Plant cell walls are complex structures composed of high-molecular-weight polysaccharides, proteins, and lignins. Among the wall polysaccharides, cellulose, a hydrogen-bonded β-1,4-1inked glucan microfibril, is the m...Plant cell walls are complex structures composed of high-molecular-weight polysaccharides, proteins, and lignins. Among the wall polysaccharides, cellulose, a hydrogen-bonded β-1,4-1inked glucan microfibril, is the main load-bearing wall component and a key precursor for industrial applications. Cellulose is synthesized by large multi-meric cellulose synthase （CesA） complexes, tracking along cortical microtubules at the plasma membrane. The only known components of these complexes are the cellulose synthase proteins. Recent studies have identified tentative interaction partners for the CesAs and shown that the migratory patterns of the CesA complexes depend on phosphorylation status. These advances may become good platforms for expanding our knowledge about cellulose synthesis in the near future. In addition, our current understanding of cellulose chain polymerization in the context of the CesA complex is discussed.展开更多
The ability of a plant cell to expand is largely defined by the physical constraints imposed by its cell wall. Accordingly, cell wall properties have to be regulated during development. The pectic polysaccharide homog...The ability of a plant cell to expand is largely defined by the physical constraints imposed by its cell wall. Accordingly, cell wall properties have to be regulated during development. The pectic polysaccharide homogalacturonan is a major component of the plant primary walls. Biosynthesis and in muro modification of homogalacturonan have recently emerged as key determinants of plant development, controlling cell adhesion, organ development, and phyllotactic patterning. This review will focus on recent findings regarding impact of homogalacturonan content and methylesterification status of this polymer on plant life. Demethyl-esterification of homogalacturonan occurs through the action of the ubiquitous enzyme ＇pectin methyl-esterase＇. We here describe various strategies developed by the plant to finely tune the methyl-esterification status of homogalacturonan along key events of the plant lifecycle.展开更多
Nexine is a conserved layer of the pollen wall. We previously reported that the nexine layer is absent in the knockout mutant of Arabidopsis TRANSPOSABLE ELEMENT SILENCING VIA AT-HOOK （TEK） gene. In this study, we i...Nexine is a conserved layer of the pollen wall. We previously reported that the nexine layer is absent in the knockout mutant of Arabidopsis TRANSPOSABLE ELEMENT SILENCING VIA AT-HOOK （TEK） gene. In this study, we investigated the molecular regulatory functions of TEK in pollen development and identified the genes encoding Arabinogalactan proteins （AGPs） as direct targets of TEK, which are essential for nexine formation. Phenotypic similarity between tek and the TEK-SRDX transgenic lines suggest that TEK plays a role in transcriptional activation in anther development. Microarray analysis identified a total of 661 genes downregulated in tek, including four genes encoding AGPs, AGP6, AGP11, AGP23, and AGP40. Electrophoretic mobility shift assays showed that TEK could directly bind the nuclear matrix attachment region （MAR） and the promoter of AGP6. Chromatin immunoprecipitation followed by PCR analysis demonstrated that TEK is enriched in the promoters of the four AGP genes. Expression of AGP6 driven by the TEK promoter in tek partially rescued both nexine formation and plant fertility. These results indicate that TEK directly reg- ulates AGP expression in the anther to control nexine layer formation. We also proposed that glycoproteins might be essential components of the nexine laver in the oollen wall.展开更多
In this review, we address the question of how the tip-growing pollen tube achieves its rapid rate of elongation while maintaining an intact cell wall. Although turgor is essential for growth to occur, the local expan...In this review, we address the question of how the tip-growing pollen tube achieves its rapid rate of elongation while maintaining an intact cell wall. Although turgor is essential for growth to occur, the local expansion rate is controlled by local changes in the viscosity of the apical wall. We focus on several different structures and underly- ing processes that are thought to be major participants including exocytosis, the organization and activity of the actin cytoskeleton, calcium and proton physiology, and cellular energetics. We think that the actin cytoskeleton, in particular the apical cortical actin fringe, directs the flow of vesicles to the apical domain, where they fuse with the plasma mem- brane and contribute their contents to the expanding cell wall. While pH gradients, as generated by a proton-ATPase located on the plasma membrane along the side of the clear zone, may regulate rapid actin turnover and new polymeri- zation in the fringe, the tip-focused calcium gradient biases secretion towards the polar axis. The recent data showing that exocytosis of new wall material precedes and predicts the process of cell elongation provide support for the idea that the intussusception of newly secreted pectin contributes to decreases in apical wall viscosity and to cell expansion. Other prime factors will be the localization and activity of the enzyme pectin methyl-esterase, and the chelation of calcium by pectic acids. Finally, we acknowledge a role for reactive oxygen species in the control of wall viscosity.展开更多
