Identification et caractérisation des acteurs moléculaires potentiellement responsables pour des propriétés mécaniques du bois de tension

The aim of this thesis was to approach the underlying molecular mechanisms responsible for the particular properties of the G-layer and the outstanding mechanical properties of tension wood (TW). Accordingly, three potential molecular players (fasciclin-like arabinogalactan protein (FLA), chitinase-like protein (CTL) and β-galactosidase (BGAL)) were chosen and studied through a phylogenetic analysis, expression analyses and most importantly characterization of RNAi transgenic poplars. This multilevel characterization revealed that CTL2 and FLAs have function in the regulation of cellulose crystallinity in TW. CTL2 was also shown to be important both for the cell wall organization and stem mechanical properties. BGAL was studied in a light of the previously reported modifications of RG-I pectin, potentially important for the mechanical properties of TW. Study of BGAL revealed that the enzyme has higher activity in TW than in opposite wood. BGAL7, whose gene was expressed specifically in TW, does not seem to be responsible for the higher BGAL activity in TW. In comparison to poplar, we analyzed the occurrence of molecular players potentially responsible for TW mechanical properties in simarouba, a tropical species, which develops different TW fiber. Arabinogalactan proteins and RG-I pectin potentially targeted by BGAL were localized in TW fibers both in poplar and simarouba and therefore may be involved in a common mechanism of tensile stress generation in different TW types. A model was finally proposed to elucidate a potential function of the studied molecular players in the regulation of G-layer properties and tensile stress generation.Le but de cette thèse était d’identifier les mécanismes moléculaires responsables des propriétés particulières de la couche G et les propriétés mécaniques remarquables du bois de tension (BT). Trois acteurs moléculaires potentiels (des protéines à arabinogalactane avec domaine fasciclin-like (FLA), une protéine chitinase-like (CTL) et une β-galactosidase (BGAL)) ont été choisis et étudiés dans: analyse phylogénétique, analyses d'expression et caractérisation de peupliers transgéniques affectés dans l’expression de chacun de ces acteurs. La caractérisation fine de ce matériel a révélé que CTL2 et les FLA jouent un rôle dans la régulation de la cristallinité de la cellulose dans le BT. CTL2 apparaît également important dans l'organisation de la paroi cellulaire et des propriétés mécaniques des tiges. BGAL a été avant proposé pour une fonction dans modification de pectine RG-I potentiellement important pour des propriétés mécaniques de BT. Le bois de tension exhibe une activité BGAL plus élevée que dans le bois opposé. L’inhibition par RNAi de l’expression de BGAL7, spécifiquement exprimée dans le BT, n’est pas responsable à lui seul de la forte activité BGAL présente dans le BT. En contrepoint à l’étude menée sur le peuplier, nous avons également évalué la présence d’acteurs moléculaires potentiellement responsables des propriétés mécaniques du BT chez le simarouba qui développe dans leur BT des fibres ayant leurs sous-couches de la paroi intermédiaire entre la G et la S2. Des protéines à arabinogalactanes ainsi que des pectines du type RG-I sont présentes dans les fibres de BT de peuplier et de simarouba et pourraient avoir une fonction dans un mécanisme commun de génération des contraintes dans le BT. Finalement, un modèle est proposé sur le rôle présumé des différents acteurs moléculaires étudié dans la régulation des propriétés de la couche G et la génération des fortes contraintes du bois de tension

Characterization of transgenic poplars potentially interesting for bioethanol production

