Effects of the mur1 mutation on xyloglucans produced by suspension-cultured Arabidopsis thaliana cells

Mutation of the Arabidopsis thaliana (L.) Heynh. gene MUR1, which encodes an isoform of GDP-D-mannose-4,6-dehydratase, affects the biosynthetic conversion of GDP-mannose to GDP-fucose. Cell walls in the aerial tissues of mur1 plants are almost devoid of alpha-L-fucosyl residues, which are partially replaced by closely related alpha-L-galactosyl residues. A line of suspension-cultured A. thaliana cells was generated from leaves of mur1 plants and the structure of the xyloglucan in the walls of these cells was structurally characterized. Xyloglucan fractions were prepared from the walls of both wild-type (WT) and mur1 cells by sequential extraction with a xyloglucan-specific endoglucanase (XEG) and aqueous KOH. Structural analysis of these fractions revealed that xyloglucan produced by cultured mur1 cells is similar, but not identical to that isolated from leaves of mur1 plants. As previously reported for mur1 leaves, the xyloglucan from cultured mur1 cells contains less than 5% of the fucose present in the xyloglucan from WT cells. Fucosylation of the xyloglucan is substantially restored when mur1 cells are grown in medium supplemented with L-fucose. Xyloglucan isolated from leaves contains more oligosaccharide subunits in which the central sidechain is terminated with a beta-D-galactosyl residue than does xyloglucan prepared from cultured cells. This was observed for both mur1 and WT plants, indicating that this correlation is independent of the mur1 mutation and that it is possible to distinguish changes due to genetic mutation from those due to the physiological state of the cells in culture. Suspension-cultured cells thus provide a convenient source of genetically altered cell wall material, facilitating the biochemical characterization of mutations that affect cell wall structure.     

The mur4 mutant of arabidopsis is partially defective in the de novo synthesis of uridine diphospho L-arabinose.

To obtain information on the synthesis and function of arabinosylated glycans, the mur4 mutant of Arabidopsis was characterized. This mutation leads to a 50% reduction in the monosaccharide L-arabinose in most organs and affects arabinose-containing pectic cell wall polysaccharides and arabinogalactan proteins. Feeding L-arabinose to mur4 plants restores the cell wall composition to wild-type levels, suggesting a partial defect in the de novo synthesis of UDP-L-arabinose, the activated sugar used by arabinosyltransferases. The defect was traced to the conversion of UDP-D-xylose to UDP-L-arabinose in the microsome fraction of leaf material, indicating that mur4 plants are defective in a membrane-bound UDP-D-xylose 4-epimerase.     

The galactose residues of xyloglucan are essential to maintain mechanical strength of the primary cell walls in Arabidopsis during growth

In land plants, xyloglucans (XyGs) tether cellulose microfibrils into a strong but extensible cell wall. The MUR2 and MUR3 genes of Arabidopsis encode XyG-specific fucosyl and galactosyl transferases, respectively. Mutations of these genes give precisely altered XyG structures missing one or both of these subtending sugar residues. Tensile strength measurements of etiolated hypocotyls revealed that galactosylation rather than fucosylation of the side chains is essential for maintenance of wall strength. Symptomatic of this loss of tensile strength is an abnormal swelling of the cells at the base of fully grown hypocotyls as well as bulging and marked increase in the diameter of the epidermal and underlying cortical cells. The presence of subtending galactosyl residues markedly enhance the activities of XyG endotransglucosylases and the accessibility of XyG to their action, indicating a role for this enzyme activity in XyG cleavage and religation in the wall during growth for maintenance of tensile strength. Although a shortening of XyGs that normally accompanies cell elongation appears to be slightly reduced, galactosylation of the XyGs is not strictly required for cell elongation, for lengthening the polymers that occurs in the wall upon secretion, or for binding of the XyGs to cellulose.     

木葡聚糖及其在植物抗逆过程中的功能研究进展

木葡聚糖(XyG)是一种存在于所有陆生植物细胞壁中的基质多糖, 是双子叶植物初生细胞壁中含量(20%-25%, w/w)最丰富的半纤维素成分。作为细胞壁的组分, XyG不仅与植物的生长发育密切相关, 还在植物抵抗各种生物和非生物逆境过程中发挥重要作用。XyG代谢相关基因主要通过改变植物细胞壁的组成以及对细胞壁进行重排进而改变细胞壁的弹性/硬度等特性, 影响植物的抗逆性。XyG及其寡糖也可能作为信号分子, 或与其它信号分子协同作用应对逆境胁迫。该文概述了XyG的结构与类型及参与XyG生物合成与降解的相关基因, 重点阐述XyG相关基因应答生物和非生物胁迫的作用机制。     

