ABSTRACT
Traditional marker-based mapping and next-generation sequencing was used to determine that the Arabidopsis (Arabidopsis thaliana) low cell wall arabinose mutant murus5 (mur5) encodes a defective allele of REVERSIBLY GLYCOSYLATED POLYPEPTIDE2 (RGP2). Marker analysis of 13 F2 confirmed mutant progeny from a recombinant mapping population gave a rough map position on the upper arm of chromosome 5, and deep sequencing of DNA from these 13 lines gave five candidate genes with GâA (CâT) transitions predicted to result in amino acid changes. Of these five, only insertional mutant alleles of RGP2, a gene that encodes a UDP-arabinose mutase that interconverts UDP-arabinopyranose and UDP-arabinofuranose, exhibited the low cell wall arabinose phenotype. The identities of mur5 and two SALK insertional alleles were confirmed by allelism tests and overexpression of wild-type RGP2 complementary DNA placed under the control of the 35S promoter in the three alleles. The mur5 mutation results in the conversion of cysteine-257 to tyrosine-257 within a conserved hydrophobic cluster predicted to be distal to the active site and essential for protein stability and possible heterodimerization with other isoforms of RGP.
Subject(s)
Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Arabidopsis/cytology , Arabinose/metabolism , Cell Wall/metabolism , Glucosyltransferases/genetics , Glucosyltransferases/metabolism , Alleles , Arabidopsis/genetics , Arabidopsis Proteins/chemistry , Arabinose/genetics , Cell Wall/genetics , Chromosome Mapping , Chromosomes, Plant , Gene Expression Regulation, Plant , Genetic Complementation Test , Glucosyltransferases/chemistry , High-Throughput Nucleotide Sequencing , Mutation , Plants, Genetically Modified , Protein Domains , Protein Folding , Protein Stability , Sequence Homology, Amino AcidABSTRACT
Plant cells are surrounded by a cell wall that plays a key role in plant growth, structural integrity, and defense. The cell wall is a complex and diverse structure that is mainly composed of polysaccharides. The majority of noncellulosic cell wall polysaccharides are produced in the Golgi apparatus from nucleotide sugars that are predominantly synthesized in the cytosol. The transport of these nucleotide sugars from the cytosol into the Golgi lumen is a critical process for cell wall biosynthesis and is mediated by a family of nucleotide sugar transporters (NSTs). Numerous studies have sought to characterize substrate-specific transport by NSTs; however, the availability of certain substrates and a lack of robust methods have proven problematic. Consequently, we have developed a novel approach that combines reconstitution of NSTs into liposomes and the subsequent assessment of nucleotide sugar uptake by mass spectrometry. To address the limitation of substrate availability, we also developed a two-step reaction for the enzymatic synthesis of UDP-l-rhamnose (Rha) by expressing the two active domains of the Arabidopsis UDP-l-Rha synthase. The liposome approach and the newly synthesized substrates were used to analyze a clade of Arabidopsis NSTs, resulting in the identification and characterization of six bifunctional UDP-l-Rha/UDP-d-galactose (Gal) transporters (URGTs). Further analysis of loss-of-function and overexpression plants for two of these URGTs supported their roles in the transport of UDP-l-Rha and UDP-d-Gal for matrix polysaccharide biosynthesis.
Subject(s)
Arabidopsis/metabolism , Golgi Apparatus/metabolism , Monosaccharide Transport Proteins/metabolism , Multigene Family , Rhamnose/metabolism , Uridine Diphosphate Glucose/metabolism , Arabidopsis/enzymology , Biological Transport , Kinetics , Molecular Sequence Data , Pectins/metabolism , Phylogeny , Proteolipids/metabolism , Subcellular Fractions/metabolism , Time FactorsABSTRACT
Xyloglucan is a polysaccharide that has important roles in the formation and function of the walls that surround growing land plant cells. Many of these plants synthesize xyloglucan that contains galactose in two different side chains (L and F), which exist in distinct molecular environments. However, little is known about the contribution of these side chains to xyloglucan function. Here, we show that Arabidopsis (Arabidopsis thaliana) mutants devoid of the F side chain galactosyltransferase MURUS3 (MUR3) form xyloglucan that lacks F side chains and contains much less galactosylated xylose than its wild-type counterpart. The galactose-depleted xyloglucan is dysfunctional, as it leads to mutants that are dwarfed with curled rosette leaves, short petioles, and short inflorescence stems. Moreover, cell wall matrix polysaccharides, including xyloglucan and pectin, are not properly secreted