IRX14 and IRX14-LIKE （IRX14L） are two closely related glycosyl transferases in the glycosyl transferase 43 （GT43） family of Arabidopsis. A T-DNA insertion mutant for IRX14 results in comparatively minor changes, s...IRX14 and IRX14-LIKE （IRX14L） are two closely related glycosyl transferases in the glycosyl transferase 43 （GT43） family of Arabidopsis. A T-DNA insertion mutant for IRX14 results in comparatively minor changes, such as irregular xylem, while a mutation for IRX14L results in no changes. However, an irx14 and irx14L double mutant severely affects growth and development, with the dwarf plants failing to produce an inflorescence stem. Plants that are homozygous for IRX14 but heterozygous for IRX14L （irx14 irx14L（±）） exhibit an intermediate phenotype, including noticeably smaller leaves, stems, and underdeveloped siliques. Additionally, the T-DNA insertion mutant for IRX14 was found to result in a drought-tolerant phenotype. Carbohydrate analysis of total cell wall extracts revealed a reduction in xylose for the irx14 and irx14 irx14L（±） mutants, consistent with a defect in glucuronoxylan biosynthesis. Immunolocalization of xylan with the LM10 antibody revealed a loss of xylan in irx14 mutants and a further reduction in the irx14 irx14L（±） mutants. IRX14L likely functions redundantly with IRX14 in glucuronoxylan biosynthesis, with IRX14 having a more important role in the process.展开更多
All plant cells are surrounded by a cell wall that determines the directionality of cell growth and protects the cell against its environment. Plant cell walls are comprised primarily of polysaccharides and represent ...All plant cells are surrounded by a cell wall that determines the directionality of cell growth and protects the cell against its environment. Plant cell walls are comprised primarily of polysaccharides and represent the largest sink for photosynthetically fixed carbon, both for individual plants and in the terrestrial biosphere as a whole. Cell wall synthesis is a highly sophisticated process, involving multiple enzymes and metabolic intermediates, intracellular trafficking of proteins and cell wall precursors, assembly of cell wall polymers into the extracellular matrix, remodeling of polymers and their interactions, and recycling of cell wall sugars. In this review we discuss how newly fixed carbon, in the form of UDP-glucose and other nucleotide sugars, contributes to the synthesis of cell wall polysaccharides, and how cell wall synthesis is influenced by the carbon status of the plant, with a focus on the model species Arabidopsis （Arabidopsis thaliana）.展开更多
Methods developed to isolate intact cells from both unripe and ripe tomato fruit pericarp parenchyma have allowed the cell biological analysis of polysaccharide epitopes at the surface of separated cells. The LM7 pect...Methods developed to isolate intact cells from both unripe and ripe tomato fruit pericarp parenchyma have allowed the cell biological analysis of polysaccharide epitopes at the surface of separated cells. The LM7 pectic homogalacturonan epitope is a marker of the junctions of adhesion planes and intercellular spaces in parenchyma systems. The LM7 epitope persistently marked the former edge of adhesion planes at the surface of cells separated from unripe and ripened tomato fruit and also from fruits with the Cnr mutation. The LM 11 xylan epitope was associated, in sections, with cell walls lining intercellular space but the epitope was not detected at the surface of isolated cells, being lost during cell isolation. The LM15 xyloglucan epitope was present at the surface of cells isolated from unripe fruit in a pattern reflecting the former edge of cell adhesion planes/intercellular space but with gaps and apparent breaks. An equivalent pattern of LM15 epitope occurrence was revealed at the surface of cells isolated by pectate lyase action but was not present in cells isolated from ripe fruit or from Cnr fruit. In contrast to wild-type cells, the LM5 galactan and LM21 mannan epitopes occurred predominantly in positions reflecting intercellular space in Cnr, suggesting a concerted alteration in cell wall microstructure in response to this mutation. Galactanase and mannanase, along with pectic homogalacturonan-degrading enzymes, were capable of releasing cells from unripe fruit parenchyma. These observations indicate that hemicellulose polymers are present in architectural contexts reflecting cell adhesion and that several cell wall polysaccharide classes are likely to contribute to cell adhesion/cell separation in tomato fruit pericarp parenchyma.展开更多
Polysaccharide-rich plant cell walls are important biomaterials that underpin plant growth, are major repositories for photosynthetically accumulated carbon, and, in addition, impact greatly on the human use of plants...Polysaccharide-rich plant cell walls are important biomaterials that underpin plant growth, are major repositories for photosynthetically accumulated carbon, and, in addition, impact greatly on the human use of plants. Land plant cell walls contain in the region of a dozen major polysaccharide structures that are mostly encompassed by cellulose, hemicelluloses, and pectic polysaccharides. During the evolution of land plants, polysaccharide diversification appears to have largely involved structural elaboration and diversification within these polysaccharide groups. Cell wall chemistry is well advanced and a current phase of cell wall science is aimed at placing the complex polysaccharide chemistry in cellular contexts and developing a detailed understanding of cell wall biology. Imaging cell wall glycomes is a challenging area but recent developments in the establishment of cell wall molecular probe panels and their use in high throughput procedures are leading to rapid advances in the molecular understanding of the spatial heterogeneity of individual cell walls and also cell wall differences at taxonomic levels. The challenge now is to integrate this knowledge of cell wall heterogeneity with an understanding of the molecular and physiological mechanisms that underpin cell wall properties and functions.展开更多
