An alternative pathway for ureide usage in legumes: enzymatic formation of a ureidoglycolate adduct in Cicer arietinum and Phaseolus vulgaris.

Ureidoglycolate is an intermediate in the degradation of the ureides, allantoin and allantoate, found in many organisms. In some leguminous plant species these compounds are used to transport recently fixed nitrogen in the root nodules to the aerial parts of the plant. In the present study, it was demonstrated that purified ureidoglycolases from chickpea (Cicer arietinum) and French bean (Phaseolus vulgaris) do not produce glyoxylate, and can use phenylhydrazine as a substrate with K(m) values of 4.0 mM and 8.5 mM, respectively. Furthermore, these enzymes catalyse the transfer of the ureidoglycolyl group to phenylhydrazine to produce ureidoglycolyl phenylhydrazide, which degrades non-enzymatically to glyoxylate phenylhydrazone and urea. This supports their former classification as ureidoglycolate urea-lyases. The enzymatic reaction catalysed by the characterized ureidoglycolases uncovered here can be viewed as a novel type of phenylhydrazine ureidoglycolyl transferase. The implications of these findings for ureide metabolism in legume nitrogen metabolism are discussed.

Nucleoside Metabolism Is Induced in Common Bean During Early Seedling Development.

Nucleoside hydrolases (NSH; nucleosidases) catalyze the cleavage of nucleosides into ribose and free nucleobases. These enzymes have been postulated as key elements controlling the ratio between nucleotide salvage and degradation. Moreover, they play a pivotal role in ureidic legumes by providing the substrate for the synthesis of ureides. Furthermore, nucleotide metabolism has a crucial role during germination and early seedling development, since the developing seedlings require high amount of nucleotide simultaneously to the mobilization of nutrient in cotyledons. In this study, we have cloned two nucleosidases genes from Phaseolus vulgaris, PvNSH1 and PvNSH2, expressed them as recombinant proteins, and characterized their catalytic activities. Both enzymes showed a broad range of substrate affinity; however, PvNSH1 exhibited the highest activity with uridine, followed by xanthosine, whereas PvNSH2 hydrolyses preferentially xanthosine and shows low activity with uridine. The study of the regulation of nucleosidases during germination and early postgerminative development indicated that nucleosidases are induced in cotyledons and embryonic axes just after the radicle emergence, coincident with the induction of nucleases activity and the synthesis of ureides in the embryonic axes, with no remarkable differences in the level of expression of both nucleosidase genes. In addition, nucleosides and nucleobase levels were determined as well in cotyledons and embryonic axes. Our results suggest that PvNSH1 and PvNSH2 play an important role in the mobilization of nutrients during this crucial stage of plant development.

Nuclease and ribonuclease activities in response to salt stress: Identification of PvRNS3, a T2/S-like ribonuclease induced in common bean radicles by salt stress.

The increase in soil salinization due to global climate change could cause large losses in crop productivity affecting, among other biological processes, to germination and seedling development. We have studied how salt stress affects nucleic acid degrading activities in radicles of common bean during seedling development. In radicles of common bean, a main nuclease of 37 kDa and two ribonucleases of 17 and 19 kDa were detected. Saline stress did not alter these three activities but induced a new ribonuclease of 16 kDa. All three ribonucleases are acidic enzymes that were inhibited by Zn. The 16 and 17 kDa ribonucleases are inhibited by guanilates. In the genome of common bean, we have identified 13 genes belonging to the T2 ribonuclease family and that are grouped in the 3 classes of T2 ribonucleases. The analysis of the expression of the 3 genes belonging to Class I (PvRNS1 to 3) and the unique gene from Class II (PvRNS4) in radicles showed that PvRNS3 is highly induced under salt stress.

Biochemical and Molecular Characterization of PvNTD2, a Nucleotidase Highly Expressed in Nodules from Phaseolus vulgaris.

Nucleotides are molecules of great importance in plant physiology. In addition to being elementary units of the genetic material, nucleotides are involved in bio-energetic processes, play a role as cofactors, and are also components of secondary metabolites and the hormone cytokinin. The common bean (Phaseolus vulgaris) is a legume that transports the nitrogen fixed in nodules as ureides, compounds synthetized from purine nucleotides. The first step in this pathway is the removal of the 5'-phosphate group by a phosphatase. In this study, a gene that codes for a putative nucleotidase (PvNTD2) has been identified in P. vulgaris. The predicted peptide contains the conserved domains for haloacid dehalogenase-like hydrolase superfamily. The protein has been overexpressed in Escherichia coli, and the purified protein showed molybdate-resistant phosphatase activity with nucleoside monophosphates as substrates, confirming that the identified gene codes for a nucleotidase. The optimum pH for the activity was 7-7.5. The recombinant enzyme did not show special affinity for any particular nucleotide, although the behaviour with AMP was different from that with the other nucleotides. The activity was inhibited by adenosine, and a regulatory role for this nucleoside was proposed. The expression pattern of PvNTD2 shows that it is ubiquitously expressed in all the tissues analysed, with higher expression in nodules of adult plants. The expression was maintained during leaf ontogeny, and it was induced during seedling development. Unlike PvNTD1, another NTD previously described in common bean, the high expression of PvNTD2 was maintained during nodule development, and its possible role in this organ is discussed.