and instead accumulate within intracellular aggregates. Near-normal growth is restored by generating mur3 mutants that produce no detectable amounts of xyloglucan. Thus, cellular processes are affected more by the presence of the dysfunctional xyloglucan than by eliminating xyloglucan altogether. To identify structural features responsible for xyloglucan dysfunction, xyloglucan structure was modified in situ by generating mur3 mutants that lack specific xyloglucan xylosyltransferases (XXTs) or that overexpress the XYLOGLUCAN L-SIDE CHAIN GALACTOSYLTRANSFERASE2 (XLT2) gene. Normal growth was restored in the mur3-3 mutant overexpressing XLT2 and in mur3-3 xxt double mutants when the dysfunctional xyloglucan was modified by doubling the amounts of galactosylated side chains. Our study assigns a role for galactosylation in normal xyloglucan function and demonstrates that altering xyloglucan side chain structure disturbs diverse cellular and physiological processes.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/metabolism , Galactose/metabolism , Galactosyltransferases/metabolism , Glucans/metabolism , Xylans/metabolism , Arabidopsis/genetics , Arabidopsis/growth & development , Arabidopsis Proteins/genetics , Cell Wall/chemistry , Galactosyltransferases/genetics , Glucans/chemistry , Inflorescence/genetics , Inflorescence/growth & development , Inflorescence/metabolism , Mutation , Pectins/metabolism , Phenotype , Plant Leaves/genetics , Plant Leaves/growth & development , Plant Leaves/metabolism , Plant Stems/genetics , Plant Stems/growth & development , Plant Stems/metabolism , Polysaccharides/metabolism , Xylans/chemistryABSTRACT
During the past few years, substantial progress has been made to understand the enzymology and regulation of nucleotide sugar interconversion reactions that are irreversible in vivo on thermodynamic grounds. Feedback inhibition of enzymes by metabolic end products appears to be a common theme but some experimental results on recombinant enzymes are difficult to interpret. Using a combination of metabolic flux analysis, enzyme assays, and bioinformatics approaches, the significance of several proposed alternate pathways has been clarified. Expression of plant nucleotide sugar interconversion enzymes in yeast has become a promising approach to understand metabolic regulation and produce valuable compounds. In a major advance for the understanding of the synthesis of arabinosylated cell wall polysaccharides, reversibly glycosylated proteins turned out to act as mutases that interconvert the pyranose and furanose forms of UDP-L-arabinose.
Subject(s)
Carbohydrate Metabolism , Nucleotides/metabolism , Plants/metabolism , Carbohydrate Dehydrogenases/genetics , Carbohydrate Dehydrogenases/metabolism , Carbohydrate Epimerases/genetics , Carbohydrate Epimerases/metabolism , Gene Expression Regulation, Enzymologic , Models, Biological , Plant Proteins/genetics , Plant Proteins/metabolism , Plants/enzymology , Plants/genetics , Recombinant Proteins/metabolismABSTRACT
UDP-sugars serve as substrates in the synthesis of cell wall polysaccharides and are themselves generated through sequential interconversion reactions from UDP-Glc (UDP-glucose) as the starting substrate in the cytosol and the Golgi apparatus. For the present study, a soluble enzyme with UDP-Xyl (UDP-xylose) 4-epimerase activity was purified approx. 300-fold from pea (Pisum sativum L.) sprouts by conventional chromatography. The N-terminal amino acid sequence of the enzyme revealed that it is encoded by a predicted UDP-Glc 4-epimerase gene, PsUGE1, and is distinct from the UDP-Xyl 4-epimerase localized in the Golgi apparatus. rPsUGE1 (recombinant P. sativum UGE1) expressed in Escherichia coli exhibited both UDP-Xyl 4-epimerase and UDP-Glc 4-epimerase activities with apparent Km values of 0.31, 0.29, 0.16 and 0.15 mM for UDP-Glc, UDP-Gal (UDP-galactose), UDP-Ara (UDP-L-arabinose) and UDP-Xyl respectively. The apparent equilibrium constant for UDP-Ara formation from UDP-Xyl was 0.89, whereas that for UDP-Gal formation from UDP-Glc was 0.24. Phylogenetic analysis revealed that PsUGE1 forms a group with Arabidopsis UDP-Glc 4-epimerases, AtUGE1 and AtUGE3, apart from a group including AtUGE2, AtUGE4 and AtUGE5. Similar to rPsUGE1, recombinant AtUGE1 and AtUGE3 expressed in E. coli showed high UDP-Xyl 4-epimerase activity in addition to their UDP-Glc 4-epimerase activity. Our results suggest that PsUGE1 and its close homologues catalyse the interconversion between UDP-Xyl and UDP-Ara as the last step in the cytosolic de novo pathway for UDP-Ara generation. Alternatively, the net flux of metabolites may be from UDP-Ara to UDP-Xyl as part of the salvage pathway for Ara.
