RESUMO
MAIN CONCLUSION: Cellulosic secondary walls evolved convergently in coralline red macroalgae, reinforcing tissues against wave-induced breakage, despite differences in cellulose abundance, microfibril orientation, and wall structure. Cellulose-enriched secondary cell walls are the hallmark of woody vascular plants, which develop thickened walls to support upright growth and resist toppling in terrestrial environments. Here we investigate the striking presence and convergent evolution of cellulosic secondary walls in coralline red algae, which reinforce thalli against forces applied by crashing waves. Despite ostensible similarities to secondary wall synthesis in land plants, we note several structural and mechanical differences. In coralline red algae, secondary walls contain three-times more cellulose (~ 22% w/w) than primary walls (~ 8% w/w), and their presence nearly doubles the total thickness of cell walls (~ 1.2 µm thick). Field emission scanning electron microscopy revealed that cellulose bundles are cylindrical and lack any predominant orientation in both primary and secondary walls. His-tagged recombinant carbohydrate-binding module differentiated crystalline and amorphous cellulose in planta, noting elevated levels of crystalline cellulose in secondary walls. With the addition of secondary cell walls, Calliarthron genicular tissues become significantly stronger and tougher, yet remain remarkably extensible, more than doubling in length before breaking under tension. Thus, the development of secondary walls contributes to the strong-yet-flexible genicular tissues that enable coralline red algae to survive along wave-battered coastlines throughout the NE Pacific. This study provides an important evolutionary perspective on the development and biomechanical significance of secondary cell walls in a non-model, non-vascular plant.
Assuntos
Parede Celular/metabolismo , Celulose/metabolismo , Alga Marinha/metabolismo , Fenômenos Biomecânicos , Parede Celular/ultraestrutura , Microfibrilas/metabolismo , Microscopia Eletrônica de Varredura , Alga Marinha/ultraestruturaRESUMO
Pycnoporus cinnabarinus laccase and a chimeric laccase-CBM were applied in softwood kraft pulp biobleaching in the presence of 1-hydroxybenzotriazole (HBT). The presence of CBM could enhance the laccase biobleaching potential as a decrease in the enzymatic charge and chlorine dioxide consumption, as well as an increase in pulp brightness were observed. Laccase/HBT treatment could be improved by increasing oxygen pressure from 1 to 3bar and pulp consistency from 5% to 10%. Conversely, under the same conditions, no improvement of laccase-CBM/HBT treatment was observed, indicating a different behavior of both systems. However, laccase-CBM/HBT treatment led to a better preservation of pulp properties. This effect was probably due to fiber surface modifications involving the action of the CBM. Transmission electron microscopy examination of pulp fibers indicated a retention of laccase-CBM inside the pulp fibers due to CBM binding and an increased external microfibrillation of the fibers due to enzymatic treatments.
Assuntos
Biotecnologia/métodos , Celulose 1,4-beta-Celobiosidase/metabolismo , Lacase/metabolismo , Lignina/metabolismo , Papel , Triazóis/metabolismo , Aspergillus niger/enzimologia , Microbiologia Industrial/métodos , Microscopia Eletrônica de Transmissão , Pycnoporus/enzimologiaRESUMO
In the cell walls of higher plants, cellulose chains are present in crystalline microfibril, with an amorphous part at the surface, or present as amorphous material. To assess the distribution and relative occurrence of the two forms of cellulose in the inflorescence stem of Arabidopsis, we used two carbohydrate-binding modules, CBM3a and CBM28, specific for crystalline and amorphous cellulose, respectively, with immunogold detection in TEM. The binding of the two CBMs displayed specific patterns suggesting that the synthesis of cellulose leads to variable nanodomains of cellulose structures according to cell type. In developing cell walls, only CBM3a bound significantly to the incipient primary walls, indicating that at the onset of its deposition cellulose is in a crystalline structure. As the secondary wall develops, the labeling with both CBMs becomes more intense. The variation of the labeling pattern by CBM3a between transverse and longitudinal sections appeared related to microfibril orientation and differed between fibers and vessels. Although the two CBMs do not allow the description of the complete status of cellulose microstructures, they revealed the dynamics of the deposition of crystalline and amorphous forms of cellulose during wall formation and between cell types adapting cellulose microstructures to the cell function.
