Tobacco nectarin I. Purification and characterization as a germin-like, manganese superoxide dismutase implicated in the defensE of floral reproductive tissues

Nectarin I, a protein that accumulates in the nectar of Nicotiana sp. , was determined to contain superoxide dismutase activity by colorimetric and in-gel assays. This activity was found to be remarkably thermostable. Extended incubations at temperatures up to 90 degrees C did not diminish the superoxide dismutase activity of nectarin I. This attribute allowed nectarin I to be purified to homogeneity by heat denaturation of the other nectar proteins. By SDS-polyacrylamide gel electrophoresis, nectarin I appeared as a 29-kDa monomer. If the protein sample was not boiled prior to loading the gel, then nectarin I migrated as 165-kDa oligomeric protein. By matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, the protomer subunit was found to be a 22.5-kDa protein. Purified nectarin I contained 0.5 atoms of manganese/monomer, and the superoxide dismutase activity of nectarin I was not inhibited by either H(2)O(2) or NaCN. Following denaturation, the superoxide dismutase activity was restored after Mn(2+) addition. Addition of Fe(2+), Cu(2+), Zn(2+), and Cu(2+)/Zn(2+) did not restore superoxide dismutase activity. The quaternary structure of the reconstituted enzyme was examined, and only tetrameric and pentameric aggregates were enzymatically active. The reconstituted enzyme was also shown to generate H(2)O(2). Putative nectarin I homologues were found in the nectars of several other plant species.     

Role of NADPH oxidase versus neutrophil proteases in antimicrobial host defense

NADPH oxidase is a crucial enzyme in mediating antimicrobial host defense and in regulating inflammation. Patients with chronic granulomatous disease, an inherited disorder of NADPH oxidase in which phagocytes are defective in generation of reactive oxidant intermediates (ROIs), suffer from life-threatening bacterial and fungal infections. The mechanisms by which NADPH oxidase mediate host defense are unclear. In addition to ROI generation, neutrophil NADPH oxidase activation is linked to the release of sequestered proteases that are posited to be critical effectors of host defense. To definitively determine the contribution of NADPH oxidase versus neutrophil serine proteases, we evaluated susceptibility to fungal and bacterial infection in mice with engineered disruptions of these pathways. NADPH oxidase-deficient mice (p47(phox-/-)) were highly susceptible to pulmonary infection with Aspergillus fumigatus. In contrast, double knockout neutrophil elastase (NE)(-/-)×cathepsin G (CG)(-/-) mice and lysosomal cysteine protease cathepsin C/dipeptidyl peptidase I (DPPI)-deficient mice that are defective in neutrophil serine protease activation demonstrated no impairment in antifungal host defense. In separate studies of systemic Burkholderia cepacia infection, uniform fatality occurred in p47(phox-/-) mice, whereas NE(-/-)×CG(-/-) mice cleared infection. Together, these results show a critical role for NADPH oxidase in antimicrobial host defense against A. fumigatus and B. cepacia, whereas the proteases we evaluated were dispensable. Our results indicate that NADPH oxidase dependent pathways separate from neutrophil serine protease activation are required for host defense against specific pathogens.     

Role of neutrophils and NADPH phagocyte oxidase in host defense against respiratory infection with virulent Francisella tularensis in mice

Francisella tularensis subspecies (subsp.) tularensis is a CDC Category A biological warfare agent and inhalation of as few as 15 bacilli can initiate severe disease. Relatively little is known about the cellular and molecular mechanisms of host defense against respiratory infection with subsp. tularensis. In this study, we examined the role of neutrophils and NADPH phagocyte oxidase in host resistance to pulmonary infection in a mouse intranasal infection model. We found that despite neutrophil recruitment to the lungs and increased concentrations of neutrophil-chemotactic chemokines (KC, MIP-2 and RANTES) in the bronchoalveolar lavage fluid following intranasal inoculation of the pathogen, neither depletion of neutrophils nor enhancement of their recruitment into the lungs had any impact on bacterial burdens or survival rate/time. Nevertheless, mice deficient in NADPH phagocyte oxidase (gp91(phox?/?)) did exhibit higher tissue and blood bacterial burdens and succumbed to infection one day earlier than wild-type C57BL/6 mice. These results imply that although neutrophils are not a major effector cell in defense against subsp. tularensis infection, NADPH phagocyte oxidase does play a marginal role.     

