Cloning, characterization and mRNA expression analysis of PVAS1, a type I asparagine synthetase gene from Phaseolus vulgaris.

A gene encoding a putative asparagine synthetase (AS; EC 6.3.5.4) has been isolated from common bean (Phaseolus vulgaris L.). A 2-kb cDNA clone of this gene (PVAS1) encodes a protein of 579 amino acids with a predicted molecular mass of 65,265 Da, an isoelectric point of 6.3, and a net charge of -9.3 at pH 7.0. The PVAS1 protein sequence conserves all the amino acid residues that are essential for glutamine-dependent AS, and PVAS1 complemented an Escherichia coli asparagine auxotroph, which demonstrates that it encodes a glutamine-dependent AS. The PVAS1 protein showed the highest similarity to soybean SAS1, and piled up with other legume ASs to form an independent dendritic group of type-I AS enzymes. Northern blot analyses revealed that the expression pattern of PVAS1 resembles that of PVAS2, another AS previously described in the common bean. Unlike PVAS2, however, PVAS1 was not expressed in the nodule and was not repressed by light, suggesting different functions for these two AS genes.

Urea is a product of ureidoglycolate degradation in chickpea. Purification and characterization of the ureidoglycolate urea-lyase.

A ureidoglycolate-degrading activity was analyzed in different organs of chickpea (Cicer arietinum). Activity was detected in all the tissues analyzed, but highest levels of specific activity were found in pods, from which it has been purified and characterized. This is the first ureidoglycolate-degrading activity that has been purified to homogeneity from any photosynthetic organism. Only one ureidoglycolate-degrading activity was found during the purification. The enzyme was purified 1,500-fold, and specific activity for the pure enzyme was 8.6 units mg(-1), which corresponds with a turnover number of 1,600 min(-1). The native enzyme has a molecular mass of 180 kD and consists of six identical or similar-sized subunits of 31 kD each. The enzyme exhibited hyperbolic, Michaelian kinetics for (-) ureidoglycolate with K(m) values of 6 and 10 microM in the presence or absence of Mn(2+), respectively. Optimum pH was between 7 and 8 and maximum activity was found at temperatures above 70 degrees C, the enzyme being extremely stable and resistant to heat denaturation. The activity was inhibited by EDTA and enhanced by several bivalent cations, thus suggesting that the enzyme is a metalloprotein. This enzyme has been characterized as a ureidoglycolate urea-lyase (EC 4.3.2.3), which catalyzes the degradation of (-) ureidoglycolate to glyoxylate and urea. This is the first time that such an activity is detected in plant tissues. A possible function for this activity and its implications in the context of nitrogen mobilization in legume plants is also discussed.

Allantoate amidinohydrolase (Allantoicase) from Chlamydomonas reinhardtii: its purification and catalytic and molecular characterization.

An allantoate-degrading enzyme has been purified to electrophoretic homogeneity for the first time from a photosynthetic organism, the unicellular green algae Chlamydomonas reinhardtii. The purification procedure included a differential protein extraction followed by conventional steps such as ammonium sulfate fractionation, gel filtration, anion exchange chromatography, and preparative electrophoresis. Under the routine assay conditions (7 mM allantoate), specific activity for the purified enzyme was 185 U/mg, which rose to 225 U/mg under kinetic considerations (saturating substrate). Therefore, a turnover number of 4.5 x 10(4) min(-1) can be deduced for the 200-kDa protein. The enzyme is a true allantoicase (EC 3.5.3.4) that catalyzes the degradation of allantoate to (-)ureidoglycolate and (+)ureidoglycolate to glyoxylate. The enzyme exhibited hyperbolic kinetic for allantoate and ureidoglycolate with K(m) values of 2 and 0.7 mM, respectively. V(max) of the reaction with allantoate as substrate was nine times higher than that with ureidoglycolate. The native enzyme has a molecular weight of 200 kDa and consists of six identical or similar-sized subunits of 34 kDa each, organized in two trimers of 100 kDa. Each subunit has five cysteine residues, four of which are involved in disulfide bonds, with a total of 12 disulfide bonds in the 200-kDa protein. Allantoate inhibits competitively the reaction with ureidoglycolate as substrate. In addition, buffers and group-specific reagents affect the activity in the same manner irrespective of the substrate used. Those results suggest that both substrates use the same active site. The effect of group-specific reagents suggest that the amino acids histidine, tyrosine, and cysteine are essentials for the allantoicase activity with both substrates.