Cilj studije, koja je dio europskog EnergyPoplar projekta, je karakterizacija novoproduciranih multitransformanata topole proizvedenih kombinatorijskim pristupom transformacije u kojem su zanimljivi geni biosintetskog puta lignina (CAD, CCR, F5H) i giberlina (GA 20-Ox) kombinirani kako bi potencijalno utjecali na kvalitetu biomase i biljni rast. Uz promijenjeni sadržaj lignina, topola može biti poželjna lignocelulozna sirovina za proizvodnju bioetanola. Novih 14 transgeničnih linija karakterizirano je na molekularnoj i fenotipskoj razini, i uspoređeno sa divljim tipom. Kombinatorijski pristup je rezultirao dobivanjem većine transformanata sa jednim insertiranim tipom gena (79%) i samo 3 transfromanta sa dva i tri insertirana gena (CAD-CCR, CCR-F5H, CAD-CCR-F5H). Potpuna represija ekspresije gena CAD i CCR nije postignuta. Nijedna kombinacija sa GA 20-ox genom nije dobivena. Transgenične linije topole, uzgajane u skoro idealnim uvjetima u stakleniku, nisu pokazale vidljivo zaostajanje u rastu, s iznimkom transformanta sa tri insertirana gena (CAD-CCR-F5H), čiji je rast bio reduciran za oko 40%. Analiza anatomije drva je ukazala na odgođenu lignifikaciju u drvu stabljike većine transfromanta, kao i snažne promjene u intenzitetu autoflourescencije kod dvostrukog transformanta (CCR-F5H). Ove promjene nisu bile povezane niti su utjecale na snažnu modifikaciju kemijskog sadržaja i strukture drva, jer je analiza Near Infrared spektroskopijom ukazala na razlike samo u trostrukom transfromantu (CAD-CCR-F5H). Posljedično, sadržaj lignina u stabljici s uklonjenom korom bio je reduciran samo u navedenom transfromantu.The aim of the study, which is part of the European Energypoplar project, was characterize newly-produced poplar multitransformants, obtained using a combinatorial transformation approach, in which interesting genes of lignin (CAD, CCR, F5H) and gibberellin synthesis pathway (GA 20-Ox) were potentially combined to affect both biomass quality and plant growth. With altered lignin composition, poplar can be a desirable lignocellulosic feedstock for bioethanol production. The 14 new transgenic poplar lines were characterized at the molecular and phenotypic level and compared with wild type. Combinatorial approach resulted in gaining mainly single gene transformants (79%) and only 3 double or triple transformants (CAD-CCR, CCR-F5H, CAD-CCR-F5H). Complete silencing of CAD and CCR genes was not achieved. No combination with GA 20-Ox was obtained. The transgenic lines, grown under the near-optimal growth conditions of the greenhouse, had no apparent growth retardation, except for the triple transformant (CAD-CCR-F5H), whose growth was reduced of 40%. Analysis of wood anatomy indicated a delay and a default in lignification in the stem, for most transgenics; strong changes in autofluorescence intensity were also observed in double transformant (CCR-F5H). This was however not associated to a strong modification of chemical composition and structure of wood, as NIR spectroscopy revealed differences only for the triple (CAD-CCR-F5H) transformant. In addition, lignin content for the debarked stem was only affected in this triple transformant

Characterization of transgenic poplars potentially interesting for bioethanol production

Cilj studije, koja je dio europskog EnergyPoplar projekta, je karakterizacija novoproduciranih multitransformanata topole proizvedenih kombinatorijskim pristupom transformacije u kojem su zanimljivi geni biosintetskog puta lignina (CAD, CCR, F5H) i giberlina (GA 20-Ox) kombinirani kako bi potencijalno utjecali na kvalitetu biomase i biljni rast. Uz promijenjeni sadržaj lignina, topola može biti poželjna lignocelulozna sirovina za proizvodnju bioetanola. Novih 14 transgeničnih linija karakterizirano je na molekularnoj i fenotipskoj razini, i uspoređeno sa divljim tipom. Kombinatorijski pristup je rezultirao dobivanjem većine transformanata sa jednim insertiranim tipom gena (79%) i samo 3 transfromanta sa dva i tri insertirana gena (CAD-CCR, CCR-F5H, CAD-CCR-F5H). Potpuna represija ekspresije gena CAD i CCR nije postignuta. Nijedna kombinacija sa GA 20-ox genom nije dobivena. Transgenične linije topole, uzgajane u skoro idealnim uvjetima u stakleniku, nisu pokazale vidljivo zaostajanje u rastu, s iznimkom transformanta sa tri insertirana gena (CAD-CCR-F5H), čiji je rast bio reduciran za oko 40%. Analiza anatomije drva je ukazala na odgođenu lignifikaciju u drvu stabljike većine transfromanta, kao i snažne promjene u intenzitetu autoflourescencije kod dvostrukog transformanta (CCR-F5H). Ove promjene nisu bile povezane niti su utjecale na snažnu modifikaciju kemijskog sadržaja i strukture drva, jer je analiza Near Infrared spektroskopijom ukazala na razlike samo u trostrukom transfromantu (CAD-CCR-F5H). Posljedično, sadržaj lignina u stabljici s uklonjenom korom bio je reduciran samo u navedenom transfromantu.The aim of the study, which is part of the European Energypoplar project, was characterize newly-produced poplar multitransformants, obtained using a combinatorial transformation approach, in which interesting genes of lignin (CAD, CCR, F5H) and gibberellin synthesis pathway (GA 20-Ox) were potentially combined to affect both biomass quality and plant growth. With altered lignin composition, poplar can be a desirable lignocellulosic feedstock for bioethanol production. The 14 new transgenic poplar lines were characterized at the molecular and phenotypic level and compared with wild type. Combinatorial approach resulted in gaining mainly single gene transformants (79%) and only 3 double or triple transformants (CAD-CCR, CCR-F5H, CAD-CCR-F5H). Complete silencing of CAD and CCR genes was not achieved. No combination with GA 20-Ox was obtained. The transgenic lines, grown under the near-optimal growth conditions of the greenhouse, had no apparent growth retardation, except for the triple transformant (CAD-CCR-F5H), whose growth was reduced of 40%. Analysis of wood anatomy indicated a delay and a default in lignification in the stem, for most transgenics; strong changes in autofluorescence intensity were also observed in double transformant (CCR-F5H). This was however not associated to a strong modification of chemical composition and structure of wood, as NIR spectroscopy revealed differences only for the triple (CAD-CCR-F5H) transformant. In addition, lignin content for the debarked stem was only affected in this triple transformant