Subject(s)
Biocatalysis , Cytosol/enzymology , Pisum sativum/enzymology , UDPglucose 4-Epimerase/metabolism , Uridine Diphosphate Sugars/metabolism , Uridine Diphosphate Xylose/metabolism , Amino Acid Sequence , Animals , Conserved Sequence , Humans , Molecular Sequence Data , Phylogeny , Seedlings/enzymology , Sequence Alignment , Substrate Specificity , UDPglucose 4-Epimerase/chemistry , UDPglucose 4-Epimerase/isolation & purificationABSTRACT
A screen was established for mutants in which the plant defence response is de-repressed. The pathogen-inducible isochorismate synthase (ICS1) promoter was fused to firefly luciferase (luc) and a homozygous transgenic line generated in which the ICS1:luc fusion is co-regulated with ICS1. This line was mutagenized and M(2) seedlings screened for constitutive ICS1:luc expression (cie). The cie mutants fall into distinct phenotypic classes based on tissue-specific localization of luciferase activity. One mutant, cie1, that shows constitutive luciferase activity specifically in petioles, was chosen for further analysis. In addition to ICS1, PR and other defence-related genes are constitutively expressed in cie1 plants. The cie1 mutant is also characterized by an increased production of conjugated salicylic acid and reactive oxygen intermediates, as well as spontaneous lesion formation, all confined to petiole tissue. Significantly, defences activated in cie1 are sufficient to prevent infection by a virulent isolate of Hyaloperonospora parasitica, and this enhanced resistance response protects petiole tissue alone. Furthermore, cie1-mediated resistance, along with PR gene expression, is abolished in a sid2-1 mutant background, consistent with a requirement for salicylic acid. A positional cloning approach was used to identify cie1, which carries two point mutations in a gene required for cell wall biosynthesis and actin organization, MUR3. A mur3 knockout mutant also resists infection by H. parasitica in its petioles and this phenotype is complemented by transformation with wild-type MUR3. We propose that perturbed cell wall biosynthesis may activate plant defence and provide a rationale for the cie1 and the mur3 knockout phenotypes.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/genetics , Galactosyltransferases/metabolism , Intramolecular Transferases/metabolism , Actins/metabolism , Alleles , Arabidopsis/immunology , Arabidopsis/metabolism , Arabidopsis Proteins/genetics , Cell Wall/genetics , Cell Wall/metabolism , Chromosome Mapping , Cloning, Molecular , Galactosyltransferases/genetics , Gene Expression Regulation, Plant , Gene Knockout Techniques , Genes, Plant , Genes, Reporter , Genetic Complementation Test , Immunity, Innate , Intramolecular Transferases/genetics , Mutagenesis , Plant Leaves/genetics , Plant Leaves/immunology , Plant Leaves/metabolism , Plants, Genetically Modified/genetics , Plants, Genetically Modified/immunology , Plants, Genetically Modified/metabolism , Point Mutation , Promoter Regions, Genetic , RNA, Plant/genetics , Reactive Oxygen Species/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Salicylic Acid/metabolismABSTRACT
The characterization of cell wall mutants of Arabidopsis thaliana, combined with biochemical approaches toward the purification and characterization of glycosyltransferases, has led to significant advances in understanding cell wall synthesis and the properties of cell walls. New insights have been gained into the formation of cellulose and the functions of the matrix polysaccharides rhamnogalacturonan-II and xyloglucan.
Subject(s)
Cell Wall/physiology , Cellulose/biosynthesis , Glucans , Glucosyltransferases/metabolism , Plants/metabolism , Xylans , Arabidopsis/genetics , Arabidopsis/growth & development , Arabidopsis/metabolism , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Fucose/metabolism , Fucosyltransferases/genetics , Fucosyltransferases/metabolism , Galactose/analogs & derivatives , Glucosyltransferases/genetics , Glycosyltransferases/genetics , Glycosyltransferases/metabolism , Mannans/biosynthesis , Mutation , Pentosyltransferases/genetics , Pentosyltransferases/metabolism , Plant Development , Plants/genetics , Polysaccharides/biosynthesis , Polysaccharides/metabolism , UDP Xylose-Protein XylosyltransferaseABSTRACT
One of the major sugars present in the plant cell wall is d-galacturonate, the dominant monosaccharide in pectic polysaccharides. Previous work indicated that one of the activated precursors necessary for the synthesis of pectins is UDP-d-galacturonate, which is synthesized from UDP-d-glucuronate by a UDP-d-glucuronate 4-epimerase (GAE). Here, we report the identification, cloning and characterization of a GAE6 from Arabidopsis thaliana. Functional analysis revealed that this enzyme converts UDP-d-glucuronate to UDP-d-galacturonate in vitro. An expression analysis of this epimerase and its five homologs in the Arabidopsis genome by quantitative RT-PCR and promoter::GUS fusions indicated differential expression of the family members in plant tissues and expression of all isoforms in the developing pollen of A. thaliana.
