Annabelle Déjardin

Wood formation in Angiosperms

Wood formation is a complex biological process, involving five major developmental steps, including (1) cell division from a secondary meristem called the vascular cambium, (2) cell expansion (cell elongation and radial enlargement), (3) secondary cell wall deposition, (4) programmed cell death, and (5) heartwood formation. Thanks to the development of genomic studies in woody species, as well as genetic engineering, recent progress has been made in the understanding of the molecular mechanisms underlying wood formation. In this review, we will focus on two different aspects, the lignification process and the control of microfibril angle in the cell wall of wood fibres, as they are both key features of wood material properties.

Purification, characterization and physiological role of sucrose synthase in the pea seed coat (Pisum sativum L.)

The seed coat is a maternal organ which surrounds the embryo and is involved in the control of its nutrition. This study with pea (Pisum sativum L.) was conducted to understand more fully the sucrose/starch interconversions occurring in the seed coat. The concentrations of soluble sugars, the starch content, and the activities of the sucrose-metabolizing enzymes, sucrose synthase (Sus; EC 2.4.1.13), alkaline and soluble acid invertase (EC 3.2.1.26) and sucrose-phosphate synthase (SPS; EC 2.4.1.14) were compared at four developmental stages during seed filling. Among the four enzymes, only Sus activity was very high and strongly correlated with the starch concentration in the seed coat. Sucrose synthase catalyses the cleavage of sucrose in the presence of UDP into UDP-glucose and fructose. Sucrose synthase was purified from pea seed coats in a three-step protocol, consisting of diethylaminoethyl-Sephacel chromatography, gel filtration and affinity chromatography. The enzyme was characterized at the biochemical and molecular levels. Sucrose synthase exhibits biochemical properties which allow it to function in the direction of both sucrose cleavage and synthesis. The mass-action ratio of its four substrates was close to the theoretical equilibrium constant at the four developmental stages we studied. A labelling experiment on seed coats has shown that Sus activity is reversible in vivo and can produce 37% of neosynthesized sucrose in the seed coat cells (minimum value). It is concluded that Sus could play a central role in the control of sucrose concentration in the seed coat cells in response to the demand for sucrose in the embryo during the development of the seed.

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

Abstract 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.

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

BackgroundPlant 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.FindingsIn 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.ConclusionsThe 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.

Different Routes for Conifer- and Sinapaldehyde and Higher Saccharification upon Deficiency in the Dehydrogenase CAD1

Down-regulation of CAD1 in poplar leads to different metabolic routes for coniferaldehyde and sinapaldehyde and alters lignin amount and structure, improving the physicochemical properties of wood for saccharification. In the search for renewable energy sources, genetic engineering is a promising strategy to improve plant cell wall composition for biofuel and bioproducts generation. Lignin is a major factor determining saccharification efficiency and, therefore, is a prime target to engineer. Here, lignin content and composition were modified in poplar (Populus tremula × Populus alba) by specifically down-regulating CINNAMYL ALCOHOL DEHYDROGENASE1 (CAD1) by a hairpin-RNA-mediated silencing approach, which resulted in only 5% residual CAD1 transcript abundance. These transgenic lines showed no biomass penalty despite a 10% reduction in Klason lignin content and severe shifts in lignin composition. Nuclear magnetic resonance spectroscopy and thioacidolysis revealed a strong increase (up to 20-fold) in sinapaldehyde incorporation into lignin, whereas coniferaldehyde was not increased markedly. Accordingly, ultra-high-performance liquid chromatography-mass spectrometry-based phenolic profiling revealed a more than 24,000-fold accumulation of a newly identified compound made from 8-8 coupling of two sinapaldehyde radicals. However, no additional cinnamaldehyde coupling products could be detected in the CAD1-deficient poplars. Instead, the transgenic lines accumulated a range of hydroxycinnamate-derived metabolites, of which the most prominent accumulation (over 8,500-fold) was observed for a compound that was identified by purification and nuclear magnetic resonance as syringyl lactic acid hexoside. Our data suggest that, upon down-regulation of CAD1, coniferaldehyde is converted into ferulic acid and derivatives, whereas sinapaldehyde is either oxidatively coupled into S′(8-8)S′ and lignin or converted to sinapic acid and derivatives. The most prominent sink of the increased flux to hydroxycinnamates is syringyl lactic acid hexoside. Furthermore, low-extent saccharification assays, under different pretreatment conditions, showed strongly increased glucose (up to +81%) and xylose (up to +153%) release, suggesting that down-regulating CAD1 is a promising strategy for improving lignocellulosic biomass for the sugar platform industry.