A deep-sequencing approach was pursued utilizing 454 and Illumina sequencing methods to discover new genes involved in xyloglucan biosynthesis, cDNA sequences were generated from developing nasturtium （Tropaeolum ma...A deep-sequencing approach was pursued utilizing 454 and Illumina sequencing methods to discover new genes involved in xyloglucan biosynthesis, cDNA sequences were generated from developing nasturtium （Tropaeolum majus） seeds, which produce large amounts of non-fucosylated xyloglucan as a seed storage polymer. In addition to known xyloglucan biosynthetic genes, a previously uncharacterized putative xyloglucan galactosyltransferase was iden- tified. Analysis of an Arabidopsis thaliana mutant line defective in the corresponding ortholog （AT5G62220） revealed that this gene shows no redundancy with the previously characterized xyloglucan galactosyltransferase, MUR3, but is required for galactosyl-substitution of xyloglucan at a different position. The gene was termed XLT2 for Xyloglucan L-side chain galactosylTransferase position 2. It represents an enzyme in the same subclade of glycosyltransferase family 47 as MUR3. A double mutant defective in both MUR3 （mur3.1） and XLT2 led to an Arabidopsis plant with xyloglucan that consists essentially of only xylosylated glucosyl units, with no further substitutions.展开更多
Galacturonosyltransferase 1 （GAUT1） is an α1,4-D-galacturonosyltransferase that transfers galacturonic acid from uridine 5＇-diphosphogalacturonic acid onto the pectic polysaccharide homogalacturonan （Sterling et ...Galacturonosyltransferase 1 （GAUT1） is an α1,4-D-galacturonosyltransferase that transfers galacturonic acid from uridine 5＇-diphosphogalacturonic acid onto the pectic polysaccharide homogalacturonan （Sterling et al., 2006）. The 25-member Arabidopsis thaliana GAUT1-related gene family encodes 15 GAUT and 10 GAUT-like （GATL） proteins with, respectively, 56-84 and 42-53% amino acid sequence similarity to GAUT1. Previous phylogenetic analyses of AtGAUTs indicated three clades： A through C. A comparative phylogenetic analysis of the Arabidopsis, poplar and rice GAUT families has sub-classified the GAUTs into seven clades： clade A-1 （GAUTs 1 to 3）; A-2 （GAUT4）; A-3 （GAUTs 5 and 6）; A-4 （GAUT7）; B-1 （GAUTs 8 and 9）; B-2 （GAUTs 10 and 11）; and clade C （GAUTs 12 to 15）. The Arabidopsis GAUTs have a distribution comparable to the poplar orthologs, with the exception of GAUT2, which is absent in poplar. Rice, however, has no orthologs of GAUTs 2 and 12 and has multiple apparent orthologs of GAUTs 1, 4, and 7 compared with eitherArabidopsis or poplar. The cell wall glycosyl residue compositions of 26 homozygous T-DNA insertion mutants for 13 of 15 Arabidopsis GAUTgenes reveal significantly and reproducibly different cell walls in specific tissues of gaut mutants 6, 8, 9, 10, 11, 12, 13, and 14 from that of wild-type Arabidopsis walls. Pectin and xylan polysaccharides are affected by the loss of GAUT function, as demonstrated by the altered galacturonic acid, xylose, rhamnose, galactose, and arabinose composition of distinct gaut mutant walls. The wall glycosyl residue compositional phenotypes observed among the gaut mutants suggest that at least six different biosynthetic linkages in pectins and/or xylans are affected by the lesions in these GAUTgenes. Evidence is also presented to support a role for GAUT11 in seed mucilage expansion and in seed wall and mucilage composition.展开更多
The biochemical and molecular processes involved in the habituation of maize cells to growth in the presence of the cellulose biosynthesis inhibitor dichlobenil （DCB） were investigated. DCB affects the synthesis of ...The biochemical and molecular processes involved in the habituation of maize cells to growth in the presence of the cellulose biosynthesis inhibitor dichlobenil （DCB） were investigated. DCB affects the synthesis of cellulose both in active and stationary growth phases and alters the expression of several CesA genes. Of these, ZmCesA5 and ZmCesA7 seem to play a major role in habituating cells to growth in the presence of DCB. As a consequence of the reduction in cellulose, the expression of several genes involved in the synthesis of hydroxycinnamates is increased, resulting in cell walls with higher levels of ferulic and p-coumaric acids. A proteomic analysis revealed that habituation to DCB is linked to modifications in several metabolic pathways. Finally, habituated cells present a reduction in glutathione S-transferase detoxifying activity and antioxidant activities. Plant cell adaptation to the disturbance of such a crucial process as cellulose biosynthesis requires changes in several metabolic networks, in order to modify cell wall architecture and metabolism, and survive in the presence of the inhibitor. Some of these modifications are described in this paper.展开更多
