Paolo Montalbini

Soluble superoxide dismutase (SOD) in susceptible and resistant host-parasite complexes of Phaseolus vulgaris and Uromyces phaseoli☆

Abstract The level of soluble superoxide dismutase (E.G.1.15.1.1) was determined in Phaseolus vulgaris leaves susceptible (cv. Pinto 111) and hypersensitive resistant (cvs K.W. 765 and K.W. 814) to Uromyces phaseoli. In all cultivars tested increased levels of extracted Superoxide dismutase (SOD) appeared to be associated with the appearance of flecks and the differentiation of the uredosori, in the susceptible cultivar, and of microscopic or macroscopic necrotic lesions in the hypersensitive cultivars. Different SOD enzymes (Mn or Cu, Zn-SOD) were selectively stimulated by compatible and incompatible host-pathogen interactions. There was an increase in activity of the Mn-enzyme in the susceptible leaves during stroma differentiation in the mesophyll. By contrast there was an increase in activity of the Cu,Zn-enzyme in the hypersensitive response of resistant leaves. The above mentioned increases were due to a host response to infection since the contribution of fungal SOD was negligible. SOD and peroxidase increased simultaneously. The increase of peroxidasr greatly exceeded that of SOD only in the hypersensitive cultivars. The infection did not change the electrophoretic behaviour of SOD enzymes.

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.

Ureides and enzymes of ureide synthesis in wheat seeds and leaves and effect of allopurinol on Puccinia recondita f. sp. tritici infection

Abstract Xanthine oxidoreductase exhibiting a superoxide-producing activity (xanthine: O2 oxidoreductase, EC 1.2.3.2), uricase (urate: O2 oxidoreductase, EC 1.7.3.3) and allantoinase (EC 3.5.2.5) as well as ureides (allantoin and allamtoic acid) were detected in leaves, roots and seeds of wheat. Allopurinol [4-hydroxypyrazolo(3,4-d)pyrimidine], an analogue of purine and a tightly binding inhibitor of xanthine oxidizing enzyme, applied as soil drench at 40–60 μM starting 2–3 days after planting reduced the concentration of ureides in leaves and strongly inhibits the biotrophic growth of Puccinia recondita f. sp. tritici. Both phenomena are considered consequence of the in vivo inhibition of xanthine oxidoreductase in wheat plants. These findings indicate that purine catabolic pathway is involved during host-parasite relationship in rust-affected wheat leaves.

Purification and some properties of xanthine dehydrogenase from wheat leaves

Abstract Wheat leaf xanthine oxidoreductase was purified to electrophoretic homogeneity. Due to the low activity in leaves, 0.5 mg of pure protein with 1–3 nkat activity was obtained from 1 kg of leaf material at the end of purification. Based on uric acid production, the pure enzyme specific activity was 1.8 and 6.5 nkat mg−1 with NAD+ and PMS as acceptors, respectively. Molecular oxygen alone did not accomplish substrate hydroxylation, indicating that the wheat leaf xanthine oxidizing enzyme is strictly a xanthine dehydrogenase (XDH) (EC 1.1.1.204., formerly EC 1.2.1.37.). The total ineffectiveness of wheat leaf XDH to utilize NADP+, FAD and ferricyanide as oxidizing substrates, the inhibition of NAD+ reduction under anaerobiosis and the atypical visible absorption spectrum characteristics (maximum at 329–332 nm, a shoulder at about 392–394 nm and low absorption peak from 480 to 510 nm) differentiate wheat leaf XDH from other similar enzymes. Inhibitor effects, particularly p-hydroxymercuribenzoate and salicylhydroxamic acid, are instead in agreement with those reported for other xanthine oxidases and dehydrogenases. Wheat leaf XDH affinity for reducing substrates was very high with Km values of 4–8 μM. Oxidizing substrates instead showed a rather high Km value for NAD+ but much lower for PMS and NBT. Wheat leaf XDH is inactivated by incubation of the enzyme with the physiological electron donors xanthine and hypoxanthine but this effect was reversed by a prolonged reaction time. Although the fluorescence spectrum emission maximum was at 329–332 nm indicating the presence of flavin in wheat leaf XDH molecule, the exact nature of prosthetic groups remains to be verified.