Subject(s)
Arabidopsis/enzymology , Carbohydrate Epimerases/metabolism , Arabidopsis Proteins/genetics , Arabidopsis Proteins/isolation & purification , Arabidopsis Proteins/metabolism , Base Sequence , Carbohydrate Epimerases/genetics , Carbohydrate Epimerases/isolation & purification , DNA Primers , Flowers/enzymology , Kinetics , Molecular Sequence Data , Phylogeny , Plant Leaves/enzymology , Plant Roots/enzymology , Plant Stems/enzymology , Polymerase Chain Reaction , Recombinant Fusion Proteins/metabolism , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
BACKGROUND: Soluble sugar levels must be closely regulated in germinating seeds to ensure an adequate supply of energy and building materials for the developing seedling. Studies on germinating cereal seeds indicate that production of sugars from starch is inhibited by increasing sugar levels. Although numerous studies have focused on the regulation of starch metabolism, very few studies have addressed the control of storage lipid metabolism by germinating oilseeds. RESULTS: Mobilization of storage lipid by germinating seeds of the model oilseed plant Arabidopsis thaliana (L.) Heynh. occurs at a greatly reduced rate in the presence of exogenous glucose or mannose, but not in the presence of equi-molar 3-O-methylglucose or sorbitol. The sugar-insensitive5-1/abscisic acid-insensitive4-101 (sis5-1/abi4-101) mutant is resistant to glucose inhibition of seed storage lipid mobilization. Wild-type seedlings become insensitive to glucose inhibition of storage lipid breakdown within 3 days of the start of imbibition. CONCLUSIONS: Growth in the presence of exogenous glucose significantly retards mobilization of seed storage lipid in germinating seeds from wild-type Arabidopsis. This effect is not solely due to the osmotic potential of the media, as substantially higher concentrations of sorbitol than of glucose are required to exert significant effects on lipid breakdown. The inhibitory effect of glucose on lipid breakdown is limited to a narrow developmental window, suggesting that completion of some critical metabolic transition results in loss of sensitivity to the inhibitory effect of glucose on lipid breakdown.
Subject(s)
Arabidopsis/drug effects , Carbohydrates/pharmacology , Lipid Metabolism , Seedlings/drug effects , Seeds/drug effects , 3-O-Methylglucose/pharmacology , Arabidopsis/genetics , Arabidopsis/metabolism , Dose-Response Relationship, Drug , Fatty Acids/metabolism , Glucose/pharmacology , Mannose/pharmacology , Mutation , Seedlings/genetics , Seedlings/metabolism , Seeds/growth & development , Seeds/metabolism , Sorbitol/pharmacology , Time FactorsABSTRACT
Cell and cell wall growth are mutually dependent processes that must be tightly coordinated and controlled. LRR-extensin1 (LRX1) of Arabidopsis thaliana is a potential regulator of cell wall development, consisting of an N-terminal leucine-rich repeat domain and a C-terminal extensin-like domain typical for structural cell wall proteins. LRX1 is expressed in root hairs, and lrx1 mutant plants develop distorted root hairs that often swell, branch, or collapse. The aberrant cell wall structures found in lrx1 mutants point toward a function of LRX1 during the establishment of the extracellular matrix. To identify genes that are involved in an LRX1-dependent developmental pathway, a suppressor screen was performed on the lrx1 mutant, and two independent rol1 (for repressor of lrx1) alleles were isolated. ROL1 is allelic to Rhamnose Biosynthesis1, which codes for a protein involved in the biosynthesis of rhamnose, a major monosaccharide component of pectin. The rol1 mutations modify the pectic polysaccharide rhamnogalacturonan I and, for one allele, rhamnogalacturonan II. Furthermore, the rol1 mutations cause a change in the expression of a number of cell wall-related genes. Thus, the lrx1 mutant phenotype is likely to be suppressed by changes in pectic polysaccharides or other cell wall components.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/cytology , Arabidopsis/genetics , Cell Wall/metabolism , Glucosyltransferases/metabolism , Plant Roots/cytology , Rhamnose/biosynthesis , Uridine Diphosphate Sugars/metabolism , Alleles , Arabidopsis/metabolism , Arabidopsis Proteins/genetics , Cell Wall/chemistry , Extracellular Matrix/chemistry , Gene Expression Profiling , Glucosyltransferases/genetics , Molecular Sequence Data , Monosaccharides/chemistry , Monosaccharides/metabolism , Mutation , PhenotypeABSTRACT
