Enzymes of ammonia assimilation and ureide biogenesis in developing pigeonpea (Cajanus cajan L.) nodules

Ammonia assimilatory and ureide biogenic enzymes were measured in the cytosol fraction of pigeonpea nodules during the period 15–120 days after sowing. The activity of enzymes involved in the initial assimilation of ammonia, i.e. glutamine synthetase, glutamate synthase, asparagine synthetase and aspartate aminotransferase, substantially increased activities during the period of plant growth and reached a maximum value around 105 days after sowing. These increases paralleled the increase in nodule mass, nitrogenase activity and ureide content in nodules. Though no regular pattern was obtained for their specific activities, yet these activities when expressed relative to the specific activity of nitrogenase were many fold higher at each stage of development. Similar increases were observed in the activities of enzymes associated with the formation of ureides from purines. In almost all cases, the activities were again maximum around 90–105 days after sowing. The specific activities of nucleotidase, nucleosidase, xanthine dehydrogenase, uricase and allantoinase, when expressed relative to the specific activity of nitrogenase at vegetative, flowering and podsetting stages were again many fold higher indicating the sufficiency of the levels of these enzymes for the biosynthesis of ureides. The data presented are consistent with the proposal that in ureide producing legumes, ammonia is initially assimilated into glutamine, aspartate, etc., which are metabolised for the denovo synthesis of purines. The purines are then utilised for the production of ureides by a group of enzymes investigated here     

Subcellular organization of ureide biogenesis from glycolytic intermediates and ammonium in nitrogen-fixing soybean nodules

Subcellular organelle fractionation of nitrogen-fixing nodules of soybean (Glycine max (L.) Merr.) indicates that a number of enzymes involved in the assimilation of ammonia into amino acids and purines are located in the proplastids. These include asparagine synthetase (EC 6.3.1.1), phosphoribosyl amidotransferase (EC 2.4.2.14), phosphoglycerate dehydrogenase (EC 1.1.1.95), serine hydroxymethylase (EC 2.1.2.1), and methylene-tetrahydrofolate dehydrogenase (EC 1.5.1.5). Of the two isoenzymes of asparate aminotransferase (EC 2.6.1.1) in the nodule, only one was located in the proplastid fraction. Both glutamate synthase (EC 1.4.1.14) and triosephosphate isomerase (EC 5.3.1.1) were associated at least in part with the proplastids. Glutamine synthetase (EC 6.3.1.2) and xanthine dehydrogenase (EC 1.2.1.37) were found in significant quantities only in the soluble fraction. Phosphoribosylpyrophosphate synthetase (EC 2.7.6.1) was found mostly in the soluble fraction, although small amounts of it were detected in other organelle fractions. These results together with recent organelle fractionation and electron microscopic studies form the basis for a model of the subcellular distribution of ammonium assimilation, amide synthesis and uredie biogenesis in the nodule.Abbreviations FH₄ tetrahydrofolic acid - PRPP 5-phospho--D-ribose 1-pyrophosphate - PRPP synthetase ribosephosphate pyrophosphokinase (phosphoribosylpyrophosphate synthetase)     

Enzymes of ammonia assimilation and ureide biosynthesis in soybean nodules: effect of nitrate

The effect of nitrate on N₂ fixation and the assimilation of fixed N₂ in legume nodules was investigated by supplying nitrate to well established soybean (Glycine max L. Merr. cv Bragg)-Rhizobium japonicum (strain 3I1b110) symbioses. Three different techniques, acetylene reduction, ¹⁵N₂ fixation and relative abundance of ureides ([ureides/(ureides + nitrate + α-amino nitrogen)] × 100) in xylem exudate, gave similar results for the effect of nitrate on N₂ fixation by nodulated roots. After 2 days of treatment with 10 millimolar nitrate, acetylene reduction by nodulated roots was inhibited by 48% but there was no effect on either acetylene reduction by isolated bacteroids or in vitro activity of nodule cytoplasmic glutamine synthetase, glutamine oxoglutarate aminotransferase, xanthine dehydrogenase, uricase, or allantoinase. After 7 days, acetylene reduction by isolated bacteroids was almost completely inhibited but, except for glutamine oxoglutarate aminotransferase, there was still no effect on the nodule cytoplasmic enzymes. It was concluded that, when nitrate is supplied to an established symbiosis, inhibition of nodulated root N₂ fixation precedes the loss of the potential of bacteroids to fix N₂. This in turn precedes the loss of the potential of nodules to assimilate fixed N₂.     