Xanthine dehydrogenase from leaves of leguminous plants: purification, characterization and properties of the enzyme.

Summary A high level of xanthine oxidoreductase purification was achieved from leaves of ten leguminous plants. Under native conditions the enzyme purified from Vicia faba, Lens esculenta, Cicer arietinum, Glycine max. and Phaseolus vulgaris appeared electrophoretically homogeneous while that from Lupinus albus, Trifolium repens and pratense, Vicia sativa , and Vigna unguiculata was accompained by one or two minor contaminating proteins. Xanthine oxidizing enzymes isolated from leaves of leguminous plants did not react with molecular oxygen at a significant rate, indicating that all of them are xanthine dehyrogenases (XDH) (EC 1.1.1.204, formerly EC 1.2.1.37.). Under (SDS)-denatured conditions one main subunit of about 130 kDa was revealed in Vicia faba and Lens esculenta XDHs while in Glycine max . and Phaseolus vulgaris XDHs there were 130 and 150 kDa subunits both occurring at about the same molar ratio. The visible absorption spectrum of the pure protein was between 312 and 390 nm, related to the molybdopterin component of the enzyme with a lower absorption from 420 to 510 nm mainly due to flavin chromophores and a negligible absorption close to 550 nm reflecting the absorption of the iron-sulphur centres. The fluorescence excitation spectrum showed peaks at 274 and 368 nm, similar to pterin and xanthopterin. Fluorescence emission spectrum was characterized by two maxima at about 400 and 460 nm, typical of flavin chromophores. Superoxide dismutase and catalase had a slight stimulatory effect on enzyme activity with NAD + as acceptor. By contrast, catalase strongly enhanced the activity when artificial acceptors such as phenazine methosulphate and methylene blue were used. The effect of inhibitors, particularly p-hydroxy-mercuribenzoate and salicylhydroxamic acid, agree with those reported for other xanthine oxidases and dehydrogenases. The affinity for reducing substrates was rather high with K m values of 8-17 μmol/L while a wide K m range from 8-19 (PMS) to 106-267 μmol/L (methylene blue) was determined for oxidizing substrates. All XDHs were inhibited under anaerobiosis and by incubating the enzyme with the physiological electron donors xanthine and hypoxanthine, but these effects were reversed by a prolonged reaction time. Serological studies with polyclonal rabbit antisera against XDHs from Vicia faba, Lens esculenta and Phaseolus vulgaris indicated that all of the enzymes purified are more or less strictly related serologically.

Effect of localized and systemic tobacco mosaic virus infection on some photochemical and enzymatic activities of isolated tobacco chloroplasts

Abstract Certain photochemical and enzymic activities of isolated chloroplast preparations were evaluated in tobacco plants susceptible (Nicotiana tabacum cv. Bright BC 60) and hypersensitive (N. tabacum cv. Havana 425) to tobacco mosaic virus (TMV) infection. The main results were as follows: 1. (i) Chloroplasts isolated from inoculated (asymptomatic) and systemically infected (with mosaic symptoms) leaves of the susceptible plants did not exhibit any change in the rates of nicotinamide adenine dinucleotide phosphate (NADP) and ferricyanide photoreduction expressed as μmol mg−1 chlorophyll. In contrast, chloroplasts isolated from inoculated leaves of the hypersensitive plants, starting at the beginning of local lesion expression (collapsed areas, 2 days after inoculation; necrotic areas, 3 to 4 days after inoculation) exhibited a strong inhibition of electron transport reaching values about 60–70% of the control. This effect was observed when water was the electron donor, irrespective of whether NADP or ferricyanide was the electron acceptor; no differences were observed when NADP reduction occurred via Photosystem I after 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) inhibition, using ascorbate and 2,6-dichlorophenol indophenol (DCPIP) as an artificial electron donor. 2. (ii) The rate of oxygen photoreduction, expressed as μmol mg−1 chlorophyll, was slightly-increased in the chloroplasts from hypersensitive leaves 1 day after inoculation and then reduced to values about 40% of the control 2–4 days after inoculation. Less oxygen photoreduction was also observed in chloroplasts from systemically infected leaves, while no variation was observed in the chloroplasts from inoculated leaves of the susceptible plants. 3. (iii) Ribulose-1,5-bisphosphate carboxylase activity (measured in chloroplast extracts) was decreased in inoculated hypersensitive leaves during differentiation of local lesions (2 to 4 days after inoculation), reaching values of 30–40% of the control. A less intense decrease of this activity was observed in uninoculated leaves of the susceptible plants displaying mosaic symptoms which did not exhibit any significant variation in photochemical activity (NADP and ferricyanide photoreduction). 4. (iv) Other enzymatic activities measured in chloroplast extracts and/or total-leaf extracts, including malate dehydrogenase nicotinamide adenine dinucleotide-dependent (NADH-dependent) glutamate oxalacetate transaminase, superoxide dismutase, and phosphoenolpyruvate carboxylase, were either unchanged or little affected by TMV infection both in susceptible and hypersensitive combinations. These results are discussed with respect to the hypothesis that the hypofunctionality of the photosynthetic apparatus, characteristic of chloroplasts from hypersensitive leaves, may contribute, in part, to the localization of infection and thence to host resistance.

