Higher plant glycosyltransferases.

SUMMARY: Uridine diphosphate (UDP) glycosyltransferases (UGTs) mediate the transfer of glycosyl residues from activated nucleotide sugars to acceptor molecules (aglycones), thus regulating properties of the acceptors such as their bioactivity, solubility and transport within the cell and throughout the organism. A superfamily of over 100 genes encoding UGTs, each containing a 42 amino acid consensus sequence, has been identified in the model plant Arabidopsis thaliana. A phylogenetic analysis of the conserved amino acids encoded by these Arabidopsis genes reveals the presence of 14 distinct groups of UGTs in this organism. Genes encoding UGTs have also been identified in several other higher plant species. Very little is yet known about the regulation of plant UGT genes or the localization of the enzymes they encode at the cellular and subcellular levels. The substrate specificities of these UGTs are now beginning to be established and will provide a foundation for further analysis of this large enzyme superfamily as well as a platform for future biotechnological applications.

Identification and biochemical characterization of an Arabidopsis indole-3-acetic acid glucosyltransferase.

Biochemical characterization of recombinant gene products following a phylogenetic analysis of the UDP-glucosyltransferase (UGT) multigene family of Arabidopsis has identified one enzyme (UGT84B1) with high activity toward the plant hormone indole-3-acetic acid (IAA) and three related enzymes (UGT84B2, UGT75B1, and UGT75B2) with trace activities. The identity of the IAA conjugate has been confirmed to be 1-O-indole acetyl glucose ester. A sequence annotated as a UDP-glucose:IAA glucosyltransferase (IAA-UGT) in the Arabidopsis genome and expressed sequence tag data bases given its similarity to the maize iaglu gene sequence showed no activity toward IAA. This study describes the first biochemical analysis of a recombinant IAA-UGT and provides the foundation for future genetic approaches to understand the role of 1-O-indole acetyl glucose ester in Arabidopsis.

Identification of glucosyltransferase genes involved in sinapate metabolism and lignin synthesis in Arabidopsis.

Sinapic acid is a major phenylpropanoid in Brassicaceae providing intermediates in two distinct metabolic pathways leading to sinapoyl esters and lignin synthesis. Glucosyltransferases play key roles in the formation of these intermediates, either through the production of the high energy compound 1-O-sinapoylglucose leading to sinapoylmalate and sinapoylcholine or through the production of sinapyl alcohol-4-O-glucoside, potentially leading to the syringyl units found in lignins. While the importance of these glucosyltransferases has been recognized for more than 20 years, their corresponding genes have not been identified. Combining sequence information in the Arabidopsis genomic data base with biochemical data from screening the activity of recombinant proteins in vitro, we have now identified five gene sequences encoding enzymes that can glucosylate sinapic acid, sinapyl alcohol, and their related phenylpropanoids. The data provide a foundation for future understanding and manipulation of sinapate metabolism and lignin biology in Arabidopsis.

Phylogenetic analysis of the UDP-glycosyltransferase multigene family of Arabidopsis thaliana.

A class of UDP-glycosyltransferases (UGTs) defined by the presence of a C-terminal consensus sequence is found throughout the plant and animal kingdoms. Whereas mammalian enzymes use UDP-glucuronic acid, the plant enzymes typically use UDP-glucose in the transfer reactions. A diverse array of aglycones can be glucosylated by these UGTs. In plants, the aglycones include plant hormones, secondary metabolites involved in stress and defense responses, and xenobiotics such as herbicides. Glycosylation is known to regulate many properties of the aglycones such as their bioactivity, their solubility, and their transport properties within the cell and throughout the plant. As a means of providing a framework to start to understand the substrate specificities and structure-function relationships of plant UGTs, we have now applied a molecular phylogenetic analysis to the multigene family of 99 UGT sequences in Arabidopsis. We have determined the overall organization and evolutionary relationships among individual members with a surprisingly high degree of confidence. Through constructing a composite phylogenetic tree that also includes all of the additional plant UGTs with known catalytic activities, we can start to predict both the evolutionary history and substrate specificities of new sequences as they are identified. The tree already suggests that while the activities of some subgroups of the UGT family are highly conserved among different plant species, others subgroups shift substrate specificity with relative ease.

