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Oxalic acid and oxalates are secondary metabolites secreted to the surrounding environment by fungi, bacteria, and plants. Oxalates are linked to a variety of processes in soil, e.g. nutrient availability, weathering of minerals, or precipitation of metal oxalates. Oxalates are also mentioned among low-molecular weight compounds involved indirectly in the degradation of the lignocellulose complex by fungi, which are considered to be the most effective degraders of wood. The active regulation of the oxalic acid concentration is linked with enzymatic activities; hence, the biochemistry of microbial biosynthesis and degradation of oxalic acid has also been presented. The potential of microorganisms for oxalotrophy and the ability of microbial enzymes to degrade oxalates are important factors that can be used in the prevention of kidney stone, as a diagnostic tool for determination of oxalic acid content, as an antifungal factor against plant pathogenic fungi, or even in efforts to improve the quality of edible plants. The potential role of fungi and their interaction with bacteria in the oxalate-carbonate pathway are regarded as an effective way for the transfer of atmospheric carbon dioxide into calcium carbonate as a carbon reservoir. [ABSTRACT FROM AUTHOR]

A biosensor is considered an integrated receptor transducer device, with the ability to convert a biological impulse into an electrical signal. The amendment of biosensors has been recognized for its great potential by many researchers, due to its numerous applications e.g., environmental management, disease diagnosis, agricultural aspects, food companies, health care, drug monitoring, and water treatment as it can be used in the detection of water quality. Moreover, technological development of the biosensor is integrated with several merits such as affordability and enhancement in medical fields in disease detection and body response; furthermore, it is easy to use, effective, and scalable. This article briefly reviews how to construct an oxalic acid (OA) biosensor by integration of tissue enzymes and peroxide simulated enzymes. OA is converted to peroxide (H2O2) and carbon dioxide (CO2) with the help of the oxalate oxidase (OxOx) present in spinach leaves as catalyst. Afterwards, with the presence of cobalt ferrite (CoFe2O4), nanoparticles (NPs) have a catalytic effect on concentrated H2O2 and chemiluminescence (CL) luminol (C8H7N3O2). Therefore, CL flow can be constructed under a biosensor to determine OA in the sample. The co-presence of tissue column and CoFe2O4, as well as a high level of relative CL intensity can be obtained. The biosensor based on H2O2 and involving inorganic nanomaterials has many advantages such as high efficiency, affordability, outstanding sensitivity, stability and selectivity, a fast response, and an extended range of linearity with a lower detection limit. In addition, optimization factors for the oxalate biosensor, limitations, and outlooks for the biosensor were also highlighted. [ABSTRACT FROM AUTHOR]

Oxalate oxidase (EC 1.2.3.4) is an oxalate-decomposing enzyme predominantly found in plants but also described in basidiomycete fungi. In this study, we investigated 23 fungi to determine their capability of oxalic acid degradation. After analyzing their secretomes for the products of the oxalic acid-degrading enzyme activity, three groups were distinguished among the fungi studied. The first group comprised nine fungi classified as oxalate oxidase producers, as their secretome pattern revealed an increase in the hydrogen peroxide concentration, no formic acid, and a reduction in the oxalic acid content. The second group of fungi comprised eight fungi described as oxalate decarboxylase producers characterized by an increase in the formic acid level associated with a decrease in the oxalate content in their secretomes. In the secretomes of the third group of six fungi, no increase in formic acid or hydrogen peroxide contents was observed but a decline in the oxalate level was found. The intracellular activity of OXO in the mycelia of Schizophyllum commune, Trametes hirsuta, Gloeophyllum trabeum, Abortiporus biennis, Cerrena unicolor, Ceriosporopsis mediosetigera, Trametes sanguinea, Ceriporiopsis subvermispora, and Laetiporus sulphureus was confirmed by a spectrophotometric assay. [ABSTRACT FROM AUTHOR]

This study describes the development of an ethylene glycol (EG) biofuel cell that combines the organic catalyst 4‐amino‐TEMPO (TEMPO‐NH2) and the enzyme oxalate oxidase (OxOx) in solution for complete EG oxidation. Cyclic voltammetric curves recorded in the presence of EG showed that the hybrid TEMPO‐NH2/OxOx catalyst provided 1.2 higher current density than the TEMPO‐NH2 catalyst. Chronoamperometric assays after successive EG injections and long‐term electrolysis at a fixed potential of 0.75 V (vs Ag/AgCl) showed that the hybrid TEMPO‐NH2/OxOx catalyst in the presence of EG promoted 14 % and 58 % increase in the current obtained with the TEMPO‐NH2 catalyst, respectively. High‐performance liquid chromatography (HPLC) results confirmed that TEMPO‐NH2 and OxOx respectively acted on EG oxidation and C−C bond cleavage, leading to complete EG oxidation, harvesting all 10 of the EG electrons and high catalytic activity. [ABSTRACT FROM AUTHOR]

Oxalic acid (OA) is a crucial pathogenic factor for Sclerotinia spp. fungi, which is closely related to Botrytis spp. fungi. Whether OA is a pathogenic factor for the causal agent of grey mould in lily, Botrytis elliptica, and the response of lily to OA are poorly understood.To address these questions, lesion tissues and deposition of calcium oxalate (CaOX) and callose were observed in diploid and tetraploid leaves of L. rosthornii after inoculation with B. elliptica. Oxalate oxidase (OXO) activity and the transcript levels of some genes related to OA degradation (LrGLP1, LrGLP2 and LrWRKY4), reactive oxygen species (ROS) production/scavenging systems (LrRBOHD, LrGST, LrPOD and LrAPX1) and pathogen‐related protein (PR) synthesis (LrCHI, LrBGL and LrPR10) were compared.After diploid and tetraploid leaves inoculation, lesion tissue and callose and CaOX were separately observed around in guard cells and stomata rather than the epidermis in the infected area. OXO activity was triggered at 2 h post‐inoculation (hpi) in both ploidy leaves, and it was higher in the latter from 12‐48 hpi. Expression of LrGLP1, LrGLP2, LrRBOHD, LrGST, LrPOD, LrCHI, LrBGL and LrPR10 was higher in tetraploids than in diploids from 24(12)–36(48) hpi.In conclusion, for B. elliptica, OA mainly chelates Ca2+ from the stomata cell wall. The strong capability to degrade OA and higher expression levels of some genes related to ROS accumulation/scavenging and PR synthesis may partially explain the relatively higher grey mould resistance of tetraploid L. rosthornii. [ABSTRACT FROM AUTHOR]