Assuntos
Arabidopsis/anatomia & histologia , Arabidopsis/química , Parede Celular/química , Celulose/análise , Celulose/biossíntese , Inflorescência/anatomia & histologia , Xilema/anatomia & histologia , Parede Celular/metabolismo , Inflorescência/química , Microfibrilas/metabolismo , Xilema/químicaRESUMO
Cinnamyl alcohol dehydrogenase (CAD) is a key enzyme involved in the last step of monolignol biosynthesis. The effect of CAD down-regulation on lignin production was investigated through a transgenic approach in maize. Transgenic CAD-RNAi plants show a different degree of enzymatic reduction depending on the analyzed tissue and show alterations in cell wall composition. Cell walls of CAD-RNAi stems contain a lignin polymer with a slight reduction in the S-to-G ratio without affecting the total lignin content. In addition, these cell walls accumulate higher levels of cellulose and arabinoxylans. In contrast, cell walls of CAD-RNAi midribs present a reduction in the total lignin content and of cell wall polysaccharides. In vitro degradability assays showed that, although to a different extent, the changes induced by the repression of CAD activity produced midribs and stems more degradable than wild-type plants. CAD-RNAi plants grown in the field presented a wild-type phenotype and produced higher amounts of dry biomass. Cellulosic bioethanol assays revealed that CAD-RNAi biomass produced higher levels of ethanol compared to wild-type, making CAD a good target to improve both the nutritional and energetic values of maize lignocellulosic biomass.
Assuntos
Oxirredutases do Álcool/genética , Biocombustíveis , Celulose/metabolismo , Regulação para Baixo/genética , Etanol/metabolismo , Lignina/biossíntese , Zea mays/genética , Oxirredutases do Álcool/deficiência , Oxirredutases do Álcool/metabolismo , Parede Celular/metabolismo , Flavonoides/química , Flavonoides/metabolismo , Fenóis/química , Fenóis/metabolismo , Caules de Planta/citologia , Caules de Planta/genética , Caules de Planta/crescimento & desenvolvimento , Caules de Planta/metabolismo , Plantas Geneticamente Modificadas , Interferência de RNA , Solubilidade , Zea mays/citologia , Zea mays/crescimento & desenvolvimento , Zea mays/metabolismoRESUMO
PREMISE: Although many highly successful weed species use a ballistic seed dispersal mechanism, little is known about the mechanics of this process. Bittercress (Cardamine hirsuta) siliques are morphologically similar to Arabidopsis siliques, but they can project their seeds up to 5 m, while Arabidopsis seeds are dispersed by gravity. Comparison of these species should enable us to determine which structures might be responsible for ballistic seed dispersal. METHODS: Sections of Arabidopsis and bittercress siliques were immunolabeled with antibodies raised against a variety of polysaccharide epitopes. RESULTS: In bittercress, the second endocarp layer (enB) of the valve had strongly asymmetrical cell wall thickenings, whereas the analogous cells in Arabidopsis were reinforced symmetrically and to a lesser extent. Additionally, an accumulation of mucilaginous pectins was found between the first and second endocarp (enA and enB) layers in the bittercress valve that was not present in Arabidopsis. However, in both species, highly de-esterified homogalacturonan was lost in the dehiscence zone (at the carpel/replum interface) as the siliques matured, thus allowing for separation of the valve at maturity. CONCLUSIONS: Ballistic seed dispersal in bittercress may involve the contraction of the outer pericarp tissue against the highly asymmetrically thickened enB cells, which are hypothesized to bend in one direction preferentially. The stress generated by the differential drying of the inner and outer layers of the valve is released suddenly as the adhesion between the cells of the dehiscence zone is lost, leading to a rapid coiling of the valve and dispersal of the seeds.