Ureide metabolism during seedling development in French bean (Phaseolus vulgaris).

French bean (Phaseolus vulgaris) is a legume that transports most of the atmospheric nitrogen fixed in its nodules to the aerial parts of the plant as ureides. Changes in ureide content and in enzymatic activities involved in their metabolism were identified in the cotyledons and embryonic axes during germination and early seedling development. Accumulation of ureides (ca. 1300 nmol per pair of cotyledons) was observed in the cotyledons of dry seeds. Throughout germination, the total amount of ureides slightly decreased to about 1200 nmol, but increased both in cotyledons and in embryonic axes after radicle emergence. In the axes, the ureides were almost equally distributed in roots, hypocotyls and epicotyls. The pattern of ureide distribution was not affected by the presence of nitrate or sucrose in the media up to 6 days after imbibition. Ureides are synthesized from purines because allopurinol (a xanthine dehydrogenase inhibitor) blocks the increase of ureides. Allantoin and allantoate-degrading activities were detected in French bean dried seeds, whereas no ureidoglycolate-degrading activity was detected. During germination, the levels of the three activities remain unchanged in cotyledons. After radicle emergence, the levels of activities in cotyledons changed. Allantoin-degrading activity increased, allantoate-degrading activity decreased and ureidoglycolate-degrading activity remained undetectable in cotyledons. In developing embryonic axes, the three activities were detected throughout germination and early seedling development. The embryonic axes are able to synthesize ureides, because those compounds accumulated in axes without cotyledons.

Relationship between ureidic/amidic metabolism and antioxidant enzymatic activities in legume seedlings.

Ureides are nitrogenous compounds with a special function in some legume under nitrogen fixing conditions, the ureidic legumes. In this group, ureides are the predominant nitrogen transport molecule from nodules to the upper part, whereas amidic legumes use amides as nitrogen transport compounds. In this study, the ureide levels have been analysed in seedlings from four ureidic and four amidic legume plants. It has been found that the differentiation among ureide and amide plants already exists in seedlings during early seedling development, with high levels of ureide and allantoinase activity in cotyledons and embryonic axes from ureide plants. Since ureides have been implicated in the response of plant to several stress, total hydrosoluble antioxidant capacity and the levels of several antioxidant activities have been determined and compared among these two legume groups. The total antioxidant capacity did not follow any differential pattern in cotyledons or embryonic axes for the analysed plants. The levels of superoxide dismutase, guaiacol peroxidase and ascorbate peroxidase in both embryonic axes and cotyledons are statistical different between amide and ureide seedlings, whereas the catalase activity was similar among these groups of plants. We discuss than amides and ureides could follow different strategies to protect against oxidation.

Tissue abundance and characterization of two purified proteins with allantoinase activity from French bean (Phaseolus vulgaris).

Allantoinase (allantoin amidohydrolase, EC 3.5.2.5) catalyses the hydrolysis of allantoin to allantoic acid, a key reaction in the biosynthesis and degradation of ureides. This activity was determined in different tissues of French bean plants (Phaseolus vulgaris L.) which were grown under nitrogen-fixing conditions. Allantoinase activity was detected in all tissues analysed, but the highest levels of specific activity were found in developing fruits, from which allantoinase has been purified to electrophoretic homogeneity and further characterized. After diethylaminoethyl (DEAE)-Sephacel chromatography, two peaks showing allantoinase activity were obtained in the chromatographic profile and the corresponding proteins were independently purified. Total allantoinase activity was purified 200-fold, indicating the relevance of this enzymatic activity in French bean developing fruits, with allantoinase representing 0.5% of total soluble protein. Both proteins with allantoinase activity are monomeric with molecular masses of 45 and 42 kDa. The specific activities of the purified proteins were 560 and 295 units mg(-1), which correspond to turnover numbers of 25,200 and 12,100 min(-1), respectively. The two proteins have very similar biochemical properties showing Michaelis-Menten kinetics for allantoin with K(m) values of about 60 mM, with high optimal temperatures; are metalloenzymes; are inhibited by compounds reacting with sulphydryl groups; and are unaffected by reducing agents. All analysed tissues exhibited the two activities responsible for allantoin degradation, although one of them was the main form in leaves (the most photosynthetic tissue) and the other protein was the main form in roots (non-photosynthetic tissue). The allantoinase activity and distribution of both proteins have been analysed during fruit development. For both proteins, the allantoinase activity and distribution pattern were the same in plants growing either under nitrogen-fixing conditions or fertilized with nitrate.

