In den Pflanzenzellwänden befindliche Polysaccharide stehen dem Prozess nur bedingt als Energiequelle zur Verfügung, da diese in einem Komplex mit Lignin verknüpft sind. Um diese Substanzen für den Biogasprozess verfügbar zu machen und demnach den Substratumsatz bzw. die Prozesseffizienz zu erhöhen, sind geeignete Stoffe oder Techniken einzusetzen bzw. zu entwickeln. In dieser Arbeit wurde zielführend der Einsatz von unterschiedlichen Enzympräparaten in drei verschiedenen Prozessstufen bei ausgewählten Energiepflanzen mit variierender Häcksellänge untersucht. Anhand von Enzymaktivitätsbestimmungen konnten Enzympräparate für die einzelnen Stufen selektiert werden. Die ausgewählten Enzyme wurden einzeln oder in Mischung während der Silierung, direkt vor dem Biogasprozess sowie während des Biogasprozesses zum Substrat dotiert und dieses nach der jeweiligen Vorbehandlung in Batch-Gärtests vergoren. Neben der Biogas- und Methanausbeute wurde zur Bewertung der Enzymleistung der Abbau an Lignocellulose sowie die Freisetzung an niedermolekularen Kohlenhydraten ermittelt. Zusätzlich wurde das Quellen der Lignocellulose mit Hilfe eines Wasserzusatzes in Form einer Vorhydrolyse als Vorbehandlungsmethode mit allgemein positivem Ergebnis geprüft. Das Ziel der verbesserten Substratumsetzung bei Mais und Roggen und folglich einer Erhöhung der Biogasproduktion wurde durch den Zusatz ausgewählter Enzympräparate erreicht. Es konnten Grundlagen bezüglich der Wirkung von Enzymen in Biogasprozessen geschaffen werden, anhand derer deutlich wurde, dass besonders die enzymatische Behandlung in den der Methanisierung vorgelagerten Prozessstufen weiterzuentwickeln ist. / Polysaccharides of plant cell walls are of limited digestibility due to their cross-linking to lignin. In order to make the molecules available for the biogas process and thus increase the substrate utilization and process efficiency appropriate substances or techniques are needed. It was therefore the aim of this work to investigate the effects of different enzyme preparations in three digestion process stages. Selected energy plants with varying degrees of particle sizes (chopping lengths) were used as digester feedstock. Enzyme preparations for the different process stages were chosen by enzyme assays. The selected enzymes were added to the feedstock during the ensiling, directly before the biogas process or during the biogas process separate or in mixtures. Pre-treated substrates were subsequently digested in batch fermentation tests. Beside the biogas and methane yield the degradation degree of lignocellulose and the release of low molecular carbohydrates were investigated for evaluating the enzyme performance. Additionally, the swelling of lignocellulose caused by addition of water in a pre-hydrolysis process was examined as a method of pre-treatment, with generally positive results. The aim of an improved substrate conversion of maize and rye and thus an enhanced biogas production by enzymatic pretreatments was achieved. Scientific fundamentals regarding the impact of enzymes on biogas processes were established. Enzymatic pretreatments in process steps before methanation showed potential for further developments.

Infection of barley (Hordeum vulgare) leaves with the rust fungus, Puccinia hordei, causes changes in the host protein synthesis. This thesis analyses these changes in the barley cultivar Triumph following inoculation of 7-day-old leaves with either a virulent or an avirulent race of P. hordei. The initial approach was to isolate membrane-bound polysomes from infected leaves, translate them in vitro and analyse the translation products. These products include the integral membrane proteins which were expected to be involved in the response of the host to the pathogen. A method based on differential centrifugation in the presence of a ribonuclease-inhibiting buffer was developed for separating membrane-bound polysomes from the rest of the cytoplasmic polysomes. Membrane-bound polysomes were found to comprise one fifth of the total polysomes in the leaves. Analysis of the translation products of membrane-bound polysomes by SDS-PAGE showed them to be of higher average molecular weight than those from free polysomes. Comparison of polypeptides produced by membrane-bound polysomes from healthy and inoculated plants showed some differences however the low yield of membrane-bound polysomes made it difficult to obtain conclusive results. Thus it was decided to isolate total polysomes by including 1% Triton X-100 in the extraction buffer. Polysomes were extracted from 12 to 72 h after inoculation. Infection caused a decline in yield of polysomes during this period when compared with healthy leaves of the same age. Polysomes isolated 16 h after inoculation with the virulent race were 20% less efficient at translation than polysomes from control leaves. In contrast polysome isolated from leaves inoculated with the avirulent race were 20% more efficient. Analysis of the labelled translation products by SDS-PAGE and fluorography showed relative increases in the synthesis of some proteins by 16 h after inoculation with either race when compared to products from healthy leaves. Protein synthesis in the infected plants was further analysed by in vivo labelling and one- and two-dimensional PAGE. The fluorographs revealed increased synthesis of a group of proteins from 58 to 116 kDa starting 12 h after inoculation with either race of P. hordei; confirming the results from the polysome translations. Two polypeptides with molecular weights of about 66 kDa were found to increase following infection only with the virulent race. By three days after inoculation with either fungal race the most obvious change in protein synthesis was a marked decrease in the synthesis of the two most prominent polypeptides with molecular weights of 15 and 51 kDa which were considered to be the subunits of ribulose bisphosphate carboxylase. The elicitor hypothesis, in attempting to explain cultivar-specific resistance in plants, postulates that resistance is controlled by the interaction of specific fungal elicitors and plant receptors and that this interaction which only occurs between resistant hosts and avirulent pathogens triggers specific gene expression leading to resistance. This hypothesis does not fit the situation in the barley-P. hordei interaction as protein synthesis showed similar changes following infection with either a virulent or an avirulent race.