Oxalate oxidase (OxO), a manganese dependent enzyme, is involved in the catalysis of oxalate oxidation to carbon dioxide with the formation of hydrogen peroxide. OxO is present in the cell wall of plants. It increases the resistivity against diseases and external stress. Oxalate is found as waste metabolites in mammals, excess accumulation of oxalate results in hyperoxaluria and urolithiasis in humans. These disorders can be diagnosed by the help of OxO in many analytical methods. The present study is aimed on isolation of OxO enzyme from Pseudomonas strain under high salt stress. The OxO enzyme was produced in bulk under selected salt stress. It was partially purified by ammonium sulphate precipitation method, dialysis and ion-exchange chromatography. The OxO enzyme on enzymatic analysis showed potent activity concerning oxalic acid substrate. The microtiter plate analysis confirmed the ability of OxO enzyme purified from Pseudomonas strain in the diagnosis of oxalate-related disorders and in the future could be used to prepare the diagnostic biosensors. [ABSTRACT FROM AUTHOR]

Taro (Colocasia esculenta) starch is known to possess unique physical and functional properties such as low amylose content, A-crystalline form, small granules, higher swelling power, etc. Due to the presence of significant amount of calcium oxalate crystals, the food industry is reluctant to explore this unique and cheap starch source for various food applications. Traditional processes utilizing various physical and chemical methods to remove oxalate content of starch inevitably change its physical and functional properties. However, using oxalate oxidase can effectively remove oxalates without altering the unique properties of starch. Hence, an attempt was made to optimize oxalate oxidase assisted starch extraction process from taro flour using response surface methodology. A central composite design comprising 20 experimental trials with 10 cube points augmented with six axial points and four replicates at the center point was applied. A mathematical model was developed to show the effect of taro flour concentration, enzyme load and incubation time on the oxalate removal. Validity of the model was experimentally verified and found that 98.3% of total oxalates can be removed under optimal conditions. This is the first report of optimization of the production of starch from taro flour using microbial oxalate oxidase. [ABSTRACT FROM AUTHOR]

Calcium oxalate (nephrolithiasis) is the most frequent type of kidney stone disease (KSD) affecting the urinary tract. In this study, the degradation and excretion of oxalate from body was carried out by utilizing oxalate oxidase producing bacterial strains. Bacterial strains were isolated from medical waste dumped soil and analyzed for its oxalate oxidase activity by using calcium oxalate, L-ascorbic acid, and oxalic acid and their ability to degrade the substrates was studied. Also, the optimized condition for the effective enzyme activity was found to be higher at pH - 5, temperature (40°C), substrates (Calcium oxalate & Oxalic acid), ZnO metal (0.01 mg/mL) and chelating agent (EDTA) of 0.01 mg/mL concentration. Further, ammonium sulphate precipitation of 50% saturation was performed and then partially purified by dialysis. The optimization for the partially purified enzyme activity was executed and the optimized conditions are temperature (400C), substrates (Oxalic acid), and pH (5). In vitro analysis of the kidney stone formation was tested for the partially purified enzyme and identified to be the potential source for the treatment of kidney stones. [ABSTRACT FROM AUTHOR]

Oxalate oxidase enzyme in monocotyledonous plants, act as a scavenger to breakdown oxalic acid into carbon dioxide and hydrogen peroxide. They perform several important functions in early plant defence responses, including induction of PR protein synthesis and systemic resistance. In the present study oxalic acid, the virulence factor of fungi, was applied to monocotyledonous plants and it was observed that it activates the innate host immune system, along with secretion of oxalate oxidase. The enzymatic activities of other host defence related enzymes like catalase, superoxide dismutase, ascorbic acid oxidase, phenolic content, phenylalanine ammonia lyase were compared between the control and oxalic acid treated (2mM) plants. [ABSTRACT FROM AUTHOR]

Four oxalate degrading endophytic bacteria were isolated from oxalate rich Colocasia esculenta tubers. Based upon the oxalate oxidase (EC 1.2.3.4) activity produced in nutrient medium, one bacterium was selected and identified as Ochrobactrum intermedium by 16S rDNA sequencing. Studies on effect of nutritional and non-nutritional parameters showed that oxalate oxidase production is inducible, requires Manganese ions in the medium, and very low fill-up volume is beneficial. Shake flask fermentation carried out with medium comprising Sucrose, Ammonium chloride, Sodium oxalate along with basal salts gave 0.5 UmL-1 oxalate oxidase activity and 0.454 Umg-1specific activity after 65h of fermentation. [ABSTRACT FROM AUTHOR]

Typha angustifolia L. (Typhaceae) is an emergent aquatic plant, and aerenchyma is formed through cell lysis in its leaves. The developing aerenchyma of T. angustifolia contains many CaOx crystals (raphides). Oxalate oxidase (OXO) (oxalate : oxygen oxidoreductase, EC1.2.3.4) can degrades calcium oxalate to carbon dioxide and hydrogen peroxide (H2O2). High level of H2O2 acts as a key inducer for different types of developmentally and environmentally programmed cell death (PCD) and can promote the formation of aerenchyma. Therefore, the objective of this study was to describe the relationship between aerenchyma formation and the degradation of CaOx crystals. Light and transmission electron microscopy (TEM) results showed that CaOx crystals occurred between PCD-susceptible cells in the early phase of aerenchyma formation, and those cells and CaOx crystals were degraded at aerenchyma maturation. Cytochemical localisation was used to detect H2O2, and H2O2 was found in crystal idioblasts. In addition, the oxalate content, H2O2 content and OXO activity were determined. The results showed that the concentration of oxalate was the highest in the third cavity formation stage and the H2O2 concentration was also highest at this stage. Meanwhile, the activity of OXO was also high in the third cavity formation stage. TpOXO was highly expressed during the CaOx crystal degradation period by quantitative real-time PCR analysis. These results show that the degradation of CaOx crystals is involved in the regulation of the PCD process of aerenchyma. This study will contribute to understanding the changes in CaOx crystals during the formation of aerenchyma in T. angustifolia. [ABSTRACT FROM AUTHOR]