Enzymes of ureide synthesis in pea and soybean

Soybean (Glycine max) and pea (Pisum sativum) differ in the transport of fixed nitrogen from nodules to shoots. The dominant nitrogen transport compounds for soybean are ureides, while amides dominate in pea. A possible enzymic basis for this difference was examined.

The level of enzymes involved in the formation of the ureides allantoin and allantoic acid from inosine 5′-monophosphate (IMP) was compared in different tissues of pea and soybean. Two enzymes, 5′-nucleotidase and uricase, from soybean nodules were found to be 50- and 25-fold higher, respectively, than the level found in pea nodules. Other purine catabolizing enzymes (purine nucleosidase, xanthine dehydrogenase, and allantoinase) were found to be at the same level in the two species. From comparison of enzyme activities in nodules with those from roots, stems, and leaves, two enzymes were found to be nodule specific, namely uricase and xanthine dehydrogenase. The level of enzymes found in the bacteroids indicated no significant contribution of Rhizobium japonicum purine catabolism in the overall formation of ureides in the soybean nodule. The presence in the nodules of purine nucleosidase and ribokinase activities makes a recirculation of the ribose moiety possible. In concert with phosphoribosylpyrophosphate synthetase, ribose becomes available for a new round of purine de novo synthesis, and thereby ureide formation.

     

Specialization for ureide biogenesis in the root nodules of black locust (Robinia pseudoacacia L.), an amide exporter

Summary Black locust (Robinia pseudoacacia L.), a member of the legume tribe Robinieae, has indeterminate root nodules and is primarily an amide exporter. However, in greenhouse-grown seedlings inoculated with rhizobia, ureide-N comprises approximately 8% of the total soluble-N in the xylem sap. Ultrastructurally, young interstitial cells (i.e., uninfected cells in the infected region near the nodule meristem) develop enlarged peroxisomes and abundant tubular ER, properties heretofore found to be characteristic only of members of the Phaseoleae, which have determinate nodules and are exporters principally of ureides. Many of the peroxisomes in the interstitial cells of black locust nodules react strongly for uricase (EC 1.7.3.3) activity in a cytochemical test employing diaminobenzidine, but some of the peroxisomes, particularly those farther back from the meristem, react weakly or not at all, even though enlarged. The ultrastructural specializations of the interstitial cells seem disproportionately large in comparison to the relatively low levels of ureides in the xylem transport stream. We suggest that similar specialization of some of the interstitial cells might be found in other legumes that export low amounts of ureides.Abbreviations DAB 3,3'diaminobenzidine - ER endoplasmic reticulum Dedicated to the memory of Professor Oswald Kiermayer     

Ultrastructural specialization for ureide production in uninfected cells of soybean root nodules

Summary Ultrastructural studies were conducted on root nodules of soybean (Glycine max) inoculated as seeds withRhizobium japonicum. The development of the large peroxisomes and abundant tubular endoplasmic reticulum (ER) characteristic of the uninfected interstitial cells was followed during nodule growth and maturation. Quantitative data on differences between the uninfected and infected cells in volumes and numbers of peroxisomes, plastids and mitochondria were analyzed statistically. The peroxisomes are 60 times greater in volume per unit cytoplasm in the uninfected cells than the small presumptive peroxisomes in the infected cells. Plastids are about equal in volume in the two types of cells. Mitochondria have 4 × the volume and 3 × the number of profiles per unit cytoplasm in the infected cells than in the uninfected. The observations are discussed in relation to published evidence that several enzymes involved in ureide production are localized in organelles of the uninfected cells. The uninfected cells are viewed as essential components in the symbiotic relationship between host and bacterium.Abbreviations DAB 3,3-diaminobenzidine - ER endoplasmic reticulum     