Fluorescent studies of living plant cells such as confocal microscopy and fluorescence lifetime imaging often suffer from a strong autofluorescent background contribution that significantly reduces the dynamic image c...Fluorescent studies of living plant cells such as confocal microscopy and fluorescence lifetime imaging often suffer from a strong autofluorescent background contribution that significantly reduces the dynamic image contrast and the quantitative access to sub-cellular processes at high spatial resolution. Here, we present a novel technique--fluorescence intensity decay shape analysis microscopy （FIDSAM） to enhance the dynamic contrast of a fluorescence image of at least one order of magnitude. The method is based on the analysis of the shape of the fluorescence intensity decay （fluorescence lifetime curve） and benefits from the fact that the decay patterns of typical fluorescence label dyes strongly differ from emission decay curves of autofluorescent sample areas. Using FIDSAM, we investigated Arabidopsis thaliana hypocotyl cells in their tissue environment, which accumulate an eGFP fusion of the plasma membrane marker protein LTI6b （LTI6b-eGFP） to low level. Whereas in conventional confocal fluorescence images, the membranes of neighboring cells can hardly be optically resolved due to the strong autofluorescence of the cell wall, FIDSAM allows for imaging of single, isolated membranes at high spatial resolution. Thus, FIDSAM will enable the sub-cellular analysis of even low-expressed fluorophoretagged proteins in living plant cells. Furthermore, the combination of FIDSAM with fluorescence lifetime imaging provides the basis to study the local physico-chemical environment of fluorophore-tagged biomolecules in living plant cells.展开更多
PDZ domain proteins in metazoans function in diverse roles, and in conjunction with PDZ domain-binding proteins form macromolecular complexes for signaling at synapses and cell junctions. Bioinformatics approaches usi...PDZ domain proteins in metazoans function in diverse roles, and in conjunction with PDZ domain-binding proteins form macromolecular complexes for signaling at synapses and cell junctions. Bioinformatics approaches using the SMART tool indicate there are only a modest number of Arabidopsis PDZ proteins. However, there are hundreds of proteins predicted to possess PDZ domain-binding motifs, suggesting that there are many PDZ domain proteins not detectable by conventional bioinformatic approaches. Our Scansite analysis of PDZ domain-binding proteins indicates that PDZ domain proteins may play key roles in cytoskeletal organization including actin microfilaments, microtubules, and nuclear cytoskeletal proteins, and in the organization of macromolecular complexes involved in cell-to-cell signaling, transport, and cell wall formation.展开更多
Oligosaccharide Mass Profiling （OLIMP） allows a fast and sensitive assessment of cell wall polymer structure when coupled with Matrix Assisted Laser Desorption Ionisation Time Of Flight Mass Spectrometry （MALDI-TOF...Oligosaccharide Mass Profiling （OLIMP） allows a fast and sensitive assessment of cell wall polymer structure when coupled with Matrix Assisted Laser Desorption Ionisation Time Of Flight Mass Spectrometry （MALDI-TOF MS）. The short time required for sample preparation and analysis makes possible the study of a wide range of plant organs, revealing a high degree of heterogeneity in the substitution pattern of wall polymers such as the cross-linking glycan xyloglucan and the pectic polysaccharide homogalacturonan. The high sensitivity of MALDI-TOF allows the use of small amounts of samples, thus making it possible to investigate the wall structure of single cell types when material is collected by such methods as laser micro-dissection. As an example, the analysis of the xyloglucan structure in the leaf cell types outer epidermis layer, entire epidermis cell layer, palisade mesophyll cells, and vascular bundles were investigated. OLIMP is amenable to in situ wall analysis, where wall polymers are analyzed on unprepared plant tissue itself without first isolating cell walls. In addition, OLIMP enables analysis of wall polymers in Golgi-enriched fractions, the location of nascent matrix polysaccharide biosynthesis, enabling separation of the processes of wall biosynthesis versus post-deposition apoplastic metabolism. These new tools will make possible a semi-quantitative analysis of the cell wall at an unprecedented level.展开更多
Structural characteristics of xyloglucan are constant in the pericarp cell walls of kiwifruit （Actinidia deliciosa） throughout fruit enlargement and maturation. Most of the xyioglucan （XG） persists in the cell wal...Structural characteristics of xyloglucan are constant in the pericarp cell walls of kiwifruit （Actinidia deliciosa） throughout fruit enlargement and maturation. Most of the xyioglucan （XG） persists in the cell walls of ripe kiwifruit. XG from the pericarp tissues of 36-h ethylene-treated kiwifruit was extracted as hemicellulose Ⅱ （HC-Ⅱ） with 4.28 M KOH containing 0.02% NaBH4, and purified using iodine precipitation and subsequent anion-exchange chromatography. This purifying protocol increased XG purity from 50 mol% in HC-Ⅱ fraction to 62 mol% in the purified XG powder. The molar ratio of glucose： xylose： galactose： fucose in the purified XG was 10： 6.9： 2.1： 0.3. Gel permeation chromatography indicated that purified XG had an average molecular-mass of 161 KDa, a value that exceeds the 95 KDa Mr determined for total polymeric sugars. Sugar linkage analysis confirmed the lack of fucose in the kiwifruit XG, but a small amount of arabinoxylan and low Mr glucomannan remained associated with this fraction.展开更多