Assuntos
Cardamine/fisiologia , Dispersão de Sementes , Sementes/fisiologia , Anticorpos , Arabidopsis/fisiologia , Fenômenos Biomecânicos , Parede Celular/fisiologia , Epitopos , Imuno-Histoquímica , Microscopia Eletrônica de Transmissão , Pectinas/análise , Células Vegetais/fisiologia , Polissacarídeos/análise , Especificidade da Espécie , Estresse MecânicoRESUMO
During cell wall formation and degradation, it is possible to detect cellulose microfibrils assembled into thicker and thinner lamellar structures, respectively, following inverse parallel patterns. The aim of this study was to analyse such patterns of microfibril aggregation and cell wall delamination. The thickness of microfibrils and lamellae was measured on digital images of both growing and degrading cell walls viewed by means of transmission electron microscopy. To objectively detect, measure and classify microfibrils and lamellae into thickness classes, a method based on the application of computerized image analysis combined with graphical and statistical methods was developed. The method allowed common classes of microfibrils and lamellae in cell walls to be identified from different origins. During both the formation and degradation of cell walls, a preferential formation of structures with specific thickness was evidenced. The results obtained with the developed method allowed objective analysis of patterns of microfibril aggregation and evidenced a trend of doubling/halving lamellar structures, during cell wall formation/degradation in materials from different origin and which have undergone different treatments.
Assuntos
Parede Celular/metabolismo , Celulose/metabolismo , Aminação , Parede Celular/ultraestrutura , Microscopia Eletrônica de TransmissãoRESUMO
Tobacco plants expressing an antisense construct for a cationic peroxidase, which down-regulated lignin content at the presumed level of polymerisation, have been further analysed. T(1) plants were derived from a large-scale screen of T(0) mutant lines, previously published, which identified lines demonstrating consistent lignin down-regulation. Of these, line 1074 which had the most robust changes in lignin distribution through several generations was shown to have accompanying down-regulation of transcription of most lignin biosynthesis genes, except cinnamoyl-CoA reductase. The consistent 20% reduction in lignin was not accompanied by significant gross changes in vascular polysaccharide content and composition, despite a modest up-regulation of transcripts of genes involved in cellulose and hemicellulose synthesis. Morphologically, 1074 plants have under-developed xylem with both fibers and vessels having thin cell walls and limited secondary wall thickening with an abnormal S2 layer. However, they were not compromised in overall growth. Nevertheless, these and other lines showed improved potential industrial utility through a threefold increase in enzymic saccharification efficiency compared with wild-type (wt). Therefore, they were profiled for further un-intended effects of transgenesis that might compromise their value for industrial or biofuel processes. Other phenotypic changes included increased leaf thickness and bifurcation at the tip of the leaf. wt-Plants had smaller chloroplasts and higher stomatal numbers than mutants. Transgenic lines also showed a variable leaf pigment distribution with light-green areas that contained measurably less chlorophyll a, b, and carotenoids. Changes in epidermal pavement cells of mutant lines were also observed after exposure to various chemicals, while wt leaves retained their structural integrity. Despite these changes, the mutant plants grew and were viable indicating that lignification patterns can be manipulated considerably through targeting polymerisation without serious deleterious effects.
Assuntos
Metabolismo dos Carboidratos , DNA Antissenso , Lignina/biossíntese , Nicotiana/enzimologia , Peroxidases/metabolismo , Folhas de Planta/metabolismo , Xilema/metabolismo , Aldeído Oxirredutases/genética , Aldeído Oxirredutases/metabolismo , Biocombustíveis , Metabolismo dos Carboidratos/genética , Carboidratos , Carotenoides/análise , Celulose/biossíntese , Celulose/genética , Clorofila/análise , Cloroplastos/metabolismo , Regulação para Baixo , Expressão Gênica , Genes de Plantas , Lignina/genética , Peroxidases/genética , Fenótipo , Folhas de Planta/genética , Plantas Geneticamente Modificadas , Nicotiana/genética , Xilema/genéticaRESUMO
A cinnamoyl-CoA reductase 1 knockout mutant in Arabidopsis thaliana was investigated for the consequences of lignin synthesis perturbation on the assembly of the cell walls. The mutant displayed a dwarf phenotype and a strong collapse of its xylem vessels corresponding to lower lignin content and a loss of lignin units of the noncondensed type. Transmission electron microscopy revealed that the transformation considerably impaired the capacity of interfascicular fibers and vascular bundles to complete the assembly of cellulose microfibrils in the S(2) layer, the S(1) layer remaining unaltered. Such disorder in cellulose was correlated with X-ray diffraction showing altered organization. Semi-quantitative immunolabeling of lignins showed that the patterns of distribution were differentially affected in interfascicular fibers and vascular bundles, pointing to the importance of noncondensed lignin structures for the assembly of a coherent secondary wall. The use of laser capture microdissection combined with the microanalysis of lignins and polysaccharides allowed these polymers to be characterized into specific cell types. Wild-type A. thaliana displayed a two-fold higher syringyl to guaiacyl ratio in interfascicular fibers compared with vascular bundles, whereas this difference was less marked in the cinnamoyl-CoA reductase 1 knockout mutant.