Specialization of the inner cortex for ureide production in soybean root nodules

Summary The possibility that cells in the inner cortex of determinate root nodules participate in ureide production from recently fixed N₂, as do the uninfected (interstitial) cells of the infected central region, has been investigated in soybean (Glycine max) inoculated as seeds withBradyrhizobium japonicum. Like the interstitial cells, cells of the three innermost cortical layers produce enlarged peroxisomes and a meshwork of tubular ER during differentiation. These changes are most pronounced in the innermost cortical layer, are successively less so in the 2nd and 3rd layers, and are usually undetectable in more distant layers. Peroxisomes in the inner three layers are stained in the DAB (3,3-diaminobenzidine) test for uricase (EC 1.7.3.3) activity, indicative of the potential for ureide formation, but peroxisomes in more distant cortical cells are not stained. A nodulespecific uricase also is demonstrable in the inner three cortical layers by immunogold labeling enhanced with silver for visualization in the light microscope. The observations suggest that with respect to ureide production the cells of the inner layers of the cortex are functionally similar to the interstitial cells of the infected region despite the apparent distinctiveness of the two regions anatomically.Abbreviations DAB 3,3-diaminobenzidine - ER endoplasmic reticulum     

Enzymes of sucrose breakdown in soybean nodules: alkaline invertase

The specific activities of acid and alkaline invertases (β-d-fructofuranoside fructohydrolase, EC 3.2.1.26), sucrose synthase (UDPglucose: d-fructose 2-α-d-glucosyltransferase, EC 2.4.1.13), hexokinase (ATP: d-hexose 6-phosphotransferase, EC 2.7.1.1), and fructokinase (ATP: d-fructose 6-phosphotransferase, EC 2.7.1.4) were determined in soybean (Glycine max L. Merr cv Williams) nodules at different stages of development and, for comparison, in roots of nonnodulated soybeans. Alkaline invertase and sucrose synthase were both involved in sucrose metabolism in the nodules, but there was only a small amount of acid invertase present. The nodules contained more phosphorylating activity with fructose than glucose. Essentially all of the alkaline invertase, sucrose synthase, and fructokinase were in the soluble fraction of nodule extracts whereas hexokinase was in the bacteroid, plant particulate, and soluble fractions.     

Cellular compartmentation of ureide biogenesis in root nodules of cowpea (Vigna unguiculata (L.) Walp.)

Cowpea (Vigna unguiculata (L.) Walp.) nodules have been investigated by means of cytochemical and immunocytochemical procedures at the ultrastructural level in order to assess the role of the uninfected cells in ureide biogenesis. Uricase activity in the nodules was shown by cytochemical methods to be localized exclusively in the numberous large peroxisomes confined to the uninfected cells; the small peroxisomes in the infected cells did not stain for uricase. Uricase was also localized in the peroxisomes of uninfected cells by immunogold techniques employing polyclonal antibodies against nodule-specific uricase of soybean. There was no labeling above background of any structures in the infected cells. The results indicate that the uninfected cells are essential for ureide biogenesis in cowpea. Although tubular endoplasmic reticulum, the presumptive site of allantoinase, increases greatly in the uninfected cells during nodule development, it virtually disappears as the nodules mature. The inconsistency between the disappearance of the tubular endoplasmic reticulum from older nodules and the high allantoinase activity reported for older plants remains to be explained.Abbreviations DAB 3,3-diaminobenzidine - ER endoplasmic reticulum - GARG goat anti-rabbit immunoglobulin G - IgG immunoglobulin G - kDa knodalton - Mr apparent molecular mass - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Ureide biogenesis and the enzymes of ammonia assimilation and ureide biosynthesis in nitrogen fixing pigeonpea (Cajanus cajan) nodules