cDNA-AFLP reveals a striking overlap in race-specific resistance and wound response gene expression profiles.

The tomato Cf-9 gene confers resistance to races of the fungal pathogen Cladosporium fulvum expressing the Avr9 gene. cDNA amplified fragment length polymorphism analysis was used to display transcripts whose expression is rapidly altered during the Avr9- and Cf-9-mediated defense response in tobacco cell cultures. Diphenyleneiodonium was used to abolish the production of active oxygen species during gene induction. Of 30,000 fragments inspected, 290 showed altered abundance, of which 263 were induced independently of active oxygen species. cDNA clones were obtained for 13 ACRE (for Avr9/Cf-9 rapidly elicited) genes. ACRE gene induction occurred in the presence of cycloheximide. Avr9 induced ACRE gene expression in leaves. Surprisingly, ACRE genes were also rapidly but transiently induced in leaves in response to other stresses. The amino acid sequences of some ACRE proteins are homologous to sequences of known proteins such as ethylene response element binding protein transcription factors, the N resistance protein, a calcium binding protein, 13-lipoxygenase, and a RING-H2 zinc finger protein. Rapid induction of ACRE genes suggests that they play a pivotal role during plant defense responses.

Resistance gene-dependent activation of a calcium-dependent protein kinase in the plant defense response.

In the Cf-9/Avr9 gene-for-gene interaction, the Cf-9 resistance gene from tomato confers resistance to the fungal pathogen Cladosporium fulvum, which expresses the corresponding pathogen-derived avirulence product Avr9. To understand R gene function and dissect the signaling mechanisms involved in the induction of plant defenses, we studied Cf-9/Avr9-dependent activation of protein kinases in transgenic Cf9 tobacco cell cultures. Using a modified in-gel kinase assay with histone as substrate, we identified a membrane-bound, calcium-dependent protein kinase (CDPK) that showed a shift in electrophoretic mobility from 68 to 70 kD within 5 min after Avr9 elicitor was added. This transition from the nonelicited to the elicited CDPK form was caused by a phosphorylation event and was verified when antibodies to CDPK were used for protein gel blot analysis. In addition, the interconversion of the corresponding CDPK forms could be induced in vitro in both directions by treatment with either phosphatase or ATP. In vitro protein kinase activity toward syntide-2 or histone with membrane extracts or gel-purified enzyme was dependent on Ca(2)+ content and was compromised by the calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) but not by its inactive isoform N-(6-aminohexyl)-1-naphthalenesulfonamide. In these assays, the CDPK activity in elicited samples, reflecting predominantly the phosphorylated 70-kD CDPK form, was greater than in nonelicited samples. Thus, Avr9/Cf-9-dependent phosphorylation and subsequent transition from the nonelicited to the elicited form correlate with the activation of a CDPK isoform after in vivo stimulation. Because that transition was not inhibited by W-7, the in vivo CDPK activation probably is not the result of autophosphorylation. Studies with pharmacological inhibitors indicated that the identified CDPK is independent of or is located upstream from a signaling pathway that is required for the Avr9-induced active oxygen species.

Functional, c-myc-tagged Cf-9 resistance gene products are plasma-membrane localized and glycosylated.