Transcriptional coordination, or co-expression, of genes may signify functional relatedness of the corresponding proteins. For example, several genes involved in secondary cell wall cellulose biosynthesis are co-expre...Transcriptional coordination, or co-expression, of genes may signify functional relatedness of the corresponding proteins. For example, several genes involved in secondary cell wall cellulose biosynthesis are co-expressed with genes engaged in the synthesis of xylan, which is a major component of the secondary cell wall. To extend these types of analyses, we investigated the co-expression relationships of all Carbohydrate-Active enZYmes （CAZy）-related genes for Arabidopsis thaliana. Thus, the intention was to transcriptionally link different cell wall-related processes to each other, and also to other biological functions. To facilitate easy manual inspection, we have displayed these interactions as networks and matrices, and created a web-based interface （http：llaranet.mpimp-golm.mpg.delcorecarb） containing downloadable files for all the transcriptional associations.展开更多
文摘Plant cell walls are complex structures composed of high-molecular-weight polysaccharides, proteins, and lignins. Among the wall polysaccharides, cellulose, a hydrogen-bonded β-1,4-1inked glucan microfibril, is the main load-bearing wall component and a key precursor for industrial applications. Cellulose is synthesized by large multi-meric cellulose synthase （CesA） complexes, tracking along cortical microtubules at the plasma membrane. The only known components of these complexes are the cellulose synthase proteins. Recent studies have identified tentative interaction partners for the CesAs and shown that the migratory patterns of the CesA complexes depend on phosphorylation status. These advances may become good platforms for expanding our knowledge about cellulose synthesis in the near future. In addition, our current understanding of cellulose chain polymerization in the context of the CesA complex is discussed.
文摘The ability of a plant cell to expand is largely defined by the physical constraints imposed by its cell wall. Accordingly, cell wall properties have to be regulated during development. The pectic polysaccharide homogalacturonan is a major component of the plant primary walls. Biosynthesis and in muro modification of homogalacturonan have recently emerged as key determinants of plant development, controlling cell adhesion, organ development, and phyllotactic patterning. This review will focus on recent findings regarding impact of homogalacturonan content and methylesterification status of this polymer on plant life. Demethyl-esterification of homogalacturonan occurs through the action of the ubiquitous enzyme ＇pectin methyl-esterase＇. We here describe various strategies developed by the plant to finely tune the methyl-esterification status of homogalacturonan along key events of the plant lifecycle.
文摘Nexine is a conserved layer of the pollen wall. We previously reported that the nexine layer is absent in the knockout mutant of Arabidopsis TRANSPOSABLE ELEMENT SILENCING VIA AT-HOOK （TEK） gene. In this study, we investigated the molecular regulatory functions of TEK in pollen development and identified the genes encoding Arabinogalactan proteins （AGPs） as direct targets of TEK, which are essential for nexine formation. Phenotypic similarity between tek and the TEK-SRDX transgenic lines suggest that TEK plays a role in transcriptional activation in anther development. Microarray analysis identified a total of 661 genes downregulated in tek, including four genes encoding AGPs, AGP6, AGP11, AGP23, and AGP40. Electrophoretic mobility shift assays showed that TEK could directly bind the nuclear matrix attachment region （MAR） and the promoter of AGP6. Chromatin immunoprecipitation followed by PCR analysis demonstrated that TEK is enriched in the promoters of the four AGP genes. Expression of AGP6 driven by the TEK promoter in tek partially rescued both nexine formation and plant fertility. These results indicate that TEK directly reg- ulates AGP expression in the anther to control nexine layer formation. We also proposed that glycoproteins might be essential components of the nexine laver in the oollen wall.
文摘In this review, we address the question of how the tip-growing pollen tube achieves its rapid rate of elongation while maintaining an intact cell wall. Although turgor is essential for growth to occur, the local expansion rate is controlled by local changes in the viscosity of the apical wall. We focus on several different structures and underly- ing processes that are thought to be major participants including exocytosis, the organization and activity of the actin cytoskeleton, calcium and proton physiology, and cellular energetics. We think that the actin cytoskeleton, in particular the apical cortical actin fringe, directs the flow of vesicles to the apical domain, where they fuse with the plasma mem- brane and contribute their contents to the expanding cell wall. While pH gradients, as generated by a proton-ATPase located on the plasma membrane along the side of the clear zone, may regulate rapid actin turnover and new polymeri- zation in the fringe, the tip-focused calcium gradient biases secretion towards the polar axis. The recent data showing that exocytosis of new wall material precedes and predicts the process of cell elongation provide support for the idea that the intussusception of newly secreted pectin contributes to decreases in apical wall viscosity and to cell expansion. Other prime factors will be the localization and activity of the enzyme pectin methyl-esterase, and the chelation of calcium by pectic acids. Finally, we acknowledge a role for reactive oxygen species in the control of wall viscosity.