D-apiose serves as the binding site for borate cross-linking of rhamnogalacturonan II (RG-II) in the plant cell wall, and biosynthesis of D-apiose involves UDP-D-apiose/UDP-D-xylose synthase catalyzing the conversion of UDP-D-glucuronate to a mixture of UDP-D-apiose and UDP-D-xylose. In this study we have analyzed the cellular effects of depletion of UDP-D-apiose/UDP-D-xylose synthases in plants by using virus-induced gene silencing (VIGS) of NbAXS1 in Nicotiana benthamiana. The recombinant NbAXS1 protein exhibited UDP-D-apiose/UDP-D-xylose synthase activity in vitro. The NbAXS1 gene was expressed in all major plant organs, and an NbAXS1-green fluorescent protein fusion protein was mostly localized in the cytosol. VIGS of NbAXS1 resulted in growth arrest and leaf yellowing. Microscopic studies of the leaf cells of the NbAXS1 VIGS lines revealed cell death symptoms including cell lysis and disintegration of cellular organelles and compartments. The cell death was accompanied by excessive formation of reactive oxygen species and by induction of various protease genes. Furthermore, abnormal wall structure of the affected cells was evident including excessive cell wall thickening and wall gaps. The mutant cell walls contained significantly reduced levels of D-apiose as well as 2-O-methyl-L-fucose and 2-O-methyl-D-xylose, which serve as markers for the RG-II side chains B and A, respectively. These results suggest that VIGS of NbAXS1 caused a severe deficiency in the major side chains of RG-II and that the growth defect and cell death was likely caused by structural alterations in RG-II due to a D-apiose deficiency.
Subject(s)
Carbon-Nitrogen Ligases/deficiency , Cell Wall/metabolism , Nicotiana/cytology , Nicotiana/enzymology , Pectins/metabolism , Uridine Diphosphate Sugars/metabolism , Xylose/metabolism , Carbon-Nitrogen Ligases/metabolism , Cell Death , Gene Silencing , Molecular Sequence Data , Plant Proteins/metabolism , Nicotiana/metabolismABSTRACT
d-Apiose is a plant-specific branched-chain monosaccharide found in rhamnogalacturonan II (RG-II), apiogalacturonan, and several apioglycosides. Within RG-II, d-apiose serves as the binding site for borate, which leads to the formation of cross-links within the wall. Biochemical studies in duckweed and parsley have established that uridine 5'-diphospho-d-apiose (UDP-d-apiose) is formed from UDP-d-glucuronate by decarboxylation and re-arrangement of the carbon skeleton, leading to ring contraction and branch formation. The enzyme catalyzing this reaction also forms UDP-d-xylose by decarboxylation of UDP-d-glucuronate, and has therefore been named UDP-d-apiose/UDP-d-xylose synthase. Using a bioinformatics approach, we identified a candidate gene (AXS1) for this enzyme in Arabidopsis and functionally expressed its cDNA in Escherichia coli. The recombinant enzyme catalyzed the conversion of UDP-d-glucuronate to a mixture of UDP-d-apiose and UDP-d-xylose with a turnover number of 0.3 min-1. AXS1 required NAD+ for enzymatic activity, and was strongly inhibited by UDP-d-galacturonate. It was highly expressed in all plant organs consistent with a function in synthesizing an essential cell wall precursor. Database searches indicated the presence of closely related sequences in a variety of crop plants. The cloning of the AXS1 gene will help to investigate the biosynthesis of RG-II, and permit insights into the mechanism by which d-apiose and other branched monosaccharides are formed.
Subject(s)
Carboxy-Lyases/genetics , Pentoses/biosynthesis , Photosynthesis/physiology , Arabidopsis Proteins/metabolism , Base Sequence , Carboxy-Lyases/isolation & purification , Carboxy-Lyases/metabolism , Chromatography, Ion Exchange , Cloning, Molecular , DNA Primers , Escherichia coli/enzymology , Escherichia coli/genetics , Kinetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Pectic cell wall polysaccharides owe their high negative charge to the presence of D-galacturonate, a monosaccharide that appears to be present only in plants and some prokaryotes. UDP-D-galacturonate, the activated form of this sugar, is known to be formed by the 4-epimerization of UDP-D-glucuronate; however, no coding regions for the epimerase catalyzing this reaction have previously been described in plants. To better understand the mechanisms by which precursors for pectin synthesis are produced, we used a bioinformatics approach to identify and functionally express a UDP-D-glucuronate 4-epimerase (GAE1) from Arabidopsis. GAE1 is predicted to be a type II membrane protein that belongs to the family of short-chain dehydrogenases/reductases. The recombinant enzyme expressed in Pichia pastoris established a 1.3:1 equilibrium between UDP-D-galacturonate and UDP-D-glucuronate but did not epimerize UDP-D-Glc or UDP-D-Xyl. Enzyme assays on cell extracts localized total UDP-D-glucuronate 4-epimerase and recombinant GAE1 activity exclusively to the microsomal fractions of Arabidopsis and Pichia, respectively. GAE1 had a pH optimum of 7.6 and an apparent Km of 0.19 mm. The recombinant enzyme was strongly inhibited by UDP-D-Xyl but not by UDP, UDP-D-Glc, or UDP-D-Gal. Analysis of Arabidopsis plants transformed with a GAE1:GUS construct showed expression in all tissues. The Arabidopsis genome contains five GAE1 paralogs, all of which are transcribed and predicted to contain a membrane anchor. This suggests that all of these enzymes are targeted to an endomembrane system such as the Golgi where they may provide UDP-D-galacturonate to glycosyltransferases in pectin synthesis.