Assuntos
Aldeído Oxirredutases/genética , Arabidopsis/enzimologia , Parede Celular/enzimologia , Inativação Gênica , Lignina/metabolismo , Arabidopsis/ultraestrutura , Metabolismo dos Carboidratos , Parede Celular/ultraestrutura , Microanálise por Sonda Eletrônica , Flores/química , Flores/citologia , Flores/enzimologia , Flores/ultraestrutura , Cromatografia Gasosa-Espectrometria de Massas , Glucose/metabolismo , Imuno-Histoquímica , Lignina/química , Espectroscopia de Ressonância Magnética , Microdissecção , Mutação/genética , Extratos Vegetais/química , Caules de Planta/química , Caules de Planta/citologia , Caules de Planta/enzimologia , Caules de Planta/ultraestrutura , Coloração e Rotulagem , Difração de Raios X , Xilose/metabolismoRESUMO
The involvement of the maize ZmMYB42 R2R3-MYB factor in the phenylpropanoid pathway and cell wall structure and composition was investigated by overexpression in Arabidopsis thaliana. ZmMYB42 down-regulates several genes of the lignin pathway and this effect reduces the lignin content in all lignified tissues. In addition, ZmMYB42 plants generate a lignin polymer with a decreased S to G ratio through the enrichment in H and G subunits and depletion in S subunits. This transcription factor also regulates other genes involved in the synthesis of sinapate esters and flavonoids. Furthermore, ZmMYB42 affects the cell wall structure and degradability, and its polysaccharide composition. Together, these results suggest that ZmMYB42 may be part of the regulatory network controlling the phenylpropanoid biosynthetic pathway.
Assuntos
Arabidopsis/citologia , Parede Celular/metabolismo , Lignina/biossíntese , Proteínas de Plantas/metabolismo , Zea mays/genética , Arabidopsis/genética , Ésteres/metabolismo , Flavonoides/biossíntese , Regulação da Expressão Gênica de Plantas , Malatos/metabolismo , Fenilpropionatos/metabolismo , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Lignified cell walls are widely considered to be key innovations in the evolution of terrestrial plants from aquatic ancestors some 475 million years ago. Lignins, complex aromatic heteropolymers, stiffen and fortify secondary cell walls within xylem tissues, creating a dense matrix that binds cellulose microfibrils and crosslinks other wall components, thereby preventing the collapse of conductive vessels, lending biomechanical support to stems, and allowing plants to adopt an erect-growth habit in air. Although "lignin-like" compounds have been identified in primitive green algae, the presence of true lignins in nonvascular organisms, such as aquatic algae, has not been confirmed. Here, we report the discovery of secondary walls and lignin within cells of the intertidal red alga Calliarthron cheilosporioides. Until now, such developmentally specialized cell walls have been described only in vascular plants. The finding of secondary walls and lignin in red algae raises many questions about the convergent or deeply conserved evolutionary history of these traits, given that red algae and vascular plants probably diverged more than 1 billion years ago.