Allantoic acid production from IMP, XMP, inosine, xanthosine, hypoxanthine, xanthine, uric acid and allantoin was investigated by incubating each of these substrates withCajanus cajan cytosol and bacteroid fractions separately in the presence and absence of NAD+ and allopurinol. Allantoic acid synthesis by bacteroid fraction could only be observed with uric acid and allantoin as substrates. Addition of NAD⁺ or allopurinol to the reaction mixtures had no effect. However, with cytosol fraction, allantoic acid was produced by each of these substrates, with maximum rate with allantoin. With NAD⁺ or with allopurinol, allantoic acid was produced only with uric acid and allantoin as substrates. NADH production with cytosol fraction could again be observed with all the substrates. Except with uric acid and allantoin, allopurinol completely inhibited NADH formation. Regardless of the presence or absence of allopurinol, none of the substrates exhibited significant activity with bacteroid fraction. Based on the activities of glutamine synthetase, glutamate synthase, glutamate dehydrogenase, aspartate aminotransferase, asparagine synthetase, nucleotidase, nucleosidase, xanthine de-hydrogenase, uricase and allantoinase and their intracellular localisation in various nodule fractions, a probable pathway for the biogenesis of ureides in pigeonpea nodules has been proposed     

Isolation and characterization of infected and uninfected cells from soybean nodules : role of uninfected cells in ureide synthesis

The distribution of organelles and associated enzymes between cells containing bacteroids and uninfected cells from nodules of Glycine max L. Merr. cv Amsoy 71 was investigated by separation of protoplasts on a sucrose step-gradient. Infected protoplasts were much larger, irregular in shape, and more dense than uninfected protoplasts. The peroxisomal enzymes, uricase and catalase, were present at much higher specific activity in the uninfected cell fraction. Allantoinase, an enzyme of the endoplasmic reticulum, had a greater specific activity in the uninfected cell fraction. Several enzymes whose products are required for purine biosynthesis, including phosphoglycerate dehydrogenase, aspartate aminotransferase, 6-phosphogluconate dehydrogenase, and glucose-6-phosphate dehydrogenase, exhibited a higher specific activity in the uninfected cell fraction. Isozymes of aspartate aminotransferase were separated on native gels and located by an activity stain. The soluble isozyme was predominantly found in the uninfected cell fraction. These data suggest that peroxisomes, containing uricase and catalase for conversion of uric acid to allantoin, are present only in the uninfected cells of soybean nodules. The uninfected cells also appear to be the site of the allantoinase reaction.     

Immunogold localization of nodule-specific uricase in developing soybean root nodules

Immunogold labeling was used to study the time of appearance and distribution of a nodule-specific form of uricase (EC 1.7.3.3) in developing nodules of soybean (Glycine max (L.) Merr.) inoculated with Bradyrhizobium japonicum. The enzyme was detected in thin sections of tissue embedded in either L R White acrylic resin or Spurr's epoxy resin, by employing a polyclonal antibody preparation active against a subunit of soybean nodule uricase. Antigenicity was better preserved in L R White resin, but ultrastructure was better maintained in Spurr's. Uricase was first detectable with protein A-gold in young, developing peroxisomes in uninfected cells, coincident with the release of Bradyrhizobium bacteroids from infection threads in adjacent infected cells. As the peroxisomes enlarged, labeling of the dense peroxisomal matrix increased. Gold particles were never observed over the paracrystalline inclusions of peroxisomes, however. Despite a close association between enlarging peroxisomes and tubular endoplasmic reticulum, uricase was not detectable in the latter. In mature nodules, labeling of uricase was limited to the large peroxisomes in uninfected cells. Small peroxisome-like bodies present in infected cells did not become labeled.Abbreviations BSA bovine serum albumin - Da dalton - ER endoplasmic reticulum - IgG immunoglobulin G     

Asparagine and ureide accumulation in nodules and shoots as feedback inhibitors of N2 fixation in soybean

