Manuel Pineda

Local inhibition of nitrogen fixation and nodule metabolism in drought-stressed soybean

Drought stress is a major factor limiting symbiotic nitrogen fixation (NF) in soybean crop production. However, the regulatory mechanisms involved in this inhibition are still controversial. Soybean plants were symbiotically grown in a split-root system (SRS), which allowed for half of the root system to be irrigated at field capacity while the other half remained water deprived. NF declined in the water-deprived root system while nitrogenase activity was maintained at control values in the well-watered half. Concomitantly, amino acids and ureides accumulated in the water-deprived belowground organs regardless of transpiration rates. Ureide accumulation was found to be related to the decline in their degradation activities rather than increased biosynthesis. Finally, proteomic analysis suggests that plant carbon metabolism, protein synthesis, amino acid metabolism, and cell growth are among the processes most altered in soybean nodules under drought stress. Results presented here support the hypothesis of a local regulation of NF taking place in soybean and downplay the role of ureides in the inhibition of NF.

Molecular analysis of ureide accumulation under drought stress in Phaseolus vulgaris L.

Under water deficit, ureidic legumes accumulate ureides in plant tissues, and this accumulation has been correlated with the inhibition of nitrogen fixation. In this work we used a molecular approach to characterize ureide accumulation under drought stress in Phaseolus vulgaris. Accumulation of ureides, mainly allantoate, was found in roots, shoots and leaves, but only a limited transient increase was observed in nodules from drought-stressed plants. We show that ureide accumulation is regulated at the transcriptional level mainly through induction of allantoinase (ALN), whereas allantoate amidohydrolase (AAH), involved in allantoate degradation, was slightly reduced, indicating that inhibition of this enzyme, key in ureide breakdown in aerial tissues, is not the main cause of allantoate accumulation. Expression of the ureide metabolism genes analysed in this study was induced by abscisic acid (ABA), suggesting the involvement of this plant hormone in ureide accumulation. Moreover, we observed that increases of ureide levels in P. vulgaris drought-stressed tissues were similar in non-nodulated, nitrate-fed plants, and in plants cultured under nitrogen-fixation conditions. Our results indicate that ureide accumulation in response to water deficit is independent from de novo synthesis of ureides in nodules, and therefore uncoupled from nitrogen fixation.

Uricase from leaves: its purification and characterization from three different higher plants

Abstract. Uricase (urate: oxygen oxidoreductase, EC␣1.7.3.3) from leaves of chickpea (Cicer arietimum L.), broad bean (Vicia faba major L.), and wheat (Triticum aestivum L.) has been purified to electrophoretic homogeneity by a procedure which includes xanthine-agarose affinity chromatography as the main step. Purification factors of 74 000–83 000 and recoveries of 80–90% were achieved. Purified preparations had specific activities between 600 and 800 nkat · mg protein−1 (turnover numbers between 4400 and 6400 min−1). The three plant uricases were found by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be tetramers of similar molecular mass (120–130 kDa) and to have identical or similar-sized subunits (32–34 kDa). They also had a similar optimum pH (9–9.5) and showed a hyperbolic kinetics with Km values from 9–24 μM. All of them showed similar responses to putative activators/inhibitors. Oxonate, xanthine and, to a lesser extent, neocuproin inhibited uricase activity, whereas allantoin, ammonium, citrulline and glutamine did not. The three leaf uricases lacked catalase activity and were not activated by cadaverine. None of the three plant enzymes cross-reacted with anti-uricase monoclonal antibodies from soybean nodules or anti-uricase polyclonal antibodies from Chlamydomonas reinhardtii or rat liver. These results are consistent with the view that uricase in plants is probably a unique enzyme which is expressed at very low level in leaves.

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 Km 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.

Purification and characterization of anl-amino-acid oxidase fromChlamydomonas reinhardtii

Anl-amino-acid oxidase (EC 1.4.3.1) that catalyzes the oxidative deamination of twelvel-amino acids has been purified 21-fold and with 14% yield to electrophoretic homogeneity fromChlamydomonas 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. ApparentKm values of the twelvel-amino acids which can act as substrates ofl-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.

Purification and molecular properties of urate oxidase from Chlamydomonas reinhardtii

Urate oxidase (urate: oxygen oxidoreductase, EC 1.7.3.3) from the unicellular green alga Chlamydomonas reinhardtii has been purified to electrophoretic and immunological homogeneity by a procedure which includes as main steps ammonium sulfate fractionation, gel filtration, ion exchange and xanthine-agarose affinity chromatography. The native enzyme has a relative molecular mass (Mr) of 124,000 and consists of four identical or similar-sized subunits of Mr 31,000 each. The enzyme has a Stokess radius of 3.87 nm, a sedimentation coefficient of 6.8 S and an f/f0 of 1.23, and exhibits its maximal absorption at 276 nm. Optimum pH was 8.5 and maximum activity was shown at 40 degrees C, with an activation energy of 53 kJ.mol-1 and a Q10 of 1.96. Absorption spectrum of native reduced enzyme showed two transient maxima at 392 and 570 nm, very similar to those of metal-urate complexes, which disappeared in the presence of cyanide. Inhibition by cyanide and neocuproin, but not by salicylhydroxamic acid, strongly suggests that copper is the metal involved in enzymatic urate oxidation. By using a sensitive photokinetic method for copper determination, a content of 4 mol of copper per mol of enzyme has been found.