A major function of the tobacco floral nectary is defense against microbial attack

 We have characterized the major nectar protein (Nectarin I) from ornamental tobacco as a superoxide dismutase that functions to generate high levels of hydrogen peroxide in nectar. Other nectar functions include an anti-polygalacturonase activity that may be due to a polygalacturonase inhibiting protein (PGIP). We also examined the expression of defense related genes in the nectary gland by two independent methods. We isolated a sample of nectary-expressed cDNAs and found that 21% of these cDNAs were defense related clones. Finally, we examined the expression of a number of specific defense-related genes by hybridization to specific cDNAs. These results demonstrated that a number of specific defense genes were more strongly expressed in the floral nectary than in the foliage. Taken together these results indicate that the floral nectary gland can have specific functions in plant defense. Received August 8, 2002; accepted January 7, 2003 Published online: June 2, 2003     

Role of inhibitors of proteolytic enzymes in plant defense against phytopathogenic microorganisms

This review analyzes the literature on various mechanisms of proteolytic enzyme inhibitors involved in plant defense against attack by phytopathogenic microorganisms. The action of proteinase inhibitors from plants upon the enzymes from pathogenic microorganisms and viruses is reviewed. Considerable attention is given to the induction of proteinase inhibitors in plants in response to the invasion of pathogens. Some aspects of application of proteinase inhibitors in biotechnology for production of transgenic plants with enhanced resistance to diseases are discussed.Translated from Biokhimiya, Vol. 69, No. 11, 2004, pp. 1600–1606.Original Russian Text Copyright © 2004 by Valueva, Mosolov.     

Involvement of nitric oxide-induced NADPH oxidase in adventitious root growth and antioxidant defense in Panax ginseng

Nitric oxide (NO) affects the growth and development of plants and also affects plant responses to various stresses. Because NO induces root differentiation, we examined whether or not it is involved in increased ROS generation. Treatments with sodium nitroprusside (SNP), an NO donor, 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO), a specific NO scavenger, and Nω-nitro-l-arginine methyl ester hydrochloride (l-NAME), an NO synthase (NOS) inhibitor, revealed that NO is involved in the adventitious root growth of mountain ginseng. Supply of an NO donor, SNP, activates NADPH oxidase activity, resulting in increased generation of O₂ ^·−, which subsequently induces growth of adventitious roots. Moreover, treatment with diphenyliodonium chloride (DPI), an NADPH oxidase inhibitor, individually or with SNP, inhibited root growth, NADPH oxidase activity, and O₂ ^·− anion generation. Supply of the NO donor, SNP, did not induce any notable isoforms of enzymes; it did, however, increase the activity of pre-existing bands of NADPH oxidase, superoxide dismutase, catalase, peroxidase, ascorbate peroxidase, and glutathione reductase. Enhanced activity of antioxidant enzymes induced by SNP supply seems to be responsible for a low level of H₂O₂ in the adventitious roots of mountain ginseng. It was therefore concluded that NO-induced generation of O₂ ^·− by NADPH oxidase seems to have a role in adventitious root growth of mountain ginseng. The possible mechanism of NO involvement in O₂ ^·− generation through NADPH oxidase and subsequent root growth is discussed.     

ATL9, a RING zinc finger protein with E3 ubiquitin ligase activity implicated in chitin- and NADPH oxidase-mediated defense responses

Pathogen associated molecular patterns (PAMPs) are signals detected by plants that activate basal defenses. One of these PAMPs is chitin, a carbohydrate present in the cell walls of fungi and in insect exoskeletons. Previous work has shown that chitin treatment of Arabidopsis thaliana induced defense-related genes in the absence of a pathogen and that the response was independent of the salicylic acid (SA), jasmonic acid (JA) and ethylene (ET) signaling pathways. One of these genes is ATL9 ( = ATL2G), which encodes a RING zinc-finger like protein. In the current work we demonstrate that ATL9 has E3 ubiquitin ligase activity and is localized to the endoplasmic reticulum. The expression pattern of ATL9 is positively correlated with basal defense responses against Golovinomyces cichoracearum, a biotrophic fungal pathogen. The basal levels of expression and the induction of ATL9 by chitin, in wild type plants, depends on the activity of NADPH oxidases suggesting that chitin-mediated defense response is NADPH oxidase dependent. Although ATL9 expression is not induced by treatment with known defense hormones (SA, JA or ET), full expression in response to chitin is compromised slightly in mutants where ET- or SA-dependent signaling is suppressed. Microarray analysis of the atl9 mutant revealed candidate genes that appear to act downstream of ATL9 in chitin-mediated defenses. These results hint at the complexity of chitin-mediated signaling and the potential interplay between elicitor-mediated signaling, signaling via known defense pathways and the oxidative burst.     

