Genome-Wide Analysis of LIM Gene Family in Populus trichocarpa, Arabidopsis thaliana, and Oryza sativa

In Eukaryotes, LIM proteins act as developmental regulators in basic cellular processes such as regulating the transcription or organizing the cytoskeleton. The LIM domain protein family in plants has mainly been studied in sunflower and tobacco plants, where several of its members exhibit a specific pattern of expression in pollen. In this paper, we finely characterized in poplar six transcripts encoding these proteins. In Populus trichocarpa genome, the 12 LIM gene models identified all appear to be duplicated genes. In addition, we describe several new LIM domain proteins deduced from Arabidopsis and rice genomes, raising the number of LIM gene models to six for both species. Plant LIM genes have a core structure of four introns with highly conserved coding regions. We also identified new LIM domain proteins in several other species, and a phylogenetic analysis of plant LIM proteins reveals that they have undergone one or several duplication events during the evolution. We gathered several LIM protein members within new monophyletic groups. We propose to classify the plant LIM proteins into four groups: αLIM1, βLIM1, γLIM2, and δLIM2, subdivided according to their specificity to a taxonomic class and/or to their tissue-specific expression. Our investigation of the structure of the LIM domain proteins revealed that they contain many conserved motifs potentially involved in their function

Expressed Sequence Tags from Poplar Wood Tissues - A Comparative Analysis from Multiple Libraries

International audienceXylogenesis involves successive developmental processes - cambial division, cell expansion and differentiation, cell death - each occurring along a gradient from the cambium to the pith of the stem. Taking advantage of the high level of organisation of wood tissues, we isolated cambial zone (CZ), differentiating xylem (DX) and mature xylem (MX) from both tension wood (TW) and opposite wood (OW) of bent poplars. Four different cDNA libraries were then constructed and used to generate 10 062 EST, reflecting the genes expressed in the different wood tissues. For the most abundant clusters, the EST distributions were compared between libraries in order to identify genes specific or over-represented at some specific developmental stages. They clearly showed a developmental shift between CZ and DX, whereas there is a continuity of development between DX and MX. CZ was mainly characterized by clusters of genes involved in cell cycle, protein synthesis and fate. Interestingly, two clusters with no assigned function were found specific to the cambial zone. In DX and MX, clusters were mostly involved in methylation of lignin precursors and microtubule cytoskeleton. In addition, in DX, EST from TW and OW were compared: five clusters of arabinogalactan proteins, one for sucrose synthase and one for fructokinase were specific or over-represented in TW. Moreover, a putative transcription factor and a cluster of unknown function were also identified in DX-TW. The informative comparison of multiple libraries prepared from wood tissues led to the identification of genes - some with still unknown functions - putatively involved in xylogenesis and tension wood formation

The PMT-driven p -coumaroylation of poplar lignins impacts lignin structure and improves wood saccharification

Transgenic poplars ( Populus tremula x Populus alba , clone INRA 717-1B4) were produced by introducing the Brachypodium distachyon Bradi2g36910 ( BdPMT1 ) gene driven by the Arabidopsis ( Arabidopsis thaliana) Cinnamate 4-Hydroxylase ( AtC4H ) promoter in the wild-type (WT) line and in a line overexpressing the Arabidopsis Ferulate 5-Hydroxylase ( AtF5H). BdPMT1 encodes a transferase which catalyzes the acylation of monolignols by p- coumaric acid (CA). Several BdPMT1 - OE/WT and BdPMT1-OE/AtF5H-OE transgenic lines were grown in the greenhouse and BdPMT1 expression in xylem was confirmed by RT-PCR. The analysis of the cell walls (CW) of poplar stems and of corresponding purified dioxan lignins (DL) revealed that the BdPMT1 -OE lignins were as p -coumaroylated as the lignins of C3 grass straws. For some transformants, CA levels even reached about 11 mg/g CW and 66 mg/g DL, which by far exceeds those of Brachypodium or wheat samples. This unprecedentedly high p -coumaroylation of poplar lignins affected neither the poplar growth, nor the stem lignin content. By contrast, the transgenic lignins were structurally modified, with an increase of terminal units with free phenolic groups. Relative to controls, this increase argues for a reduced polymerization degree of BdPMT1 -OE lignins and makes them more soluble in cold NaOH solution. The p -coumaroylation of poplar samples, up to the levels of C3 grasses, improved the saccharification yield of alkali-pretreated poplar CW. These results establish that the genetically-driven p -coumaroylation of lignins is a promising strategy to make wood lignins more susceptible to the alkaline treatments that can be used during the industrial processing of lignocellulosics

Kinetic assessment of global gene expression changes during tension wood formation in poplar

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Poplar genes encoding fasciclin-like arabinogalactan proteins are highly expressed in tension wood

International audience• Fifteen poplar cDNA encoding fasciclin-like arabinogalactan proteins (PopFLAs) were finely characterized, whereas the presence of arabinogalactan proteins (AGPs) was globally assessed during wood formation. • PopFLAs transcript accumulation was analysed through EST distribution in cDNA libraries, semi-quantitative RT-PCR, microarray experiment and Northern blot analysis. Similarly, AGPs contents were globally quantified by rocket electrophoresis. AGPs accumulation was further examined by Western blotting and immunocytolocalization. • Ten PopFLAs were specifically expressed in tension wood (TW) and not expressed in the cambial zone. Rocket electrophoresis revealed important AGPs accumulation in TW xylem. An anti-AGPs specific antibody recognized two proteins preferentially present in the cell wall-bound fraction from TW. Immunocytochemistry revealed a strong labelling close to the inner part of the G-layer of TW fibres. • PopFLAs are expressed in xylem and many are up-regulated in TW. It is suggested that some PopFLAs accumulating at the inner side of the G-layer may have a specific function in the building of this layer. PopFLAs expression may therefore be linked to the specific mechanical properties of TW