Distribution and characterization of recrystallization inhibitor activity in plant and lichen species from the UK and maritime Antarctic.

Extracts from a range of evolutionarily diverse plant and lichen species from the UK and maritime Antarctic have been assayed for inhibition of ice recrystallization. Approximately 25% of overwintering UK species and all Antarctic species exhibited antifreeze activity when exposed to low temperature. Preliminary characterization of the active extracts has demonstrated that the molecules co-opted to antifreeze activity by different species are biochemically diverse.

Isolation and characterization of a novel antifreeze protein from carrot (Daucus carota).

A modified assay for inhibition of ice recrystallization which allows unequivocal identification of activity in plant extracts is described. Using this assay a novel, cold-induced, 36 kDa antifreeze protein has been isolated from the tap root of cold-acclimated carrot (Daucus carota) plants. This protein inhibits the recrystallization of ice and exhibits thermal-hysteresis activity. The polypeptide behaves as monomer in solution and is N-glycosylated. The corresponding gene is unique in the carrot genome and induced by cold. The antifreeze protein appears to be localized within the apoplast.

Fusicoccin, 14-3-3 proteins, and defense responses in tomato plants.

Fusicoccin (FC) is a fungal toxin that activates the plant plasma membrane H+-ATPase by binding with 14-3-3 proteins, causing membrane hyperpolarization. Here we report on the effect of FC on a gene-for-gene pathogen-resistance response and show that FC application induces the expression of several genes involved in plant responses to pathogens. Ten members of the FC-binding 14-3-3 protein gene family were isolated from tomato (Lycopersicon esculentum) to characterize their role in defense responses. Sequence analysis is suggestive of common biochemical functions for these tomato 14-3-3 proteins, but their genes showed different expression patterns in leaves after challenges. Different specific subsets of 14-3-3 genes were induced after treatment with FC and during a gene-for-gene resistance response. Possible roles for the H+-ATPase and 14-3-3 proteins in responses to pathogens are discussed.

Biochemical characterization of tomato annexin p35. Independence of calcium binding and phosphatase activities.

Tomato annexin p35 has been cloned and used in a site-directed mutagenesis study to explore the phospholipid binding and catalytic properties of the protein in detail. Analysis of the cDNA sequence of p35 reveals that the annexin has only two typical endonexin folds, corresponding to repeats I and IV. Expression of recombinant p35 in Escherichia coli confirmed both phospholipid binding and a nucleotide phosphatase activity that could be inhibited on interaction of the recombinant annexin with phospholipids. Site-directed mutagenesis in which the acidic residues Glu-68 (repeat I), and Asp-297 (repeat IV) were changed to Asn, generated two mutant forms, E68N and D297N, respectively. Both mutant forms of the annexin continued to express catalytic activity. Changing repeat I had little effect on phospholipid binding, whereas the change to repeat IV abolished this property. These data show that, in this plant annexin, repeat IV plays a more critical role in calcium-dependent phospholipid binding than repeat I, and that the catalytic and phospholipid binding activity of the protein can be separated experimentally.

Short chain oligogalacturonides induce ethylene production and expression of the gene encoding aminocyclopropane 1-carboxylic acid oxidase in tomato plants.

Oligogalacturonic acids (OGAs), derived from plant cell wall pectin, have been implicated in a number of signal transduction pathways involved in growth, development and defense responses of higher plants. This study investigates the size range of OGAs capable of inducing ethylene synthesis in tomato plants, and demonstrates that in contrast with many other effects, only short chain OGAs are active. Oligomers across a range of DP from 2-15 were separated and purified to homogeneity by QAE-Sephadex anion exchange chromatography using a novel elution system. The OGAs were applied to tomato plants and assayed for their ability to induce ethylene gas release and changes in steady state levels of mRNA encoding the ethylene forming enzyme aminocyclopropane-1-carboxylic acid oxidase (ACO). The study demonstrated that only OGAs in the size range of DP4-6 were active both in eliciting ACO expression and in the production of ethylene.