Developmental effects on ureide levels are mediated by tissue-specific regulation of allantoinase in Phaseolus vulgaris L.

The ureides allantoin and allantoate are key molecules in the transport and storage of nitrogen in ureide legumes. In shoots and leaves from Phaseolus vulgaris plants using symbiotically fixed nitrogen as the sole nitrogen source, ureide levels were roughly equivalent to those of nitrate-supported plants during the whole vegetative stage, but they exhibited a sudden increase at the onset of flowering. This rise in the level of ureides, mainly in the form of allantoate, was accompanied by increases in allantoinase gene expression and enzyme activity, consistent with developmental regulation of ureide levels mainly through the tissue-specific induction of allantoate synthesis catalysed by allantoinase. Moreover, surprisingly high levels of ureides were also found in non-nodulated plants fertilized with nitrate, at both early and late developmental stages. The results suggest that remobilized N from lower leaves is probably involved in the sharp rise in ureides in shoots and leaves during early pod filling in N(2)-fixing plants and in the significant amounts of ureides observed in non-nodulated plants.

Nitrogen stress and the expression of asparagine synthetase in roots and nodules of soybean (Glycine max)

The difficulty of assaying asparagine synthetase (AS) (EC 6.3.5.4) activity in roots of soybean has been circumvented by measuring expression of the AS genes. Expression of three soybean asparagine synthetase (SAS) genes (SAS1, SAS2 and SAS3) was observed in roots of non-nodulated soybean plants cultivated on nitrate. Expression of these genes was reduced to very low levels within days after submitting the plants to a N-free medium. The subsequent return to a complete medium (containing nitrate) restored expression of all three AS genes. Roots of nodulated plants, where symbiotic nitrogen fixation was the exclusive source of N (no nitrate present), showed very weak expression of all three AS genes, but on transfer to a nitrate-containing medium, strong expression of these genes was observed within 24 h. In nodules, all three genes were expressed in the absence of nitrate. Under conditions that impair nitrogen fixation (nodules submerged in aerated hydroponics), only SAS1 expression was reduced. However, in the presence of nitrate, an inhibitor of N(2) fixation, SAS1 expression was maintained. High and low expressions of AS genes in the roots were associated with high and low ratios of Asn/Asp transported to the shoot through xylem. It is concluded that nitrate (or one of its assimilatory products) leads to the induction of AS in roots of soybean and that this underlies the variations found in xylem sap Asn/Asp ratios. Regulation of nodule AS expression is quite different from that of the root, but nodule SAS1, at least, appears to involve a product of N assimilation rather than nitrate itself.

UPTAKE AND METABOLISM OF ALLANTOIN AND ALLANTOATE BY CELLS OF CHLAMYDOMONAS REINHARDTII (CHLOROPHYCEAE)

The green alga Chlamydomonas reinhardtii can use the ureides allantoin and allantoate as sole nitrogen sources. Once the uptake systems for allantoin and allantoate were induced, the uptake and growth rates were identical for the two ureides. However, the enzymatic activities involved in the degradation of the two ureides (allantoinase and allantoicase) were regulated differently. Allantoinase seems to be constitutive, since it was detected in all the nitrogen sources studied, while allantoicase behaved as an inducible enzyme, since it was present only in cells cultured in ureides or any metabolic precursor of these compounds. Neither allantoinase nor allantoicase activities were repressed by ammonium in the presence of ureides. Allantoicase activity was not induced under nitrogen starvation conditions, while it was induced in cells that had been cultured with allantoin or allantoate in the dark. Allantoin uptake showed a pattern similar to that of allantoate under all nutritional and environmental condit...

Purification and substrate inactivation of xanthine dehydrogenase from Chlamydomonasreinhardtii

Xanthine dehydrogenase (XDH) from the unicellular green alga Chlamydomonas reinhardtii has been purified to electrophoretic homogeneity by a procedure which includes several conventional steps (gel filtration, anion exchange chromatography and preparative gel electrophoresis). The purified protein exhibited a specific activity of 5.7 units/mg protein (turnover number = 1.9 .10(3) min-1) and a remarkable instability at room temperature. Spectral properties were identical to those reported for other xanthine-oxidizing enzymes with absorption maxima in the 420-450 nm region and a shoulder at 556 nm characteristic of molybdoflavoproteins containing iron-sulfur centers. Chlamydomonas XDH was irreversibly inactivated upon incubation of enzyme with its physiological electron donors xanthine and hypoxanthine, in the absence of NAD+, its physiological electron acceptor. As deduced from spectral changes in the 400-500 nm region, xanthine addition provoked enzyme reduction which was followed by inactivation. This irreversible inactivation also took place either under anaerobic conditions or whenever oxygen or any of its derivatives were excluded. Adenine, 8-azaxanthine and acetaldehyde which could act as reducing substrates of XDH were also able to inactivate it upon incubation. The same inactivating effect was observed with NADH and NADPH, electron donors for the diaphorase activity associated with xanthine dehydrogenase. In addition, partial activities of XDH were differently affected by xanthine incubation. We conclude that xanthine dehydrogenase inactivation by substrate is due to an irreversible process affecting mainly molybdenum center and that sequential and uninterrupted electron flow from xanthine to NAD+ is essential to maintain the enzyme in its active form.