Caractérisation fonctionnelle de deux facteurs de transcription MYB R2R3 : Rôle dans la formation du bois chez les angiospermes

Le xylème secondaire (appelé bois chez les arbres), est un tissu vasculaire caractérisé par la présence d'un composé phénolique caractéristique, la lignine qui confère hydrophobicité et résistance mécanique aux parois. La différenciation du xylème est un processus complexe qui fait intervenir plusieurs centaines de gènes dont l'expression doit être strictement régulée dans l'espace et dans le temps. Cette coordination spatiotemporelle très fine est assurée au niveau transcriptionnel par des facteurs de transcription. Certains membres de la famille MYB sont par exemple connus pour réguler l'expression des gènes de la voie de biosynthèse des phénylpropanoides, incluant la lignine. Le but de mes travaux était de caractériser fonctionnellement deux facteurs de transcription MYB, EgMYB1 et EgMYB2, isolés à partir d'une banque d'ADNc de xylème d'Eucalyptus gunnii. La majeure partie de mes résultats concerne la caractérisation d'EgMYB1 qui phylogénétiquement, fait partie du sous-groupe 4 des MYB R2R3. Ce sous-groupe comprend plusieurs répresseurs des gènes du métabolisme phénolique. Des expériences de co-expression in vivo dans le tabac appuient l'hypothèse qu'EgMYB1 pourrait agir en tant que régulateur négatif de l'expression des gènes de la voie de biosynthèse de la lignine. Ce rôle potentiel en accord avec l'expression préférentielle d'EgMYB1 dans le xylème de racines et de tiges d'Eucalyptus a été vérifié in planta chez des peupliers transgéniques. En réponse à la surexpression d'EgMYB1, le contenu en lignine du xylème secondaire des tiges est réduit. On note également une diminution du nombre de fibres de phloème, et des taux de transcrits des gènes de la biosynthèse de la lignine. De plus, des analyses transcriptomiques réalisées à partir d'ARNs de xylème et d'écorce de tiges de peupliers surexprimant EgMYB1 met en évidence des classes de gènes sous-exprimés ou surexprimés. De nombreuses similitudes sont retrouvées avec les classes de gènes dérégulées chez les mutants affectés dans une des étapes de la voie de biosynthèse de la lignine, renforçant le rôle initialement proposé pour EgMYB1. Les peupliers surexprimant EgMYB1 montrent également d'intéressants phénotypes foliaires qui ont été étudiés. La caractérisation fonctionnelle d'EgMYB2 réalisée chez des tabacs transgéniques surexpresseurs, nous a amenés à proposer un rôle d'activateur transcriptionnel de la voie de biosynthèse des lignines. Les résultats présentés dans cette thèse, apportent des indications claires quant aux rôles opposés des facteurs de transcription EgMYB1 et EgMYB2 dans la lignification lors de la formation du bois.Lignin is one of the major characteristics of plant secondary cell walls. In trees, it accumulates mostly during the differentiation of secondary xylem (wood). It provides hydrophobicity to vessels elements and imparts mechanical resistance of the stem. Transcriptional regulation of gene expression during the different stages of xylem cell differentiation is complex; it is thought to involve many molecular actors to enable tight coordination of cellular events. R2R3 MYB transcription factors play a role in this process by regulating the biosynthesis of phenolic compounds including lignin. The goal of my research was to functionally characterize the R2R3-MYB transcription factors, EgMYB2 and EgMYB1, isolated from a Eucalyptus gunnii xylem cDNA library. Most of my results are focused on the characterization of EgMYB1. Phylogenetically, EgMYB1 was classified as part of the R2R3 MYB subgroup 4 containing repressors of phenylpropanoid biosynthesis genes. Together, its expression profile in Eucalyptus and co-infiltration in tobacco results suggested that EgMYB1 could act as a negative regulator of lignin biosynthesis gene expression during wood formation. This putative role was further studied in planta using transgenic poplars over-expressing EgMYB1. Stems of the transgenic plants contained less lignin in the secondary xylem, fewer phloem fibers and lignin biosynthesis genes were down regulated compared to controls. Transcriptomic analysis showed that similar classes of genes were differentially expressed between the transgenic over-expressing EgMYB1 poplars and lignin biosynthesis mutants, consistent with its role as a repressor of the lignin biosynthesis pathway. We also investigated the intriguing foliar phenotypes of poplars over-expressing EgMYB1. Functional characterization of EgMYB2 achieved in transgenic tobacco plants over-expressing this transcription factor, led us to propose a role of activator of the lignin biosynthetic pathway during xylogenesis. The data presented herein provide clear indications as to the antagonist roles of EgMYB2 and EgMYB1 in lignin biosynthesis during wood formation

