Asunto(s)
Acetiltransferasas , Proteínas de Arabidopsis , Arabidopsis , Glucanos , Mananos , Acetiltransferasas/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Pared Celular/metabolismo , Glucanos/metabolismo , Mananos/metabolismo , Pectinas , XilanosRESUMEN
The molecular basis of cell-cell adhesion in woody tissues is not known. Xylem cells in wood particles of hybrid poplar (Populus tremula × P. alba cv. INRA 717-1B4) were separated by oxidation of lignin with acidic sodium chlorite when combined with extraction of xylan and rhamnogalacturonan-I (RG-I) using either dilute alkali or a combination of xylanase and RG-lyase. Acidic chlorite followed by dilute alkali treatment enables cell-cell separation by removing material from the compound middle lamellae between the primary walls. Although lignin is known to contribute to adhesion between wood cells, we found that removing lignin is a necessary but not sufficient condition to effect complete cell-cell separation in poplar lines with various ratios of syringyl:guaiacyl lignin. Transgenic poplar lines expressing an Arabidopsis thaliana gene encoding an RG-lyase (AtRGIL6) showed enhanced cell-cell separation, increased accessibility of cellulose and xylan to hydrolytic enzyme activities, and increased fragmentation of intact wood particles into small cell clusters and single cells under mechanical stress. Our results indicate a novel function for RG-I, and also for xylan, as determinants of cell-cell adhesion in poplar wood cell walls. Genetic control of RG-I content provides a new strategy to increase catalyst accessibility and saccharification yields from woody biomass for biofuels and industrial chemicals.
Asunto(s)
Adhesión Celular , Pectinas/química , Populus , Madera/citología , Pared Celular , Lignina , Plantas Modificadas Genéticamente , Polisacárido Liasas/genéticaRESUMEN
BACKGROUND: Cell elongation is mainly limited by the extensibility of the cell wall. Dicotyledonous primary (growing) cell walls contain cellulose, xyloglucan, pectin and proteins, but little is known about how each polymer class contributes to the cell wall mechanical properties that control extensibility. RESULTS: We present evidence that the degree of pectin methyl-esterification (DE%) limits cell growth, and that a minimum level of about 60% DE is required for normal cell elongation in Arabidopsis hypocotyls. When the average DE% falls below this level, as in two gibberellic acid (GA) mutants ga1-3 and gai, and plants expressing pectin methyl-esterase (PME1) from Aspergillus aculeatus, then hypocotyl elongation is reduced. CONCLUSION: Low average levels of pectin DE% are associated with reduced cell elongation, implicating PMEs, the enzymes that regulate DE%, in the cell elongation process and in responses to GA. At high average DE% other components of the cell wall limit GA-induced growth.
Asunto(s)
Arabidopsis/metabolismo , Hipocótilo/metabolismo , Pectinas/metabolismo , Arabidopsis/citología , Arabidopsis/enzimología , Arabidopsis/crecimiento & desarrollo , Hidrolasas de Éster Carboxílico/metabolismo , División Celular , Esterificación , Hipocótilo/citología , Hipocótilo/crecimiento & desarrollo , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
Seed coat development in Arabidopsis thaliana involves a complex pathway where cells of the outer integument differentiate into a highly specialized cell type after fertilization. One aspect of this developmental process involves the secretion of a large amount of pectinaceous mucilage into the apoplast. When the mature seed coat is exposed to water, this mucilage expands to break the primary cell wall and encapsulate the seed. The mucilage-modified2 (mum2) mutant is characterized by a failure to extrude mucilage on hydration, although mucilage is produced as normal during development. The defect in mum2 appears to reside in the mucilage itself, as mucilage fails to expand even when the barrier of the primary cell wall is removed. We have cloned the MUM2 gene and expressed recombinant MUM2 protein, which has beta-galactosidase activity. Biochemical analysis of the mum2 mucilage reveals alterations in pectins that are consistent with a defect in beta-galactosidase activity, and we have demonstrated that MUM2 is localized to the cell wall. We propose that MUM2 is involved in modifying mucilage to allow it to expand upon hydration, establishing a link between the galactosyl side-chain structure of pectin and its physical properties.
Asunto(s)
Adhesivos/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Semillas/metabolismo , beta-Galactosidasa/metabolismo , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/genética , Carbonatos/química , Pared Celular/metabolismo , Aparato de Golgi/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Microscopía Confocal , Microscopía Electrónica de Rastreo , Datos de Secuencia Molecular , Pectinas/química , Pectinas/metabolismo , Plantas Modificadas Genéticamente , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Semillas/genética , Semillas/ultraestructura , Espectroscopía Infrarroja por Transformada de Fourier , beta-Galactosidasa/genéticaRESUMEN
Guard cell walls combine exceptional strength and flexibility in order to accommodate the turgor pressure-driven changes in size and shape that underlie the opening and closing of stomatal pores. To investigate the molecular basis of these exceptional qualities, we have used a combination of compositional and functional analyses in three different plant species. We show that comparisons of FTIR spectra from stomatal guard cells and those of other epidermal cells indicate a number of clear differences in cell-wall composition. The most obvious characteristics are that stomatal guard cells are enriched in phenolic esters of pectins. This enrichment is apparent in guard cells from Vicia faba (possessing a type I cell wall) and Commelina communis and Zea mays (having a type II wall). We further show that these common defining elements of guard cell walls have conserved functional roles. As previously reported in C. communis, we show that enzymatic modification of the pectin network in guard cell walls in both V. faba and Z. mays has profound effects on stomatal function. In all three species, incubation of epidermal strips with a combination of pectin methyl esterase and endopolygalacturonase (EPG) caused an increase in stomatal aperture on opening. This effect was not seen when strips were incubated with EPG alone indicating that the methyl-esterified fraction of homogalacturonan is key to this effect. In contrast, arabinanase treatment, and incubation with feruloyl esterase both impeded stomatal opening. It therefore appears that pectins and phenolic esters have a conserved functional role in guard cell walls even in grass species with type II walls, which characteristically are composed of low levels of pectins.