AXY8 encodes an α-fucosidase, underscoring the importance of apoplastic metabolism on the fine structure of Arabidopsis cell wall polysaccharides

An Arabidopsis thaliana mutant with an altered structure of its hemicellulose xyloglucan (XyG; axy-8) identified by a forward genetic screen facilitating oligosaccharide mass profiling was characterized. axy8 exhibits increased XyG fucosylation and the occurrence of XyG fragments not present in the wild-type plant. AXY8 was identified to encode an α-fucosidase acting on XyG that was previously designated FUC95A. Green fluorescent protein fusion localization studies and analysis of nascent XyG in microsomal preparations demonstrated that this glycosylhydrolase acts mainly on XyG in the apoplast. Detailed structural analysis of XyG in axy8 gave unique insights into the role of the fucosidase in XyG metabolism in vivo. The genetic evidence indicates that the activity of glycosylhydrolases in the apoplast plays a major role in generating the heterogeneity of XyG side chains in the wall. Furthermore, without the dominant apoplastic glycosylhydrolases, the XyG structure in the wall is mainly composed of XXXG and XXFG subunits.     

A unique beta1,3-galactosyltransferase is indispensable for the biosynthesis of N-glycans containing Lewis a structures in Arabidopsis thaliana

In plants, the only known outer-chain elongation of complex N-glycans is the formation of Lewis a [Fuc alpha1-4(Gal beta1-3)GlcNAc-R] structures. This process involves the sequential attachment of beta1,3-galactose and alpha1,4-fucose residues by beta1,3-galactosyltransferase and alpha1,4-fucosyltransferase. However, the exact mechanism underlying the formation of Lewis a epitopes in plants is poorly understood, largely because one of the involved enzymes, beta1,3-galactosyltransferase, has not yet been identified and characterized. Here, we report the identification of an Arabidopsis thaliana beta1,3-galactosyltransferase involved in the biosynthesis of the Lewis a epitope using an expression cloning strategy. Overexpression of various candidates led to the identification of a single gene (named GALACTOSYLTRANSFERASE1 [GALT1]) that increased the originally very low Lewis a epitope levels in planta. Recombinant GALT1 protein produced in insect cells was capable of transferring beta1,3-linked galactose residues to various N-glycan acceptor substrates, and subsequent treatment of the reaction products with alpha1,4-fucosyltransferase resulted in the generation of Lewis a structures. Furthermore, transgenic Arabidopsis plants lacking a functional GALT1 mRNA did not show any detectable amounts of Lewis a epitopes on endogenous glycoproteins. Taken together, our results demonstrate that GALT1 is both sufficient and essential for the addition of beta1,3-linked galactose residues to N-glycans and thus is required for the biosynthesis of Lewis a structures in Arabidopsis. Moreover, cell biological characterization of a transiently expressed GALT1-fluorescent protein fusion using confocal laser scanning microscopy revealed the exclusive location of GALT1 within the Golgi apparatus, which is in good agreement with the proposed physiological action of the enzyme.     

In silico prediction of proteins related to xyloglucan fucosyltransferases in Solanaceae genomes

Two independent studies have shown that the cell wall of pollen tubes from tobacco and tomato species contained fucosylated xyloglucan (XyG). These findings are intriguing as many reports have shown that XyG of somatic cells of these species is not fucosylated but instead is arabinosylated. In order to produce fucosylated XyG, plants must express a functional galactoside α-2-fucosyltransferase. Here, using a bioinformatics approach, we show that several candidate genes coding for XyG fucosyltransferases are present in the genome of coffee and several Solanaceae species including tomato, tobacco, potato, eggplant and pepper. BLAST and protein alignments with the 2 well-characterized XyG fucosyltransferases from Arabidopsis thaliana and Pisum sativum revealed that at least 6 proteins from different Solanaceae species and from coffee displayed the 3 conserved motifs required for XyG fucosyltransferase activity.     

