Le lin est une angiosperme annuelle dicotylédone connue pour ses fibres cellulosiques longues aux propriétés remarquables. Ces fibres se distinguent des cellules du bois par leur faible teneur en lignine et la distribution de ce polymère au sein des couches pariétales formant la fibre. Afin de mieux comprendre les facteurs contrôlant le taux de lignine dans les différents tissus de lin, nous avons entrepris une étude détaillée de la lignification au moyen d?approches microscopiques et biochimiques. Dans un premier temps, nos travaux ont souligné des similarités (lignine gaïacyle, liaisons condensées, détection du motif dibenzodioxocine et de la coniférine) entre la lignine de lin et celle des conifères. Une approche biochimique a montré que l?activité oxydasique majeure dépendrait des peroxydases. La partielle purification et des caractérisations initiales (électrophorèses natives, IEF, affinité de substrat) des peroxydases pariétales ont mis en évidence des différences importantes entre les isoformes du xylème, et celles des tissus externes (riches en fibres) de la tige. L?utilisation du chlorure de cérium a permis de préciser la distribution subcellulaire de l?H2O2 (catalyseur de polymérisation) dans la tige du lin au stade floraison. Dans les fibres longues, l?H2O2 n?a pu être détecté que dans la région médiane de la tige. En revanche l?H2O2 était présente dans toutes les cellules du xylème examinées. Des observations en ESEM et en microscopie confocale suggèrent que les fibres longues de lin forment un réseau dense qui limite la pénétration de protéines. L?ensemble de ces résultats souligne les différences entre le xylème et fibres longues pouvant expliquer en partie la faible lignification des fibres externes. / The angiosperm flax is an annual dicotyledon grown for its cellulose-rich bast fibres that show interesting properties. These fibres are characterized by a low lignin level in the different layers of the fibre cell wall that distinguishes them from xylem cells. In order to obtain a better understanding of the factors controlling lignin levels in different flax stem tissues we have undertaken a detailed study of lignification using microscopic and biochemical approaches. Initial results underlined similarities (guaiacyl lignin, condensed bonds, presence of dibenzodioxocine and coniferin) between flax lignin and that of conifers. A biochemical approach showed that the major oxidase activity was provided by peroxidases. Partial purification and initial characterization (native electrophoresis, IEF, substrate affinities) of cell wall peroxidases indicated important differences between the isoforms present in xylem and outer (fibre-rich) stem tissues. The use of cesium chloride allowed us to determine the cellular locations of H2O2 (polymerisation catalyst) in flax stems at the flowering stage. In bast fibres, H2O2 was only detected in the median region of the stem. In contrast H2O2 was observed in all xylem cells examined. Observation by ESEM and confocal microscopy suggested that the cell walls of flax bast fibres form a dense network that limits protein penetration. Altogether, these results highlight a number of differences between flax xylem and bast fibres that could partially explain the low lignification of the external bast fibres.

Copper (Cu) significantly inhibits the growth of radish (Raphanus sativus) seedlings, even at the concentration of 1 £gM. As far as the relationship between the growth of seedlings and peroxidase (POD) activity was concerned, the reduction of radish seedlings was correlated with the induction of cationic and anionic PODs. The data show that the increase of cationic PODs (pI 8.6 and pI 9.3) and anionic PODs (pI 5.1 and pI 3.5) activities was correlated with the rise of lignin contents in Cu-treated tissues. In our investigation, among the radish root PODs, the cationic pI 8.6 POD isozyme displayed a high affinity (Km of 57.9 £gM) for syringaldazine (an analog of lignin monomer) and the similar value of catalytic efficiency jointly with the anionic pI 5.1 POD, 0.14 £gM-1S-1 and 0.12 £gM-1S-1, respectively. The results suggest that the increase of cationic POD (pI 8.6) induced by Cu treatment might be responsible for the lignification in radish roots.