Effect of tobacco mosaic virus infection of levels of soluble superoxide dismutase (SOD) in nicotiana tabacum and nicotiana glutinosa leaves

Abstract The activity of superoxide dismutase (SOD: E.C. 1.15.1.1) was evaluated on Nicotiana tabacum and Nicotiana glutinosa leaf tissue after Tobacco Mosaic Virus (TMV) infection. Significant increase in extracted SOD appeared to be directly related to the appearance of necrotic and systemic symptoms in hypersensitive ( N. glutinosa and N. tabacum cv. Havana 425) and susceptible ( N. tabacum cv. Bright BC 60) leaves, respectively. SOD activity did not change significantly during the replication of TMV in the inoculated susceptible leaves up to 4 days after inoculation. Both cyanide-insensitive (2 days after inoculation) and sensitive (3–4 days after inoculation) enzymes increased during the expression of the hypersensitivity. Only cyanide-sensitive enzyme increased in systematically infected leaves. SOD and peroxidase increased simultaneously and the enhancement of peroxidase was higher than that of SOD. The values of peroxidase greatly exceeded that of SOD only in the hypersensitive leaves during local lesion differentiation. In N. tabacum leaves 4 clear SOD bands were separated by polyacrylamide gel electrophoresis: 3 cyanide-sensitive (Cu,Zn enzyme) and 1 cyanide-insensitive, while N. glutinosa had 3 bands: 2 cyanide-sensitive and 1 cyanide-insensitive. The cyanide-insensitive band, both in N. tabacum and N. glutinosa , was sensitive to H 2 O 2 and insensitive to chloroform-ethanol treatment and thus supposed to be Fe enzyme. The infection did not induce change in the electrophoretic pattern of SOD enzymes. In summary, our results indicate that the pathogenic alteration caused by TMV infection both in the compatible and in the incompatible combinations are characterized by an induction of SOD activity, particularly cyanide-sensitive Cu,Zn-SOD. The connection between the induction of SOD and a possible activation of O 2 − production in the hypersensitive tissue following TMV infection is discussed.

Isolation and characterization of uricase from bean leaves and its comparison with uredospore enzymes