文摘IRX14 and IRX14-LIKE （IRX14L） are two closely related glycosyl transferases in the glycosyl transferase 43 （GT43） family of Arabidopsis. A T-DNA insertion mutant for IRX14 results in comparatively minor changes, such as irregular xylem, while a mutation for IRX14L results in no changes. However, an irx14 and irx14L double mutant severely affects growth and development, with the dwarf plants failing to produce an inflorescence stem. Plants that are homozygous for IRX14 but heterozygous for IRX14L （irx14 irx14L（±）） exhibit an intermediate phenotype, including noticeably smaller leaves, stems, and underdeveloped siliques. Additionally, the T-DNA insertion mutant for IRX14 was found to result in a drought-tolerant phenotype. Carbohydrate analysis of total cell wall extracts revealed a reduction in xylose for the irx14 and irx14 irx14L（±） mutants, consistent with a defect in glucuronoxylan biosynthesis. Immunolocalization of xylan with the LM10 antibody revealed a loss of xylan in irx14 mutants and a further reduction in the irx14 irx14L（±） mutants. IRX14L likely functions redundantly with IRX14 in glucuronoxylan biosynthesis, with IRX14 having a more important role in the process.
文摘All plant cells are surrounded by a cell wall that determines the directionality of cell growth and protects the cell against its environment. Plant cell walls are comprised primarily of polysaccharides and represent the largest sink for photosynthetically fixed carbon, both for individual plants and in the terrestrial biosphere as a whole. Cell wall synthesis is a highly sophisticated process, involving multiple enzymes and metabolic intermediates, intracellular trafficking of proteins and cell wall precursors, assembly of cell wall polymers into the extracellular matrix, remodeling of polymers and their interactions, and recycling of cell wall sugars. In this review we discuss how newly fixed carbon, in the form of UDP-glucose and other nucleotide sugars, contributes to the synthesis of cell wall polysaccharides, and how cell wall synthesis is influenced by the carbon status of the plant, with a focus on the model species Arabidopsis （Arabidopsis thaliana）.
文摘Methods developed to isolate intact cells from both unripe and ripe tomato fruit pericarp parenchyma have allowed the cell biological analysis of polysaccharide epitopes at the surface of separated cells. The LM7 pectic homogalacturonan epitope is a marker of the junctions of adhesion planes and intercellular spaces in parenchyma systems. The LM7 epitope persistently marked the former edge of adhesion planes at the surface of cells separated from unripe and ripened tomato fruit and also from fruits with the Cnr mutation. The LM 11 xylan epitope was associated, in sections, with cell walls lining intercellular space but the epitope was not detected at the surface of isolated cells, being lost during cell isolation. The LM15 xyloglucan epitope was present at the surface of cells isolated from unripe fruit in a pattern reflecting the former edge of cell adhesion planes/intercellular space but with gaps and apparent breaks. An equivalent pattern of LM15 epitope occurrence was revealed at the surface of cells isolated by pectate lyase action but was not present in cells isolated from ripe fruit or from Cnr fruit. In contrast to wild-type cells, the LM5 galactan and LM21 mannan epitopes occurred predominantly in positions reflecting intercellular space in Cnr, suggesting a concerted alteration in cell wall microstructure in response to this mutation. Galactanase and mannanase, along with pectic homogalacturonan-degrading enzymes, were capable of releasing cells from unripe fruit parenchyma. These observations indicate that hemicellulose polymers are present in architectural contexts reflecting cell adhesion and that several cell wall polysaccharide classes are likely to contribute to cell adhesion/cell separation in tomato fruit pericarp parenchyma.
文摘Polysaccharide-rich plant cell walls are important biomaterials that underpin plant growth, are major repositories for photosynthetically accumulated carbon, and, in addition, impact greatly on the human use of plants. Land plant cell walls contain in the region of a dozen major polysaccharide structures that are mostly encompassed by cellulose, hemicelluloses, and pectic polysaccharides. During the evolution of land plants, polysaccharide diversification appears to have largely involved structural elaboration and diversification within these polysaccharide groups. Cell wall chemistry is well advanced and a current phase of cell wall science is aimed at placing the complex polysaccharide chemistry in cellular contexts and developing a detailed understanding of cell wall biology. Imaging cell wall glycomes is a challenging area but recent developments in the establishment of cell wall molecular probe panels and their use in high throughput procedures are leading to rapid advances in the molecular understanding of the spatial heterogeneity of individual cell walls and also cell wall differences at taxonomic levels. The challenge now is to integrate this knowledge of cell wall heterogeneity with an understanding of the molecular and physiological mechanisms that underpin cell wall properties and functions.