Asunto(s)
Commelina/fisiología , Pectinas/metabolismo , Hojas de la Planta/química , Hojas de la Planta/fisiología , Vicia faba/fisiología , Zea mays/fisiología , Commelina/química , Commelina/citología , Vicia faba/química , Vicia faba/citología , Zea mays/química , Zea mays/citologíaRESUMEN
Four potato cellulose synthase (CesA) homologs (StCesA1, 2, 3 and 4) were isolated by screening a cDNA library made from developing tubers. Based on sequence comparisons and the fact that all four potato cDNAs were isolated from this single cDNA-library, all four StCesA clones are likely to play a role in primary cell wall biosynthesis. Several constructs were generated to modulate cellulose levels in potato plants in which the granule-bound starch synthase promoter was used to target the modification to the tubers. The StCesA3 was used for up- and down-regulation of the cellulose levels by sense (SE-StCesA3) and antisense (AS-StCesA3) expression of the complete cDNA. Additionally, the class-specific regions (CSR) of all four potato cellulose synthase genes were used for specific down-regulation (antisense) of the corresponding CesA genes (csr1, 2, 3 and 4). None of the transformants showed an overt developmental phenotype. Sections of tubers were screened for altered cell wall structure by Fourier Transform Infrared microspectroscopy (FTIR) and exploratory Principal Component Analysis (PCA), and those plants discriminating from WT plants were analysed for cellulose content and monosaccharide composition. Several transgenic lines were obtained with mainly decreased levels of cellulose. These results show that the cellulose content in potato tubers can be reduced down to 40% of the WT level without affecting normal plant development, and that constructs based on the CSR alone are specific and sufficient to down-regulate cellulose biosynthesis.
Asunto(s)
Pared Celular/metabolismo , Celulosa/biosíntesis , Glucosiltransferasas/genética , ARN sin Sentido/biosíntesis , Solanum tuberosum/metabolismo , Celulosa/metabolismo , Colorimetría , ADN Complementario/genética , Expresión Génica , Glucosiltransferasas/metabolismo , Monosacáridos/química , Familia de Multigenes , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente , ARN sin Sentido/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Solanum tuberosum/enzimología , Solanum tuberosum/genética , Espectroscopía Infrarroja por Transformada de Fourier , Transformación GenéticaRESUMEN
Pectin is a class of complex cell wall polysaccharides with multiple roles during cell development. Assigning specific functions to particular polysaccharides is in its infancy, in part, because of the limited number of mutants and transformants available with modified pectic polymers in their walls. Pectins are also important polymers with diverse applications in the food and pharmaceutical industries, which would benefit from technology for producing pectins with specific functional properties. In this report, we describe the generation of potato (Solanum tuberosum L. cv Posmo) tuber transformants producing pectic rhamnogalacturonan I (RGI) with a low level of arabinosylation. This was achieved by the expression of a Golgi membrane-anchored endo-alpha-1,5-arabinanase. Sugar composition analysis of RGI isolated from transformed and wild-type tubers showed that the arabinose content was decreased by approximately 70% in transformed cell walls compared with wild type. The modification of the RGI was confirmed by immunolabeling with an antibody recognizing alpha-1,5-arabinan. This is the first time, to our knowledge, that the biosynthesis of a plant cell wall polysaccharide has been manipulated through the action of a glycosyl hydrolase targeted to the Golgi compartment.
Asunto(s)
Aparato de Golgi/metabolismo , Pectinas/biosíntesis , Solanum tuberosum/genética , Pared Celular/metabolismo , Regulación Enzimológica de la Expresión Génica , Glicósido Hidrolasas/genética , Glicósido Hidrolasas/metabolismo , Pectinas/antagonistas & inhibidores , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Sialiltransferasas/genética , Sialiltransferasas/metabolismo , Solanum tuberosum/enzimologíaRESUMEN
Rhamnogalacturonan (RG) I is a branched pectic polysaccharide in plant cell walls. Rhamnogalacturonan lyase (eRGL) from Aspergillus aculeatus is able to cleave the RG I backbone at specific sites. Transgenic potato (Solanum tuberosum L.) plants were made by the introduction of the gene encoding eRGL, under the control of the granule-bound starch synthase promoter. The eRGL protein was successfully expressed and translated into an active form, demonstrated by eRGL activity in the tuber extracts. The transgenic plants produced tubers with clear morphological alterations, including radial swelling of the periderm cells and development of intercellular spaces in the cortex. Sugar compositional analysis of the isolated cell walls showed a large reduction in galactosyl and arabinosyl residues in transgenic tubers. Immunocytochemical studies using the LM5 (galactan) and LM6 (arabinan) antibodies also showed a large reduction in galactan and arabinan side-chains of RG I. Most of the remaining LM5 epitopes were located in the expanded middle lamella at cell corners of eRGL tubers, which is in contrast to their normal location in the primary wall of wild type tubers. These data suggest that RG I has an important role in anchoring galactans and arabinans at particular regions in the wall and in normal development of the periderm.