Nucleases activities during French bean leaf aging and dark-induced senescence.

During leaf senescence resources are managed, with nutrients mobilized from older leaves to new sink tissues. The latter implies a dilemma in terms of resource utilization, the leaf senescence should increase seed quality whereas delay in senescence should improve the seed yield. Increased knowledge about nutrient recycling during leaf senescence could lead to advances in agriculture and improved seed quality. Macromolecules mobilized during leaf senescence include proteins and nucleic acids. Although nucleic acids have been less well studied than protein degradation, they are possible reservoirs of nitrogen and phosphorous. The present study investigated nuclease activities and gene expression patterns of five members of the S1/P1 family in French bean (Phaseolus vulgaris L. cv.)Page: 2 during leaf senescence. An in-gel assay was used to detect nuclease activity during natural and dark-induced senescence, with single-stranded DNA (ssDNA) used as a substrate. The results revealed two nucleases (glycoproteins), with molecular masses of 34 and 39kDa in the senescent leaves. The nuclease activities were higher at a neutral than at an acidic pH. EDTA treatment inhibited the activities of the nucleases, and the addition of zinc resulted in the recovery of these activities. Both the 34 and 39kDa nucleases were able to use RNA and double-stranded DNA (dsDNA) as substrates, although their activities were low when dsDNA was used as a substrate. In addition, two ribonucleases with molecular masses of 14 and 16kDa, both of which could only utilize RNA as a substrate, were detected in the senescent leaves. Two members of the S1/P1 family, PVN2 and PVN5, were expressed under the experimental conditions, suggesting that these two genes were involved in senescence. The nuclease activity of the glycoproteins and gene expression were similar under both natural senescence and dark-induced senescence conditions.

Identification and characterization of a gene encoding for a nucleotidase from Phaseolus vulgaris.

Nucleotidases are phosphatases that catalyze the removal of phosphate from nucleotides, compounds with an important role in plant metabolism. A phosphatase enzyme, with high affinity for nucleotides monophosphate previously identified and purified in embryonic axes from French bean, has been analyzed by MALDI TOF/TOF and two internal peptides have been obtained. The information of these peptide sequences has been used to search in the genome database and only a candidate gene that encodes for the phosphatase was identified (PvNTD1). The putative protein contains the conserved domains (motif I-IV) for haloacid dehalogenase-like hydrolases superfamily. The residues involved in the catalytic activity are also conserved. A recombinant protein overexpressed in Escherichia coli has shown molybdate resistant phosphatase activity with nucleosides monophosphate as substrate, confirming that the identified gene encodes for the phosphatase with high affinity for nucleotides purified in French bean embryonic axes. The activity of the purified protein was inhibited by adenosine. The expression of PvNTD1 gene was induced at the specific moment of radicle protrusion in embryonic axes. The gene was also highly expressed in young leaves whereas the level of expression in mature tissues was minimal.

Purification and identification of a nuclease activity in embryo axes from French bean.

Plant nucleases are involved in nucleic acid degradation associated to programmed cell death processes as well as in DNA restriction, repair and recombination processes. However, the knowledge about the function of plant nucleases is limited. A major nuclease activity was detected by in-gel assay with whole embryonic axes of common bean by using ssDNA or RNA as substrate, whereas this activity was minimal in cotyledons. The enzyme has been purified to electrophoretic homogeneity from embryonic axes. The main biochemical properties of the purified enzyme indicate that it belongs to the S1/P1 family of nucleases. This was corroborated when this protein, after SDS-electrophoresis, was excised from the gel and further analysis by MALDI TOF/TOF allowed identification of the gene (PVN1) that codes this protein. The gene that codes the purified protein was identified. The expression of PVN1 gene was induced at the specific moment of radicle protrusion. The inclusion of inorganic phosphate to the imbibition media reduced the level of expression of this gene and the nuclease activity suggesting a relationship with the phosphorous status in French bean seedlings.

Identification of a novel phosphatase with high affinity for nucleotides monophosphate from common bean (Phaseolus vulgaris).