文摘A deep-sequencing approach was pursued utilizing 454 and Illumina sequencing methods to discover new genes involved in xyloglucan biosynthesis, cDNA sequences were generated from developing nasturtium （Tropaeolum majus） seeds, which produce large amounts of non-fucosylated xyloglucan as a seed storage polymer. In addition to known xyloglucan biosynthetic genes, a previously uncharacterized putative xyloglucan galactosyltransferase was iden- tified. Analysis of an Arabidopsis thaliana mutant line defective in the corresponding ortholog （AT5G62220） revealed that this gene shows no redundancy with the previously characterized xyloglucan galactosyltransferase, MUR3, but is required for galactosyl-substitution of xyloglucan at a different position. The gene was termed XLT2 for Xyloglucan L-side chain galactosylTransferase position 2. It represents an enzyme in the same subclade of glycosyltransferase family 47 as MUR3. A double mutant defective in both MUR3 （mur3.1） and XLT2 led to an Arabidopsis plant with xyloglucan that consists essentially of only xylosylated glucosyl units, with no further substitutions.
文摘Galacturonosyltransferase 1 （GAUT1） is an α1,4-D-galacturonosyltransferase that transfers galacturonic acid from uridine 5＇-diphosphogalacturonic acid onto the pectic polysaccharide homogalacturonan （Sterling et al., 2006）. The 25-member Arabidopsis thaliana GAUT1-related gene family encodes 15 GAUT and 10 GAUT-like （GATL） proteins with, respectively, 56-84 and 42-53% amino acid sequence similarity to GAUT1. Previous phylogenetic analyses of AtGAUTs indicated three clades： A through C. A comparative phylogenetic analysis of the Arabidopsis, poplar and rice GAUT families has sub-classified the GAUTs into seven clades： clade A-1 （GAUTs 1 to 3）; A-2 （GAUT4）; A-3 （GAUTs 5 and 6）; A-4 （GAUT7）; B-1 （GAUTs 8 and 9）; B-2 （GAUTs 10 and 11）; and clade C （GAUTs 12 to 15）. The Arabidopsis GAUTs have a distribution comparable to the poplar orthologs, with the exception of GAUT2, which is absent in poplar. Rice, however, has no orthologs of GAUTs 2 and 12 and has multiple apparent orthologs of GAUTs 1, 4, and 7 compared with eitherArabidopsis or poplar. The cell wall glycosyl residue compositions of 26 homozygous T-DNA insertion mutants for 13 of 15 Arabidopsis GAUTgenes reveal significantly and reproducibly different cell walls in specific tissues of gaut mutants 6, 8, 9, 10, 11, 12, 13, and 14 from that of wild-type Arabidopsis walls. Pectin and xylan polysaccharides are affected by the loss of GAUT function, as demonstrated by the altered galacturonic acid, xylose, rhamnose, galactose, and arabinose composition of distinct gaut mutant walls. The wall glycosyl residue compositional phenotypes observed among the gaut mutants suggest that at least six different biosynthetic linkages in pectins and/or xylans are affected by the lesions in these GAUTgenes. Evidence is also presented to support a role for GAUT11 in seed mucilage expansion and in seed wall and mucilage composition.
文摘The biochemical and molecular processes involved in the habituation of maize cells to growth in the presence of the cellulose biosynthesis inhibitor dichlobenil （DCB） were investigated. DCB affects the synthesis of cellulose both in active and stationary growth phases and alters the expression of several CesA genes. Of these, ZmCesA5 and ZmCesA7 seem to play a major role in habituating cells to growth in the presence of DCB. As a consequence of the reduction in cellulose, the expression of several genes involved in the synthesis of hydroxycinnamates is increased, resulting in cell walls with higher levels of ferulic and p-coumaric acids. A proteomic analysis revealed that habituation to DCB is linked to modifications in several metabolic pathways. Finally, habituated cells present a reduction in glutathione S-transferase detoxifying activity and antioxidant activities. Plant cell adaptation to the disturbance of such a crucial process as cellulose biosynthesis requires changes in several metabolic networks, in order to modify cell wall architecture and metabolism, and survive in the presence of the inhibitor. Some of these modifications are described in this paper.