Enzyme therapeutics have great potential for the treatment of systemic disorders such as urolithiasis and nephrocalcinosis, which are caused by the excessive accumulation of oxalate. However, exogenous enzymes have short half-lives in vivo and elicit high immunogenicity, which largely limit the therapeutic outcomes. Herein, we report a delivery strategy whereby therapeutic enzymes are encapsulated within a thin zwitterionic polymer shell to form enzyme nanocapsules. The strategy is exemplified by the encapsulation of oxalate oxidase (OxO) for the treatment of hyperoxaluria, because as-synthesized OxO nanocapsules have a prolonged blood circulation half-life and elicit reduced immunogenicity. Our design of enzyme nanocapsules that enable the systemic delivery of therapeutic enzymes can be extended to various biomedical applications. [ABSTRACT FROM AUTHOR]

Being sessile organisms, plants are continuously challenged by phytopathogenic fungi, contributing the largest share in loss due to plant disease. Plants naturally possess a well-developed and programmed protein-based defense system, capable of producing antimicrobial cationic peptides to ward off pathogen attack. Numerous genes encoding antifungal proteins have been isolated, cloned, sequenced and transgenically expressed against multiple phytopathogenic fungi successfully. Genetic engineering technology has been widely utilized to produce transgenic plants with enhanced resistance against pathogens. Pathogenesis-related proteins (PR-proteins) is a group of the most important inducible defense-related antifungal proteins, including defensins, thionins, osomtin-like proteins, thaumatin-like proteins, chitinases, glucanases, oxalate oxidase or oxalate oxidase-like proteins and lipid transfer proteins. Transgenic plants have been developed by imparting the artificial expression of genes encoding antifungal PR-proteins. The expression of transgenes belonging to a single group of PR-proteins or synergistic action of transgenes from different groups has greatly uplifted the level of defense response in plants against fungi. Transgenic expression of antifungal PR-proteins has led to remarkably enhanced resistance in transgenic plants. In this review, we have summarized the role of PR-proteins in plant defense against fungi and 15 years of success achieved so far to generate a variety of transgenic plants resistant against fungi through overexpression of transgenes from different groups of PR-proteins. [ABSTRACT FROM AUTHOR]

Kochia sieversiana (Pall.) C.A. Mey. is a forage plant that can grow in extremely alkalinized grasslands at pH 10 or higher. Accumulation of a large amount of oxalic acid (OxA) is a primary characteristic of K. sieversiana. In our study, seedlings of K. sieversiana were exposed to the following conditions: non-stress, salinity (200 mM, a molar ratio of NaCl and NaSO 1:1), and alkali stress (200 mM, a molar ratio of NaHCO and NaCO 1:1). Growth, water content, content of organic acids (including OxA), Na, and K, and activities of some OxA metabolism-related enzymes were determined. Results show that glycolate oxidase was the key enzyme for OxA synthesis; however, the carboxylation of phospho enolpyruvate (PEP) by PEP carboxylase (PEPC) probably played a minor role in the OxA-synthetic pathway. The pathway of L-ascorbic acid catabolism was not the main source of OxA accumulation, and the activity of oxalate oxidase (OxO) involved in OxA decomposition was not a limiting factor for inner OxA accumulation. Taken together, accumulation of a large amount of OxA are not related to the degradation and secretion function of OxO but largely depend upon its synthetic function. [ABSTRACT FROM AUTHOR]

Oxalate oxidase (EC 1.2.3.4) catalyzes the oxidative cleavage of oxalate to carbon dioxide with the reduction of molecular oxygen to hydrogen peroxide. Oxalate oxidase found its application in clinical assay for oxalate in blood and urine. This study describes the purification and biochemical characterization of an oxalate oxidase produced from an endophytic bacterium, Ochrobactrum intermedium CL6. The cell-free fermentation broth was subjected to two-step enzyme purification, which resulted in a 58.74-fold purification with 83% recovery. Specific activity of the final purified enzyme was 26.78 U mg−1 protein. The enzyme displayed an optimum pH and temperature of 3.8 and 80°C, respectively, and high stability at 4–80°C for 6 h. The enzymatic activity was not influenced by metal ions and chemical agents (K+, Na+, Zn2+, Fe3+, Mn2+, Mg2+, glucose, urea, lactate) commonly found in serum and urine, with Cu2+ being the exception. The enzyme appears to be a metalloprotein stimulated by Ca2+ and Fe2+. Its Km and Kcat for oxalate were found to be 0.45 mM and 85 s−1, respectively. This enzyme is the only known oxalate oxidase which did not show substrate inhibition up to a substrate concentration of 50 mM. Thermostability, kinetic properties, and the absence of substrate inhibition make this enzyme an ideal candidate for clinical applications. [ABSTRACT FROM AUTHOR]

The success of the necrotrophic fungus Sclerotinia sclerotiorum is largely dependent on its major virulence factor, oxalic acid ( OA). Virulence is lost in transgenic plants that express OA degrading enzymes, e.g. oxalate oxidase (OxO). The histopathology of S. sclerotiorum infection and OA accumulation was examined in a transgenic soybean line over-expressing OxO (OxO- OE) and its isogenic parent ( WT). In situ flower inoculation showed that the OxO- OE plants were highly resistant to the pathogen while the WT parents were susceptible. This difference in resistance was not apparent in the floral tissues, as aggressive hyphal activity was similar on both hosts, showing that high OxO activity and low OA accumulation in OxO- OE was not a deterrent. However, the process of fungal infection on excised leaf tissue differed on the two hosts. Primary lesions developed and showed similar severe ultrastructural damage on both hosts but rapid lesion expansion (colonization) proceeded only on the WT, concomitant with OA accumulation. Oxalic acid rose in OxO- OE 1 day post-inoculation and did not change over the following 3 days, showing that colonization can be blocked by maintaining low levels of OA. However, OxO degradation of OA did not deter initial host penetration and primary lesion formation. This shows that OA, the major virulence factor of S. sclerotiorum, is critical for host colonization but may not be required during primary lesion formation, suggesting that other factors are contributing to the establishment of the primary lesion. [ABSTRACT FROM AUTHOR]

Field trials were conducted from 2006 to 2008 at the Tidewater Agricultural Research and Extension Center (TAREC) in Suffolk, Virginia to determine whether Blight Blocker transgenic peanut lines showed possible increased or decreased susceptibility to common peanut pathogens. Disease susceptibility was evaluated for seven transgenic lines containing a barley oxalate oxidase gene and their corresponding parental cultivars (Perry, Wilson, NC 7). In addition to Sclerotinia blight, the peanut diseases evaluated included: i) early leaf spot caused by Cercospora arachidicola, ii) Cylindrocladium black rot caused by Cylindrocladium parasiticum, iii) southern stem rot caused by Sclerotium rolfsii, iv) tomato spotted wilt virus, and v) aflatoxin levels in seeds caused by Aspergillus flavus or A. parasiticus. Results demonstrated that the susceptibility of Blight Blocker transgenic lines to common peanut pathogens was similar to that of non-transgenic cultivars, while transgenic lines provided resistance to Sclerotinia blight caused by S. minor. Transgenic lines consistently provided high levels of resistance to S. minor in all three years, however, the barley oxalate oxidase had little or no effect on the disease susceptibility to other organisms on peanut. The results of this research should provide additional evidence needed to petition for deregulation of Blight Blocker peanut lines. [ABSTRACT FROM AUTHOR]