Inhibition of N₂ fixation under water deficits has been hypothesized to result from N feedback within the plant involving ureides and/or asparagine (Asn). This study was undertaken to investigate ureide and Asn accumulation in shoots and nodules in response to treatments inhibiting nodule activity (acetylene reduction assay, ARA) such as Asn, ureide, or polyethylene glycol application to the nutrient solution of plants, boric acid on leaves, and imposition of a water deficit. ARA inhibition and nodule concentration of Asn and ureide were correlated to the ureide treatment applied (3–15 m M applied in the nutrient solution). Supplying Asn (3–9 m M applied in the nutrient solution) also induced an increase in nodule Asn and ureide concentration, which was associated with ARA inhibition. Spraying boric acid on leaves also inhibited ARA in parallel to an increase in shoot ureide and nodule Asn concentration while nodule ureide remained unchanged. By contrast, polyethylene glycol (PEG) inhibited ARA in parallel to an increase in nodule Asn and ureide concentration while shoot ureide was unchanged. The decline in ARA in response to water deficit was associated with an increase in nodule ureide, Asn and aspartate (Asp), although the increases in Asn and Asp were less than for ureides. Altogether, the results of these experiments indicated that Asn cannot be the only compound involved in the feedback inhibition of ARA. Instead ureide and Asn are probably both involved, either directly by accumulation of products that fail to be exported, or by feedback from the shoot due to an N -compound supply that exceeds shoot requirements.     

Enzymes of carbohydrate and amino acid metabolism in developing and mature nodules of white clover

The enzymic potential for the metabolism of carbohydrate (photosyntheticproducts) and amino acids (the assimilation of was determined by enzyme assay and by immunodetectionin developing and mature nodules of white clover. White nodulesappeared within 6 d of placing stolon tip cuttings in wet sand.The protein content and enzyme activities of these young nodulesincreased by 10-fold within 9 d. The expression of leghaemoglobinand nitrogenase components 1 and 2 were just detectable in nodulesat 7 d. All other enzymes measured were found in young rootsand white nodules as well as in mature red nodules. However,the expression of sucrose synthase and glutamine synthetase(the key enzymes of sucrose metabolism and assimilation, respectively) were greatly enhancedin nodules compared with roots. Nodule protein content, leghaemoglobinlevels and enzyme activities peaked at approximately 50 d aftertaking stolon cuttings, and then declined by about 50% by 80d. The results are discussed in the context of carbon and nitrogenfluxes in legume nodules. Key words: White clover, legume nodules, carbohydrate metabolism, amino acid metabolism, enzymes     

Localization of enzymes of ureide biosynthesis in peroxisomes and microsomes of nodules

The intracellular location of enzymes involved in the synthesis of the ureides, allantoin and allantoic acid, was investigated in nodules of Glycine max L. Merr. Cellular organelles were separated on isopycnic sucrose density gradients. Xanthine dehydrogenase activity (270 nanomoles per min per gram fresh weight) was totally soluble, whereas approximately 15% of the total uricase and catalase activities (1 and 2000 micromoles per minute per gram fresh weight, respectively) was in the fraction containing intact peroxisomes. Allantoinase activity (680 nanomoles per minute per gram fresh weight) was associated with the microsomal fraction, which apparently originates from the endoplasmic reticulum.     

Structure and possible ureide degrading function of the ubiquitous urease of soybean

Ubiquitous soybean urease, as opposed to the seed-specific urease, designates the seemingly identical ureolytic activities of suspension cultures and leaves. It also appears to be the basal urease in developing seeds of a variety, Itachi, which lacks the seed-specific urease (Polacco, Winkler 1984 Plant Physiol 74: 800-804). On native polyacrylamide gels the ureolytic activities in crude extracts of these three tissues comigrate as determined by assays of gel slices. At this level of resolution the ubiquitous urease also migrates with or close to the fast (trimeric) form of the seed-specific urease.

The ubiquitous urease was purified approximately 100-fold from suspension cultures of two cultivars (Itachi and Prize) as well as from developing seeds of Itachi. These partially purified preparations allowed visualization of native urease on polyacrylamide gels by activity staining and of urease subunits on denaturing lithium dodecyl sulfate gels by electrophoretic transfer to nitrocellulose and immunological detection (“Western Blot”). The ubiquitous urease holoenzyme migrates slightly less rapidly than the fast seed urease in native gels; its subunit migrates slightly less rapidly than the 93.5 kilodaltons subunit of either the fast or slow (hexameric) seed enzyme. The ubiquitous urease elutes from an agarose A-0.5 meter column with the fast form of the seed urease species suggesting that the ubiquitous urease, like the fast seed urease, exists as a trimeric holoenzyme. The soybean cultivar, Prize, produces the hexameric seed urease; yet its ubiquitous urease (from leaf and suspension culture) is trimeric.