Expression analysis of <it>LIM </it>gene family in poplar, toward an updated phylogenetic classification

Abstract Background Plant LIM domain proteins may act as transcriptional activators of lignin biosynthesis and/or as actin binding and bundling proteins. Plant LIM genes have evolved in phylogenetic subgroups differing in their expression profiles: in the whole plant or specifically in pollen. However, several poplar PtLIM genes belong to uncharacterized monophyletic subgroups and the expression patterns of the LIM gene family in a woody plant have not been studied. Findings In this work, the expression pattern of the twelve duplicated poplar PtLIM genes has been investigated by semi quantitative RT-PCR in different vegetative and reproductive tissues. As in other plant species, poplar PtLIM genes were widely expressed in the tree or in particular tissues. Especially, PtXLIM1a, PtXLIM1b and PtWLIM1b genes were preferentially expressed in the secondary xylem, suggesting a specific function in wood formation. Moreover, the expression of these genes and of the PtPLIM2a gene was increased in tension wood. Western-blot analysis confirmed the preferential expression of PtXLIM1a protein during xylem differentiation and tension wood formation. Genes classified within the pollen specific PLIM2 and PLIM2-like subgroups were all strongly expressed in pollen but also in cottony hairs. Interestingly, pairs of duplicated PtLIM genes exhibited different expression patterns indicating subfunctionalisations in specific tissues. Conclusions The strong expression of several LIM genes in cottony hairs and germinating pollen, as well as in xylem fibers suggests an involvement of plant LIM domain proteins in the control of cell expansion. Comparisons of expression profiles of poplar LIM genes with the published functions of closely related plant LIM genes suggest conserved functions in the areas of lignin biosynthesis, pollen tube growth and mechanical stress response. Based on these results, we propose a novel nomenclature of poplar LIM domain proteins.</p

Expression analysis of LIM gene family in poplar, toward an updated phylogenetic classification.

International audienceUNLABELLED: ABSTRACT: BACKGROUND: Plant LIM domain proteins may act as transcriptional activators of lignin biosynthesis and/or as actin binding and bundling proteins. Plant LIM genes have evolved in phylogenetic subgroups differing in their expression profiles: in the whole plant or specifically in pollen. However, several poplar PtLIM genes belong to uncharacterized monophyletic subgroups and the expression patterns of the LIM gene family in a woody plant have not been studied. FINDINGS: In this work, the expression pattern of the twelve duplicated poplar PtLIM genes has been investigated by semi quantitative RT-PCR in different vegetative and reproductive tissues. As in other plant species, poplar PtLIM genes were widely expressed in the tree or in particular tissues. Especially, PtXLIM1a, PtXLIM1b and PtWLIM1b genes were preferentially expressed in the secondary xylem, suggesting a specific function in wood formation. Moreover, the expression of these genes and of the PtPLIM2a gene was increased in tension wood. Western-blot analysis confirmed the preferential expression of PtXLIM1a protein during xylem differentiation and tension wood formation. Genes classified within the pollen specific PLIM2 and PLIM2-like subgroups were all strongly expressed in pollen but also in cottony hairs. Interestingly, pairs of duplicated PtLIM genes exhibited different expression patterns indicating subfunctionalisations in specific tissues. CONCLUSIONS: The strong expression of several LIM genes in cottony hairs and germinating pollen, as well as in xylem fibers suggests an involvement of plant LIM domain proteins in the control of cell expansion. Comparisons of expression profiles of poplar LIM genes with the published functions of closely related plant LIM genes suggest conserved functions in the areas of lignin biosynthesis, pollen tube growth and mechanical stress response. Based on these results, we propose a novel nomenclature of poplar LIM domain proteins

Identification <em> in vivo</em> des ADN cibles de facteurs de transcription impliqués dans la formation du bois de tension

National audienceLorsqu’un arbre feuillu est incliné en raison d’une contrainte gravitationnelle ou mécanique (vent, sol en pente), il met en place sur la partie supérieure des tiges inclinées, un bois de réaction, appelé bois de tension permettant aux tiges de retrouver une position d’équilibre (bien souvent verticale). Au niveau anatomique, le bois de tension se caractérise par la présence de fibres particulières aux parois très épaisses, appelées fibres G. La paroi des fibres G présente une couche surnuméraire, pauvre en lignines, très riche en cellulose dont les microfibrilles sont orientées quasi parallèlement à l’axe de la fibre. Le bois de tension représente un bon système biologique pour décrypter certains mécanismes importants de la formation du bois. Les facteurs de transcription (FT) jouent un rôle central dans la régulation de ces mécanismes. L’objectif de ces travaux est d’identifier les séquences d’ADN cibles de certains FT choisis (Myb1, Myb21, VND7), potentiellement importants pour la formation du bois. Pour cela, la technique ChIP-SEQ (Immunoprécipitation de chromatine suivie par un séquençage à très haut-débit) a été développée afin d’identifier in vivo ces ADN cibles. Cette technique, bien que souvent employée en biologie animal [1] [2], est encore peu utilisée en biologie végétal. Suite à la ChIP, une analyse qPCR permet de mesurer l’enrichissement des cibles connues afin de vérifier l’efficacité de l’immunoprécipitation, avant de séquencer les échantillons. Actuellement les premiers essais de ChIP, analysés en qPCR, ont permis de valider la méthode, et ont mis en évidence l’enrichissement en un promoteur cible connu pour un des FT choisis. L’avancée des résultats sera présentée lors du congrès