Renal urolithiasis is a pathological condition common to a multitude of genetic, physiological and nutritional disorders, ranging from general hyperoxaluria to obesity. The concept of quickly dissolving renal uroliths via chemolysis, especially calcium-oxalate kidney stones, has long been a clinical goal, but yet to be achieved. Over the past 25 years, there has been a serious effort to examine the prospects of using plant and microbial oxalate-degrading enzymes known to catabolize oxalic acid and oxalate salts. While evidence is emerging that bacterial probiotics can reduce recurrent calcium-oxalate kidney stone disease by lowering systemic hyperoxaluria, the possible use of free oxalate-degrading enzyme therapy remains a challenge with several hurdles to overcome before reaching clinical practice. [ABSTRACT FROM AUTHOR]

Oxalate oxidase (OxOx), a well known enzyme catalyzes the cleavage of oxalate to carbon dioxide with reduction of dioxygen to hydrogen peroxide, however its catalytic process is not well understood. To define the substrate binding site, interaction of Fe ions with OxOx was systemically investigated using biochemical method, circular dichrosim spectroscopy, microscale thermophoresis, and computer modeling. We demonstrated that Fe is a non-competitive inhibitor with a milder binding affinity to OxOx, and the secondary structure of the OxOx was slightly altered upon its binding. On the basis of the structural properties of the OxOx and its interaction with Fe ions, two residue clusters of OxOx were assigned as potential Fe binding sites, the mechanism of the inhibition of Fe was delineated. Importantly, the residues that interact with Fe ions are involved in the substrate orienting based on computer docking. Consequently, the interaction of OxOx with Fe highlights insight into substrate binding site in OxOx. [ABSTRACT FROM AUTHOR]

Germin and germin-like proteins (GLPs) are a broad and diverse family of developmentally regulated proteins widely distributed in plants. Oryza sativa L. harbours a large family of GLPs and serves as a good model for their study. In the present study, a germin-like protein gene ( OsRGLP1) of rice origin was characterized by its heterologous expression in tobacco. The real-time PCR established almost a uniform expression of OsRGLP1 in leaves, stem, and roots of T Nicotiana tabacum cv. Samsun. Although no morphological difference was apparent between T transgenic and wild-type plants, leaves of mature transgenic plants showed necrotic lesions associated with an elevated content of HO, which was evidenced by in situ 3,3′-diaminobenzidine staining. A significantly higher activity of heat resistant superoxide dismutase (SOD) was observed in the transgenic plants as compared to the wild-type. The SOD activity in the transgenic plants was insensitive to potassium cyanide and sensitive to HO. [ABSTRACT FROM AUTHOR]

Previous work on the adaptation of maize (Zea mays L.) primary root growth to water stress showed that cell elongation is maintained in the apical region of the growth zone but progressively inhibited further from the apex. Cell wall proteomic analysis suggested that levels of apoplastic reactive oxygen species (ROS), particularly hydrogen peroxide (H2O2), may be modified in a region-specific manner within the growth zone of water-stressed roots. Apoplastic ROS may have wall loosening or tightening effects and may also have other growth regulatory functions. To gain an understanding of how apoplastic ROS levels change under water stress, cerium chloride staining was used in conjunction with transmission electron microscopy to examine the spatial distribution of apoplastic H2O2. The results revealed that apoplastic H2O2 levels increased specifically in the apical region of the growth zone under water stress, correlating spatially with the maintenance of cell elongation. The basal regions of the growth zone of water-stressed roots and the entire growth zone of well-watered roots exhibited relatively low levels of apoplastic H2O2. The increase in apoplastic H2O2 in the apical region under water stress probably resulted, at least in part, from a pronounced increase in oxalate oxidase activity in this region. By contrast, well-watered roots showed negligible oxalate oxidase activity throughout the growth zone. The results show that changes in apoplastic ROS levels in the root growth zone under water-deficit conditions are regulated in a spatially-specific manner, suggesting that this response may play an important role in maize root adaptation to water stress. [ABSTRACT FROM AUTHOR]

Barley oxalate oxidase (OxO), a manganese-containing protein, is largely employed for determination of oxalate in various biologic materials. The present report describes in vitro effects of nanoparticles (NPs) of three metal oxides, i.e., zinc oxide (ZnO), copper oxide (CuO), and manganese oxide (MnO), on the activity and stability of OxO purified from barley roots. The transmission electron microscopy and X-ray diffraction studies of these NPs revealed their very fine crystalline structure with the dimeter in the range 30-70, 50-60, and 20-60 nm for ZnO NPs, CuO NPs, and MnO NPs, respectively. The addition of suspension of these three NPs into assay mixture of enzyme individually, led to the adsorption of OxO over their surface, as confirmed by Fourier transform infrared spectra and UV-Vis spectroscopic studies. Compared to free enzyme, MnO NPs-bound enzyme showed improved activity (35 % stimulation at 2.5 mg/ml concentration), while ZnO NPs- and CuO NPs-bound enzyme had no substantial improvement. The kinetic properties of individually NPs-bound enzyme were studied and compared with those of free enzyme. The MnO NPs-bound enzyme also showed marked improvement in its storage and thermal stability compared to free enzyme. [ABSTRACT FROM AUTHOR]

Oxalic acid plays major role in the pathogenesis by Sclerotinia sclerotiorum; it lowers the pH of nearby environment and creates the favorable condition for the infection. In this study we examined the degradation of oxalic acid through oxalate oxidase and biocontrol of Sclerotinia sclerotiorum. A survey was conducted to collect the rhizospheric soil samples from Indo-Gangetic Plains of India to isolate the efficient fungal strains able to tolerate oxalic acid. A total of 120 fungal strains were isolated from root adhering soils of different vegetable crops. Out of 120 strains a total of 80 isolates were able to grow at 10 mM of oxalic acid whereas only 15 isolates were grow at 50 mM of oxalic acid concentration. Then we examined the antagonistic activity of the 15 isolates against Sclerotinia sclerotiorum. These strains potentially inhibit the growth of the test pathogen. A total of three potential strains and two standard cultures of fungi were tested for the oxalate oxidase activity. Strains S7 showed the maximum degradation of oxalic acid (23 %) after 60 min of incubation with fungal extract having oxalate oxidase activity. Microscopic observation and ITS (internally transcribed spacers) sequencing categorized the potential fungal strains into the Aspergillus, Fusarium and Trichoderma. Trichoderma sp. are well studied biocontrol agent and interestingly we also found the oxalate oxidase type activity in these strains which further strengthens the potentiality of these biocontrol agents. [ABSTRACT FROM AUTHOR]