The Cf-9 resistance gene from tomato confers resistance to races of the fungal pathogen Cladosporium fulvum that express the corresponding avirulence gene, Avr9. Avr9 encodes a secreted peptide. To investigate Cf-9 function, we tagged the Cf-9 protein with a triple myc epitope at either the amino- or carboxy-terminus of the mature protein. Tobacco plants carrying these constructs activate a defence response to Avr9 peptide. The Cf-9 sequence predicts a protein of 94 kDa, with 22 glycosylation sites. Using c-myc antibodies, c-myc : Cf-9 protein was detected as a unique band with a molecular size of 160 kDa. The band shifted to approximately 105 kDa after glucosidase treatment, indicating that Cf-9 protein is highly glycosylated. Plasma membranes were isolated using two-phase partitioning, and c-myc : Cf-9 was enriched in these fractions, indicating that Cf-9 is a plasma membrane protein. This was confirmed by silver-enhanced immunogold labelling of tobacco protoplasts carrying the amino-terminal c-myc tag; a higher labelling density was observed on the surface of protoplasts derived from c-myc : Cf-9 tobacco compared to untransformed control. The presence of Cf-9 in the plasma membrane is consistent with its role in conferring recognition of the extracellular Avr9 peptide.

Early signalling events in the Avr9/Cf-9-dependent plant defence response.

Abstract Resistance of tomato to the leaf mould fungus Cladosporium fulvum is controlled by the interaction between a plant-encoded resistance gene (Cf-9) and pathogen-encoded avirulence (Avr9) gene. Our objective is to understand the underlying molecular mechanisms that transmit the Cf-9/Avr9-dependent pathogen perception event and activate the plant defence response. Our approach toward the understanding of Cf-function is based on the analysis of early Cf-9/Avr9-mediated responses and signalling events. Because Cf-9 transgenically expressed in tobacco retains its specificity and activity to the Avr9 elicitor, signalling experiments were conducted in the heterologous system using these transgenic lines or derived Cf9 tobacco cell cultures. Among the earliest responses to the Avr9/Cf-9 elicitation event were rapid changes in ion-fluxes, the synthesis of active oxygen species (AOS), probably catalysed by a plant NADPH-oxidase, and the transient activation of two MAP kinases. These kinases were identified as WIPK (wounding-induced protein kinase) and SIPK (salicylic-acid induced kinase) from tobacco. Studies with pharmacological inhibitors suggested that the MAP kinases are located in an independent signalling pathway from the Avr9/Cf-9-dependent synthesis of AOS. SIPK and WIPK were involved in pathogen-related elicitation processes as well as in abiotic stress responses. This indicates that the plant defence is triggered via a signalling network that shares components with pathways originating from abiotic environmental stress stimuli.

Rapid Avr9- and Cf-9 -dependent activation of MAP kinases in tobacco cell cultures and leaves: convergence of resistance gene, elicitor, wound, and salicylate responses.

The Cf-9 resistance (R) gene from tomato confers resistance to the fungal pathogen Cladosporium fulvum expressing the corresponding, pathogen-derived avirulence gene product Avr9. To understand how an initial R/Avr recognition event is transmitted and triggers the induction of plant defenses, we investigated early Avr9/Cf-9-dependent activation of protein kinases in transgenic tobacco expressing the Cf-9 gene. We identified two protein kinases of 46 and 48 kD, using myelin basic protein as substrate, that became rapidly activated in a strictly gene-for-gene manner within 2 to 5 min after Avr9 elicitation in both Cf9 tobacco plants and derived cell cultures. Studies with pharmacological inhibitors and effectors revealed that Ca2+ influx and a phosphorylation event(s) are required for kinase activation, but neither enzyme is involved in the Avr9-dependent synthesis of active oxygen species. The activation of both kinases is achieved via post-translational mechanisms, and the activation but not inactivation step includes tyrosine phosphorylation. Using specific antibodies, we found that the 46- and 48-kD kinases were similiar to WIPK (for wound-induced protein kinase) and SIPK (for salicylic acid-induced protein kinase), two previously characterized mitogen-activated protein (MAP) kinases from tobacco. In addition, Cf9 tobacco plants and cell cultures showed an Avr9-dependent accumulation of the WIPK transcript. Cf9 tobacco suspension cultures are thus a unique system in which to analyze the earliest events in R gene function. These data indicate that (1) the R/Avr-mediated induction of plant defense is accomplished via several parallel signaling mechanisms, and (2) R/Avr-dependent signal transduction pathways are interlinked at MAP kinases with responses of plants not only to non-race-specific elicitors but also to abiotic stimuli, such as wounding and mechanical stress.