Signaling from an altered cell wall to the nucleus mediates sugar-responsive growth and development in Arabidopsis thaliana

Sugars such as glucose function as signal molecules that regulate gene expression, growth, and development in plants, animals, and yeast. To understand the molecular mechanisms of sugar responses, we isolated and characterized an Arabidopsis thaliana mutant, high sugar response8 (hsr8), which enhances sugar-responsive growth and gene expression. Light-grown hsr8 plants exhibited increased starch and anthocyanin and reduced chlorophyll content in response to glucose treatment. Dark-grown hsr8 seedlings showed glucose-hypersensitive hypocotyl elongation and development. The HSR8 gene, isolated using map-based cloning, was allelic to the MURUS4 (MUR4) gene involved in arabinose synthesis. Dark-grown mur1 and mur3 seedlings also exhibited similar sugar responses to hsr8/mur4. The sugar-hypersensitive phenotypes of hsr8/mur4, mur1, and mur3 were rescued by boric acid, suggesting that alterations in the cell wall cause hypersensitive sugar-responsive phenotypes. Genetic analysis showed that sugar-hypersensitive responses in hsr8 mutants were suppressed by pleiotropic regulatory locus1 (prl1), indicating that nucleus-localized PRL1 is required for enhanced sugar responses in hsr8 mutant plants. Microarray analysis revealed that the expression of many cell wall-related and sugar-responsive genes was altered in mur4-1, and the expression of a significant proportion of these genes was restored to wild-type levels in the mur4-1 prl1 double mutant. These findings reveal a pathway that signals changes in the cell wall through PRL1 to altered gene expression and sugar-responsive metabolic, growth, and developmental changes.     

Cell Wall Metabolism in Ripening Fruit (VI. Effect of the Antisense Polygalacturonase Gene on Cell Wall Changes Accompanying Ripening in Transgenic Tomatoes)

Cell walls of tomato (Lycopersicon esculentum Mill.) fruit, prepared so as to minimize residual hydrolytic activity and autolysis, exhibit increasing solubilization of pectins as ripening proceeds, and this process is not evident in fruit from transgenic plants with the antisense gene for polygalacturonase (PG). A comparison of activities of a number of possible cell wall hydrolases indicated that antisense fruit differ from control fruit specifically in their low PG activity. The composition of cell wall fractions of mature green fruit from transgenic and control (wild-type) plants were indistinguishable except for trans-1,2-diaminocyclohexane-N,N,N[prime],N[prime]-tetraacetic acid (CDTA)-soluble pectins of transgenic fruit, which had elevated levels of arabinose and galactose. Neutral polysaccharides and polyuronides increased in the water-soluble fraction of wild-type fruit during ripening, and this was matched by a decline in Na2CO3-soluble pectins, equal in magnitude and timing. This, together with compositional analysis showing increasing galactose, arabinose, and rhamnose in the water-soluble fraction, mirrored by a decline of these same residues in the Na2CO3-soluble pectins, suggests that the polyuronides and neutral polysaccharides solubilized by PG come from the Na2CO3-soluble fraction of the tomato cell wall. In addition to the loss of galactose from the cell wall as a result of PG activity, both antisense and control fruit exhibit an independent decline in galactose in both the CDTA-soluble and Na2CO3-soluble fractions, which may play a role in fruit softening.     

High-throughput mapping of cell-wall polymers within and between plants using novel microarrays

We describe here a methodology that enables the occurrence of cell-wall glycans to be systematically mapped throughout plants in a semi-quantitative high-throughput fashion. The technique (comprehensive microarray polymer profiling, or CoMPP) integrates the sequential extraction of glycans from multiple organs or tissues with the generation of microarrays, which are probed with monoclonal antibodies (mAbs) or carbohydrate-binding modules (CBMs) with specificities for cell-wall components. The profiles generated provide a global snapshot of cell-wall composition, and also allow comparative analysis of mutant and wild-type plants, as demonstrated here for the Arabidopsis thaliana mutants fra8, mur1 and mur3. CoMPP was also applied to Physcomitrella patens cell walls and was validated by carbohydrate linkage analysis. These data provide new insights into the structure and functions of plant cell walls, and demonstrate the potential of CoMPP as a component of systems-based approaches to cell-wall biology.     

Functional nonequivalency of actin isovariants in Arabidopsis.

Plants encode at least two ancient and divergent classes of actin, reproductive and vegetative, and each class produces several subclasses of actin isovariants. To gain insight into the functional significance of the actin isovariants, we generated transgenic Arabidopsis lines that expressed a reproductive actin, ACT1, under the control of the regulatory sequences of a vegetative actin gene, ACT2. In the wild-type plants, ACT1 is predominantly expressed in the mature pollen, growing pollen tubes, and ovules, whereas ACT2 is constitutively and strongly expressed in all vegetative tissues and organs, but not in pollen. Misexpression of ACT1 in vegetative tissues causes dwarfing of plants and altered morphology of most organs, and the effects are in direct proportion to protein expression levels. Similar overexpression of ACT2 has little effect. Immunolocalization of actin in leaf cells from transgenic plants with highest levels of ACT1 protein revealed massive polymerization, bundling, and reorganization of actin filaments. This phenomenon suggests that misexpression of ACT1 isovariant in vegetative tissues affects the dynamics of actin and actin-associated proteins, in turn disrupting the organization of actin cytoskeleton and normal development of plants.     