Poaceae (or Gramineae) belong to the grass family and is one of the largest families among flowering plants on land. They include some of the most important cereal crops such as rice (Oryza sativa), barley (Hordeum vulgare), wheat (Triticum aestivum), maize (Zea mays), and sorghum (Sorghum bicolor). The characteristic bushy appearance of grass plants, including cereal crops, is formed by the activities of axillary meristems (AMs) generated in the leaf axil. These give rise to tillers from the basal nodes which recapitulate secondary growth axis and AMs are formed during vegetative development. On transition to flowering the apical meristem transforming to an inflorescence meristem (IM) which produces branches from axillary meristem. These IM gives rise to branches that ultimately bear florets. Vegetative branching/tillering determines plant biomass and influences the number of inflorescences per plant. While inflorescence branching determines the number of florets and hence seeds. Thus the overall activity of axillary meristems plays a key role in determining plant architecture during both vegetative and reproductive stages. In Arabidopsis, research on the plant specific transcription factor LEAFY (LFY) has pioneered our understanding of its regulatory functions during transition from vegetative to reproductive development and its role in specifying a floral meristem (FM) identity to the newly arising lateral meristems. In the FM LFY activates other FM genes and genes for floral organ patterning transcription factors. LFY is strongly expressed throughout the young floral meristems from the earliest stages of specification but is completely absent from the IM (Weigel et al., 1992). LFY expression can also be detected at low levels in the newly emerging leaf primordia during the vegetative phase, and these levels gradually increase until the floral transition (Blazquez et al., 1997; Hempel et al., 1997). In rice, the LFY ortholog-RFL/APO2 is expressed predominantly in very young branching panicles/ inflorescence meristems (Kyozuka et al., 1998; Prasad et al., 2003) while in the vegetative phase RFL is expressed at axils of leaves (Rao et al., 2008). In rice FMs expression is restricted to primordia of lodicules, stamens, carpels and ovules (Ikeda-Kawakatsu et al., 2012). Knockdown of RFL activity or loss of function mutants show delayed flowering and poor panicle branching with reduced number of florets and lower fertility (Rao et al., 2008, Ikeda-Kawakatsu et al., 2012). In some genotypes reduced vegetative axillary branching is also compromised (Rao et al., 2008). On the other hand RFL overexpression leads to the early flowering, attributing a role as an activator for the transition of vegetative meristems to inflorescence meristems (Rao et al., 2008). Thus, RFL shows a distinct developmental expression profile, has unique mutant phenotypes as compared to Arabidopsis LFY thus indicating a divergence in functions. We have used various functional genomics approaches to investigate regulatory networks controlledby RFL in the vegetative axillary meristems and in branching panicles with florets. These regulatory effects influence tillering and panicle branching, thus contributing to rice plant architecture. RFL functions in axillary meristem Vegetative AMs are secondary shoot meristems whose outgrowth determines plant architecture. In rice, AMs form tillers from basal nodes and mutants with altered tillering reveal that an interplay between transcription factors and the phytohormones - auxin, strigolactone underpins this process. We probed the relationship between RFL and other factors that control AM development. Our findings indicate that the derangements in AM development that occur on RFL knockdown arise from its early effects during specification of these meristems and also later effects during their outgrowth of AM as a tiller. Overall, the derailments of both steps of AM development lead to reduced tillering in plants with reduced RFL activity. Our studies on the gene expression status for key transcription factor genes, genes for strigolactone pathway and for auxin transporters gave an insight on the interplay between RFL, LAX1 and strigolactone signalling. Expression levels of LAX1 and CUC genes, that encode transcription factors with AM specification functions, were modulated upon RFL knockdown and on induction of RFL:ΔGR fusion protein. Thus our findings imply a likely, direct activating role for RFL in AM development that acts in part, through attaining appropriate LAX1 expression levels. Our data place meristem specification transcription factors LAX1 and CUC downstream to RFL. Arabidopsis LFY has a predominant role in conferring floral meristem (FM) identity (Weigel et al., 1992; Wagner, 2009; Irish, 2010; Moyroud et al., 2010). Its functions in axillary meristems were not known until recently. The latter functions were uncovered with the new LFYHARA allele with only partial defects in floral meristem identity (Chahtane et al., 2013). This mutant allele showed LFY can promote growth of vegetative AMs through its direct target REGULATOR OF AXILLARY MERISTEMS1 (RAX1), a R2R3 myb domain factor (Chahtane et al., 2013). These functions for Arabidopsis LFY and RAX1 in AMs development are parallel to and redundant with the pathway regulated by LATERAL SUPPRESSOR (LAS) and REGULATOR OF AXILLARY MERISTEM FORMATION1 (ROX1) (Yang et al., 2012; Greb et al., 2003). Interestingly, ROX1 is orthologous to rice LAX1 and our data show LAX1 expression levels in rice panicles and in culms with vegetative AMs is dependent on the expression status of RFL. Thus, we speculate that as compared to Arabidopsis AM development, in rice the LFY-dependent and LFY-independent regulatory pathways for AMs development are closely linked. In Arabidopsis, CUC2 and CUC3 genes in