Common bean (Phaseolus vulgaris) seedlings accumulate ureides derived from purines after germination. The first step in the conversion of purines to ureides is the removal of the 5'-phosphate group by a phosphatase that has not been established yet. Two main phosphatase activities were detected in the embryonic axes of common bean using inosine monophosphate as substrate in an in-gel assay. Both activities differed in their sensitive to the common phosphatase inhibitor molybdate, with the molybdate-resistant as the first enzyme induced after radicle protrusion. The molybdate-resistant phosphatase has been purified to electrophoretic homogeneity and this is the first enzyme which shows this resistance purified and characterized from plant tissues. The native enzyme was a monomer of 55 kDa and it showed highest activity with nucleotides as substrates, with the K(m) values in the micromolar range. Among nucleotides, the highest specific constant (V(max)/K(m)) was observed for adenosine monophosphate. Furthermore, the enzyme was inhibited by nucleosides, the products of the enzymatic reaction, with maximum effect for adenosine. Common bean seedlings imbibed in the presence of adenosine monophosphate in vivo showed the highest molybdate-resistant phosphatase activity in the axes in addition to increased ureide content. The data presented suggests that purified phosphatase is involved in nucleotide metabolism in embryonic axes from common bean.

Biochemical characterisation of an allantoate-degrading enzyme from French bean (Phaseolus vulgaris): the requirement of phenylhydrazine.

In tropical legumes like French bean (Phaseolus vulgaris) or soybean (Glycine max), most of the atmospheric nitrogen fixed in nodules is used for synthesis of the ureides allantoin and allantoic acid, the major long distance transport forms of organic nitrogen in these species. The purpose of this investigation was to characterise the allantoate degradation step in Phaseolus vulgaris. The degradation of allantoin, allantoate and ureidoglycolate was determined "in vivo" using small pieces of chopped seedlings. With allantoate and ureidoglycolate as substrates, the determination of the reaction products required the addition of phenylhydrazine to the assay mixture. The protein associated with the allantoate degradation has been partially purified 22-fold by ultracentrifugation and batch separation with DEAE-Sephacel. This enzyme was specific for allantoate and could not use ureidoglycolate as substrate. The activity was completely dependent on phenylhydrazine, which acts as an activator at low concentrations and decreases the affinity of the enzyme for the substrate at higher concentrations. The optimal pH for the activity of the purified protein was 7.0 and the optimal temperature was 37 degrees C. The activity was completely inhibited by EDTA and only manganese partially restored the activity. The level of activity was lower in extracts obtained from leaves and fruits of French bean grown with nitrate than in plants actively fixing nitrogen and, therefore, relying on ureides as nitrogen supply. This is the first time that an allantoate-degrading activity has been partially purified and characterised from a plant extract. The allosteric regulation of the enzyme suggests a critical role in the regulation of ureide degradation.

Degradation of ureidoglycolate in French bean (Phaseolus vulgaris) is catalysed by a ubiquitous ureidoglycolate urea-lyase.

A ureidoglycolate-degrading activity was analysed in different tissues of French bean (Phaseolus vulgaris L.) plants during development. Activity was detected in all the tissues analysed, although values were very low in seeds before germination and in cotyledons. After radicle emergence, the activity increased due to high activity present in the axes. The highest levels of specific activity were found in developing fruits, from which the enzyme was purified and characterised. This is the first ureidoglycolate-degrading activity that has been purified to homogeneity from a ureide legume. The enzyme was purified 280 fold, and the specific activity for the pure enzyme was 4.4 units mg(-1), which corresponds to a turnover number of 1,055 min(-1). The native enzyme has a molecular mass of 240 kDa and consists of six identical or similar-sized subunits each of 38 kDa. The activity of the purified enzyme was completely dependent on manganese and asparagine. The enzyme exhibited hyperbolic, Michaelian kinetics for ureidoglycolate with a K(m) value of 3.9 mM. This enzyme has been characterised as a ureidoglycolate urea-lyase (EC 4.3.2.3).

Update on ureide degradation in legumes.

Warm season N2-fixing legumes move fixed N from the nodules to the aerial portions of the plant primarily in the form of ureides, allantoin and allantoate, oxidation products of purines synthesized de novo in the nodule. Ureides are also products of purine turnover in senescing tissues, such as seedling cotyledons. A combination of biochemical and molecular approaches in both crop and model species has shed new light on the metabolic pathways involved in both the synthesis and degradation of allantoin. Improved understanding of ureide biochemistry includes two 'additional' enzymatic steps in the conversion of uric acid to allantoin in the nodule and the mechanism of allantoin and allantoate breakdown in leaf tissue. Ureide accumulation and metabolism in leaves have also been implicated in the feedback inhibition of N2-fixation under water limitation. Sensitivity to water deficit differs among soybean cultivars. Manganese supplementation has been shown to modify relative susceptibility or tolerance to this process in a cultivar-dependent manner. A discussion of the potential roles for ureides and manganese in the feedback inhibition of N2-fixation under water limitation is presented. The existing data are examined in relation to potential changes in both aerial carbon and nitrogen supply under water deficit.