文摘Fluorescent studies of living plant cells such as confocal microscopy and fluorescence lifetime imaging often suffer from a strong autofluorescent background contribution that significantly reduces the dynamic image contrast and the quantitative access to sub-cellular processes at high spatial resolution. Here, we present a novel technique--fluorescence intensity decay shape analysis microscopy （FIDSAM） to enhance the dynamic contrast of a fluorescence image of at least one order of magnitude. The method is based on the analysis of the shape of the fluorescence intensity decay （fluorescence lifetime curve） and benefits from the fact that the decay patterns of typical fluorescence label dyes strongly differ from emission decay curves of autofluorescent sample areas. Using FIDSAM, we investigated Arabidopsis thaliana hypocotyl cells in their tissue environment, which accumulate an eGFP fusion of the plasma membrane marker protein LTI6b （LTI6b-eGFP） to low level. Whereas in conventional confocal fluorescence images, the membranes of neighboring cells can hardly be optically resolved due to the strong autofluorescence of the cell wall, FIDSAM allows for imaging of single, isolated membranes at high spatial resolution. Thus, FIDSAM will enable the sub-cellular analysis of even low-expressed fluorophoretagged proteins in living plant cells. Furthermore, the combination of FIDSAM with fluorescence lifetime imaging provides the basis to study the local physico-chemical environment of fluorophore-tagged biomolecules in living plant cells.
文摘PDZ domain proteins in metazoans function in diverse roles, and in conjunction with PDZ domain-binding proteins form macromolecular complexes for signaling at synapses and cell junctions. Bioinformatics approaches using the SMART tool indicate there are only a modest number of Arabidopsis PDZ proteins. However, there are hundreds of proteins predicted to possess PDZ domain-binding motifs, suggesting that there are many PDZ domain proteins not detectable by conventional bioinformatic approaches. Our Scansite analysis of PDZ domain-binding proteins indicates that PDZ domain proteins may play key roles in cytoskeletal organization including actin microfilaments, microtubules, and nuclear cytoskeletal proteins, and in the organization of macromolecular complexes involved in cell-to-cell signaling, transport, and cell wall formation.
文摘Oligosaccharide Mass Profiling （OLIMP） allows a fast and sensitive assessment of cell wall polymer structure when coupled with Matrix Assisted Laser Desorption Ionisation Time Of Flight Mass Spectrometry （MALDI-TOF MS）. The short time required for sample preparation and analysis makes possible the study of a wide range of plant organs, revealing a high degree of heterogeneity in the substitution pattern of wall polymers such as the cross-linking glycan xyloglucan and the pectic polysaccharide homogalacturonan. The high sensitivity of MALDI-TOF allows the use of small amounts of samples, thus making it possible to investigate the wall structure of single cell types when material is collected by such methods as laser micro-dissection. As an example, the analysis of the xyloglucan structure in the leaf cell types outer epidermis layer, entire epidermis cell layer, palisade mesophyll cells, and vascular bundles were investigated. OLIMP is amenable to in situ wall analysis, where wall polymers are analyzed on unprepared plant tissue itself without first isolating cell walls. In addition, OLIMP enables analysis of wall polymers in Golgi-enriched fractions, the location of nascent matrix polysaccharide biosynthesis, enabling separation of the processes of wall biosynthesis versus post-deposition apoplastic metabolism. These new tools will make possible a semi-quantitative analysis of the cell wall at an unprecedented level.
基金supported by the Postdoctoral Fellowship of Venture Business Laboratory in Hiroshima University and of the Japanese Society for Promotion of Science to Dr. XJ Li (P05190)
文摘Structural characteristics of xyloglucan are constant in the pericarp cell walls of kiwifruit （Actinidia deliciosa） throughout fruit enlargement and maturation. Most of the xyioglucan （XG） persists in the cell walls of ripe kiwifruit. XG from the pericarp tissues of 36-h ethylene-treated kiwifruit was extracted as hemicellulose Ⅱ （HC-Ⅱ） with 4.28 M KOH containing 0.02% NaBH4, and purified using iodine precipitation and subsequent anion-exchange chromatography. This purifying protocol increased XG purity from 50 mol% in HC-Ⅱ fraction to 62 mol% in the purified XG powder. The molar ratio of glucose： xylose： galactose： fucose in the purified XG was 10： 6.9： 2.1： 0.3. Gel permeation chromatography indicated that purified XG had an average molecular-mass of 161 KDa, a value that exceeds the 95 KDa Mr determined for total polymeric sugars. Sugar linkage analysis confirmed the lack of fucose in the kiwifruit XG, but a small amount of arabinoxylan and low Mr glucomannan remained associated with this fraction.
文摘Transcriptional coordination, or co-expression, of genes may signify functional relatedness of the corresponding proteins. For example, several genes involved in secondary cell wall cellulose biosynthesis are co-expressed with genes engaged in the synthesis of xylan, which is a major component of the secondary cell wall. To extend these types of analyses, we investigated the co-expression relationships of all Carbohydrate-Active enZYmes （CAZy）-related genes for Arabidopsis thaliana. Thus, the intention was to transcriptionally link different cell wall-related processes to each other, and also to other biological functions. To facilitate easy manual inspection, we have displayed these interactions as networks and matrices, and created a web-based interface （http：llaranet.mpimp-golm.mpg.delcorecarb） containing downloadable files for all the transcriptional associations.