Subject(s)
Arabidopsis Proteins/genetics , Arabidopsis/enzymology , Carbohydrate Epimerases/genetics , Galactose/biosynthesis , Hexuronic Acids/metabolism , Amino Acid Sequence , Arabidopsis/genetics , Arabidopsis Proteins/chemistry , Carbohydrate Epimerases/chemistry , Cloning, Molecular , Enzyme Stability , Flowers/enzymology , Molecular Sequence Data , Plant Leaves/enzymology , Plant Roots/enzymologyABSTRACT
GDP-D-mannose 4,6-dehydratase catalyzes the first step in the de novo synthesis of GDP-L-fucose, the activated form of L-fucose, which is a component of glycoconjugates in plants known to be important to the development and strength of stem tissues. We have determined the three-dimensional structure of the MUR1 dehydratase isoform from Arabidopsis thaliana complexed with its NADPH cofactor as well as with the ligands GDP and GDP-D-rhamnose. MUR1 is a member of the nucleoside-diphosphosugar modifying subclass of the short-chain dehydrogenase/reductase enzyme family, having homologous structures and a conserved catalytic triad of Lys, Tyr, and Ser/Thr residues. MUR1 is the first member of this subfamily to be observed as a tetramer, the interface of which reveals a close and intimate overlap of neighboring NADP(+)-binding sites. The GDP moiety of the substrate also binds in an unusual syn conformation. The protein-ligand interactions around the hexose moiety of the substrate support the importance of the conserved triad residues and an additional Glu side chain serving as a general base for catalysis. Phe and Arg side chains close to the hexose ring may serve to confer substrate specificity at the O2 position. In the MUR1/GDP-D-rhamnose complex, a single unique monomer within the protein tetramer that has an unoccupied substrate site highlights the conformational changes that accompany substrate binding and may suggest the existence of negative cooperativity in MUR1 function.
Subject(s)
Arabidopsis Proteins/chemistry , Hydro-Lyases/chemistry , Arabidopsis Proteins/biosynthesis , Arabidopsis Proteins/isolation & purification , Binding Sites , Catalytic Domain , Conserved Sequence , Crystallization , Crystallography, X-Ray , Guanosine Diphosphate/chemistry , Guanosine Diphosphate Sugars/chemistry , Hydro-Lyases/biosynthesis , Hydro-Lyases/isolation & purification , Hydrogen Bonding , Ligands , Macromolecular Substances , Models, Molecular , NADP/chemistry , Protein Conformation , Protein Structure, Tertiary , Recombinant Proteins/biosynthesis , Recombinant Proteins/isolation & purification , Substrate SpecificityABSTRACT
Plant cell walls are composed of a large number of complex polysaccharides, which contain at least 13 different monosaccharides in a multitude of linkages. This structural complexity of cell wall components is paralleled by a large number of predicted glycosyltransferases in plant genomes, which can be grouped into several distinct families based on conserved sequence motifs (B. Henrissat, G.J. Davies [2000] Plant Physiol 124: 1515-1519). Despite the wealth of genomic information in Arabidopsis and several crop plants, the biochemical functions of these coding regions have only been established in a few cases. To lay the foundation for the genetic and biochemical characterization of putative glycosyltransferase genes, we conducted a phylogenetic and expression analysis on 10 predicted coding regions (AtGT11-20) that are closely related to the MUR3 xyloglucan galactosyltransferase of Arabidopsis. All of these proteins contain the conserved sequence motif pfam 03016 that is the hallmark of the beta-d-glucuronosyltransferase domain of exostosins, a class of animal enzymes involved in the biosynthesis of the extracellular polysaccharide heparan sulfate. Reverse transcriptase-polymerase chain reaction and promoter:beta-glucuronidase studies indicate that all AtGT genes are transcribed. Although six of the 10 AtGT genes were expressed in all major plant organs, the remaining four genes showed more restricted expression patterns that were either confined to specific organs or to highly specialized cell types such as hydathodes or pollen grains. T-DNA insertion mutants in AtGT13 and AtGT18 displayed reductions in the Gal content of total cell wall material, suggesting that the disrupted genes encode galactosyltransferases in plant cell wall synthesis.