On computing tree and path decompositions with metric constraints on the bags

We here investigate on the complexity of computing the \emph{tree-length} and the \emph{tree-breadth} of any graph $G$, that are respectively the best possible upper-bounds on the diameter and the radius of the bags in a tree decomposition of $G$. \emph{Path-length} and \emph{path-breadth} are similarly defined and studied for path decompositions. So far, it was already known that tree-length is NP-hard to compute. We here prove it is also the case for tree-breadth, path-length and path-breadth. Furthermore, we provide a more detailed analysis on the complexity of computing the tree-breadth. In particular, we show that graphs with tree-breadth one are in some sense the hardest instances for the problem of computing the tree-breadth. We give new properties of graphs with tree-breadth one. Then we use these properties in order to recognize in polynomial-time all graphs with tree-breadth one that are planar or bipartite graphs. On the way, we relate tree-breadth with the notion of \emph{$k$-good} tree decompositions (for $k=1$), that have been introduced in former work for routing. As a byproduct of the above relation, we prove that deciding on the existence of a $k$-good tree decomposition is NP-complete (even if $k=1$). All this answers open questions from the literature.Comment: 50 pages, 39 figure

Connected Tropical Subgraphs in Vertex-Colored Graphs

International audienceA subgraph of a vertex-colored graph is said to be tropical whenever it contains each color of the graph. In this work we study the problem of finding a minimal connected tropical subgraph. We first show that this problem is NP-Hard for trees, interval graphs and split graphs, but polynomial when the number of colors is logarithmic in terms of the order of the graph (i.e. FPT). We then provide upper bounds for the order of the minimal connected tropical subgraph under various conditions. We finally study the problem of finding a connected tropical subgraph in a randomly vertex-colored random graph

Plant Extracellular Vesicles and Nanovesicles: Focus on Secondary Metabolites, Proteins and Lipids with Perspectives on Their Potential and Sources

While human extracellular vesicles (EVs) have attracted a big deal of interest and have been extensively characterized over the last years, plant-derived EVs and nanovesicles have earned less attention and have remained poorly investigated. Although a series of investigations already revealed promising beneficial health effects and drug delivery properties, adequate (pre)clinical studies are rare. This fact might be caused by a lack of sources with appropriate qualities. Our study introduces plant cell suspension culture as a new and well controllable source for plant EVs. Plant cells, cultured in vitro, release EVs into the growth medium which could be harvested for pharmaceutical applications. In this investigation we characterized EVs and nanovesicles from distinct sources. Our findings regarding secondary metabolites indicate that these might not be packaged into EVs in an active manner but enriched in the membrane when lipophilic enough, since apparently lipophilic compounds were associated with nanovesicles while more hydrophilic structures were not consistently found. In addition, protein identification revealed a possible explanation for the mechanism of EV cell wall passage in plants, since cell wall hydrolases like 1,3-β-glucosidases, pectinesterases, polygalacturonases, β-galactosidases and β-xylosidase/α-L-arabinofuranosidase 2-like are present in plant EVs and nanovesicles which might facilitate cell wall transition. Further on, the identified proteins indicate that plant cells secrete EVs using similar mechanisms as animal cells to release exosomes and microvesicles

Estimates of n-widths of Sobolev's classes on compact globally symmetric spaces of rank one

AbstractEstimates of Kolmogorov's and linear n-widths of Sobolev's classes on compact globally symmetric spaces of rank 1 (i.e. on Sd, Pd(R), Pd(C), Pd(H), P16(Cay)) are established. It is shown that these estimates have sharp orders in different important cases. New estimates for the (p,q)-norms of multiplier operators Λ={λk}k∈N are given. We apply our results to get sharp orders of best polynomial approximation and n-widths

EgMYB2, a new transcriptional activator from Eucalyptus xylem, regulates secondary cell wall formation and lignin biosynthesis

International audienceEgMYB2, a member of a new subgroup of the R2R3 MYB family of transcription factors, was cloned from a library consisting of RNA from differentiating Eucalyptus xylem. EgMYB2 maps to a unique locus on the Eucalyptus grandis linkage map and co-localizes with a quantitative trait locus (QTL) for lignin content. Recombinant EgMYB2 protein was able to bind specifically the cis-regulatory regions of the promoters of two lignin biosynthetic genes, cinnamoyl-coenzyme A reductase (CCR) and cinnamyl alcohol dehydrogenase (CAD), which contain MYB consensus binding sites. EgMYB2 was also able to regulate their transcription in both transient and stable expression assays. Transgenic tobacco plants over-expressing EgMYB2 displayed phenotypic changes relative to wild-type plants, among which were a dramatic increase in secondary cell wall thickness, and an alteration of the lignin profiles. Transcript abundance of genes encoding enzymes specific to lignin biosynthesis was increased to varying extents according to the position of individual genes in the pathway,whereas core phenylpropanoid geneswere not significantly affected. Together these results suggest a role for EgMYB2 in the co-ordinated control of genes belonging to the monolignol-specific pathway, and therefore in the biosynthesis of lignin and the regulation of secondary cell wall formation

Molecular investigation of Tuscan sweet cherries sampled over three years: gene expression analysis coupled to metabolomics and proteomics.