Copper-treated soybean ( Glycine max ) seedling shows significant inhibition in soybean root growth, and enhancement in POD activity. Cu is an efficient catalyst in the formation of several reactive oxygen species and free radical. The increase in POD activity induced by Cu might remove excess hydrogen peroxide serving a detoxifying role during Cu-treatment. The increase of cationic ( pI 8.9, pI 8.3 and pI 7.7 ) PODs and anionic ( pI 6.5, pI 5.6 and pI 4.4 ) PODs activities is accompanied by a rise of lignin contents in Cu-treated tissues. We suggest that the increase in cationic ( pI 8.9, pI 8.3 and pI 7.7 ) and anionic ( pI 6.5, pI 5.6 and pI 4.4 ) PODs induced by Cu is responsible for lignin synthesis in soybean roots during Cu treatment.

Diss. (sammanfattning) Umeå : Sveriges lantbruksuniv., 2008. / Härtill 4 uppsatser.

Ce projet de thèse s’inscrit dans un contexte de changement climatique et de remplacement des énergies fossiles, où la réduction des apports en eau et l’optimisation de la valorisation de la biomasse sont deux enjeux majeurs des systèmes de productions durables. La dégradabilité de la biomasse est principalement limitée par la dégradabilité des parois et afin de l’améliorer, il est nécessaire de comprendre quels facteurs sont impliqués dans cette limitation de dégradabilité. Plusieurs études ont montré que la dégradabilité pariétale est impactée par la composition et la structure de la paroi mais aussi par la distribution des tissus lignifiés au sein des organes. Pour faire la part entre l’impact de la biochimie et celui de l’histologie sur la dégradabilité dans différentes conditions d’irrigation, des outils haut-débit de quantifications biochimiques et histologiques ont été développés et dédiés à l’étude d’entrenoeuds portant l’épi principal. Les études ont porté sur un panel de diversité génétique de maïs et d’une population de lignées recombinantes, cultivés durant plusieurs années dans des conditions d’irrigation contrastées dans le sud de la France. Nos résultats mettent en évidence que le déficit hydrique induit une augmentation de la dégradabilité pariétale, accompagnée par une diminution de la teneur en lignines pariétales et par une induction préférentielle d’une lignification corticale, plus p-coumaroylée. De façon originale, nous avons aussi cartographié 90 QTLs de caractères histologiques dans les différentes conditions d’irrigation sur le génome du maïs. Plus particulièrement, une large région entre le bin 1.07 et le bin 1.11 est impliquée dans les variations observées du nombre de faisceaux vasculaires et de la surface de section des entrenoeuds. De façon globale, de nombreux QTLs de composition pariétale de l’entrenoeud co-localisent avec ceux obtenus au niveau de la plante entière sans épis chez la même population. Enfin nous avons pu démontrer que le choix de l’entrenoeud portant l’épi principal est judicieux pour représenter à la fois les caractéristiques histologiques des tiges entières et biochimiques de la biomasse lignocellulosique des plantes entières. Ainsi, les caractéristiques histologiques et biochimiques des entrenoeuds de maïs sont proposées comme des cibles de choix pour sélectionner des lignées de maïs résilientes au déficit hydrique. / This PhD project encompass in a context of global warming and replacement of fossil resources, where both water supply decrease and biomass valorisation optimisation are two major issues for providing sustainable systems of production. Biomass degradability is mainly limited by cell wall degradability and in order to improve it, it is necessary to understand what are the factors involved in the limitation of the degradability. Several studies have shown that cell wall degradability is impacted by the structure and the composition of the cell wall but also by the distribution of the lignified tissues within the organs. To decipher the impact of the biochemistry and from the one of the histology on degradability under different watering conditions, high-throughput quantifications tools for histology and biochemistry have been developed and dedicated to the study of the internode carrying the main ear. The studies were conducted on a maize genetic diversity panel and a recombinant inbred lines population, cultivated during several years under contrasted irrigation conditions in South of France. Our results highlight that water deficit induce an increase of the cell wall degradability, associated to a decrease of the cell wall lignin content and a preferential induction of a cortical lignification, more p-coumaroylated. In original ways, we also detected 90 QTLs for histological traits under the different irrigations scenarios on the maize genome. More particularly, a large region between bin 1.07 and bin 1.11 is involved in the observed variations of the bundle number and the stem section area of the internodes. More generally, numerous QTLs of cell wall composition of the internode co-localised with the ones detected for the whole plant without ear level in the same population. Finally we were able to demonstrate that the choice of the internode carrying the main ear is judicious to represent both histological characteristics from the whole stem and biochemical characteristics from the lignocellulosic biomass of the whole plant. Thus, histological and biochemical traits in maize internode are proposed as particular targets to select lines resilient to water deficit.