Assuntos
Evolução Biológica , Parede Celular , Lignina/química , Rodófitas , Alga Marinha , Proteínas de Algas/química , Proteínas de Algas/metabolismo , Parede Celular/química , Parede Celular/ultraestrutura , Lignina/classificação , Lignina/metabolismo , Estrutura Molecular , Filogenia , Rodófitas/química , Rodófitas/citologia , Alga Marinha/química , Alga Marinha/citologiaRESUMO
Xyloglucan endotransglucosylase/hydrolases (XTHs; EC 2.4.1.207 and/or EC 3.2.1.151) are enzymes involved in the modification of cell wall structure by cleaving and, often, also re-joining xyloglucan molecules in primary plant cell walls. Using a pool of antibodies raised against an enriched cell wall protein fraction, a new XTH cDNA in maize, ZmXTH1, has been isolated from a cDNA expression library obtained from the elongation zone of the maize root. The predicted protein has a putative N-terminal signal peptide and possesses the typical domains of this enzyme family, such as a catalytic domain that is homologous to that of Bacillus macerans beta-glucanase, a putative N-glycosylation motif, and four cysteine residues in the central and C terminal regions of the ZmXTH1 protein. Phylogenetic analysis of ZmXTH1 reveals that it belongs to subgroup 4, so far only reported from Poaceae monocot species. ZmXTH1 has been expressed in Pichia pastoris (a methylotrophic yeast) and the recombinant enzyme showed xyloglucan endotransglucosylase but not xyloglucan endohydrolase activity, representing the first enzyme belonging to subgroup 4 characterized in maize so far. Expression data indicate that ZmXTH1 is expressed in elongating tissues, modulated by culture conditions, and induced by gibberellins. Transient expression assays in onion cells reveal that ZmXTH1 is directed to the cell wall, although weakly bound. Finally, Arabidopsis thaliana plants expressing ZmXTH1 show slightly increased xyloglucan endohydrolase activity and alterations in the cell wall structure and composition.
Assuntos
Arabidopsis/genética , Arabidopsis/metabolismo , Glicosiltransferases/metabolismo , Proteínas de Plantas/metabolismo , Zea mays/enzimologia , Sequência de Aminoácidos , Parede Celular , Regulação da Expressão Gênica de Plantas/fisiologia , Genoma de Planta , Glicosiltransferases/genética , Dados de Sequência Molecular , Filogenia , Proteínas de Plantas/genética , Plantas Geneticamente ModificadasRESUMO
In view of tracing the fate of cellulose fine elements added to a suspension of cellulose fibers, a novel method for specific labeling of polysaccharides in a composite material was developed. The purpose was to visualize a given cellulose material within a cellulose mixture. The method consists of generating aldehyde groups in the chain by mild periodic acid oxidation followed by biotinylation of the carbonyls. Once added to the composite, the biotinylated molecules are labeled with streptavidin conjugated to a fluorescent probe for confocal microscopy, or streptavidin-gold for electron microscopy observations. In the present work, the fate of fresh fines (never-dried) and dead fines (dried) when they were added to a purified suspension of fibers was followed by observation of the labeling in confocal and electron microscopy. The differential mode of interaction of fresh fines and dead fines with the fibers was correlated to the mechanical characteristics measured on the corresponding papers. The versatility of the new labeling method and its possible generalization to other polysaccharides incorporated to a polysaccharide or nonpolysaccharide material should be of potential interest for the study of composite microstructure.
Assuntos
Materiais Biocompatíveis/química , Biotinilação , Celulose/análise , Celulose/química , Aldeídos/química , Biotina/química , Corantes Fluorescentes/química , Ouro/química , Métodos , Microscopia Confocal , Microscopia Eletrônica de Varredura , Oxirredução , Ácido Periódico/química , Estreptavidina/químicaRESUMO
BACKGROUND AND AIMS: Plants growing in altered gravity conditions encounter changes in vascular development and cell wall deposition. The aim of this study was to investigate xylem anatomy and arrangement of cellulose microfibrils in vessel walls of different organs of soybean seedlings grown in Space. METHODS: Seeds germinated and seedlings grew for 5 d in Space during the Foton-M2 mission. The environmental conditions, other than gravity, of the ground control repeated those experienced in orbit. The seedlings developed in space were compared with those of the control test on the basis of numerous anatomical and ultrastructural parameters such as number of veins, size and shape of vessel lumens, thickness of cell walls and deposition of cellulose microfibrils. KEY RESULTS: Observations made with light, fluorescence and transmission electron microscopy, together with the quantification of the structural features through digital image analysis, showed that the alterations due to microgravity do not occur at the same level in the various organs of soybean seedlings. The modifications induced by microgravity or by the indirect effect of space-flight conditions, became conspicuous only in developing vessels at the ultrastructural level. The results suggested that the orientation of microfibrils and their assembly in developing vessels are perturbed by microgravity at the beginning of wall deposition, while they are still able to orient and arrange in thicker and ordered structures at later stages of secondary wall deposition. CONCLUSIONS: The process of proper cell-wall building, although not prevented, is perturbed in Space at the early stage of development. This would explain the almost unaltered anatomy of mature structures, accompanied by a slower growth observed in seedlings grown in Space than on Earth.