Subject(s)
Arabidopsis/enzymology , Arabidopsis/genetics , Galactosyltransferases/genetics , Genes, Plant , Amino Acid Sequence , Base Sequence , Cell Wall/chemistry , DNA, Bacterial/genetics , DNA, Plant/genetics , Gene Expression Profiling , Molecular Sequence Data , Multigene Family , Mutagenesis, Insertional , Phylogeny , Plants, Genetically Modified , Sequence Homology, Amino AcidABSTRACT
l-Fucose (l-Fuc) is a monosaccharide constituent of plant cell wall polysaccharides and glycoproteins. The committing step in the de novo synthesis of l-Fuc is catalyzed by GDP-d-mannose 4,6-dehydratase, which, in Arabidopsis, is encoded by the GMD1 and GMD2 (MUR1) genes. To determine the functional significance of this genetic redundancy, the expression patterns of both genes were investigated via promoter-beta-glucuronidase fusions and immunolocalization of a Fuc-containing epitope. GMD2 is expressed in most cell types of the root, with the notable exception of the root tip where strong expression of GMD1 is observed. Within shoot organs, GMD1::GUS expression is confined to stipules and pollen grains leading to fucosylation of the walls of these cell types in the mur1 mutant. These results suggest that GMD2 represents the major housekeeping gene for the de novo synthesis of GDP-l-Fuc, whereas GMD1 expression is limited to a number of specialized cell types. We conclude that the synthesis of GDP-l-Fuc is controlled in a cell-autonomous manner by differential expression of two isoforms of the same enzyme.
Subject(s)
Arabidopsis/enzymology , Gene Expression Regulation, Plant , Hydro-Lyases/genetics , Amino Acid Sequence , Arabidopsis/cytology , Arabidopsis/genetics , Base Sequence , Cloning, Molecular , DNA Primers , Escherichia coli/enzymology , Escherichia coli/genetics , Flowers/enzymology , Gene Expression Regulation, Enzymologic , Glucuronidase/genetics , Hydro-Lyases/chemistry , Isoenzymes/chemistry , Isoenzymes/genetics , Molecular Sequence Data , Plant Leaves/enzymology , Plant Roots/enzymology , Plant Stems/enzymology , Plants, Genetically Modified/enzymology , RNA, Plant/genetics , RNA, Plant/isolation & purification , Sequence Alignment , Sequence Homology, Amino AcidABSTRACT
The mur4 mutant of Arabidopsis shows a 50% reduction in the monosaccharide L-Ara in leaf-derived cell wall material because of a partial defect in the 4-epimerization of UDP-D-Xyl to UDP-L-Ara. To determine the genetic lesion underlying the mur4 phenotype, the MUR4 gene was cloned by a map-based procedure and found to encode a type-II membrane protein with sequence similarity to UDP-D-Glc 4-epimerases. Enzyme assays of MUR4 protein expressed in the methylotropic yeast Pichia pastoris indicate that it catalyzes the 4-epimerization of UDP-D-Xyl to UDP-L-Ara, the nucleotide sugar used by glycosyltransferases in the arabinosylation of cell wall polysaccharides and wall-resident proteoglycans. Expression of MUR4-green fluorescent protein constructs in Arabidopsis revealed localization patterns consistent with targeting to the Golgi, suggesting that the MUR4 protein colocalizes with glycosyltransferases in the biosynthesis of arabinosylated cell wall components. The Arabidopsis genome encodes three putative proteins with >76% sequence identity to MUR4, which may explain why mur4 plants are not entirely deficient in the de novo synthesis of UDP-L-Ara.