Purification and characterization of N-glycanase, a concanavalin A binding protein from jackbean (Canavalia ensiformis).

Removal of the N-glycan from the concanavalin A (Con A) glycoprotein precursor is a key step in its conversion into an active lectin. N-Glycanase (EC 3.5.1.52), the enzyme from jackbean catalysing this process, has been purified to homogeneity as judged by native PAGE. One of the purification steps is binding of the enzymic activity to Con A-Sepharose and its elution by methyl alpha-mannoside. On SDS/PAGE the principal components were found to be 78 kDa, 74 kDa, 54 kDa, 32 kDa and 30 kDa polypeptides. These did not react with Con A on an affinity blot. Cleveland mapping indicated that some of these polypeptides had related primary structures. The enzyme has a broad pH optimum in the region of 5.0.

A novel tomato gene that rapidly responds to wound- and pathogen-related signals.

The expression of a novel defence-related gene from tomato which responds rapidly to wound- and pathogen-related signals has been characterised. The gene, which encodes a protein with homology to glucosyl transferase enzymes, is expressed within 15 min of mechanical damage to tomato leaves, and responds to signals which differ from those on the systemin/jasmonic acid pathway typical of well-characterised wound-induced genes of tomato. Furthermore, expression of the gene is also rapidly and specifically induced during a resistance response elicited by the application of Avr9 avirulence peptide to tomato plants carrying the corresponding Cf9 resistance gene. Whilst expression can also be induced by the application of exogenous salicylic acid and related analogues to tomato plants, several lines of evidence suggest that elevated salicylic acid is not a causal signal in planta during either the wound or pathogen resistance response.

The wound response of tomato plants: analysis of local and long-range signalling events.

The wound response of tomato plants has been used extensively to characterize events involved in signal transduction pathways. Leaf injury leads to the up-regulation of defence-related genes, such as those encoding proteinase inhibitors at the wound site and elsewhere in distant, unwounded tissues of the plant. Signalling molecules encompassing glycans, lipids and peptides, as well as 'classic' plant hormones, all play a role in the regulation of the wound response. The nature of the systemic signal remains ill-defined, with chemical and physical candidates, and various long-range routes of intercellular transport involving the xylem and the phloem, as well as short-range diffusion through the apoplast.

Post-translational peptide bond formation during concanavalin A processing in vitro.

Post-translational processing of concanavalin A (Con A) is complex, involving deglycosylation, proteolytic cleavage on the carboxy group side of asparagine residues and formation of a peptide bond de novo. This has been studied with the 125I-labelled Con A glycoprotein precursor as a substrate for processing in vitro. Extracts of immature jackbean cotyledons and the commercially available purified preparation of asparaginylendo-peptidase were able to catalyse the above processes. The processing resulted in the conversion of the 33.5 kDa inactive glycoprotein precursor into an active lectin. Processing activity was maximal at approx. pH 5.5. Evidence to support processing at authentic sites was obtained by observation of the release of 125I at positions in the sequence where tyrosine residues were present.

Tomato annexins p34 and p35 bind to F-actin and display nucleotide phosphodiesterase activity inhibited by phospholipid binding.