Abstract Uricase (urate: oxygen oxidoreductase; EC 1.7.3.3) from bean (Phaseolus vulgaris) leaves and uredospores of two different rust fungi (Uromyces phaseoli and Uromyces fabae) has been purified to electrophoretic homogeneity by a procedure which includes xanthine–agarose affinity chromatography as the main step. Pure preparations had similar specific activities (2–6 U mg−1) with turnover numbers from 250 to 750 min−1, and all enzymes were tetramers consisting of identical or similar-sized subunits of 32–33 kDa. They also exhibited similar optimum pH (around 9.0), showed hyperbolic kinetics with Km values of 15–34 μM and behaved similarly against a number of putative activators/inhibitors, all of them being inhibited only by oxonate and xanthine. However, leaf and uredospore uricases differed in the strength of binding to DEAE-cellulose since leaf uricase did not bind to the exchanger and that from U. fabae bound stronger than that of U. phaseoli. Uredospore uricases showed complete antigenic independence against anti-uricase polyclonal antibodies from bean leaves and anti-uricase monoclonal antibodies from soybean nodules. Cross-reaction was observed between leaf uricase and nodule monoclonal antibodies and between nodule enzyme and leaf polyclonal antibodies. These results confirm the homogeneity of plant uricases and demonstrate that fungal obligate parasites have their own uricase, which is similar to the plant enzyme in many molecular and kinetic properties but different in DEAE-cellulose binding characteristics and immunological properties.

Effect of rust infection on levels of uricase, allantoinase and ureides in susceptible and hypersensitive bean leaves☆

Abstract The enzymatic activities involved in two sequential steps of purine catabolism and ureide biosynthesis, i.e. uricase, catalase and allantoinase, were determined in susceptible and hypersensitive bean leaf tissue after infection with Uromyces phaseoli. Uricase increased to approx. two times more than the control value 20–22 h after inoculation of the hypersensitive cv. K.W. 765 (reaction grade 2++) close to pathogen penetration and first haustoria formation and, 1 day later, in the susceptible cvs Golden Gate Wax, Pinto 111 (reaction grade 6) and in the hypersensitive cv. K.W. 814 (reaction grade 1). Afterwards, the highest increases in uricase activity (up to 10 times the control), mainly localized in fractions of leaf homogenate sedimenting at 2000 g and 15000 g, were detected in c.v. K.W. 765 during lesion development, indicating that a peroxisome-associated enzyme was involved. Lower increases (up to two to three times the control) were observed in the susceptible cultivars during stroma differentiation (flecks) and uredospore formation. Catalase linked to the particulate fractions increased in susceptible cultivars to the same extent as uricase from 2 to 6 days after inoculation. Also in cv. K.W. 765 it increased during lesion development, but very much less than uricase, with the consequence that a possible toxic action, due to unscavenged H2O2 production, could arise during hypersensitivity expression. An enhancement of allantoinase two or three times the control values during stroma differentiation was detected in susceptible cultivars. By contrast, allantoinase activity was unchanged during local lesion formation in cv. K.W. 765, indicating that the activation of this enzyme is specifically involved during the establishment of a biotrophic relationship between Phaseolus vulgaris and Uromyces phaseoli. In the susceptible cultivar, an increase in allantoic acid during the late stages of infection (uredospores differentiation) was observed. Both allantoin and allantoic acid were enhanced during lesion development in the hypersensitive cultivar. The results are consistent with the interpretation that the enhanced purine catabolism in rust-infected bean leaves is connected both to a nutritional benefit for the parasite in the compatible relationship, and to a toxic effect for the host in the incompatible one.

Allopurinol Metabolites and Xanthine Accumulation in Allopurinol-Treated Tobacco

Summary The metabolic conversion products of allopurinol were analyzed in tobacco plants allopurinol-treated by root absorption. The data presented indicate that two transformation proceses occur within the plant: i) oxidation to oxipurinol and xanthine accumulation, and ii) ribosyl transfer reactions with synthesis of ribonucleoside derivatives. While oxipurinol was detectable at 2 days after allopurinol application, 6 days were needed to detect ribonucleosides. Both proceses also took place when allopurinol and oxipurinol were applied by leaf infiltration. This is the first report of in vivo metabolic ribsosidation of allopurinol and oxipurinol in plants, well-known in human organisms and bacterial cells. in vivo treatment of tobacco tisues by allopurinol and the above mentioned allopurinol derivatives indicates that oxipurinol is the active compound in inhibiting tobacco xanthine oxidase. In contrast, in vitro experiments on xanthine oxidase immobilized on polyacrylamide gel rods after electrophoresis of leaf extract showed that only allopurinol was an effective tobacco xanthine oxidase inhibitor.