Purification of a novel flavoprotein involved in the thyroid NADPH oxidase. Cloning of the porcine and human cdnas

Hydrogen peroxide is the final electron acceptor for the biosynthesis of thyroid hormone catalyzed by thyroperoxidase at the apical surface of thyrocytes. Pig and human thyroid plasma membrane contain a Ca(2+)-dependent NAD(P)H oxidase that generates H(2)O(2) by transferring electrons from NAD(P)H to molecular oxygen. We purified from pig thyroid plasma membrane a flavoprotein which constitutes the main, if not the sole, component of the thyroid NAD(P)H oxidase. Microsequences permitted the cloning of porcine and human full-length cDNAs encoding, respectively, 1207- and 1210-amino acid proteins with a predicted molecular mass of 138 kDa (p138(Tox)). Human and porcine p138(Tox) have 86.7% identity. The strongest similarity was to a predicted polypeptide encoded by a Caenorhabditis cDNA and with rbohA, a protein involved in the Arabidopsis NADPH oxidase. p138(Tox) shows also similarity to the p65(Mox) and to the gp91(Phox) in their C-terminal region and have consensus sequences for FAD- and NADPH-binding sites. Compared with gp91(Phox), p138(Tox) shows an extended N-terminal containing two EF-hand motifs that may account for its calcium-dependent activity, whereas three of four sequences implicated in the interaction of gp91(Phox) with the p47(Phox) cytosolic factor are absent in p138(Tox). The expression of porcine p138(Tox) mRNA analyzed by Northern blot is specific of thyroid tissue and induced by cyclic AMP showing that p138(Tox) is a differentiation marker of thyrocytes. The gene of human p138(Tox) has been localized on chromosome 15q15.     

JFC1, a novel tandem C2 domain-containing protein associated with the leukocyte NADPH oxidase

We have employed a yeast two-hybrid system to screen a B lymphoblast-derived cDNA library, searching for regulatory components of the NADPH oxidase. Using as bait the C-terminal half of p67(phox), which contains both Src homology 3 domains, we have cloned JFC1, a novel human 62-kDa protein. JFC1 possesses two C2 domains in tandem. The C2A domain shows homology with the C2B domain of synaptotagmins. JFC1 mRNA was abundantly expressed in bone marrow and leukocytes. The expression of JFC1 in neutrophils was restricted to the plasma membrane/secretory vesicle fraction. We confirmed JFC1-p67(phox) association by affinity chromatography. JFC1-containing beads pulled down both p67(phox) and p47(phox) subunits from neutrophil cytosol, but when the recombinant proteins were used, only p67(phox) bound to JFC1, indicating that JFC1 binds to the cytosolic complex via p67(phox) without affecting the interaction between p67(phox) and p47(phox). In contrast to synaptotagmins, JFC1 was unable to bind to inositol 1,3,4,5-tetrakisphosphate but did bind to phosphatidylinositol 3,4,5-trisphosphate and to a lesser extent to phosphatidylinositol 3,4-diphosphate. From the data presented here, it is proposed that JFC1 is acting as an adaptor protein between phosphatidylinositol 3-kinase products and the oxidase cytosolic complex.     

Aerotolerance of strictly anaerobic microorganisms and factors of defense against oxidative stress: a review

Effects of aerobic conditions on strictly anaerobic microorganisms belonging to diverse taxa (clostridia, acetogenic bacteria, lactic acid bacteria, bacteroids, sulfate-reducing bacteria, and methanogenic archaea) and differing considerably in their oxygen resistance have been reviewed, with emphasis on the role of aerotolerance in the ecology of anaerobes. Consideration is given to components of nutritive media for anaerobe culturing, which decrease the toxic effects of oxygen and there by contribute significantly to maintenance and storage of industrial cultures of strictly anaerobic microorganisms. Physiological and biochemical factors are described, accounting for the relative resistance of many strict anaerobes to oxygen and products of incomplete reduction thereof. Specific attention is given to regulation of enzymes of antioxidative defense, operating in the cells of strict anaerobes under the conditions of oxidative stress caused by oxygen, superoxide anion, or hydrogen peroxide.     