This study describes the influence of copper nanoparticles (CNPs) on activity and kinetic properties of a Cu2+ requiring sorghum oxalate oxidase (OxO). CNPs were synthesised by citrate-induced reduction of CuCl2 and their size (range 13–58 nm) was determined by transmission electron microscopy. The CNPs were characterised by scanning electron microscopy and X-ray diffraction studies. These CNPs enhanced OxO activity by 30%. Thermal and storage stability were increased, while Km value for oxalate decreased in the presence of CNPs. CNPs protected OxO against chelation by diethyldithiocarbamate, a Cu2+ specific chelator. The analytic use of OxO in the presence of CNP for determination of oxalate in food stuff is demonstrated. [ABSTRACT FROM AUTHOR]

American chestnut [Castanea dentata (Marshall) Borkhausen], a canopy dominant species across wide swaths of eastern North America, was reduced to an understory shrub after introduction of the blight fungus [Cryphonectria parasitica (Murrill) Barr] in the early 1900s. Restoration of American chestnut by using bioteehnology is promising, but the imprecise nature of transgenesis may inadvertently alter tree phenotype, thus potentially impacting ecologically dependent organisms. We quantified effects of genetic engineering and fungal inoculation of trees on insect herbivores by using transgenic American chestnuts expressing an oxalate oxidase gene and wild-type American and Chinese (C. mollissima Blume) chestnuts. Of three generalist folivores bioassayed, only gypsy moth [Lymantvia dispar (L.)] was affected by genetic modification, exhibiting faster growth on transgenic than on wild-type chestnuts, whereas growth of polyphemus moth [Antheraea polyphemus (Cramer)] differed between wild-type species, and fall webworm [Hyphantria cunea (Drury)] performed equally on all trees. Inoculation of chestnuts with blight fungus had no effect on the growth of two herbivores assayed (polyphemus moth and fall webworm). Enhanced fitness of gypsy moth on genetically modified trees may hinder restoration efforts if this invasive herbivore's growth is improved because of transgene expression. [ABSTRACT FROM AUTHOR]

Germin-like proteins (GLPs) play diversified roles in plant development and defense response. Here, we identified 36 expressed sequence tags (ESTs) encoding GLPs from peanut ( Arachis hypogaea L.). After assembly, these ESTs were integrated into eight unigenes named AhGLP1 to AhGLP8, of which, three ( AhGLP1-3) were comprised 14, ten, and seven EST clones, respectively, whereas the remaining ones were associated with one single clone. The length of the deduced amino acid (AA) residues ranged from 208 to 223 AAs except for AhGLP6 and AhGLP8, which were incomplete at the carboxyl terminus. All of the AhGLPs contained a possible N-terminal signal peptide that was 17 to 24 residues in length excluding AhGLP7, where there is likely a non-cleavable amino terminus. Phylogenetic analysis showed that these AhGLPs were classified into three subfamilies. Southern blot analysis indicated that AhGLP1 and AhGLP2 likely have multiple copies in the peanut genome. The recombinant mature AhGLP1 and AhGLP2 proteins were successfully expressed in Escherichia coli. The purified AhGLP2 has superoxide dismutase (SOD) activity in enzymatic assay, but not oxalate oxidase activity. The SOD activity of AhGLP2 was stable up to 70°C and resistant to hydrogen peroxide, suggesting that AhGLP2 might be a manganese-containing SOD. Furthermore, AhGLP2 could confer E. coli resistance to oxidative damage caused by paraquat, suggesting that the AhGLP2 likely protects peanut plants from reactive oxygen metabolites. Thus, information provided in this study indicates the diverse nature of the peanut GLP family and suggests that some of AhGLPs might be involved in plant defense response. [ABSTRACT FROM AUTHOR]

Background: Germin and germin-like proteins constitute a ubiquitous family of plant proteins. A role of some family members in defense against pathogen attack had been proposed based on gene regulation studies and transgenic approaches. Soybean (G. max L. Merr.) germin genes had not been characterized at the molecular and functional levels. Results: In the present study, twenty-one germin gene members in soybean cultivar 'Maple Arrow' (partial resistance to Sclerotinia stem rot of soybean) were identified by in silico identification and RACE method (GmGER 1 to GmGER 21). A genome-wide analyses of these germin-like protein genes using a bioinformatics approach showed that the genes located on chromosomes 8, 1, 15, 20, 16, 19, 7, 3 and 10, on which more disease-resistant genes were located on. Sequence comparison revealed that the genes encoded three germin-like domains. The phylogenetic relationships and functional diversity of the germin gene family of soybean were analyzed among diverse genera. The expression of the GmGER genes treated with exogenous IAA suggested that GmGER genes might be regulated by auxin. Transgenic tobacco that expressed the GmGER 9 gene exhibited high tolerance to the salt stress. In addition, the GmGER mRNA increased transiently at darkness and peaked at a time that corresponded approximately to the critical night length. The mRNA did not accumulate significantly under the constant light condition, and did not change greatly under the SD and LD treatments. Conclusions: This study provides a complex overview of the GmGER genes in soybean. Phylogenetic analysis suggested that the germin and germin-like genes of the plant species that had been founded might be evolved by independent gene duplication events. The experiment indicated that germin genes exhibited diverse expression patterns during soybean development. The different time courses of the mRNAs accumulation of GmGER genes in soybean leaves appeared to have a regular photoperiodic reaction in darkness. Also the GmGER genes were proved to response to abiotic stress (such as auxin and salt), suggesting that these paralogous genes were likely involved in complex biological processes in soybean. [ABSTRACT FROM AUTHOR]

