Breeding grasses for capacity to biofuel production or silage feeding value: an updated list of genes involved in maize secondary cell wall biosynthesis and assembly

In the near future, maize, sorghum, or switchgrass stovers and cereal straws will be a significant source of carbohydrates for sustainable biofuel production, in addition to the current use of grass silage in cattle feeding. However, cell wall properties, including the enzymatic degradability of structural polysaccharides in industrial fermenters or animal rumen, is greatly influenced by the embedding of cell wall carbohydrates in lignin matrix, and the linkages between lignins, p-hydroxycinnamic acids, and arabinoxylans. Breeding for higher and cheaper biofuel or silage production will thus be based on the discovery of genetic traits involved in each cell wall component biosynthesis and deposition in each lignified tissue. Due to its considerable genetic and genomic backgrounds, maize is the relevant model species for identifying traits underlying cell wall degradability variations in grasses. Maize genes involved or putatively involved in the biosynthesis of cell wall phenolic compounds, cell wall carbohydrates and regulation factors were therefore searched for using data available in grass, Arabidopsis, and woody species (mostly poplar and eucalyptus). All maize ortholog genes were searched for using protein sequences and a “blastp” strategy against data available in the www.maizesequence.org database. Genes were also mapped in silico considering their physical position in the same database. Finally, 409 candidate genes putatively involved in secondary cell wall biosynthesis and assembly were shown in the maize genome, out of which 130 were related to phenolic compound biosynthesis, 81 were related to cell wall carbohydrate biosynthesis, and 198 were involved in more or less known regulation mechanisms. Most probable candidate genes involved in regulation and assembly of secondary cell wall belonged to the MYB (45 genes) and NAC (38 genes) families, but also included zinc finger and HDZipIII encoding genes. While genes involved in ferulic acid cross-linkages with other cell wall components were little known, several families putatively involved in (arabino)-xylan chain biosynthesis and in feruloyl transfer were shown, including especially arabinosyl-CoA-acyltransferases, feruloyl-AX b-1,2-xylosyl transferases, and xylan-O-3-arabinosyl transferases. This candidate gene list, which focused on genes and orthologs known to be involved in cell wall component biosynthesis and regulation, cannot be considered as exhaustive. Other genes, whose role in cell wall lignification and deposition have not yet been defined, should very likely be added to the list of candidates required for secondary cell wall assembly. Genes encoding proteins of still unknown function should also be added to the list, as several of the latter are probably involved in lignified tissue biosynthesis and deposition

Comparative expression of cell wall related genes in four maize RILs and one parental line of variable lignin content and cell wall degradability

A comparison of gene expression in maize between the parental line F271 and four RILs derived from the cross F288 x F271 was investigated based on hybridization on the 17,555 probes Affymetrix micro-array, targeting nearly one third of the genes present in maize genomes. The parental line had unfavorable alleles for cell wall degradability traits at the major QTL position in bin 6.06, while the set of RILs had both the favorable allele and high cell wall degradability. 360 genes were differentially expressed in the four RIL in comparison to F271, including nine genes underlying the major QTL position and 36 underlying two other QTL positions. However, their proposed function (whenever is described) do not allow us to firmly consider their involvement in the observed variation of cell wall related traits. Only a few genes involved in monolignol biosynthesis or polymerization located elsewhere in the genome were differentially expressed between the four RILs and F271, corroborating with the fact that these genes are probably not involved in major determinants of cell wall degradability in the studied set of lines. Among the investigated regulation factors, three ZmMYB, one NAC and one C3HC4 zinc finger were differentially expressed between the four RILs and F271, but they were not located in bin 6.06. Notwithstanding, the obtained results especially strengthened the probable involvement of these genes in maize secondary wall assembly and/ or lignification

Comparative expression of cell wall related genes in four maize RILs and one parental line of variable lignin content and cell wall degradability

A comparison of gene expression in maize between the parental line F271 and four RILs derived from the cross F288 x F271 was investigated based on hybridization on the 17,555 probes Affymetrix micro-array, targeting nearly one third of the genes present in maize genomes. The parental line had unfavorable alleles for cell wall degradability traits at the major QTL position in bin 6.06, while the set of RILs had both the favorable allele and high cell wall degradability. 360 genes were differentially expressed in the four RIL in comparison to F271, including nine genes underlying the major QTL position and 36 underlying two other QTL positions. However, their proposed function (whenever is described) do not allow us to firmly consider their involvement in the observed variation of cell wall related traits. Only a few genes involved in monolignol biosynthesis or polymerization located elsewhere in the genome were differentially expressed between the four RILs and F271, corroborating with the fact that these genes are probably not involved in major determinants of cell wall degradability in the studied set of lines. Among the investigated regulation factors, three ZmMYB, one NAC and one C3HC4 zinc finger were differentially expressed between the four RILs and F271, but they were not located in bin 6.06. Notwithstanding, the obtained results especially strengthened the probable involvement of these genes in maize secondary wall assembly and/ or lignification