addition to their role in shoot meristem formation and organ separation play a role in AM development possibly by defining a boundary for the emerging AM. These functions for the Arabidopsis CUC genes are routed through their effects on LAS and also by mechanisms independent of LAS (Hibara et al., 2006; Raman et al., 2008). These data show modulation in RFL activity using the inducible RFL:∆GR protein leads to corresponding expression changes in CUC1/CUC2 and CUC3 genes expression in culm tissues. Thus, during rice AM development the meristem functions of RFL and CUC genes are related. Consequent to specification of AM the buds are kept dormant. Bud outgrowth is influenced by auxin and strigolactone signalling pathways. We investigated the transcript levels, in rice culms of genes involved in strigolactone biosynthesis and perception and found the strigolactone biosynthesis gene D10 and hormone perception gene are significantly upregulated in RFL knockdown plants. Further, bioassays were done for strigolactone levels, where we used arbuscular mycorrhiza colonization assay as an indicator for strigolactone levels in wild type plants and in RFL knockdown plants. These data validate higher strigolactone signalling in RFL knockdown plants. To probe the relationship between RFL and the strigolactone pathway we created plants knocked down for both RFL and D3. For comparison of the tillering phenotype of these double knockdown plants we created plants with D3 knockdown alone. We observed reduced tillering in plants with knockdown of both RFL and D3 as compared to the tiller number in plants with knockdown of D3 alone. These data suggest that RFL acts upstream to D3 of control bud outgrowth. As effects of strigolactones are influenced by auxin transport we studied expression of OsPIN1 and OsPIN3 in RFL knockdown plants. Their reduced expression was correlated with auxin deficiency phenotypes of the roots in RFL knockdown plants. These data in conjunction with observations on OsPIN3 the gene expression modulation by the induction of RFL:∆GR allow us to speculate on a relationship between RFL, auxin transport and strigolactones with regard to bud outgrowth. We propose that the low tillering phenotype of RFL knockdown plants arises from weakened PATS, consequent to low levels of PIN1 and PIN3, coupled with moderate increase in strigolactones. Taken together, our findings suggest functions for RFL during AM specification and tiller bud outgrowth. RFL functions in panicle branching Prior studies on phenotypes of RFL knockdown or loss of function mutants suggested roles for RFL in transition to flowering, inflorescence meristem development, emergence of lateral organs and floral organ development (Rao et al., 2008; Ikeda-Kawakatsu et al., 2012). It has been speculated that RFL acts to suppress the transition from inflorescence meristem to floral meristem through its interaction with APO1 (Ikeda-Kawakatsu et al., 2012). The downstream genes regulated by RFL in these processes have not yet been elucidated. To identify direct targets of RFL in developing panicles we adopted ChIP-seq coupled with studies on gene expression modulation on induction of RFL. For the former we raised polyclonal anti-sera and chromatin from branching panicles with few florets. For gene expression modulation studies, we created transgenics with a T-DNA construct where an artificial miRNA against 3’UTR specifically knocked endogenous RFL and the same T-DNA had a second expression cassette for generation of a chemically inducible RFL-ΔGR protein that is not targeted by amiR RFL. Our preliminary ChIP-seq data in the wild type panicle tissues hints that RFL binds to hundreds of loci across the genome thus providing first glimpse of direct targets of RFL in these tissues. These data, while preliminary, were manually curated to identify likely targets that function in flowering, we summarize here some key findings. Our study indicates a role of RFL in flowering transition by activating genes like OsSPL14 and OsPRMT6a. Recent studies indicate that OsSPL14 directly binds to the promoter of OsMADS56 or FTL1, the rice homologs of SOC1 and FT to promote flowering (Lu et al., 2013). As RFL knockdown plants show highly reduced expression of OsMADS50/SOC1 and for RFT1 (Rao et al., 2008), and we show here RFL can bind and induce OsSPL14 expression we suggest the RFL¬OsSPL14 module can contribute to the transition of the SAM to flowering. Further, OsSPL14 in the young panicles directly activates DENSE AND ERECT PANICLE1 (DEP1) to control panicle length (Lu et al., 2013). Thus RFL-OsSPL14-DEP1 module could explain the role of RFL in controlling panicle architecture (Rao et al., 2008; Ikeda-Kawakatsu et al., 2012). Thus RFL plays a role in floral transition and this function is conserved across several LFY homologs. Our data ChIP-seq in the wild type tissue and gene expression modulation studies in transgenics also give molecular evidences for the role of RFL in suppression of floral fate. The direct binding of RFL to OsMADS17, OsYABBY3, OsMADS58 and HD-ZIP-IV loci and the changes in their transcript levels on induction of RFL support this hypothesis. Once the transition from SAM to FM takes place, we speculate RFL represses the conversion of inflorescence branch meristems to floral fate by negatively regulating OsYABBY3, HD-ZIP class IV and OsMADS17 that can promote differentiation. These hypotheses indicate a diverged function for RFL in floral fate repression. Arabidopsis LFY is known to activate the expression of AGAMOUS (AG), whose orthologs in rice are OsMADS3 and OsMADS58. Our studies confirm conservation with regard to RFL binding to cis elements at OsMADS58 locus that is homologous to Arabidopsis AG. But importantly we show altered consequences of this binding on gene expression. We find RFL can