Quantification de la puissance motrice des arbres

National audienc

Physiological and degenerative loading of bovine intervertebral disc in a bioreactor: A finite element study of complex motions

Intervertebral disc (IVD) degeneration and regenerative therapies are commonly studied in organ-culture experiments with uniaxial compressive loading. Recently, in our laboratory, we established a bioreactor system capable of applying loads in six degrees-of-freedom (DOF) to bovine IVDs, which replicates more closely the complex multi-axial loading of the IVD in vivo. However, the magnitudes of loading that are physiological (able to maintain cell viability) or mechanically degenerative are unknown for load cases combining several DOFs. This study aimed to establish physiological and degenerative levels of maximum principal strains and stresses in the bovine IVD tissue and to investigate how they are achieved under complex load cases related to common daily activities. The physiological and degenerative levels of maximum principal strains and stresses were determined via finite element (FE) analysis of bovine IVD subjected to experimentally established physiological and degenerative compressive loading protocols. Then, complex load cases, such as a combination of compression + flexion + torsion, were applied on the FE-model with increasing magnitudes of loading to discover when physiological and degenerative tissue strains and stresses were reached. When applying 0.1 MPa of compression and ±2–3° of flexion and ±1–2° of torsion the investigated mechanical parameters remained at physiological levels, but with ±6–8° of flexion in combination with ±2–4° of torsion, the stresses in the outer annulus fibrosus (OAF) exceeded degenerative levels. In the case of compression + flexion + torsion, the mechanical degeneration likely initiates at the OAF when loading magnitudes are high enough. The physiological and degenerative magnitudes can be used as guidelines for bioreactor experiments with bovine IVDs.ISSN:1751-6161ISSN:1878-018

Chemonucleolysis combined with dynamic loading for inducing degeneration in bovine caudal intervertebral discs

Chemonucleolysis has become an established method of producing whole organ culture models of intervertebral disc (IVD) degeneration. However, the field needs more side-by-side comparisons of the degenerative effects of the major enzymes used in chemonucleolysis towards gaining a greater understanding of how these organ culture models mimic the wide spectrum of characteristics observed in human degeneration. In the current work we induced chemonucleolysis in bovine coccygeal IVDs with 100 µL of papain (65 U/mL), chondroitinase ABC (chABC, 5 U/mL), or collagenase II (col’ase, 0.5 U/mL). Each enzyme was applied in a concentration projected to produce moderate levels of degeneration. After 7 days of culture with daily dynamic physiological loading (0.02–0.2 MPa, 0.2 Hz, 2 h), the cellular, biochemical and histological properties of the IVDs were evaluated in comparison to a PBS-injected control. Papain and collagenase, but not chABC, produced macroscopic voids in the tissues. Compared to day 0 intact IVDs, papain induced the greatest magnitude glycosaminoglycan (GAG) loss compared to chABC and col’ase. Papain also induced the greatest height loss (3%), compared to 0.7%, 1.2% and 0.4% for chABC, col’ase, and PBS, respectively. Cell viability in the region adjacent to papain and PBS-injection remained at nearly 100% over the 7-day culture period, whereas it was reduced to 60%–70% by chABC and col’ase. Generally, enzyme treatment tended to downregulate gene expression for major ECM markers, type I collagen (COL1), type II collagen (COL2), and aggrecan (ACAN) in the tissue adjacent to injection. However, chABC treatment induced an increase in COL2 gene expression, which was significant compared to the papain treated group. In general, papain and col’ase treatment tended to recapitulate aspects of advanced IVD degeneration, whereas chABC treatment captured aspects of early-stage degeneration. Chemonucleolysis of whole bovine IVDs is a useful tool providing researchers with a robust spectrum of degenerative changes and can be utilized for examination of therapeutic interventions.ISSN:2296-418