Morphological features of distribution of branches of the ethmoid arteries on from the shape of the orbit

To study the topography of the lattice of the arteries of the labyrinth and their relationship to the form of the orbit was carried out anthropometric measurements by the method of V. N. Shevkunenko, A. M. Eselevich, T. V. Zolotareva, G. N. Toporov. To determine the shape of the orbit was measured by the following parameters: 1 - the height of the entrance to the eye socket - the distance between the upper and lower walls of the orbit; 2 - entrance width - the distance between the inner and outer walls of the orbit; 3 - the depth - the distance between the inner boundary of the entrance into the orbit to the optic canal

Water Deficit-Responsive QTLs for Cell Wall Degradability and Composition in Maize at Silage Stage

The use of lignocellulosic biomass for animal feed or biorefinery requires the optimization of its degradability. Moreover, biomass crops need to be better adapted to the changing climate and in particular to periods of drought. Although the negative impact of water deficit on biomass yield has often been mentioned, its impact on biomass quality has only been recently reported in a few species. In the present study, we combined the mapping power of a maize recombinant inbred line population with robust near infrared spectroscopy predictive equations to track the response to water deficit of traits associated with biomass quality. The population was cultivated under two contrasted water regimes over 3 consecutive years in the south of France and harvested at silage stage. We showed that cell wall degradability and β-O-4-linked H lignin subunits were increased in response to water deficit, while lignin and p-coumaric acid contents were reduced. A mixed linear model was fitted to map quantitative trait loci (QTLs) for agronomical and cell wall-related traits. These QTLs were categorized as “constitutive” (QTL with an effect whatever the irrigation condition) or “responsive” (QTL involved in the response to water deficit) QTLs. Fifteen clusters of QTLs encompassed more than two third of the 213 constitutive QTLs and 13 clusters encompassed more than 60% of the 149 responsive QTLs. Interestingly, we showed that only half of the responsive QTLs co-localized with constitutive and yield QTLs, suggesting that specific genetic factors support biomass quality response to water deficit. Overall, our results demonstrate that water deficit favors cell wall degradability and that breeding of varieties that reconcile improved drought-tolerance and biomass degradability is possible

Natural Variation in Arabidopsis thaliana as a Tool for Highlighting Differential Drought Responses

To test whether natural variation in Arabidopsis could be used to dissect out the genetic basis of responses to drought stress, we characterised a number of accessions. Most of the accessions belong to a core collection that was shown to maximise the genetic diversity captured for a given number of individual accessions in Arabidopsis thaliana. We measured total leaf area (TLA), Electrolyte Leakage (EL), Relative Water Content (RWC), and Cut Rosette Water Loss (CRWL) in control and mild water deficit conditions. A Principal Component Analysis revealed which traits explain most of the variation and showed that some accessions behave differently compared to the others in drought conditions, these included Ita-0, Cvi-0 and Shahdara. This study relied on genetic variation found naturally within the species, in which populations are assumed to be adapted to their environment. Overall, Arabidopsis thaliana showed interesting phenotypic variations in response to mild water deficit that can be exploited to identify genes and alleles important for this complex trait

Laser heating for the study of the mechanisms involved in nuclear fuel during accidental conditions