suppress the expression of OsMADS58 which we speculate can promote a meristematic fate. Further, we also present the abnormal upregulation of floral organ fate genes on RFL downregulation. These data too indicate functions of RFL, are in part, distinct from the role of Arabidopsis LFY where it works in promoting floral meristem specification and development. These inferences are supported by our data that rice gene homologs for AP1, AP3 and SEP3 are not directly regulated by RFL, unlike their direct regulation by Arabidopsis LFY during flower development. We also report the expression levels of LAX1, FZP, OsIDS1 and OsMADS34 genes involved in meristem phase change and IM branching are RFL dependent. This is consistent with its role in the suppression of determinacy, thereby extending the IM activity for branch formation. But as yet we do not know if these effects are direct. Together, our data report direct targets of RFL that contribute to its functions in meristem regulation, flowering transition, and suppression of floral organ development. Overall, our preliminary data on RFL chromatin occupancy combined with our detailed studies on the modulation of gene expression provides evidence for targets and pathways unique to the rice RFL during inflorescence development. Comparative analysis of genes downstream to RFL in vegetative tillers Vs panicles Tillers and panicle branches arise from the axillary meristems at vegetative and reproductive stages, respectively, of a rice plant and overall contribute to the plant architecture. Some regulatory factors control branching in both these tissues - for example, MOC1 and LAX1. Mutants at these loci affect tillers and panicle branch development thus indicating common mechanisms control lateral branch primordia development (Li et al., 2003; Komatsu et al., 2003; Oikawa and Kyozuka, 2009). Knockdown of RFL activity or loss-of-function mutants cause significantly reduced panicle branching and in few instances, reduction in vegetative axillary branching (Rao et al., 2008; Ikeda- Kawakatsu et al., 2012). We took up the global expression profiling of RFL knockdown plants compared to wild type plants in the axillary meristem and branching panicle tissue. These data provide a useful list of potential targets of RFL in axillary meristem and branching panicle tissue. The comparative analysis of the genes affected in the two tissues indicates only a subset of genes is affected by RFL in both the vegetative axillary meristems and branching panicle. These genes include transcription factors (OsSPL14, Zn finger domain protein, and bHLH domain protein), hormone signalling molecules (GA2 ox9) and cell signalling (LRR protein) as a set of genes activated by RFL in both tissues. On the other hand, these comparative expression profiling studies also show distinct set of genes deregulated by RFL knockdown in these two tissues therefore implicating RFL functions have a tissue-specific context. The genes deregulated only in axillary meristem tissue only include D3- involved in the perception of strigolactone, OsMADS34 speculated to have a role in floral transition and RCN1 involved in transition to flowering. On the other hand, the genes – CUC1, OsMADS3, OsMADS58 involved in organ development and floral meristem determination were found to be deregulated only in panicle tissues of RFL knockdown plants. These data point towards presence of distinct mechanisms for the development of AMs as tillers versus the development of panicle axillary as rachis branches. Overall, these data implicate genes involved in transition to flowering, axillary meristem development and floral meristem development are controlled by RFL in different meristems to thereby control plant architecture and transition to flowering.

In angiosperms, specialized reproductive structures are borne in flowers to ensure their reproductive success. After the vegetative growth, plants undergo reproductive phase change to produce flowers. Floral meristems (FMs) are generated on the flanks of inflorescence and groups of specialized stem cells in the FM differentiate into four whorls of organs of a flower. In dicots, floral meristem successively gives rise to sepals, petals, stamens and carpels; after which it terminates. The fate of organs formed on FM is under the control of genetic regulators, key among which are members of MADS box transcription factor family. Their individual and combined act confers distinct identities to floral organs. Grass flowers are highly modified in structure. Rice flower, a model for grasses, is borne on a short branch called spikelet and they together from the basic structural units of the rice infloresences known as panicle. The outer whorl organs of a grass floret are bract-like structures known as lemma and palea to dicot sepals is highly dibated (see Chapter 1). In grass florets, petal homologs are a pair of highly reduced, fleshy bracts known as lodicules, while stamen and carpel homologs occupy the same position and share the same functions as their dicot counterparts. Aside from these distinct outer whorl organs, the florets are subtended by two pairs of bracts known as empty glumes and rudimentary glumes. The genetic regulators that control their unique identities and those that perform conserved functions are very intriguing and central questions in plant developmental biology. Using various contemporary and complementary technologies, we have analysed the molecular functions and downstream pathways of a MADS box transcription factor, OsMADSI during the rice floret meristem specification and organ development. Further by reverse genetics and overexpression studies, we have also functionally characterized two target genes of OsMADSI, OsETTINI and OsETTINI2 to understand their roles downstream to OsMADSI during the rice floret development.