Subject(s)
Arabidopsis Proteins/genetics , Arabidopsis/genetics , Arabinose/biosynthesis , Carbohydrate Epimerases/genetics , Golgi Apparatus/enzymology , Amino Acid Sequence , Arabidopsis/enzymology , Arabidopsis Proteins/metabolism , Arabinose/chemistry , Carbohydrate Epimerases/metabolism , Cloning, Molecular , DNA, Complementary/chemistry , DNA, Complementary/genetics , Gene Expression Regulation, Enzymologic , Gene Expression Regulation, Plant , Genetic Complementation Test , Green Fluorescent Proteins , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Molecular Sequence Data , Mutation , Pichia/genetics , Pichia/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Sequence Alignment , Sequence Analysis, DNA , Sequence Homology, Amino AcidABSTRACT
Cell walls of the Arabidopsis mutant mur2 contain less than 2% of the wild-type amount of fucosylated xyloglucan because of a point mutation in the fucosyltransferase AtFUT1. The mur2 mutation eliminates xyloglucan fucosylation in all major plant organs, indicating that Arabidopsis thaliana fucosyltransferase 1 (AtFUT1) accounts for all of the xyloglucan fucosyltransferase activity in Arabidopsis. Despite this alteration in structure, mur2 plants show a normal growth habit and wall strength. In contrast, Arabidopsis mur1 mutants that are defective in the de novo synthesis of l-fucose exhibit a dwarfed growth habit and decreased wall strength [Reiter, W. D., Chapple, C. & Somerville, C. R. (1993) Science 261, 1032-1035]. Because the mur1 mutation affects several cell wall polysaccharides, whereas the mur2 mutation is specific to xyloglucan, the phenotypes of mur1 plants appear to be caused by structural changes in fucosylated pectic components such as rhamnogalacturonan-II. The normal growth habit and wall strength of mur2 plants casts doubt on hypotheses regarding roles of xyloglucan fucosylation in facilitating xyloglucan-cellulose interactions or in modulating growth regulator activity.
Subject(s)
Fucosyltransferases/metabolism , Glucans , Polysaccharides/metabolism , Xylans , Arabidopsis/enzymology , Arabidopsis/genetics , Cell Line, Transformed , Cell Wall , Cloning, Molecular , Fucose/metabolism , Fucosyltransferases/genetics , Humans , MutagenesisABSTRACT
The mechanical properties of plant organs depend upon anatomical structure, cell-cell adhesion, cell turgidity, and the mechanical properties of their cell walls. By testing the mechanical responses of Arabidopsis mutants, it is possible to deduce the contribution that polymers of the cell wall make to organ strength. We developed a method to measure the tensile parameters of the expanded regions of turgid or plasmolyzed dark-grown Arabidopsis hypocotyls and applied it to the fucose biosynthesis mutant mur1, the xyloglucan glycosyltransferase mutants mur2 and mur3, and the katanin mutant bot1. Hypocotyls from plants grown in the presence of increasing concentrations of dichlorobenzonitrile, an inhibitor of cellulose synthesis, were considerably weakened, indicating the validity of our approach. In order of decreasing strength, the hypocotyls of mur2 > bot1 and mur1 > mur3 were each found to have reduced strength and a proportionate reduction in modulus compared with wild type. The tensile properties of the hypocotyls and of the inflorescence stems of mur1 were rescued by growth in the presence of high concentrations of borate, which is known to cross-link the pectic component rhamnogalacturonan II. From comparison of the mechanical responses of mur2 and mur3, we deduce that galactose-containing side chains of xyloglucan make a major contribution to overall wall strength, whereas xyloglucan fucosylation plays a comparatively minor role. We conclude that borate-complexed rhamnogalacturonan II and galactosylated xyloglucan contribute to the tensile strength of cell walls.
Subject(s)
Arabidopsis/physiology , Cell Wall/physiology , Glucans , Microfibrils/physiology , Pectins/metabolism , Polysaccharides/metabolism , Xylans , Arabidopsis/genetics , Borates/metabolism , Cell Adhesion , Hypocotyl/physiology , Mutation , Reproducibility of Results , Stress, Mechanical , Tensile StrengthABSTRACT
The monoclonal antibody, CCRC-M1, which recognizes a fucose (Fuc)-containing epitope found principally in the cell wall polysaccharide xyloglucan, was used to determine the distribution of this epitope throughout the mur1 mutant of Arabidopsis. Immunofluorescent labeling of whole seedlings revealed that mur1 root hairs are stained heavily by CCRC-M1, whereas the body of the root remains unstained or only lightly stained. Immunogold labeling showed that CCRC-M1 labeling within the mur1 root is specific to particular cell walls and cell types. CCRC-M1 labels all cell walls at the apex of primary roots 2 d and older and the apices of mature lateral roots, but does not bind to cell walls in lateral root initials. Labeling with CCRC-M1 decreases in mur1 root cells that are undergoing rapid elongation growth such that, in the mature portions of primary and lateral roots, only the walls of pericycle cells and the outer walls of epidermal cells are labeled. Growth of the mutant on Fuc-containing media restores wild-type labeling, where all cell walls are labeled by the CCRC-M1 antibody. No labeling was observed in mur1 hypocotyls, shoots, or leaves; stipules are labeled. CCRC-M1 does label pollen grains within anthers and pollen tube walls. These results suggest the Fuc destined for incorporation into xyloglucan is synthesized using one or the other or both isoforms of GDP-D-mannose 4,6-dehydratase, depending on the cell type and/or developmental state of the cell.