Heterology expression of the Arabidopsis C-repeat/dehydration response element binding factor 1 gene confers elevated tolerance to chilling and oxidative stresses in transgenic tomato

In an attempt to improve stress tolerance of tomato (Lycopersicon esculentum) plants, an expression vector containing an Arabidopsis C-repeat/dehydration responsive element binding factor 1 (CBF1) cDNA driven by a cauliflower mosaic virus 35S promoter was transferred into tomato plants. Transgenic expression of CBF1 was proved by northern- and western-blot analyses. The degree of chilling tolerance of transgenic T(1) and T(2) plants was found to be significantly greater than that of wild-type tomato plants as measured by survival rate, chlorophyll fluorescence value, and radical elongation. The transgenic tomato plants exhibited patterns of growth retardation; however, they resumed normal growth after GA(3) (gibberellic acid) treatment. More importantly, GA(3)-treated transgenic plants still exhibited a greater degree of chilling tolerance compared with wild-type plants. Subtractive hybridization was performed to isolate the responsive genes of heterologous Arabidopsis CBF1 in transgenic tomato plants. CATALASE1 (CAT1) was obtained and showed activation in transgenic tomato plants. The CAT1 gene and catalase activity were also highly induced in the transgenic tomato plants. The level of H(2)O(2) in the transgenic plants was lower than that in the wild-type plants under either normal or cold conditions. The transgenic plants also exhibited considerable tolerance against oxidative damage induced by methyl viologen. Results from the current study suggest that heterologous CBF1 expression in transgenic tomato plants may induce several oxidative-stress responsive genes to protect from chilling stress.     

Rapid structural phenotyping of plant cell wall mutants by enzymatic oligosaccharide fingerprinting

Various biochemical, chemical, and microspectroscopic methods have been developed throughout the years for the screening and identification of mutants with altered cell wall structure. However, these procedures fail to provide the insight into structural aspects of the cell wall polymers. In this paper, we present various methods for rapidly screening Arabidopsis cell wall mutants. The enzymatic fingerprinting procedures using high-performance anion-exchange-pulsed-amperometric detection liquid chromatography, fluorophore-assisted carbohydrate electrophoresis, and matrix-assisted laser-desorption ionization time of flight (MALDI-TOF) mass spectrometry (MS) were exemplified by the structural analysis of the hemicellulose xyloglucan. All three techniques are able to identify structural alterations of wall xyloglucans in mur1, mur2, and mur3, which in comparison with the wild type have side chain defects in their xyloglucan structure. The quickest analysis was provided by MALDI-TOF MS. Although MALDI-TOF MS per se is not quantitative, it is possible to reproducibly obtain relative abundance information of the various oligosaccharides present in the extract. The lack of absolute quantitation by MALDI-TOF MS was compensated for with a xyloglucan-specific endoglucanase and simple colorimetric assay. In view of the potential for mass screening using MALDI-TOF MS, a PERL-based program was developed to process the spectra obtained from MALDI-TOF MS automatically. Outliers can be identified very rapidly according to a set of defined parameters based on data collected from the wild-type plants. The methods presented here can easily be adopted for the analysis of other wall polysaccharides. MALDI-TOF MS offers a powerful tool to screen and identify cell wall mutants rapidly and efficiently and, more importantly, is able to give initial insights into the structural composition and/or modification that occurs in these mutants.     

The interconversion of UDP-arabinopyranose and UDP-arabinofuranose is indispensable for plant development in Arabidopsis