Dans un Réacteur à Eau Pressurisé (REP), la modélisation du comportement du combustible en situation accidentelle comme l'Accident par Insertion de Réactivité (Reactivity Initiated Accident - RIA) ou l'Accident par Perte de Réfrigérant Primaire (APRP) est nécessaire pour prédire le comportement du combustible et pour déterminer les critères de sureté du parc en exploitation. Dans le cas du RIA, la validation des codes de simulation repose sur les données expérimentales obtenues lors d'essais intégraux en réacteur de recherches comme Cabri en France ou le NSRR au Japon. Bien que les données ainsi obtenues sont cruciales et permettent de valider les codes de simulation, ces essais ne peuvent bien évidemment pas être multipliés ou paramétrés facilement. De ce fait, certains mécanismes comme la fragmentation du combustible et le relâchement des gaz de fission pendant le transitoire restent difficiles à précisément reproduire. Or, ce relâchement des gaz de fission et sa cinétique peuvent potentiellement conduire à la rupture de la gaine par ballonnement et à une dispersion d'une partie de l'inventaire du crayon combustible dans la cuve du réacteur. Dans ce contexte, ces travaux de thèse aborderont le développement et l'utilisation d'une technique de chauffage par laser originale, basée sur l’utilisation de lasers de hautes-puissances (> kW), capable d'induire des transitoires thermiques d'intérêts sur des échantillons de céramique nucléaire (UO2 appauvri) pour l'étude du combustible nucléaire en conditions accidentelles de type RIA. Associé à ces développements expérimentaux, nous avons développé au cours de ces travaux la modélisation du comportement thermo-mécanique de l’UO2 durant ces chauffes en prenant en compte la spécificité de l'interaction laser-UO2 et le chargement thermique en résultant. Nous avons pour ce faire développé un modèle numérique basé sur la méthode des éléments finis utilisant le solveur thermo-mécanique Cast3m du CEA. En parallèle, la préparation d'échantillons dits « lames minces » nous ont permis de déterminer les propriétés optiques de la céramique afin de décrire le plus fidèlement possible le chargement laser responsable de l'élévation de température dans l'échantillon. Nous verrons que nous pouvons exprimer le terme source de chaleur résultant du chargement laser soit par une description analytique soit de manière statistique par une étape préalable de simulation Monte-Carlo. Le comportement mécanique de l'UO2 est quant à lui décrit par une loi de comportement mécanique que nous avons développé pour rendre compte du comportement de la céramique à hautes-vitesses de sollicitations (>1.s-1) caractéristiques de ces chauffes laser. Cette loi d'endommagement décrit la perte de rigidité du matériau en fonction du mode de chargement (à dominante de traction ou de compression) et de la température. Son application sur des chauffes laser dites annulaires mène à une bonne estimation de l'instant et de la localisation de la fracturation. De plus l’analyse du chargement mécanique et de la répartition des contraintes dans l’échantillon met en évidence un chargement mécanique représentatif du cas prototypique en réacteur de recherche.Enfin nous présenterons des chauffes laser visant à reproduire l'intégralité de la phase de montée en température d'un transitoire RIA et discuterons des perspectives de ce travail et notamment de l’implémentation possible de cette technique de chauffe laser en Laboratoire de Haute-Activité.In a Pressurized Water Reactor (PWR), the modeling of fuel behavior in accident situations such as Reactivity Initiated Accident (RIA) or Loss of Coolant Accident (LOCA) is necessary to predict the fuel behavior and to determine the safety criteria of the operating park. In the case of the RIA, the validation of the simulation codes is based on experimental data obtained during full-scale tests in research reactors such as Cabri in France or NSRR in Japan. Although the data obtained this way are crucial and allow the validation of the simulation codes, these tests can obviously not be multiplied or parameterized easily. Therefore, certain mechanisms such as fuel fragmentation and the release of fission gases during the transient remain difficult to reproduce completely. This release of fission gases and its kinetics can potentially lead to the rupture of the cladding by ballooning and to a dispersion of part of the fuel rod inventory in the reactor vessel. In this context, this thesis will address the development and use of an original laser heating technique, based on the use of high-powered lasers (> kW), capable of inducing thermal transients of interest on nuclear ceramic samples (depleted UO2) for the study of nuclear fuel in accidental RIA type conditions. Associated with these experimental developments, we have developed during this work the modeling of the thermo-mechanical behavior of UO2 during these heating experiments by taking into account the specificity of the laser-UO2 interaction and the resulting thermal loading. We have developed for this purpose a numerical model based on the finite element method using the CEA’s Cast3m thermo-mechanical solver. In parallel, the preparation of samples called "thin slides" allowed us to determine the optical properties of the ceramic in order to describe as accurately as possible the laser loading responsible for the temperature rise in the sample. We will see that we can express the heat source term resulting from the laser loading either by an analytical description or statistically by a preliminary step of Monte-Carlo simulation.The mechanical behavior of UO2 is described by a mechanical behavior law that we have developed to account for the behavior of the ceramic at high strain rates (>1.s-1) characteristic of these laser heating experiments. This damage law describes the loss of stiffness of the material as a function of the loading mode (tension or compression dominated) and temperature. Its application to so called annular laser heating experiments leads to a fairly good estimate of the time and location of fracturing. Moreover, the analysis of the mechanical loading and of the stress distribution in the sample highlights a mechanical loading representative of the prototypical case in a research reactor. Finally, we will present laser heating experiments aiming to reproduce the entire temperature rise phase of a RIA transient and will discuss the perspectives of this work and the possible implementation of this laser heating technique in a High-Activity Laboratory