Assuntos
Meio Ambiente Extraterreno , Glycine max/crescimento & desenvolvimento , Plântula/crescimento & desenvolvimento , Plântula/ultraestrutura , Xilema/crescimento & desenvolvimento , Ausência de PesoRESUMO
BACKGROUND: Cellulose Binding Domains (CBD) were conjugated with fluorescein isothiocyanate (FITC). The surface concentration of the Binding Domains adsorbed on cellulose fibres was determined by fluorescence image analysis. RESULTS: For a CBD-FITC concentration of 60 mg/L, a coating fraction of 78% and 110% was estimated for Portucel and Whatman fibres, respectively. For a saturating CBD concentration, using Whatman CF11 fibres, a surface concentration of 25.2 x 10-13 mol/mm2 was estimated, the equivalent to 4 protein monolayers. This result does not imply the existence of several adsorbed protein layers. CONCLUSION: It was verified that CBDs were able to penetrate the fibres, according to confocal microscopy and TEM-immunolabelling analysis. The surface concentration of adsorbed CBDs was greater on amorphous fibres (phosphoric acid swollen) than on more crystalline ones (Whatman CF11 and Sigmacell 20).
Assuntos
Celulases/metabolismo , Celulose/metabolismo , Fluoresceína-5-Isotiocianato/metabolismo , Celulases/genética , Celulose/ultraestrutura , Fluorescência , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Ligação Proteica , Estrutura Terciária de ProteínaRESUMO
Cinnamoyl-CoA reductase 1 (CCR1, gene At1g15950) is the main CCR isoform implied in the constitutive lignification of Arabidopsis thaliana. In this work, we have identified and characterized two new knockout mutants for CCR1. Both have a dwarf phenotype and a delayed senescence. At complete maturity, their inflorescence stems display a 25-35% decreased lignin level, some alterations in lignin structure with a higher frequency of resistant interunit bonds and a higher content in cell wall-bound ferulic esters. Ferulic acid-coniferyl alcohol ether dimers were found for the first time in dicot cell walls and in similar levels in wild-type and mutant plants. The expression of CCR2, a CCR gene usually involved in plant defense, was increased in the mutants and could account for the biosynthesis of lignins in the CCR1-knockout plants. Mutant plantlets have three to four-times less sinapoyl malate (SM) than controls and accumulate some feruloyl malate. The same compositional changes occurred in the rosette leaves of greenhouse-grown plants. By contrast and relative to the control, their stems accumulated unusually high levels of both SM and feruloyl malate as well as more kaempferol glycosides. These findings suggest that, in their hypolignified stems, the mutant plants would avoid the feruloyl-CoA accumulation by its redirection to cell wall-bound ferulate esters, to feruloyl malate and to SM. The formation of feruloyl malate to an extent far exceeding the levels reported so far indicates that ferulic acid is a potential substrate for the enzymes involved in SM biosynthesis and emphasizes the remarkable plasticity of Arabidopsis phenylpropanoid metabolism.