L-Ara, an important constituent of plant cell walls, is found predominantly in the furanose rather than in the thermodynamically more stable pyranose form. Nucleotide sugar mutases have been demonstrated to interconvert UDP-Larabinopyranose (UDP-Arap) and UDP-L-arabinofuranose (UDP-Araf) in rice (Oryza sativa). These enzymes belong to a small gene family encoding the previously named Reversibly Glycosylated Proteins (RGPs). RGPs are plant-specific cytosolic proteins that tend to associate with the endomembrane system. In Arabidopsis thaliana, the RGP protein family consists of five closely related members. We characterized all five RGPs regarding their expression pattern and subcellular localizations in transgenic Arabidopsis plants. Enzymatic activity assays of recombinant proteins expressed in Escherichia coli identified three of the Arabidopsis RGP protein family members as UDP-L-Ara mutases that catalyze the formation of UDP-Araf from UDP-Arap. Coimmunoprecipitation and subsequent liquid chromatography-electrospray ionization-tandem mass spectrometry analysis revealed a distinct interaction network between RGPs in different Arabidopsis organs. Examination of cell wall polysaccharide preparations from RGP1 and RGP2 knockout mutants showed a significant reduction in total L-Ara content (12–31%) compared with wild-type plants. Concomitant downregulation of RGP1 and RGP2 expression results in plants almost completely deficient in cell wall–derived L-Ara and exhibiting severe developmental defects.     

Subcompartment localization of the side chain xyloglucan-synthesizing enzymes within Golgi stacks of tobacco suspension-cultured cells

Xyloglucan is the dominant hemicellulosic polysaccharide of the primary cell wall of dicotyledonous plants that plays a key role in plant development. It is well established that xyloglucan is assembled within Golgi stacks and transported in Golgi-derived vesicles to the cell wall. It is also known that the biosynthesis of xyloglucan requires the action of glycosyltransferases including α-1,6-xylosyltransferase, β-1,2-galactosyltransferase and α-1,2-fucosyltransferase activities responsible for the addition of xylose, galactose and fucose residues to the side chains. There is, however, a lack of knowledge on how these enzymes are distributed within subcompartments of Golgi stacks. We have undertaken a study aiming at mapping these glycosyltransferases within Golgi stacks using immunogold-electron microscopy. To this end, we generated transgenic lines of tobacco (Nicotiana tabacum) BY-2 suspension-cultured cells expressing either the α-1,6-xylosyltransferase, AtXT1, the β-1,2-galactosyltransferase, AtMUR3, or the α-1,2-fucosyltransferase AtFUT1 of Arabidopsis thaliana fused to green-fluorescent protein (GFP). Localization of the fusion proteins within the endomembrane system was assessed using confocal microscopy. Additionally, tobacco cells were high pressure-frozen/freeze-substituted and subjected to quantitative immunogold labelling using anti-GFP antibodies to determine the localization patterns of the enzymes within subtypes of Golgi cisternae. The data demonstrate that: (i) all fusion proteins, AtXT1-GFP, AtMUR3-GFP and AtFUT1-GFP are specifically targeted to the Golgi apparatus; and (ii) AtXT1-GFP is mainly located in the cis and medial cisternae, AtMUR3-GFP is predominantly associated with medial cisternae and AtFUT1-GFP mostly detected over trans cisternae suggesting that initiation of xyloglucan side chains occurs in early Golgi compartments in tobacco cells.     

Enzymatic fingerprinting of Arabidopsis pectic polysaccharides using polysaccharide analysis by carbohydrate gel electrophoresis (PACE)

Plant cell wall polysaccharides vary in quantity and structure between different organs and during development. However, quantitative analysis of individual polysaccharides remains challenging, and relatively little is known about any such variation in polysaccharides in organs of the model plant Arabidopsis thaliana. We have analysed plant cell wall pectic polysaccharides using polysaccharide analysis by carbohydrate gel electrophoresis. By highly specific enzymatic digestion of a polysaccharide in a cell wall preparation, a unique fingerprint of short oligosaccharides was produced. These oligosaccharides gave quantitative and structural information on the original polysaccharide chain. We analysed enzyme-accessible polygalacturonan (PGA), linear β(1,4) galactan and linear α(1,5) arabinan in several organs of Arabidopsis: roots, young leaves, old leaves, lower and upper inflorescence stems, seeds and callus. We found that this PGA constitutes a high proportion of cell wall material (CWM), up to 15% depending on the organ. In all organs, between 60 and 80% of the PGA was highly esterified in a blockwise fashion, and surprisingly, dispersely esterified PGA was hardly detected. We found enzyme-accessible linear galactan and arabinan are both present as a minor polysaccharide in all the organs. The amount of galactan ranged from ~0.04 to 0.25% of CWM, and linear arabinan constituted between 0.015 and 0.1%. Higher levels of galactan correlated with expanding tissues, supporting the hypothesis that this polysaccharide is involved in wall extension. We show by analysis of mur4 that the methods and results presented here also provide a basis for studies of pectic polysaccharides in Arabidopsis mutants.