An amperometric oxalate biosensor was developed and optimized by immobilizing a Cl- and [image omitted] insensitive oxalate oxidase purified from sorghum leaves, on carbon paste electrode. Enzymatically produced H2O2 from oxalate was split into 2H+ + O2 + 2e- at 60 mV and flow of electron (current) was measured. The biosensor showed optimum response at pH 4.5, 35°C and within 45 sec. The current measured (mA) by biosensor was linear for oxalate up to 20 µg/ml. The biosensor was employed for determination of oxalate in urine (from both apparently healthy individuals and urinary stone formers) and different brands of beer. The percent recoveries of added oxalate in urine of healthy and stone formers were 96.6% and 98.2%, respectively. Within and between batch coefficients of variation (CV) for oxalate determination were <7.5% and <8.5% in urine of healthy persons and <5.0% and <6.5% in the urine of stone formers, respectively. There was a good correlation (r = 0.91) between urinary oxalate values by present method and the most commonly used enzymatic colorimetric method. The enzyme electrode was used for 150-times during the span of 35 days, when stored in a reaction buffer at 4°C. [ABSTRACT FROM AUTHOR]

The effect of cadmium (Cd) on the expression and activity of NADPH oxidase, peroxidase and oxalate oxidase as well as on the expression of aquaporins and dehydrins was studied in barley root tip. The root tip represented intact apical part of the barley root containing the root cap, meristems and elongation zone. Except stress induced by Cd, barley root tips were analysed after their exposure to phytotoxic concentration of mercury (Hg)-, hydrogen peroxide (H2O2)- or polyethylene glycol (PEG)-induced water stress in order to compare the Cd-induced changes with changes induced by these other stress factors. Cd, Hg, H2O2 and with some exceptions also PEG treatments caused similar alterations in the gene expression of reactive oxygen species (ROS)-generating and water deficiency-related genes, and in the activity of ROS-generating enzymes. These evidences support our opinion that ROS accumulation and water imbalance are the common symptoms of these stress factors and that the elevated production of H2O2 plays, probably as a signal molecule, a key role in the induction of plant responses to abiotic stresses in barley root tip. On the other hand, H2O2 at permanent high concentration is probably the main toxic factor during stress conditions. [ABSTRACT FROM AUTHOR]

Oxalate oxidase (OxO, EC 1.2.3.4.) was purified to homogeneity from wheat ( Triticum aestivum) seedlings by sequential thermal treatment, ultrafiltration, Sephadex G-100 gel filtration and affinity chromatography with concanavalin A. The enzyme was purified 66.11-fold with a recovery of 21.97%. It showed a subunit molecular mass of 32.6 kDa on SDS-PAGE and a native molecular mass of 170 kDa on Sephadex G-150 filtration, suggesting that it is a pentamer. The wheat OxO had a maximum activity at pH 3.5. Its K m for oxalate was 0.21 mM. Chemical modification revealed that cysteine, lysine and carboxylate residues were essential for OxO activity, whereas arginine, serine, threonine and tryptophane residues were not essential. [ABSTRACT FROM AUTHOR]

Rice oxalate oxidase genes (OXO) may play a role in resistance to Magnaporthe oryzae. Genome analyses showed four tandemly duplicated OXO genes, OsOXO1– OsOXO4, which mapped to a blast resistance QTL in chromosome 3. These genes have >90% nucleotide and amino acid identity, but they have unique gene structures, conserved motifs, and phylogeny compared to the 70 other members of the cupin superfamily in the Nipponbare genome, which were divided into several classes. In resistant and susceptible Vandana/Moroberekan advanced backcross lines, only OsOXO4 was expressed during rice– M. oryzae interactions, and its expression increased earlier in resistant than susceptible lines. The earlier expression of OsOXO4 in resistant lines correlated with a 26-bp promoter insertion containing an additional copy of the bacterial responsive nodulation cis-element. Our results showed that OsOXO1– 4 are in a separate class of rice cupin genes and supports a role for the promoter variant of OsOXO4 in resistance to M. oryzae. [ABSTRACT FROM AUTHOR]

Germin and germin-like proteins (GLPs) are encoded by a family of genes found in all plants. They are part of the cupin superfamily of biochemically diverse proteins, a superfamily that has a conserved tertiary structure, though with limited similarity in primary sequence. The subgroups of GLPs have different enzyme functions that include the two hydrogen peroxide-generating enzymes, oxalate oxidase (OxO) and superoxide dismutase. This review summarizes the sequence and structural details of GLPs and also discusses their evolutionary progression, particularly their amplification in gene number during the evolution of the land plants. In terms of function, the GLPs are known to be differentially expressed during specific periods of plant growth and development, a pattern of evolutionary subfunctionalization. They are also implicated in the response of plants to biotic (viruses, bacteria, mycorrhizae, fungi, insects, nematodes, and parasitic plants) and abiotic (salt, heat/cold, drought, nutrient, and metal) stress. Most detailed data come from studies of fungal pathogenesis in cereals. This involvement with the protection of plants from environmental stress of various types has led to numerous plant breeding studies that have found links between GLPs and QTLs for disease and stress resistance. In addition the OxO enzyme has considerable commercial significance, based principally on its use in the medical diagnosis of oxalate concentration in plasma and urine. Finally, this review provides information on the nutritional importance of these proteins in the human diet, as several members are known to be allergenic, a feature related to their thermal stability and evolutionary connection to the seed storage proteins, also members of the cupin superfamily. [ABSTRACT FROM AUTHOR]

Sclerotinia sclerotiorum causes a highly destructive disease in oilseed rape ( Brassica napus). Oxalic acid (OA) secreted by the pathogen is a key pathogenicity factor. Oxalate oxidase (OXO) can oxidize OA into CO2 and H2O2. In this study, we show that transgenic oilseed rape (sixth generation lines) constitutively expressing wheat ( Triticum aestivum) OXO displays considerably increased OXO activity and enhanced resistance to S. sclerotiorum (with up to 90.2 and 88.4% disease reductions compared with the untransformed parent line and a resistant control, respectively). Upon application of exogenous OA, the pH values in transgenic plants were maintained at levels slightly lower than 5.58 measured prior to OA treatment, whereas the pH values in untransformed plants decreased rapidly and were markedly lower than 5.63 measured prior to OA treatment. Following pathogen inoculation, H2O2 levels were higher in transgenic plants than in untransformed plants. These results indicate that the enhanced resistance of the OXO transgenic oilseed rape to Sclerotinia is probably mediated by OA detoxification. We believe that enhancing the OA metabolism of oilseed rape in this way will be an effective strategy for improving resistance to S. sclerotiorum. [ABSTRACT FROM AUTHOR]