Involvement of plasma-membrane NADPH oxidase in abscisic acid- and water stress-induced antioxidant defense in leaves of maize seedlings

The roles of the plasma-membrane (PM) NADPH oxidase in abscisic acid (ABA)- and water stress-induced antioxidant defense were investigated in leaves of maize ( Zea mays L.) seedlings. Treatment by exogenous ABA (100 micro M ABA) or osmotic stress (-0.7 MPa induced by polyethylene glycol) significantly increased the activity of the PM NADPH oxidase, the production of leaf O(2)(-), the activities of several antioxidant enzymes (superoxide dismutase, catalase, ascorbate peroxidase and glutathione reductase), and the contents of antioxidant metabolites (ascorbate and reduced glutathione). Pretreatment with three different inhibitors of NADPH oxidase (diphenylene iodonium, imidazole and pyridine) or an inhibitor of ABA biosynthesis (tungstate) reduced the increase in the activity of the PM NADPH oxidase and the production of leaf O(2)(-), and the capacity of antioxidant defense systems mediated by ABA. The inhibitory effects above caused by tungstate were reversed by exogenous ABA. These data indicate that NADPH oxidase is involved in the ABA-induced production of active oxygen species (AOS), and our results depict a minimal chain of events initiated by water stress-induced ABA accumulation, which then triggers the production of AOS by membrane-bound NADPH oxidase, resulting in the induction of antioxidant defense systems against oxidative damage in plants.     

Morphological study of floral nectaries in Euonymus and the probable origin of the echinate fruit surface

A conspicuous nectary disk is common but has a distinguishing morphology in the cosmopolitan genus Euonymus. Our study focuses on the morphology of floral nectaries in 21 representatives of Euonymus and Glyptopetalum. Two main types of nectaries were documented:a mix of inter-and extrastaminal nectaries existed between the corolla and the stigma, while the intrastaminal nectaries were distributed between the stigma and the stamen bases. The main route of nectar release in Euonymus is via modified stomata, and different nectarostomata locations were observed:in depressions, slightly raised above the epidermal surface or at the same level as the epidermis. Floral nectaries in E. sect. Echinococcus species developed into the protrusions on the fruit surface at the later stage. The development of nectaries not only explained the mystery of the origin of the echinate fruit surface, but also showed that differences in fruit surface might be inappropriate for use in infrageneric classification. These discoveries inform morphological observations of floral nectaries in Euonymus.     

Studies on the NADPH oxidase of phagocytes. Production of a monoclonal antibody which blocks the enzymatic activity of pig neutrophil NADPH oxidase

We describe in this paper a monoclonal antibody to pig NADPH oxidase which inhibits enzymatic activity. This antibody, designated 1H8.2, was selected from a group of monoclonal antibodies produced against active preparations of purified NADPH oxidase and which showed selectivity of binding. 1H8.2 is an IgM restricted in binding to pig NADPH oxidase and showing higher binding to NADPH oxidase purified from phorbol myristate acetate-stimulated than from resting neutrophils. The antibody inhibits by about 90% the oxidase activity at 20-50 micrograms/ml. Inhibition is due to a decrease of the Vmax of the oxidase, and the Km is not affected. Incubation of the NADPH oxidase with 1H8.2 in the presence of concentrations of NADPH up to 25-fold the Km does not prevent the inhibition. Together with the evidence that the antibody does not inhibit the neutrophil superoxide dismutase-insensitive NADPH cytochrome c reductase and the liver NADPH-cytochrome c reductase this observation indicates that the 1H8.2 does not bind to an epitope belonging to the NADPH-binding site. Experiments of immunoprecipitation of iodinated membrane proteins and of immunoaffinity purification showed that 1H8.2 recognizes a heterodimer of apparent molecular mass of 16/18 and 14 kDa. These polypeptides can be involved in the NADPH oxidase activity or represent still unrecognized molecules able to modulate its function.     

NADPH dehydrogenase activity of p67PHOX, a cytosolic subunit of the leukocyte NADPH oxidase

The leukocyte NADPH oxidase catalyzes the one-electron reduction of oxygen to O2- at the expense of NADPH. It is a multicomponent enzyme comprising a membrane-bound flavocytochrome (cytochrome b558) and at least four cytosolic components: p47PHOX, p67PHOX, p40PHOX, and Rac, a small GTPase. All the oxidase components except p40PHOX are required for enzyme activity. Many aspects of their function, however, are unclear. Using the electron acceptor ferricyanide, we found that recombinant p67PHOX from baculovirus-infected Sf9 cells could mediate the dehydrogenation of NADPH. NADPH dehydrogenation was not dependent on FAD and was insensitive to superoxide dismutase. Several control experiments showed that NADPH dehydrogenation was accomplished by p67PHOX, not by a trace contaminant in the p67PHOX preparation. The NADPH dehydrogenase activity of p67PHOX was proportional to enzyme concentration, and showed saturation kinetics with NADPH (Km 92 +/- 5 microM), but was inhibited at high concentrations of ferricyanide. NADH was also used as a substrate by p67PHOX (Km 123 +/- 38 microM). Taken together, these results show that p67PHOX is able to mediate pyridine nucleotide dehydrogenation. These findings raise the possibility that p67PHOX might participate directly in electron transfer between NADPH and the oxidase flavin.