Molecular, genetic and physiological analysis of Cladosporium resistance gene function in tomato.

Characterization of the DNA sequence of 4 tomato leaf mould disease resistance genes (Cf-2, Cf-4, Cf-5 and Cf-9) leads to the prediction that they encode C-terminally membrane anchored glycopeptides with many extracytoplasmic leucine rich repeats (LRRs). The N terminal LRRs are variable between the Cf-genes, suggesting a role in specificity, and the C terminal LRRs are more conserved, suggesting a role in signal transduction. Genetic analysis has revealed several Rcr genes that are required for Cf-gene function; their isolation will help us understand how Cf-genes work. Cf-9 confers responsiveness to pathogen-encoded Avr9 peptide on introduction to tobacco. Tobacco suspension cultures carrying the Cf-9 gene produce reactive oxygen species in response to Avr9 peptide, whereas untransformed cultures do not. The significance of these observations is discussed.

A continuous spectrophotometric assay for ureidoglycolase activity with lactate dehydrogenase or glyoxylate reductase as coupling enzyme.

A spectrophotometric assay for ureidoglycolase activity (both ureidoglycolate lyase and hydrolase), based on the reduction of glyoxylate to glycolate catalyzed by glyoxylate reductase or lactate dehydrogenase with the stoichiometric and continuous NADH oxidation, is described. The assay has been optimized for the amount of coupling enzyme, reagent concentrations, buffers, and the nonenzymatic degradation of ureidoglycolate. Under optimal assay conditions, ureidoglycolase activity can be followed with either lactate dehydrogenase or glyoxylate reductase as coupling enzyme and reaction can be started by addition of substrate or enzyme extract. Once the reaction was started, NADH oxidation was linear with time after a lag phase of 1-2 min. This linear NADH oxidation was directly proportional to enzyme concentration in the assay mixture until changes in absorbance of 0.12 per minute. This method is easy and reliable for the accurate determination of ureidoglycolase activity in crude and purified extracts from Chlamydomonas reinhardtii cells and no notable interferences have been detected. Since lactate dehydrogenase is much cheaper and has a lower Km for its substrate than glyoxylate reductase and can be used as supplied, its use as the coupling enzyme of choice is recommended.

Purification and characterization of an L-amino-acid oxidase from Chlamydomonas reinhardtii.

An L-amino-acid oxidase (EC 1.4.3.1) that catalyzes the oxidative deamination of twelve L-amino acids has been purified 21-fold and with 14% yield to electrophoretic homogeneity from Chlamydomonas reinhardtii cells by ammonium-sulfate fractionation, gel filtration through Sephacryl and Superose, anion-exchange chromatography and preparative electrophoresis in polyacrylamide gels. The native enzyme is a protein of 470 kDa and consists of eight identical or similarsized subunits of 60 kDa each. Optimum pH and temperature were 8.2 and 55° C, respectively, with a Q10 (45-55° C) of 1.7 and an activation energy of 45 kJ · mol(-1). Its absorption spectrum showed, in the visible region, maxima at 360 and 444 nm, characteristic of a flavoprotein with a calculated flavin content of 7.7 mol FAD per mol of native enzyme. Apparent K m values of the twelve L-amino acids which can act as substrates of L-amino-acid oxidase ranged between 31 µM for phenylalanine and 176 µM for methionine. The effect of several specific group reagents, chelating agents and bivalent cations on enzyme activity has also been studied.