efficiency. Finally, our results also indicate that there may be great opportunity for intra-specific comparisons to unravel specific issues that cannot be fully addressed by interspecific comparisons.

We examined the relationships between xylem resistance to cavitation and 16 structural and functional traits across eight unrelated Populus deltoides x Populus nigra genotypes grown under two contrasting water regimes. The xylem water potential inducing 50% loss of hydraulic conductance (Psi(50)) varied from -1.60 to -2.40 MPa. Drought-acclimated trees displayed a safer xylem, although the extent of the response was largely genotype dependent, with Psi(50) being decreased by as far as 0.60 MPa. At the tissue level, there was no clear relationship between xylem safety and either xylem water transport efficiency or xylem biomechanics; the only structural trait to be strongly associated with Psi(50) was the double vessel wall thickness, genotypes exhibiting a thicker double wall being more resistant. At the leaf level, increased cavitation resistance was associated with decreased stomatal conductance, while no relationship could be identified with traits associated with carbon uptake or bulk leaf carbon isotope discrimination, a surrogate of intrinsic water-use efficiency. At the whole-plant level, increased safety was associated with higher shoot growth potential under well-irrigated regime only. We conclude that common trade-offs between xylem resistance to cavitation and other physiological traits that are observed across species may not necessarily hold true at narrower scales.

We examined the relationships between xylem resistance to cavitation and 16 structural and functional traits across eight unrelated Populus deltoides x Populus nigra genotypes grown under two contrasting water regimes. The xylem water potential inducing 50% loss of hydraulic conductance (Psi(50)) varied from -1.60 to -2.40 MPa. Drought-acclimated trees displayed a safer xylem, although the extent of the response was largely genotype dependant, with Psi(50) being decreased by as far as 0.60 MPa. At the tissue level, there was no clear relationship between xylem safety and either xylem water transport efficiency or xylem biomechanics; the only structural trait to be strongly associated with Psi(50) was the double vessel wall thickness, genotypes exhibiting a thicker double wall being more resistant. At the leaf level, increased cavitation resistance was associated with decreased stomatal conductance, while no relationship could be identified with traits associated with carbon uptake or bulk leaf carbon isotope discrimination, a surrogate of intrinsic water-use efficiency. At the whole-plant level, increased safety was associated with higher shoot growth potential under well-irrigated regime only. We conclude that common trade-offs between xylem resistance to cavitation and other physiological traits that are observed across species may not necessarily hold true at narrower scales.

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.

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International audience; The course of the infection and development of the biotrophic fungus Melampsora larici-populina on leaf tissue from the hybrid poplar Populus deltoides × P. nigra ‘Ogy’ was monitored at the histological level. Leaf disks were inoculated with one of two rust physiological races (E1 and E2), resulting in interactions that were either incompatible (race E1) or compatible (race E2). In the compatible interaction, the fungus rapidly colonized the leaf without inducing any apparent host response. Symptoms appeared on the leaf several days after inoculation just prior to spore dissemination. The incompatible interaction was characterized by the early collapse and disorganization of cytoplasm of infected cells 17 h after inoculation and within 2 h after the appearance of the first haustoria. Resistance to M. larici-populina was mediated through a hypersensitive response, since it was extremely localized and involved only the few cells that were in the immediate vicinity of each infected cell.

International audience; In stems of woody angiosperms responding to mechanical stress, imposed for instance by tilting the stem or formation of a branch, tension wood (TW) forms above the affected part, while anatomically distinct opposite wood (OW) forms below it. In poplar TW the S3 layer of the secondary walls is substituted by a "gelatinous layer" that is almost entirely composed of cellulose and has much lower hemicellulose contents than unstressed wood. However, changes in xylan contents (the predominant hemicelluloses), their interactions with other wall components and the mechanisms involved in TW formation have been little studied. Therefore, in the study reported here we determined the structure and distribution of xylans, cloned the genes encoding the xylan remodeling enzymes beta-xylosidases (PtaBXLi), and examined their expression patterns during tension wood, normal wood and opposite wood xylogenesis in poplar. We confirm that poplar wood xylans are substituted solely by 4-O-methylglucuronic acid in both TW and OW. However, although glucuronoxylans are strongly represented in both primary and secondary layers of OW, no 4-O-methylGlcA xylan was found in G-layers of TW. Four full-length BXL cDNAs encoding putative beta-xylosidases were cloned. One, PtaBXL1, for which xylosidase activity was confirmed by heterologous expression in Escherichia coli, exhibited a wood-specific expression pattern in TW. In conclusion, xylan as PtaBXL1, encoding beta4-xylosidase activity, are down-regulated in TW.