The pH dependence of the ureolytic activity of seed coats of both seed urease-negative (Itachi) and seed urease-positive (Williams) cultivars suggests that this activity is exclusively the ubiquitous urease. Its relatively higher levels in seed coats than in embryos of Itachi suggests that the ubiquitous urease is involved in degradation of urea derived from ureides. Consistent with a ureide origin for urea is the observation that addition of a urease inhibitor, phenylphosphordiamidate, to extracts of developing Itachi seeds (seed coat plus embryo) results in accumulation of urea from allantoic acid.

     

Asparagine formation in soybean nodules

¹⁵NH₄⁺ and [¹⁵N](amide)-glutamine externally supplied to detached nodules from soybean plants (cv. Tamanishiki) were incorporated within nodule tissues by vacuum infiltration and metabolized to various nitrogen compounds during 60 minutes of incubation time. In the case of ¹⁵NH₄⁺ - feeding, the ¹⁵N abundance ratio was highest in the amide nitrogen of glutamine, followed by glutamate and the amide nitrogen of asparagine. In ¹⁵N content (micrograms excess ¹⁵N), the amide nitrogen of asparagine was most highly enriched after 60 minutes. ¹⁵NH₄⁺ was also appreciably assimilated into alanine.     

Allantoinase activity and ureide content of mesophyll and paraveinal mesophyll of soybean leaves

Paraveinal mesophyll (PVM) is a specialized soybean (Glycine max Merr.) leaf tissue which represents a significant biochemical compartment. Stereological measurements showed that PVM makes up 23% of the mesophyll volume in nodulated soybean. To get an indication of the extent of involvement of PVM in ureide metabolism, physical characteristics, distribution of allantoinase activity and ureide content were determined in isolated PVM protoplasts (PVMP) and mesophyll protoplasts (MP). PVMP were larger and contained less chlorophyll and protein than MP. PVMP had twice as much allantoinase activity per protoplast but only half as much allantoinase activity when expressed on a volume basis as compared to MP. Total leaf ureide concentration was high and nearly equally distributed between MP and PVMP. PVMP had a higher ureide content per protoplast, a higher concentration of allantoic acid and a lower ratio of allantoin to allantoic acid. These results suggest that both tissues have the capacity to assimilate allantoin in vivo. The data are discussed with reference to the relative access of the two mesophyll tissues to incoming ureides.     

Asparagine biosynthesis in soybean nodules

Two auxin-induced endo-1,4-β-glucanases (EC 3.2.1.4) were purified from pea (Pisum sativum L. var. Alaska) epicotyls and used to degrade purified pea xyloglucan. Hydrolysis yielded nonasaccharide (glucose/xylose/galactose/fucose, 4:3:1:1) and heptasaccharide (glucose/xylose, 4:3) as the products. The progress of hydrolysis, as monitored viscometrically (with amyloid xyloglucan) and by determination of residual xyloglucan-iodine complex (pea) confirmed that both pea glucanases acted as endohydrolases versus xyloglucan. K_m values for amyloid and pea xyloglucans were approximately the same as those for cellulose derivatives, but V_max values were lower for the xyloglucans. Auxin treatment of epicotyls in vivo resulted in increases in net deposits of xyloglucan and cellulose in spite of a great increase (induction) of endogenous 1,4-β-glucanase activity. However, the average degree of polymerization of the resulting xyloglucan was much lower than in controls, and the amount of soluble xyloglucan increased. When macromolecular complexes of xyloglucan and cellulose (cell wall ghosts) were treated in vitro with pea 1,4-β-glucanase, the xyloglucan component was preferentially hydrolyzed and solubilized. It is concluded that xyloglucan is the main cell wall substrate for pea endo-1,4-β-glucanase in growing tissue.