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

Identification of the laccase-like multicopper oxidase gene family of sweet cherry (Prunus avium L.) and expression analysis in six ancient Tuscan varieties

Laccase-like multicopper oxidases (LMCOs) are versatile enzymes used as biocatalysts performing the oxidation of different substrates of industrial relevance, with or without the intervention of a mediator. They have attracted a lot of interest for biotechnological applications in light of their ecofriendliness: they indeed oxidize the substrate(s) by coupling the four electron reduction of the final acceptor, molecular oxygen (O2), to water. Plant LMCOs represent a still poorly studied, important class of oxidoreductases controlling e.g. the post-harvest quality of fruits and enabling the tailoring of designer energy crops. We here sought to identify the LMCOs in Prunus avium L., whose fruits are rich in bioactive molecules, but are also highly perishable. The goal was to analyze them using bioinformatics (phylogenetic and in silico structural analyses) and to perform a targeted expression study on a subset of genes in six ancient varieties from Tuscany, all threatened by genetic erosion. These sweet cherry varieties contain higher amount of bioactive molecules, as compared to commercial counterparts. The results shown demonstrate strikingly different gene expression patterns in the six ancient varieties (‘Benedetta’, ‘Carlotta’, ‘Crognola’, ‘Maggiola’, ‘Morellona’, ‘Moscatella’) belonging to the Tuscan Regional Bank of Germplasm, as compared to a widely used commercial one (‘Durone’). The motivation of this study is the economic importance of P. avium and the involvement of LMCOs in post-harvest fruit parameters, like color. The results presented pave the way to follow-up researches on LMCOs of sweet cherry exploring post-harvest fruit parameters (e.g. anthocyanin stability responsible for pericarp browning and the preservation of the appealing red color), as well as developmental processes, like stony pit formation

Tuscan Varieties of Sweet Cherry Are Rich Sources of Ursolic and Oleanolic Acid: Protein Modeling Coupled to Targeted Gene Expression and Metabolite Analyses

The potential of six ancient Tuscan sweet cherry (Prunus avium L.) varieties as a source of health-promotingpentacyclictriterpenesishereevaluatedbymeansofatargetedgeneexpressionand metabolite analysis. By using a sequence homology criterion, we identify five oxidosqualene cyclase genes (OSCs) and three cytochrome P450s (CYP85s) that are putatively involved in the triterpene production pathway in sweet cherries. We performed 3D structure prediction and induced-fit docking using cation intermediates and reaction products for some OSCs to predict their function. We show that the Tuscan varieties have different amounts of ursolic and oleanolic acids and that these variations are related to different gene expression profiles. This study stresses the interest of valorizing ancient fruits as alternative sources of functional molecules with nutraceutical value. It also provides information on sweet cherry triterpene biosynthetic genes, which could be the object of follow-up functional studies

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

Characterization of MdMYB68, a suberin master regulator in russeted apples

IntroductionApple russeting is mainly due to the accumulation of suberin in the cell wall in response to defects and damages in the cuticle layer. Over the last decades, massive efforts have been done to better understand the complex interplay between pathways involved in the suberization process in model plants. However, the regulation mechanisms which orchestrate this complex process are still under investigation. Our previous studies highlighted a number of transcription factor candidates from the Myeloblastosis (MYB) transcription factor family which might regulate suberization in russeted or suberized apple fruit skin. Among these, we identified MdMYB68, which was co-expressed with number of well-known key suberin biosynthesis genes.MethodTo validate the MdMYB68 function, we conducted an heterologous transient expression in Nicotiana benthamiana combined with whole gene expression profiling analysis (RNA-Seq), quantification of lipids and cell wall monosaccharides, and microscopy.ResultsMdMYB68 overexpression is able to trigger the expression of the whole suberin biosynthesis pathway. The lipid content analysis confirmed that MdMYB68 regulates the deposition of suberin in cell walls. Furthermore, we also investigated the alteration of the non-lipid cell wall components and showed that MdMYB68 triggers a massive modification of hemicelluloses and pectins. These results were finally supported by the microscopy.DiscussionOnce again, we demonstrated that the heterologous transient expression in N. benthamiana coupled with RNA-seq is a powerful and efficient tool to investigate the function of suberin related transcription factors. Here, we suggest MdMYB68 as a new regulator of the aliphatic and aromatic suberin deposition in apple fruit, and further describe, for the first time, rearrangements occurring in the carbohydrate cell wall matrix, preparing this suberin deposition

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