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.

Des plants de Populus tremula Michx. × Populus alba L. clone 717-1B4 ont été cultivés en système de culture hydroponique pour obtenir une activité cambiale dans un milieu nutritif variable. La croissance secondaire de la tige a été caractérisée par une mesure automatique de la croissance radiale et des observations histologiques. Les résultats montrent le premier enregistrement de l'activité cambiale en hydroponie chez le peuplier. En outre, nous démontrons que les carences nutritives peuvent être étudiées en privant progressivement les plants de solution nutritive. Le système se prête à la mesure de la conductance stomatique, de la croissance primaire de la tige, de la croissance foliaire et de la croissance radiale. Dans cette étude nous observons que les croissances primaire et secondaire sont touchées par les carences nutritives. Ce système de culture en hydroponie est exploitable pour l'élucidation de l'impact des facteurs environnementaux, physiologiques et moléculaires sur l'activité cambiale et la formation du bois., Populus tremula Michx. × Populus alba L. clone 717-1B4 plants were grown in a hydroponic system in an effort to detect cambial activity in a changing nutrient environment. The secondary growth of the stem was determined by automated measurement of radial growth, as well as by histological study. This is the first time poplar cambial activity has been recorded in a hydroponic system. Further, we demonstrate that nutrient limitations can be tested with progressive deprivation of liquid medium. The system lends itself to measurements of stomatal conductance, primary stem growth, leaf growth, and radial stem growth. In this study we found that primary and secondary growth were affected by nutrient solution limitations. This hydroponic system will be valuable in elucidating the impact of environmental, physiological, and molecular factors on cambial activity and wood formation.

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; In this work, a cDNA encoding a defensin from Picea glauca, named PgD1, was isolated. The corresponding PgD1 cDNA encodes a putative protein of 83 amino acids with a calculated molecular mass of 8.9 kDa. The mature protein of 50 amino acids is characterized by the presence of specific highly conserved residues common to all plant defensins. PgD1 is developmentally expressed during seed germination and is also up-regulated by wounding and by jasmonic acid treatment, suggesting a role in both the constitutive and induced defense mechanisms. The recombinant protein was found to cause extensive growth inhibition of three fungal pathogens (Cylindrocladium floridanum, Fusarium oxysporum and Nectria galligena) at 2.5 μM.

International audience; • Fifteen poplar cDNA encoding fasciclin-like arabinogalactan proteins (PopFLAs) were finely characterized, whereas the presence of arabinogalactan proteins (AGPs) was globally assessed during wood formation. • PopFLAs transcript accumulation was analysed through EST distribution in cDNA libraries, semi-quantitative RT-PCR, microarray experiment and Northern blot analysis. Similarly, AGPs contents were globally quantified by rocket electrophoresis. AGPs accumulation was further examined by Western blotting and immunocytolocalization. • Ten PopFLAs were specifically expressed in tension wood (TW) and not expressed in the cambial zone. Rocket electrophoresis revealed important AGPs accumulation in TW xylem. An anti-AGPs specific antibody recognized two proteins preferentially present in the cell wall-bound fraction from TW. Immunocytochemistry revealed a strong labelling close to the inner part of the G-layer of TW fibres. • PopFLAs are expressed in xylem and many are up-regulated in TW. It is suggested that some PopFLAs accumulating at the inner side of the G-layer may have a specific function in the building of this layer. PopFLAs expression may therefore be linked to the specific mechanical properties of TW.

International audience; Hardwood trees are able to reorient their axes owing to tension wood differentiation. Tension wood is characterised by important ultrastructural modifications, such as the occurrence in a number of species, of an extra secondary wall layer, named gelatinous layer or G-layer, mainly constituted of cellulose microfibrils oriented nearly parallel to the fibre axis. This G-layer appears directly involved in the definition of tension wood mechanical properties. This review gathers the data available in the literature about lignification during tension wood formation. Potential roles for lignin in tension wood formation are inferred from biochemical, anatomical and mechanical studies, from the hypotheses proposed to describe tension wood function and from data coming from new research areas such as functional genomics.

We describe in this study punchless , a nonpathogenic mutant from the rice blast fungus M. grisea, obtained by plasmid-mediated insertional mutagenesis. As do most fungal plant pathogens, M. grisea differentiates an infection structure specialized for host penetration called the appressorium. We show that punchless differentiates appressoria that fail to breach either the leaf epidermis or artificial membranes such as cellophane. Cytological analysis of punchless appressoria shows that they have a cellular structure, turgor, and glycogen content similar to those of wild type before penetration, but that they are unable to differentiate penetration pegs. The inactivated gene, PLS1 , encodes a putative integral membrane protein of 225 aa (Pls1p). A functional Pls1p-green fluorescent protein fusion protein was detected only in appressoria and was localized in plasma membranes and vacuoles. Pls1p is structurally related to the tetraspanin family. In animals, these proteins are components of membrane signaling complexes controlling cell differentiation, motility, and adhesion. We conclude that PLS1 controls an appressorial function essential for the penetration of the fungus into host leaves.