The authors have proposed previously that intracellular degradation of oxalic acid via formate to CO2 occurs in the white-rot fungus Ceriporiopsis subvermispora. The formate degradation is catalyzed by NAD-dependent formate dehydrogenase (CsFDH). In this study, two cDNAs named CsFDH1 and CsFDH2 encoding CsFDH were cloned. Each cDNA consisting of 1077 bp encodes a mature protein composed of 358 amino acid residues. The amino acid sequences of the deduced CsFDH1 and CsFDH2 showed 99% identity to each other. The predicted molecular mass for each was 39.3 kDa, which was similar to that of CsFDH purified from the vegetative mycelia of Ceriporiopsis subvermispora (purified-CsFDH). The recombinant CsFDH1 and CsFDH2 expressed by Escherichia coli showed FDH activity with similar characteristics to the purified CsFDH. However, the amount of CsFDH1 transcript from the vegetative mycelia was 236–691 times greater than that of CsFDH2. Therefore, the results strongly suggest that CsFDH1, as compared with CsFDH2, predominantly contributes to the production of the purified CsFDH. [ABSTRACT FROM AUTHOR]

A reusable strip of oxalate oxidase was prepared by immobilizing covalently partially purified amaranthus leaf oxalate oxidase on to alkyl amine glass beads affixed on a plastic strip. The immobilized enzyme gave a conjugation yield of 48 mg/g support with 87% retention of initial activity of free enzyme. The strip showed maximum activity at pH 3.5 when incubated at 40° for 15 min. A method for discrete analysis of oxalate in urine was developed employing this enzyme strip. The method is based on measurement of hydrogen peroxide generated from urinary/plasma oxalate by strip bound oxalate oxidase using a colour reaction consisting of 4-aminophenazone, phenol and horseradish peroxidase as chomogenic system. The minimum detection limit of the method was 0.1 mM. The recovery of added oxalate was 96.5% in plasma and 98% in urine. Within and between assay coefficient of variation were <6% and <5%, respectively in plasma and urine. The method provides enormous ease in handling of immobilized enzyme during its reuse and is unaffected by chloride ions found in biological fluids. [ABSTRACT FROM AUTHOR]

The cDNAs encoding three germin-like proteins (PsGER1, PsGER2a, and PsGER2b) were isolated from Pisum sativum. The coding sequence of PsGER1 transiently expressed in tobacco leaves gave a protein with superoxide dismutase activity but no detectable oxalate oxidase activity according to in-gel activity stains. The transient expression of wheat germin gf-2.8 oxalate oxidase showed oxalate oxidase but no superoxide dismutase activity under the same conditions. The superoxide dismutase activity of PsGER1 was resistant to high temperature, denaturation by detergent, and high concentrations of hydrogen peroxide. In salt-stressed pea roots, a heat-resistant superoxide dismutase activity was observed with an electrophoretic mobility similar to that of the PsGER1 protein, but this activity was below the detection limit in non-stressed or H2O2-stressed pea roots. Oxalate oxidase activity was not detected in either pea roots or nodules. Following in situ hybridization in developing pea nodules, PsGER1 transcript was detected in expanding cells just proximal to the meristematic zone and also in the epidermis, but to a lesser extent. PsGER1 is the first known germin-like protein with superoxide dismutase activity to be associated with nodules. It shared protein sequence identity with the N-terminal sequence of a putative plant receptor for rhicadhesin, a bacterial attachment protein. However, its primary location in nodules suggests functional roles other than as a rhicadhesin receptor required for the first stage of bacterial attachment to root hairs. [ABSTRACT FROM PUBLISHER]

In vitro and in vivo responses of transgenic and non-transgenic potato (Solanum tuberosum L.) genotypes to salinity were investigated. A gene responsible for the production of oxalate oxidase enzyme, whose expression has been associated with plant stress tolerance, was used. Transgenic potato plants expressing the oxalate oxidase enzyme were produced using Agrobacterium-mediated transformation. The transgenic nature of the plants was verified by their antibiotic (kanamycin) resistance and then in vitro enzyme assays. Four independent transgenic genotypes obtained from 2 cultivars, Maris Bard and Desiree, were tested at 5 levels of NaCl (0%, 0.25%, 0.50%, 0.75% and 1.00% w/v) in vitro. The same genotypes were also tested at 3 levels of NaCl (0%, 0.50% and 0.75% w/v) under glasshouse conditions (in vivo). The results revealed that the growth of all genotypes was significantly inhibited by increasing salt treatments under both in vitro and in vivo conditions. Transgenic genotypes showed a relatively higher salinity tolerance than the non-transgenic genotypes in vitro. On the other hand, in the glasshouse, the results were less consistent but some transgenic genotypes showed superior tuber yield characteristics to the non-transgenics under salt stress. [ABSTRACT FROM AUTHOR]

Sustainable ecosystems can be designed to eliminate environmental toxins and reduce pathogen loads through the direct and indirect consequences of plant and microbial activities. We present an approach to the bioremediation of disturbed environments, focusing on petroleum hydrocarbon (PHC) contaminants. Treatment consists of incorporating a plant-based amendment to enhance ecosystem productivity and physiochemical degradation followed by the establishment of plants to serve as oxidizers and foundations for microbial communities. Promising amendments for widespread use are entire plants of the water fern Azolla and seed meal of Brassica napus (rapeseed). An inexpensive byproduct from the manufacture of biodiesel and lubricants, rapeseed meal is high in nitrogen (6% wt/wt), stimulates >100-fold increases in populations of resident Streptomyces species, and suppresses fungal infection of roots subsequently cultivated in the amended soil. Synergistic enzymatic and chemical activities of plant and microbial metabolism in root zones transform and degrade soil contaminants. Emphasis is given to mechanisms that enable PHC functionalization via reactive molecular species. [ABSTRACT FROM AUTHOR]

The impact of aluminium stress on activities of enzymes of the oxidative metabolism: superoxide dismutase (SOD), ascorbate peroxidase (APX), peroxidase (POD), NADH peroxidase (NADH-POD) and oxalate oxidase (OXO) was studied in barley ( Hordeum vulgare L. cv. Alfor) root tips. SOD appeared to be involved in detoxification mechanisms at highly toxic Al doses and after long Al exposure. POD and APX, H2O2 consuming enzymes, were activated following similar patterns of expression and exhibiting significant correlation between their elevated activities and root growth inhibition. The signalling role of NADH-POD in oxidative stress seems to be more probable than that of OXO, which might be involved in Al toxicity mechanism. [ABSTRACT FROM AUTHOR]