La composition des cellulosomes (complexes multi-enzymatiques impliqués dans la dégradation des polysaccharides de la paroi végétale) produits par Clostridium cellulolyticum varie en fonction du substrat de croissance. En particulier, l’expression d’un regroupement de 14 gènes prédits comme codants pour des hémicellulases (appelés xyl-doc) est induite par la présence de paille et non de cellulose. L’hypothèse a été faite que le système à deux composants putatif, codé par les deux gènes en amont des gènes xyl-doc, est impliqué dans cette régulation. Mes résultats montrent que le régulateur de réponse (appelé XydR) est impliqué dans l’activation de la transcription des gènes xyl-doc et d’un gène additionnel codant pour une protéine de fonction inconnue. Cette protéine possède cependant un module de liaison aux sucres prédit comme ciblant les hémicelluloses. Les régions promotrices, incluant les sites potentiels de liaison de XydR, ont été identifiées en amont des gènes régulés et un lien transcriptionnel entre tous les gènes xyl-doc a été mis en évidence.Un deuxième objectif de mon travail a été d’identifier le signal inducteur présent dans la paille susceptible d’être capté par le senseur apparenté à XydR. Il a été montré que la transcription des gènes cibles est spécifiquement induite par l’arabinose et le xylose qui sont les résidus glucidiques les plus abondants dans les hémicelluloses et donc relargués lors de leur dégradation.Finalement, des études biochimiques des produits de certains des gènes régulés ont montré qu’au moins trois des gènes codaient pour des produits impliqués dans la dégradation des hémicelluloses. / The composition of the cellulosomes (multi enzymatic complexes involved in the degradation of plant cell wall polysaccharides) produced by Clostridium cellulolyticum differs according to the growth substrate. In particular, the expression of a cluster of 14 hemicellulase-encoding genes (called xyl-doc) is induced by the presence of straw and not of cellulose. The hypothesis was made that the putative two-component regulatory system, encoded by the genes localized upstream of xyl-doc, was involved in this regulation.My results provided evidence that the response regulator (called XydR) is involved in the activation of the transcription of xyl-doc genes and of an additional gene encoding a protein of unknown function harboring a carbohydrate binding module predicted to target hemicelluloses. Promoter regions, including XydR binding sites, have been identified upstream of the regulated genes and the transcriptional link between all xyl-doc genes has been demonstrated. A second aim of my work has been to identify the inducing signal present in straw that could be sensed by the cognate sensor of XydR. It was shown that the transcription of the target genes is specifically induced by arabinose and xylose which are the most abundant sugar residues present in hemicellulose and thus released by its degradation.Finally, biochemical studies of the products of some of the regulated genes demonstrated that at least three genes encoded products involved in hemicellullose degradation.

Aujourd’hui, la biologie des systèmes est confrontée aux défis de l’analyse de l’énorme quantité de données biologiques et à la taille des réseaux métaboliques pour des analyses à grande échelle. Bien que plusieurs méthodes aient été développées au cours des dernières années pour résoudre ce problème, ce sujet reste un domaine de recherche en plein essor. Cette thèse se concentre sur l’analyse des propriétés structurales, le calcul des modes élémentaires de flux et la détermination d’ensembles de coupe minimales du graphe formé par ces réseaux. Dans notre recherche, nous avons collaboré avec des biologistes pour reconstruire un réseau métabolique de taille moyenne du métabolisme cellulaire de la plante, environ 90 noeuds et 150 arêtes. En premier lieu, nous avons fait l’analyse des propriétés structurelles du réseau dans le but de trouver son organisation. Les réactions points centraux de ce réseau trouvés dans cette étape n’expliquent pas clairement la structure du réseau. Les mesures classiques de propriétés des graphes ne donnent pas plus d’informations utiles. En deuxième lieu, nous avons calculé les modes élémentaires de flux qui permettent de trouver les chemins uniques et minimaux dans un réseau métabolique, cette méthode donne un grand nombre de solutions, autour des centaines de milliers de voies métaboliques possibles qu’il est difficile de gérer manuellement. Enfin, les coupes minimales de graphe, ont été utilisés pour énumérer tous les ensembles minimaux et uniques des réactions qui stoppent les voies possibles trouvées à la précédente étape. Le nombre de coupes minimales a une tendance à ne pas croître exponentiellement avec la taille du réseau a contrario des modes élémentaires de flux. Nous avons combiné l’analyse de ces modes et les ensembles de coupe pour améliorer l’analyse du réseau. Les résultats montrent l’importance d’ensembles de coupe pour la recherche de la structure hiérarchique du réseau à travers modes de flux élémentaires. Nous avons étudié un cas particulier : qu’arrive-t-il si on stoppe l’entrée de glucose ? En utilisant les coupes minimales de taille deux, huit réactions ont toujours été trouvés dans les modes élémentaires qui permettent la production des différents sucres et métabolites d’intérêt au cas où le glucose est arrêté. Ces huit réactions jouent le rôle du squelette / coeur de notre réseau. En élargissant notre analyse aux coupes minimales de taille 3, nous avons identifié cinq réactions comme point de branchement entre différent modes. Ces 13 réactions créent une classification hiérarchique des modes de flux élémentaires fixés et nous ont permis de réduire considérablement le nombre de cas à étudier (approximativement divisé par 10) dans l’analyse des chemins réalisables