25 ref.;Etat et perspectives de la populiculture; National audience

Background Due to its peculiar properties, tension wood formation constitutes a remarkable adaptation mechanism, that makes possible for the tree to reorientate its axes (stem and branches) in response to environnemental cues. In poplar, tension wood fibres harbour an extra cell wall layer, the G-layer, responsible for the peculiar mechanical properties of tension wood. This G-layer is very thick, most likely devoid of lignins and strongly enriched in highly cristalline cellulose. In addition, cellulose microfibril orientation is almost parallel to the fibre axis. We aim to identify molecular actors responsible for the tensioning of cellulose microfibrils and we choose as candidate, molecules containing complex carbohydrates, such as pectin and the glycosylated part of arabinogalactan proteins. Indeed, a wide array of different carbohydrates has been recently evidenced in the G-layer, suggesting the occurrence of complex polysaccharides other than cellulose within this layer (1, 2).

International audience; ln response to a mechanical stress, trees are able to reorientate their axes thanks to the differentiation of a special wood named tension wood. ln poplar, tension wood fibres are mainly characterized by an additional secondary layer, named G-layer mostly constituted of highly crystalline cellulose microfibrils orientated almost parallel to the fibre axis. These specific features account for the particular mechanical properties of tension wood. ln order to understand the mechanisms underlying G-layer formation, different cDNA libraries have been prepared from cambium, developing and mature xylem sam pied in wood area with or without tension wood. 10,062 EST were sequenced from these libraries and annotated. Genes specifically or preferentially expressed during tension wood formation were identified from EST distribution in the different libraries. ln addition, a poplar microarray was used to determine global gene expression pattern during tension wood formation. Among other genes, many fasciclin-like arabinogalactan proteins (PopFLAs) appeared highly expressed in the differentiating xylem, once secondary cell wall synthesis occurred. Ten of these PopFLAs were specifically expressed in tension wood. Further immunocytochemical studies indicated that some PopFLAs accumulated at the inner side of the G layer suggesting a specific function for these proteins in the building of the G layer.

Le bois de tension (BT) est produit par les arbres feuillus en réponse à des contraintes mécaniques. Il présente des caractéristiques anatomiques, physico-chimiques et mécaniques particulières qui confèrent aux arbres un potentiel d’adaptation vis-à-vis de la contrainte subie. Traditionnellement dans la production de bois d’oeuvre, sa présence en trop grande proportion dans les grumes est une caractéristique indésirable car elle peut induire des déformations ou des fentes importantes des sciages. Néanmoins, comme le bois de tension se caractérise par une très faible teneur en lignine et une forte teneur en cellulose, il présente un intérêt pour une valorisation du bois en bioéthanol de 2ème génération. Quelle que soit la valorisation envisagée, il apparait crucial de pouvoir quantifier la teneur en bois de tension dans le bois. Cependant, les techniques actuelles, principalement basées sur de la coloration différentielle, de l’analyse histologique ou une inspection visuelle (Badia et al., 2005), sont couteuses et laborieuses, et de fait elles ne permettent pas d’évaluer cette propriété dans un nombre important d’échantillons. La spectrométrie infrarouge est une technique à haut-débit prometteuse pour évaluer les propriétés physico-chimiques du bois. Sa mise en oeuvre nécessite la construction préalable d’un modèle de calibration pour la propriété ciblée. Comme le bois de tension présente des propriétés physico-chimiques particulières, l’évaluation de sa teneur dans un échantillon de bois doit être possible par spectrométrie infrarouge. L’objectif de cette étude est de tester cette hypothèse sur des échantillons de peuplier et à partir d’une gamme de pourcentage de BT artificiellement créée.

National audience; La formation du bois est un processus complexe, orchestré dans le temps et dans l’espace, faisant intervenir de nombreux métabolismes et par conséquent, sous le contrôle d’un grand nombre de gènes. Les outils de la génomique, particulièrement adaptés pour ce type d'étude, ont été développés au cours de ces dernières années et complètent la boîte à outils déjà disponible chez le peuplier, espèce modèle largement utilisée dans la communauté scientifique travaillant sur la biologie des arbres.

National audience

International audience