Assuntos
Aldeído Oxirredutases/genética , Proteínas de Arabidopsis/genética , Arabidopsis/genética , Mutação , Aldeído Oxirredutases/metabolismo , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Ácidos Cumáricos/metabolismo , Cromatografia Gasosa-Espectrometria de Massas , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Lignina/metabolismo , Malatos/metabolismo , Fenótipo , Fenilpropionatos/metabolismo , Plantas Geneticamente Modificadas , Transdução de Sinais/efeitos da radiação , Espectroscopia de Infravermelho com Transformada de Fourier , Raios UltravioletaRESUMO
Cinnamoyl-CoA reductase (CCR) catalyzes the penultimate step in monolignol biosynthesis. We show that downregulation of CCR in transgenic poplar (Populus tremula x Populus alba) was associated with up to 50% reduced lignin content and an orange-brown, often patchy, coloration of the outer xylem. Thioacidolysis, nuclear magnetic resonance (NMR), immunocytochemistry of lignin epitopes, and oligolignol profiling indicated that lignin was relatively more reduced in syringyl than in guaiacyl units. The cohesion of the walls was affected, particularly at sites that are generally richer in syringyl units in wild-type poplar. Ferulic acid was incorporated into the lignin via ether bonds, as evidenced independently by thioacidolysis and by NMR. A synthetic lignin incorporating ferulic acid had a red-brown coloration, suggesting that the xylem coloration was due to the presence of ferulic acid during lignification. Elevated ferulic acid levels were also observed in the form of esters. Transcript and metabolite profiling were used as comprehensive phenotyping tools to investigate how CCR downregulation impacted metabolism and the biosynthesis of other cell wall polymers. Both methods suggested reduced biosynthesis and increased breakdown or remodeling of noncellulosic cell wall polymers, which was further supported by Fourier transform infrared spectroscopy and wet chemistry analysis. The reduced levels of lignin and hemicellulose were associated with an increased proportion of cellulose. Furthermore, the transcript and metabolite profiling data pointed toward a stress response induced by the altered cell wall structure. Finally, chemical pulping of wood derived from 5-year-old, field-grown transgenic lines revealed improved pulping characteristics, but growth was affected in all transgenic lines tested.
Assuntos
Aldeído Oxirredutases/genética , Parede Celular/química , Regulação para Baixo/genética , Lignina/química , Lignina/metabolismo , Populus/enzimologia , Populus/genética , Carboidratos , Parede Celular/ultraestrutura , Cromatografia Líquida de Alta Pressão , Fluorescência , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Imuno-Histoquímica , Fenóis/análise , Fenótipo , Plantas Geneticamente Modificadas , Populus/citologia , Populus/ultraestrutura , Solubilidade , Espectroscopia de Infravermelho com Transformada de Fourier , Xilema/citologia , Xilema/crescimento & desenvolvimento , Xilema/ultraestruturaRESUMO
Extractability and recovery of cellulose from cell walls influences many industrial processes and also the utilisation of biomass for energy purposes. The utility of genetic manipulation of lignin has proven potential for optimising such processes and is also advantageous for the environment. Hemicelluloses, particularly secondary wall xylans, also influence the extractability of cellulose. UDP-glucuronate decarboxylase produces UDP-xylose, the precursor for xylans and the effect of its down-regulation on cell wall structure and cellulose extractability in transgenic tobacco has been investigated. Since there are a number of potential UDP-glucuronate decarboxylase genes, a 490bp sequence of high similarity between members of the family, was chosen for general alteration of the expression of the gene family. Sense and antisense transgenic lines were analysed for enzyme activity using a modified and optimised electrophoretic assay, for enzyme levels by western blotting and for secondary cell wall composition. Some of the down-regulated antisense plants showed high glucose to xylose ratios in xylem walls due to less xylose-containing polymers, while arabinose and uronic acid contents, which could also have been affected by any change in UDP-xylose provision, were unchanged. The overall morphology and stem lignin content of the modified lines remained little changed compared with wild-type. However, there were some changes in vascular organisation and reduction of xylans in the secondary walls was confirmed by immunocytochemistry. Pulping analysis showed a decreased pulp yield and a higher Kappa number in some lines compared with controls, indicating that they were less delignified, although the level of residual alkali was reduced. Such traits probably indicate that lignin was less available for removal in a reduced background of xylans. However, the viscosity was higher in most antisense lines, meaning that the cellulose was less broken-down during the pulping process. This is one of the first studies of a directed manipulation of hemicellulose content on cellulose extractability and shows both positive and negative outcomes.