dans le réseau métabolique. La combinaison de ces deux outils nous a permis d’approcher plus efficacement l’étude de la production des différents métabolites d’intérêt par la cellule de plante hétérotrophique. / Nowadays, systems biology are facing the challenges of analysing the huge amount of biological data and large-scale metabolic networks. Although several methods have been developed in recent years to solve this problem, it is existing hardness in studying these data and interpreting the obtained results comprehensively. This thesis focuses on analysis of structural properties, computation of elementary flux modes and determination of minimal cut sets of the heterotrophic plant cellmetabolic network. In our research, we have collaborated with biologists to reconstructa mid-size metabolic network of this heterotrophic plant cell. This network contains about 90 nodes and 150 edges. First step, we have done the analysis of structural properties by using graph theory measures, with the aim of finding its owned organisation. The central points orhub reactions found in this step do not explain clearly the network structure. The small-world or scale-free attributes have been investigated, but they do not give more useful information. In the second step, one of the promising analysis methods, named elementary flux modes, givesa large number of solutions, around hundreds of thousands of feasible metabolic pathways that is difficult to handle them manually. In the third step, minimal cut sets computation, a dual approach of elementary flux modes, has been used to enumerate all minimal and unique sets of reactions stopping the feasible pathways found in the previous step. The number of minimal cut sets has a decreasing trend in large-scale networks in the case of growing the network size. We have also combined elementary flux modes analysis and minimal cut sets computation to find the relationship among the two sets of results. The findings reveal the importance of minimal cut sets in use of seeking the hierarchical structure of this network through elementary flux modes. We have set up the circumstance that what will be happened if glucose entry is absent. Bi analysis of small minimal cut sets we have been able to found set of reactions which has to be present to produce the different sugars or metabolites of interest in absence of glucose entry. Minimal cut sets of size 2 have been used to identify 8 reactions which play the role of the skeleton/core of our network. In addition to these first results, by using minimal cut sets of size 3, we have pointed out five reactions as the starting point of creating a new branch in creationof feasible pathways. These 13 reactions create a hierarchical classification of elementary flux modes set. It helps us understanding more clearly the production of metabolites of interest inside the plant cell metabolism.

5 Abstract The thesis is focused on the role of regulator of G-protein signaling NtRGS1 in control of growth and cell proliferation of tobacco cell line BY-2. The protein NtRGS1 is an important candidate for being plant G-protein coupled receptor. Heterotrimeric G-proteins are involved in key signaling mechanisms in eukaryotic cells. Basic principles of this type of signaling are well conserved between plants and animals and related higher taxa. Outstanding difference of plant G-protein system is altered enzymatic activity of Gα subunit of the G-protein heterotrimer. These alterations correlate with chimeric structure and function of investigated NtRGS1 protein. The interaction of Gα and NtRGS1 is absolutely essential for running of heterotrimeric G-protein signaling in plants. Truncated versions of NtRGS1 fused to GFP were crated in the aim of protein characterization. The truncated proteins were investigated in respect of analysis of the role of NtRGS1 domains in protein targeting. Dynamic changes in NtRGS1 and selected truncated versions induced by experimental application of nutrition, especially sugars were described. Expression if Gα and NtRGS1 were investigated simultaneously. Influence of modulation of Gα and NtRGS1 expression on growth parameters of tobacco cell line BY-2 were described. Key words:...

The sensitivity, speed, and reproducibility of modern mass spectrometers enable in-depth new functional looks into the cellular proteome. Thousands of proteins can be detected in a single sample. In Co-Fractionation Mass Spectrometry (CF-MS) method, the input sample is fractionated by any biochemical method of choice. The reduced complexity of each fractionated sample leads to better proteome coverage. The separation profiles provide functional information on the proteins. This application has been used to predict organelle localization based on co-purification with marker proteins. More recently, CF-MS is being used to measure the apparent masses and determine the localization of soluble or membrane-associated protein complexes. This Ph.D. dissertation focuses on the extension of the boundary of CF-MS application to learn how protein complex evolution and protein complex composition have been accomplished. In the first part of this dissertation, the data will be presented on the degree to which variation in protein oligomerization across plant species is present, how proteomics in phylogenetic analysis (phyloproteomics/evolutionary proteomics) helps understand the evolutionary changes, and how oligomerization drives neofunctionalization during plant evolution. The latter part will describe that CF-MS coupled with multiple orthogonal chromatographic separations increases the resolving power of the profiling technique, enabling the composition of protein complexes to be predicted in the subaleurone layers of rice endosperm. Lots of novel protein complexes involved in RNA binding protein, translation, and the tissue-species metabolism will be discussed.