Non-cellulosic polysaccharide distribution during G-layer formation in poplar tension wood fibers: abundance of rhamnogalacturonan I and arabinogalactan proteins but no evidence of xyloglucan

AbstractMain conclusionRG-I and AGP, but not XG, are associated to the building of the peculiar mechanical properties of tension wood. Hardwood trees produce tension wood (TW) with specific mechanical properties to cope with environmental cues. Poplar TW fibers have an additional cell wall layer, the G-layer responsible for TW mechanical properties. We investigated, in two poplar hybrid species, the molecules potentially involved in the building of TW mechanical properties. First, we evaluated the distribution of the different classes of non-cellulosic polysaccharides during xylem fiber differentiation, using immunolocalization. In parallel, G-layers were isolated and their polysaccharide composition determined. These complementary approaches provided information on the occurrence of non-cellulosic polysaccharides during G-fiber differentiation. We found no evidence of the presence of xyloglucan (XG) in poplar G-layers, whereas arabinogalactan proteins (AGP) and rhamnogalacturonan type I pectins (RG-I) were abundant, with an apparent progressive loss of RG-I side chains during G-layer maturation. Similarly, the intensity of immunolabeling signals specific for glucomannans and glucuronoxylans varies during G-layer maturation. RG-I and AGP are best candidate matrix components to be responsible for TW mechanical properties.

Is the G-Layer a Tertiary Cell Wall?

HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. Is the G-Layer a Tertiary Cell Wall? Bruno Clair, Annabelle Dejardin, Gilles Pilate, Tancrède Alméras

Lessons from 25 Years of GM Tree Field Trials in Europe and Prospects for the Future

It is common agronomic practice to perform a formal evaluation of the behaviour of new varieties under natural field conditions. Accordingly, shortly after the optimization of genetic engineering techniques on trees, a number of field trials were set up to assess GM trees modified for different genes. Here, we review the work that has been done in this arena in Europe over the last 25 years, and summarize what we learned from these experiments. GM tree field trials remain the exception rather than the rule in Europe. Several trials have been destroyed by anti-GMO activists and it is becoming increasingly difficult to obtain authorization for a GM tree field trial. These increasing constraints on GM tree trials within Europe are both surprising and counter-productive as we learned a lot from the past 25 years of experiments and the results were promisingly positive: (1) Phenotypic effects resulting from transgene expression in GM trees grown in the field appears to be stable, albeit variable; (2) most field studies have validated earlier observations made under greenhouse conditions, although in some cases the modification of target traits was less obvious in fluctuating field environments, and in a few cases had severe growth and developmental penalties; (3) non-target effects were consistently within the range of natural variation. Overall, the European GM tree field trials failed to exemplify any significant tangible risks. Based on this evidence, it seems appropriate that Europe should now move forward beyond small confined trials to larger scale experiments better fitted to a broader context of evaluation and environmental assessment.

Scientific Research Related to Genetically Modified Trees

Over the last decade, we have witnessed impressive advances in tree molecular biology and the consolidation of tree genomics. We have essentially moved from a small portfolio of genes focusing on a specific genetic trait to large databases including thousands of genes and their respective expression profiles. In 2006, we saw the publication of the first genomic sequence of a tree, the model tree species Populus trichocarpa. Though, not surprisingly, much progress has been made with Populus, impressive research results have also been realized in more recalcitrant coniferous species such as pines and spruces.

The effect of altered lignin composition on mechanical properties of CINNAMYL ALCOHOL DEHYDROGENASE (CAD) deficient poplars

AbstractMain conclusionCAD-deficient poplars enabled studying the influence of altered lignin composition on mechanical properties. Severe alterations in lignin composition did not influence the mechanical properties. Wood represents a hierarchical fiber-composite material with excellent mechanical properties. Despite its wide use and versatility, its mechanical behavior has not been entirely understood. It has especially been challenging to unravel the mechanical function of the cell wall matrix. Lignin engineering has been a useful tool to increase the knowledge on the mechanical function of lignin as it allows for modifications of lignin content and composition and the subsequent studying of the mechanical properties of these transgenics. Hereby, in most cases, both lignin composition and content are altered and the specific influence of lignin composition has hardly been revealed. Here, we have performed a comprehensive micromechanical, structural, and spectroscopic analysis on xylem strips of transgenic poplar plants, which are downregulated for cinnamyl alcohol dehydrogenase (CAD) by a hairpin-RNA-mediated silencing approach. All parameters were evaluated on the same samples. Raman microscopy revealed that the lignin of the hpCAD poplars was significantly enriched in aldehydes and reduced in the (relative) amount of G-units. FTIR spectra indicated pronounced changes in lignin composition, whereas lignin content was not significantly changed between WT and the hpCAD poplars. Microfibril angles were in the range of 18°–24° and were not significantly different between WT and transgenics. No significant changes were observed in mechanical properties, such as tensile stiffness, ultimate stress, and yield stress. The specific findings on hpCAD poplar allowed studying the specific influence of lignin composition on mechanics. It can be concluded that the changes in lignin composition in hpCAD poplars did not affect the micromechanical tensile properties.