Cu-treated peanut seedlings not only showed a significant inhibition in peanut root growth, but also a decrease in endogenous indole-3-acetic acid (IAA) contents . The decline of IAA content in Cu-treated tissues is accompanied by an increase in cationic peroxidase activity. In addition, the increase in cationic peroxidase (pI 8.5) activity is correlated with an increase in cationic peroxidase transcripts. Cu might suppress the growth of peanut roots by inducing the synthesis of the cationic peroxidase isozyme that degrades endogenous IAA, causing the retardation of peanut root growth. The increase in activity of anionic peroxidases (pI 3.5) is also correlated with the rise in lignin content in Cu-treated roots. We suggest that the increase in anionic peroxidases (pI 3.5) induced by Cu might be responsible for lignin synthesis in peanut roots, and remove excess hydrogen peroxide, thus serving a detoxifying role during Cu treatment.

Synthetic auxin, naphthaleneacetic acid ( NAA ), promoted rooting in soybean ( Glycine max ) hypocotyls. The activities of anionic peroxidases (PODs, pI 3.5 and pI 3.7 ) and cationic PODs ( pI 8.3, pI 9.1 and pI 9.4 ) in soybean hypocotyls were significantly inhibited by exogenous NAA during the induction of adventitious roots. In addition, the reduction in the activity of PODs ( pI 4.4, pI 8.3 and pI 9.4 ) were correlated with a reduction of their corresponding transcription activity. The PODs patterns and transcription in the NAA-treated hypocotyls demonstrated the specific effect of NAA on POD ioszymes, indicating that NAA might suppress PODs genes expression during the adventitious root formation. The decline of PODs activity in the NAA-treated hypocotyls was accompanied by the decrease of lignin contents. We suggest that both of anionic and cationic PODs might be in part responsible for lignin synthesis in soybean hypocotyls.

Diss. (sammanfattning) Umeå : Sveriges lantbruksuniv., 2007. / Härtill 4 uppsatser.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第19036号 / 農博第2114号 / 新制||農||1031(附属図書館) / 学位論文||H27||N4918(農学部図書室) / 31987 / 京都大学大学院農学研究科森林科学専攻 / (主査)教授 髙部 圭司, 教授 髙野 俊幸, 教授 矢﨑 一史 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