Assuntos
Carboxiliases/metabolismo , Celulose/isolamento & purificação , DNA Antissenso/farmacologia , Nicotiana/metabolismo , Plantas Geneticamente Modificadas/enzimologia , Polissacarídeos/metabolismo , Sequência de Bases , Carboxiliases/genética , Celulose/análise , Celulose/química , Regulação para Baixo/efeitos dos fármacos , Dados de Sequência Molecular , Nicotiana/enzimologia , Xilema/químicaRESUMO
In adaptation to their function the walls of plant cell display tissue-specific variations of composition according to their developmental stage, cell type and stress of various origin. It is therefore important to obtain a precise analytical data describing the cell wall composition with respect to these different factors. In the present work, laser capture microdissection (LCM) was used for isolating different tissues from the stem of Urtica dioica L. at a semi-preparative scale. The technique was associated for the first time to a one-pot sequential cell wall preparation and hydrolysis for the carbohydrate analysis of each cell type. The results demonstrate that the combination of LCM and micro-analytical methods can provide individual cell type composition and should improve our knowledge of the biochemical diversity of cell walls in plants. This approach will be of potential interest for the understanding of the effects of stress or genetic engineering on the composition of the cell walls.
Assuntos
Carboidratos/análise , Parede Celular/ultraestrutura , Lasers , Microdissecção/métodos , Urtica dioica/citologia , Parede Celular/química , Parede Celular/metabolismo , Cotilédone/química , Cotilédone/ultraestrutura , Hidrólise , Urtica dioica/química , Urtica dioica/ultraestruturaRESUMO
In the context of our research on cell wall formation and maturation in flax (Linum usitatissimum L) bast fibers, we (1) confirmed the presence of lignin in bast fibers and (2) quantified and characterized the chemical nature of this lignin at two developmental stages. Histochemical methods (Weisner and Maüle reagents and KMnO(4)-staining) indicating the presence of lignin in bast fibers at the light and electron microscope levels were confirmed by chemical analyses (acetyl bromide). In general, the lignin content in flax bast fibers varied between 1.5% and 4.2% of the dry cell wall residues (CWRs) as compared to values varying between 23.7% and 31.4% in flax xylem tissues. Immunological and chemical analyses (thioacidolysis and nitrobenzene oxidation) indicated that both flax xylem- and bast fiber-lignins were rich in guaiacyl (G) units with S/G values inferior to 0.5. In bast fibers, the highly sensitive immunological probes allowed the detection of condensed guaiacyl-type (G) lignins in the middle lamella, cell wall junctions, and in the S1 layer of the secondary wall. In addition, lower quantities of mixed guaiacyl-syringyl (GS) lignins could be detected throughout the secondary cell wall. Chemical analyses suggested that flax bast-fiber lignin is more condensed than the corresponding xylem lignin. In addition, H units represented up to 25% of the monomers released from bast-fiber lignin as opposed to a value of 1% for the corresponding xylem tissue. Such an observation indicates that the structure of flax bast-fiber lignin is significantly different from that of the more typical 'woody plant lignin', thereby suggesting that flax bast fibers represent an interesting system for studying an unusual lignification process.
Assuntos
Linho/química , Lignina/análise , Caules de Planta/química , Parede Celular , Linho/citologia , Lignina/ultraestrutura , Caules de Planta/citologia , Caules de Planta/ultraestruturaRESUMO
Hardwood trees are able to reorient their axes owing to tension wood differentiation. Tension wood is characterised by important ultrastructural modifications, such as the occurrence in a number of species, of an extra secondary wall layer, named gelatinous layer or G-layer, mainly constituted of cellulose microfibrils oriented nearly parallel to the fibre axis. This G-layer appears directly involved in the definition of tension wood mechanical properties. This review gathers the data available in the literature about lignification during tension wood formation. Potential roles for lignin in tension wood formation are inferred from biochemical, anatomical and mechanical studies, from the hypotheses proposed to describe tension wood function and from data coming from new research areas such as functional genomics.