peer reviewedSweet cherry (Prunus avium L.) is a stone fruit widely consumed and appreciated for its organoleptic properties, as well as its nutraceutical potential. We here investigated the characteristics of six non-commercial Tuscan varieties of sweet cherry maintained at the Regional Germplasm Bank of the CNR-IBE in Follonica (Italy) and sampled ca. 60 days post-anthesis over three consecutive years (2016-2017-2018). We adopted an approach merging genotyping and targeted gene expression profiling with metabolomics. To complement the data, a study of the soluble proteomes was also performed on two varieties showing the highest content of flavonoids. Metabolomics identified the presence of flavanols and proanthocyanidins in highest abundance in the varieties Morellona and Crognola, while gene expression revealed that some differences were present in genes involved in the phenylpropanoid pathway during the 3 years and among the varieties. Finally, proteomics on Morellona and Crognola showed variations in proteins involved in stress response, primary metabolism and cell wall expansion. To the best of our knowledge, this is the first multi-pronged study focused on Tuscan sweet cherry varieties providing insights into the differential abundance of genes, proteins and metabolites

Diversity under threat: Connecting genetic diversity and threat mapping to set conservation priorities for Juglans regia L. populations in Central Asia

Central Asia is an important center of diversity for common walnut (Juglans regia L.). We characterized the genetic diversity of 21 wild and cultivated populations across Kyrgyzstan, Tajikistan and Uzbekistan. A complete threat assessment was performed evaluating the short-term threats from overexploitation, overgrazing, landslides, and fragmentation as well as long-term threats from climate change. Based on key indicators of genetic diversity and threat magnitude, we developed an approach to identify wild populations for priority conservation and defined appropriate measures to conserve their genetic diversity based on the specific threat magnitudes. We propose (i) ex-situ conservation in areas with ‘severe threat level’ due to climate change and (ii) in-situ conservation in areas with ‘minor threat level’ due to climate change. In addition, we suggest (iii) assisted natural regeneration in areas where ‘minor threat levels’ from climate change coincide with ‘severe threat levels’ from one or more short-term threats. Our research shows high levels of genetic diversity as well as high threat levels in the walnut populations examined across three countries. Overgrazing and overexploitation were identified as the two most important threats whereas climate changed turned out to be of minor concern in the near future. Four out of the five populations selected for priority conservation are severely threatened: Kara-Alma (KGP3) and Sariosiyo (UZP4) by overexploitation and overgrazing, Vanj (TJP2) by landslides and fragmentation and Bostanlyk_2 (UZP2) by overexploitation. Only the Baljuvon population (TJP4) in Tajikistan shows minor threat levels in the short and long term. Thus, for all five priority populations the most appropriate conservation approach appears to be in-situ conservation. Spontaneous natural regeneration should be sufficient to ensure the long-term survival of the Baljuvon population (TJP4), while for the other four priority populations assisted natural regeneration may be needed. This network of five sites represents the minimum number of conservation areas for wild walnut in the three countries that should be established to capture a significant amount of the species genetic diversity. Conservation efforts could be extended to home gardens, which are a valuable genetic reservoir for the wild diversity found in the region

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

Extracorporeal Membrane Oxygenation for Severe Acute Respiratory Distress Syndrome associated with COVID-19: An Emulated Target Trial Analysis.

RATIONALE: Whether COVID patients may benefit from extracorporeal membrane oxygenation (ECMO) compared with conventional invasive mechanical ventilation (IMV) remains unknown. OBJECTIVES: To estimate the effect of ECMO on 90-Day mortality vs IMV only Methods: Among 4,244 critically ill adult patients with COVID-19 included in a multicenter cohort study, we emulated a target trial comparing the treatment strategies of initiating ECMO vs. no ECMO within 7 days of IMV in patients with severe acute respiratory distress syndrome (PaO2/FiO2 <80 or PaCO2 ≥60 mmHg). We controlled for confounding using a multivariable Cox model based on predefined variables. MAIN RESULTS: 1,235 patients met the full eligibility criteria for the emulated trial, among whom 164 patients initiated ECMO. The ECMO strategy had a higher survival probability at Day-7 from the onset of eligibility criteria (87% vs 83%, risk difference: 4%, 95% CI 0;9%) which decreased during follow-up (survival at Day-90: 63% vs 65%, risk difference: -2%, 95% CI -10;5%). However, ECMO was associated with higher survival when performed in high-volume ECMO centers or in regions where a specific ECMO network organization was set up to handle high demand, and when initiated within the first 4 days of MV and in profoundly hypoxemic patients. CONCLUSIONS: In an emulated trial based on a nationwide COVID-19 cohort, we found differential survival over time of an ECMO compared with a no-ECMO strategy. However, ECMO was consistently associated with better outcomes when performed in high-volume centers and in regions with ECMO capacities specifically organized to handle high demand. This article is open access and distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives License 4.0 (http://creativecommons.org/licenses/by-nc-nd/4.0/)