O rápido declínio na digestibilidade da parede celular, devido à lignificação, dificulta o uso eficiente das gramíneas de clima tropical. Objetivou-se detectar e sequenciar o cDNA completo dos principais genes responsáveis pelo processo de biossíntese da lignina, CCoAOMT, C4H e PAL nas gramíneas Brachiaria brizantha Stapf. cv. Marandu e Panicum maximum Jacq. cv. Tanzânia. As amostras das espécies de plantas forrageiras foram coletadas do Campo Agrostológico e levadas, imediatamente, para o laboratório para a extração do RNA total. Após tratamento do RNA total com o kit GeneRacer (Invitrogen), obteve-se o comprimento total 3\' e 5\' do cDNA das forrageiras B. brizantha e P. maximum. Os primers específicos foram delineados com base nas sequências de B. brizantha, P. maximum e milho, depositadas no GenBank, e foram aneladas com os primers 3 e 5 do kit GeneRacer para obtenção do comprimento total do gene. As amplificações foram feitas através PCR qualitativo e os produtos foram purificados, clonados e seqüenciados em ambas direções. Foram encontrados alguns problemas com a otimização na reação de seqüenciamento e obteve-se 67,25 % e 82,22% do gene CCoAOMT, 13,29% e 26,16% do gene PAL e 13,22% do gene C4H em B. brizantha e P. maximum, respectivamente. O sequenciamento dos genes codificantes das enzimas envolvidas no processo de lignificação, apresentaram alta similaridade com as sequências nucleotídicas do milho, e foi possível identificar e classificá-los de acordo com as classes que compõem cada gene. B. brizantha e P. maximum apresentaram 89,6% com CCoAOMT 1 e 88,3% com CCoAOMT 3, 96% e 95% com PAL1 e 96% com C4H1, respectivamente. Ao analisar as similaridades das sequências nucleotídicas dos fragmentos sequenciados, para os genes CCoAOMT, PAL e C4H, entre as demais gramíneas amplamente estudadas, encontrou-se 91% e 91,14% CCoAOMT do arroz, 79,6% e 82% CCoAOMT do trigo, 94,33 e 94,2% CCoAOMT do sorgo, 87 e 86% CCoAOMT da aveia, 90% e 94,25% PAL do arroz, 82% e 93,33% PAL do trigo, 92 % e 89,5% PAL da aveia, 94,33 e 95% PAL do sorgo, 97% e 91% PAL cana-de-açúcar, 90% e 93% PAL da cevada, 76% C4H do arroz, 94% C4H do sorgo, 89% C4H do trigo e 87% C4H da cevada, em B. brizantha e P. maximum, respectivamente. A altíssima similaridade encontrada entre os fragmentos dos genes da via dos fenilpropanóides em B. brizantha e P. maximum, principalmente com o milho e arroz que possuem seu genoma em avançado estudo e conhecimento, possibilitam o uso do banco de dados destas espécies para estudos futuros de biologia molecular. / The rapid decline in digestibility of cell wall, due to lignification, hinders the efficient use of tropical grasses. The objective was to detect and sequence the complete cDNA of the major genes responsible for lignin biosynthesis process, CCoAOMT, PAL and C4H in grasses Brachiaria brizantha Stapf. cv. Marandu and Panicum maximum Jacq. cv. Tanzania. Samples of fodder plant species were collected from the field agrostology and taken immediately to the laboratory for extraction of total RNA. After treatment of total RNA with the Generac kit (Invitrogen), we obtained the full length 3\'and 5\' cDNA of forage B. brizantha and P. maximum. The specific primers were designed based on sequences of B. brizantha, P. maximum and corn, deposited in GenBank, and were ringed with primers 3 \'and 5\' of the Generac kit to obtain the total length of the gene. The amplifications were performed by qualitative PCR and the products were purified, cloned and sequenced in both directions. We found some problems with optimization in the sequencing reaction and obtained 67.25% and 82.22% of the CCoAOMT gene, 13.29% and 26.16% of PAL gene and 13.22% of the C4H gene in B. brizantha and P. maximum, respectively. The sequencing of genes encoding enzymes involved in lignification, showed high similarity with the nucleotide sequences of maize, and it was possible to identify and classify them according to the classes that make up each gene. B. brizantha and P. maximum 89.6% presented with CCoAOMT 1 and 88.3% with CCoAOMT 3, 96% and 95% to 96% with PAL1 and C4H1, respectively. By analyzing the similarities of the nucleotide sequences of the fragments sequenced, the genes for CCoAOMT, PAL and C4H, among the other grasses studied extensively, met 91% and 91.14% of the rice CCoAOMT, 79.6% and 82% of the CCoAOMT wheat, 94.33% and 94.2 CCoAOMT sorghum, 87 and 86% oat CCoAOMT, 90% and 94.25% of the rice PAL, 82 PAL% and 93.33% for wheat, 92% and 89.5 PAL% oats, 95% and 94.33 PAL sorghum, 97% and 91% PAL cane sugar, 90% and 93% PAL barley, rice 76% C4H, C4H 94% sorghum, 89% the C4H C4H wheat and barley 87% in B. brizantha and P. maximum, respectively. The high similarity found among the fragments of the genes of the phenylpropanoid pathway in B. brizantha and P. maximum, especially with corn and rice that have their genome in advanced study and knowledge can enable the use of the database of these species for future studies of molecular biology.