Le développement des plantes nécessite la coordination des mécanismes de différenciation des cellules méristématiques en cellules hautement spécialisées: la division, la croissance et la formation de la géométrie cellulaire. La différenciation et la morphogenèse cellulaires sont étroitement liées et régulées par les propriétés mécaniques de la paroi cellulaire. Les mécanismes conduisant à l’émergence de diverses formes et fonctions des tissus végétaux sont complexes et encore peu compris. Ma thèse de doctorat approfondie les principes mécaniques à la base de la formation des cellules épidermiques ondulées. Je me suis également penché sur l’étude des avantages mécaniques que confèrent les motifs imbriqués. Les cellules épidermiques sont constituées de deux parois cellulaires périclines parallèles reliées par des parois anticlines. Aux jonctions, les cellules épidermiques forment des cavités et des saillies imbriquées les unes aux autres. Des images en 3D, prises en microscopie confocale, de cotylédons marqués par des fluorophores spécifiques à la cellulose montrent une déposition accrue de cellulose au niveau des cavités des parois périclines s’étendant le long des parois anticlines. Le marquage des cotylédons par COS488 démontre également une plus grande abondance de pectines dé-estérifiées aux mêmes sites. J'ai développé des modèles par éléments finis de la déformation de la paroi cellulaire et simulé les disparités biochimiques en alternant les régions plus rigides à travers et au long des parois périclines des deux côtés d'une paroi anticline. Le modèle montre que les parois rigidifiées non déformables se développent en cavités lorsque la pression interne étire la paroi cellulaire. Le modèle suggère également la présence de contraintes mécaniques plus élevées au niveau des saillies. Les résultats du modèle indiquent qu'une boucle de rétroaction positive entre la contrainte et la rigidité de la paroi cellulaire générerait les formes ondulées à partir de différences infinitésimales de rigidité ou de contrainte de la paroi cellulaire. En outre, le modèle suggère que des événements de flambage stochastiques peuvent initier la morphogenèse des cellules. On a longtemps émis l'hypothèse que le motif imbriqué de cellules épidermiques améliore l'adhérence cellule-cellule et donc la résistance de traction de l'épiderme. L'étirage des feuilles d'Arabidopsis de type sauvage ou du mutant any1 (caractérisé par une réduction de l'ondulation cellulaire) n'a montré aucun détachement cellulaire en cas de rupture du tissu. J'ai émis l'hypothèse que les jonctions des cellules ondulantes renforcent la résistance de l'épiderme contre la propagation de fissures. J'ai observé une grande anisotropie dans la réponse mécanique à la rupture de l'épiderme d'oignon selon l’orientation des cellules. Les fissures qui suivent l’alignement des cellules se propagent sans trop de résistance, entraînant une rupture fragile du tissu. Ceci découlerait de la propagation de la ligne de rupture par suite du détachement des cellules. Les fissures se propagent difficilement lorsqu’elles sont perpendiculaires à l'axe principal des cellules. En fracturant des feuilles dont les cellules épidermiques sont ondulées, j'ai remarqué que les fissures se propageaient, par intermittence, à la fois au niveau des jonctions de la cellule et de la paroi cellulaire. J'ai émis l'hypothèse que ce motif de fracture d'épiderme à cellules ondulées se caractérisait par une augmentation de la résistance à la fracture. Pour n’étudier que les effets de la géométrie des cellules sur cette résistance, j’ai éliminé le rôle que jouerait l’anisotropie des matériaux en concevant des modèles physiques macroscopiques de l'épiderme. J’ai gravé au laser des motifs cellulaires sur du poly-méthacrylate de méthyle. De cette façon, le matériau isotrope permettait d'étudier uniquement l'effet de la géométrie cellulaire. Alors que la fracturation des spécimens de contrôle sans gravure et des spécimens avec des cellules gravées longitudinalement ont démontré une rupture fragile, une fracturation transversale aux rangées cellulaires, dans les modèles mimant des cellules d’oignon ou des cellules ondulées de cotylédons d’Arabidopsis, a montré une résistance accrue à la fracture. En conclusion, je démontre que la forme ondulée des cellules épidermiques est le résultat d’une distribution alternée de la rigidité dans la paroi cellulaire, un processus qui pourrait être initié par une anisotropie de stress stochastique due au flambement. De plus, ces formes cellulaires augmentent la résistance à la rupture de l'épiderme végétal en le protégeant contre la propagation des fissures; un mécanisme de défense ingénieux pour les surfaces les plus exposées. / Plant development entails cell division, cell growth and shaping, and the differentiation of meristematic cells into highly specialized cell types. Differentiation and cell shape are closely linked and involve the regulation of the mechanical properties of the cell wall. The mechanisms leading to the generation of the diverse array of shapes and functionalities found in plant tissues are perplexing and poorly understood. In my Ph.D. research, I investigated the mechanical principles underlying the formation of wavy leaf pavement cells. Further, I studied the putative mechanical advantage that emerges from the interlocking patterns. Epidermal pavement cells consist of two parallel periclinal walls connected by vertical anticlinal walls. At the borders, wavy pavement cells make interlocking indentations and protrusions. 