Annexins are a family of proteins found in a range of eukaryotic cell types. They share a characteristic amino acid sequence and a Ca(2+)-dependent affinity for specific phospholipids. In plants, proteins with common properties and significant homology with annexins have been identified in a number of species and implicated in diverse cellular functions known to be modulated by Ca2+. This study describes several novel biochemical properties of the tomato annexins p34 and p35 that are relevant to our understanding of their functions in the plant. First, the annexins were found to bind to actin in a calcium- and pH-dependent interaction that was specific for F-actin and not G-actin. Second, an enzyme activity defined as a nucleotide phosphodiesterase activity was found associated with the purified annexin preparation. Selective immunoprecipitation of p34 and p35 strongly suggests that the enzyme activity is a property of the annexins and constitutes 60% of the total soluble activity found in root extracts capable of hydrolyzing free ATP. The substrate specificity of the enzyme within in vitro assays is broad. ATP is the preferred substrate, but nearly identical rates of hydrolysis of GTP and substantial hydrolysis of other nucleotide tri- and diphosphates are observed. The enzyme activity was found to be a property of both p34 and p35, although the specific activity was routinely higher for p34. Third, the enzyme activity of the annexins was not affected by F-actin binding but could be abolished by the specific Ca(2+)-dependent interaction of the annexins with phospholipids. Our results showed that p34 and p35 account for substantial enzyme activity in tomato root cells. This activity was exhibited when the proteins were either in soluble form or attached to actin filaments. Enzyme activity was not exhibited when the annexins were bound to phospholipids. These properties suggest a role for the proteins in mediating Ca(2+)-dependent events involving interactions of the cytoskeleton and cellular membranes.

Signalling events in the wound response of tomato plants.

The wound response of tomato plants provides a useful experimental system to analyse local and systemic signalling events. Wounding one region on the plant leads to changes in the expression of genes at the local site of damage and elsewhere in unwounded tissues. A wound stimulus is thus converted to signal(s) that are transduced locally and signals that are involved in establishing long-distance spread of the initial stimulus. Data from studies at Leeds will be integrated into a wider discussion of the available evidence, to work towards an integrated model for understanding signalling events in the wound response.

Local and systemic signals in the wound response.

An injury applied to one site on a plant can lead to changes in gene expression in the region of the wound and in distant unwounded regions of the organism. This implies that local and systemic signals must operate to link the initial stimulus to the wound-induced effects. Current evidence for the involvement of chemical and physical signalling mechanisms is critically reviewed, drawing on data from studies of the wound response in planta, mutant analyses and the use of bioassays.

Temporal and spatial regulation of a novel gene in barley embryos.

The temporal and spatial pattern of expression of a novel barley gene is described. The gene has been identified through the differential screening of a cDNA library constructed to poly(A)+ RNA of zygotic embryos. Transcripts corresponding to the cDNA, pZE40, become abundant in the non-axial tissues of the developing embryo within 8-10 days after anthesis, when steady-state levels are high in the scutellum, coleoptile and coleorhiza, with the exception of the scutellar epithelium. This expression pattern is maintained throughout maturation of the embryo until levels eventually decline as the grain desiccates. On germination, there is a transient re-appearance of mRNA to pZE40, with accumulation specifically restricted to the scutellum of the seedling. In situ hybridization has enabled the detection of transcripts elsewhere in the barley plant, in highly localized groups of cells. The timing and cell specificity of expression suggests the gene product is involved in the synthesis and/or transport of metabolites.

The glycoprotein precursor of concanavalin A is converted to an active lectin by deglycosylation.

We have previously shown that concanavalin A is synthesized as a glycoprotein precursor that is unable to bind to sugars and is processed through six intermediate forms before assembly of the mature active lectin. Since processing involves removal of the N-glycan, four proteolytic steps and a religation, the precise event that leads to carbohydrate binding activity was not known. We have now purified the glycoprotein precursor from microsomal membranes and show that deglycosylation in vitro is sufficient alone to convert the precursor to an active carbohydrate binding protein. This is the first demonstration of a novel role for N-glycans and N-glycanases in the regulation of protein activity.

The pattern of plant annexin gene expression.

Peptide sequence data derived from a plant annexin, P34 [Smallwood, Keen & Bowles (1990) Biochem. J. 270, 157-161] was used to design amplimers for PCR. A unique fragment of 95 bp, amplified from tomato (Lycopersicon esculertum) genomic DNA, was used in Northern analyses and demonstrated a differential pattern of expression in vegetative tissues of tomato, potato (Solanum tuberosum) and barley (Hordeum vulgare). The tissue-specific abundance of the annexin transcript was found to correlate closely with abundance of annexin protein as revealed by their partial purification and analysis with antisera specific for annexins isolated from tomato suspension-culture cells.