3D confocal micrographs of cotyledons stained with cellulose-specific fluorophores revealed a significant accumulation of cellulose at the sites of indentation on the periclinal walls extending down the anticlinal walls. Staining the cotyledon samples with COS488 also suggested a higher abundance of de-esterified pectin at these sites. I developed finite element models of the cell wall deformation and simulated the biochemical inhomogeneities by assigning alternately stiffened regions across and along the periclinal walls on two sides of an anticlinal wall. It was observed that the non-deforming stiffened regions develop into sites of indentations when the internal pressure stretches the cell wall. The model also suggested higher stresses to associate with the neck regions. The model results indicate that a positive feedback loop between stress and cell wall stiffness could generate wavy shapes starting from infinitesimally small differences in cell wall stiffness or stress. Further, the model suggests that stochastic buckling events can initiate the cell shaping process. It has been long hypothesized that the interlocking pattern of pavement cells improves cell-cell adhesion and thus the tensile strength of the epidermis. Stretching to rupture the leaf samples of wild-type Arabidopsis or any1 mutant with reduced cell waviness did not show any cell detachment upon failure. However, I hypothesized the undulating cell borders could enhance the resistance of the epidermis against the propagation of damage. I observed a considerable anisotropy in the tear behavior of onion epidermis parallel and perpendicular to the cells’ main axis. Tears along the cell lines propagated without much resistance resulting in brittle failure of the tissue. This was observed to originate from tears propagating by cell detachment. Perpendicular to the cells’ main axis, tears had considerable difficulty in propagating. Fracturing the leaf samples with wavy epidermal cells, I noticed the cracks propagated in both the cell borders and the cell wall intermittently. I hypothesized that this pattern of fracture in the epidermis with wavy cells indicates an increase in the fracture toughness. To untangle the influence of material anisotropy from the cell geometry on fracture toughness, I designed macroscopic physical models of the epidermis by laser engraving the cell patterns on polymethylmethacrylate. This way, the isotropic material would allow studying only the effect of cell geometry. While fracturing the control specimens with no engraving and the specimens with longitudinally placed cells demonstrated a brittle fracture, fractures transverse to cell lines in the onion cell patterns or across the Arabidopsis cotyledon wavy cell pattern showed an increased fracture toughness. I suggest the wavy shape of pavement cells in the epidermis results from the alternate placement of stiffer regions in the cell wall, a process that can initiate from a stochastic stress anisotropy due to buckling. Further, these shapes increase the fracture toughness of the plant epidermis protecting it against the spread of damage; an ingenious defense mechanism at the most exposed surfaces.

The cell wall provides a complex and rigid structure to the plant for support, protection from environmental factors, and transport. It is mainly composed of polysaccharides, proteins, and lignin. Arabinoxylan (AX), pectin (P), and cellulose (C) are the main components of cereal cell walls and are particularly concentrated in the bran portion of the grain. Cereal arabinoxylans create networks in plant cell walls in which other cell wall polysaccharides are imbedded forming complex matrices. These networks give an insolubility profile to plant cell wall. A previous study in our lab showed that soluble crosslinked arabinoxylan with relatively high residual ferulic acid from corn bran provided advantageous <i>in vitro </i>human fecal fermentation products and promoted butyrogenic gut bacteria. In the present work, arabinoxylan was isolated from corn bran with a mild sodium hydroxide concentration to keep most of its ferulic acid content. Highly ferulated corn bran arabinoxylan was crosslinked to create an insoluble network to mimic the cereal grain cell wall matrices. Firstly, arabinoxylan film (Cax-F), pectin film (P-F), the film produced by embedding pectin into arabinoxylan networks (CaxP-F), and cellulose embedding arabinoxylan matrices (CaxC-F), and embedding the mixture of cellulose and pectin into arabinoxylan networks (CaxCP-F) were fabricated into simulated plant cell wall materials. Water solubility of films in terms of monosaccharide content was examined and revealed that Cax-F was insoluble, and P-F was partially insoluble, and nanosized pectin and cellulose were partially entrapped inside the crosslinked-arabinoxylan matrices. In a further study, these films were used in an <i>in vitro </i>human fecal fermentation assay to understand how gut microbiota access and utilize the different simulated plant cell walls to highlight the role of each plant cell wall component during colonic fermentation. <i>In vitro </i>fecal samples, obtained from three healthy donors were used to ferment the films (Cax-F, P-F, CaxP-F, CaxC-F, and CaxCP-F) and controls (free form of cell wall components -Cax, P and C). The fabricated films that were compositionally similar to cell walls were fermented more slowly than the free polysaccharides (Cax and P). Besides, CaxP-F produced the highest short chain fatty acids (SCFA) amount among the films after 24 hour <i>in vitro </i>fecal fermentation. Regarding specific SCFA, butyrate molar ratio of all films was significantly higher than the free, soluble Cax and P. 16S rRNA gene sequencing explained the differences of the butyrate proportion derived from specific butyrogenic bacteria. Particularly, some bacteria, especially in a butyrogenic genera from Clostridium cluster XIVa, were increased in arabinoxylan films forms compared to the native free arabinoxylan polysaccharide. However, no changes were observed between P and P-F in terms of both end products (SCFA) and microbiota compositions. Moreover, CaxP-F promoted the butyrogenic bacteria in fecal samples compared with pectin alone, arabinoxylan alone, and the arabinoxylan film. Differences in matrix insolubility of the film, which was high for the covalently linked arabinoxylan films, but low for the non-covalent ionic-linked pectin film, appears to play an important role in targeting Clostridial bacterial groups. Overall, the cell wall-like films were useful to understand which bacteria degrade them related to their physical form and location of the fiber polymers. This study showed how fabricated model plant cell wall films influence specificity and competitiveness of some gut bacteria and suggest that fabricated materials using natural fibers might be used for targeted support of certain gut bacteria and bacterial groups.