Two bursts of H2O2 production have been detected by in situ 3,3′-diaminobenzidine (DAB) staining after cutting of Lolium perenne L. leaf blades. The first burst, which occurred immediately after wounding was inhibited by Na-diethydithiocarbamate (DIECA), a Cu/Zn–superoxide dismutase (SOD) inhibitor. The second burst, which was initiated several hours later, coincided with the induction of oxalate oxidase (G-OXO) activity detected in vitro or visualized in situ by the α-chloronaphtol assay. Four genes encoding G-OXO have been identified from cDNA obtained from wounded L. perenne L . leaf blades. Comparison of protein sequences revealed more than 91% homology in the coding region between G-OXOs of the true cereals and G-OXOs of ryegrass, which is a Gramineae belonging to the tribe of Festucaceae. The wound-dependent increase of G-OXO activity in floated cut leaf blades was the result of differential induction of the four g-oxo genes. The involvement of G-OXOs in wound-induced H2O2 production coincided with the presence in leaf tissues of oxalate throughout the period of increase of G-OXO synthesis. Moreover, expression of g-oxo genes was enhanced by an exogenous supply of H2O2 or methyljasmonate (MeJa). Expression of the four g-oxo genes was also induced after in planta stinging of leaf blades. The pattern of their expression in planta was identical to that occuring in senescing leaf sheaths. These results emphasize the importance of G-OXOs in H2O2 production in oxalate-producing plant species such as ryegrass. G-OXOs might be crucial during critical events in the life of plants such as cutting and senescence by initiating H2O2-mediated defences against pathogens and foraging animals. [ABSTRACT FROM AUTHOR]

A wheat germin gene, with oxalate oxidase (OxO) activity, can be used as a sensitive reporter gene in both monocot and dicot transformations. Detection of H2O2 generated from OxO oxidation of oxalate provides simple, rapid detection of gene expression. Inexpensive substrates are required for both assays. OxO activity, could be detected histochemically in minutes, without chlorophyll clearing procedures. This assay was used to optimize transformation procedures and to track stable transgene expression in breeding populations over many generations. A simple spectrophotometric quantitative enzyme activity assay was used to select lines with various levels of transgene expression and to monitor transgene silencing phenomena. The quantitative OxO assay can also be used as an internal DNA delivery standard with a second reporter gene used in gene expression studies. The simplicity of the assay is ideal for screening large populations to identify primary transgenics, for monitoring transgene segregation in large populations in field studies and for assessing stability of transgene expression over numerous generations. [ABSTRACT FROM AUTHOR]

A rice cDNA library was screened using OSK3 protein kinase as bait in a yeast two-hybrid system. The gene encoding oxalate oxidase was one of the positive clones interacting with OSK3 protein kinase. The interactions were verified by detecting expression of the reporter gene lacZ. The results suggest that oxalate oxidase is a downstream element in the disease resistance signal cascade mediated by OSK3 protein kinase in rice. [ABSTRACT FROM PUBLISHER]

Potato (cv. Bintje) was transformed with a gene encoding an oxalate oxidase from wheat under the control of the CaMV35S promoter. Transgenic potato plants produced high constitutive levels of H2O2 as visualized by 4-chloro-l-naphtol staining. The resistance of these plants was tested against Phytophthora infestans. An increased level of resistance to the disease was marked by a reduced number of lesions as well as by a decreased number of sporangia formed per lesion. In addition, oxalate oxidase overexpressing plants also exhibited improved resistance to Streptomyces reticuliscabiei, the causal agent of netted scab. Increased expression of oxalate oxidase had no effect on the interaction with Erwinia carotovora. These experiments show that overexpression of oxalate oxidase represents a potentially interesting approach for protection of potato to pathogens. [ABSTRACT FROM AUTHOR]

A method for determination of oxidation of phenolic compounds by intact wheat seedlings using o-phenylenediamine (OPD) was developed. The reaction is initiated by the addition of oxalic acid to the incubation medium. It is suggested that an endogenous peroxidase and hydrogen peroxide formed during oxidation of oxalic acid by endogenous oxalate oxidase are involved in OPD oxidation. Treatment of plants with chitooligosaccharides (1-10 mg/liter) with acetylation degree of 65% and molecular masses of 5-10 kD significantly activated OPD oxidation by wheat seedlings. [ABSTRACT FROM AUTHOR]

Germin-like proteins (GLPs) are ubiquitous plant proteins encoded by diverse multigene families. It is not known whether they share germin's unusual biochemical properties and oxalate oxidase activity. Using specific antibodies, we have studied three GLPs (AtGER1, AtGER2 and AtGER3) in Arabidopsis thaliana (L.) Heynh. as well as in transgenic tobacco (Nicotiana tabacum L.) plants overexpressing these proteins. Like wheat (Triticum aestivum L.) germin, these Arabidopsis GLPs are associated with the extracellular matrix (ECM) and they also seem to exist as two glycosylated isoforms. However, none of them is an oxalate oxidase. Although GLPs display several conserved features, each has its specific characteristics. Both AtGER2 and AtGER3 are oligomeric proteins that share germin's resistance to pepsin and to dissociation by heat and SDS. In contrast, AtGER1 seems to exist as a monomer. The GLPs may interact with the ECM in a variety of ways, since each is efficiently extracted by different conditions. In addition, germins and GLPs all bind Cibacron Blue, a dye often but not exclusively used for the purification of enzymes having nucleotide cofactors. In the case of AtGER2, binding to the dye is so tight that it almost allows a one-step purification of this protein. The variety of sequences, expression patterns and biochemical features indicates that GLPs could be a class of receptors localized in the ECM and involved in physiological and developmental processes as well as stress response. [ABSTRACT FROM AUTHOR]

We have identified a limited number of proteins secreted into the nectar of tobacco plants. Nectarin I is the most highly expressed nectar protein and has a monomer molecular mass of 29 kDa. The other major nectar proteins are expressed at lower levels and have monomer molecular masses of 41, 54, and 65 kDa respectively. Nectarin I was purified and antiserum was raised against the protein. Under nondenaturing conditions, Nectarin I has an apparent molecular mass of >120 kDa. The expression of Nectarin I was restricted to nectary tissues and to a much lower level in the ovary. No Nectarin I was found in petals, stems, leaves, or roots or other floral tissues. The expression of Nectarin I was also developmentally regulated. It is expressed in nectary tissues only while nectar is being actively secreted. Subsequently, the N-terminus of purified Nectarin I was sequenced. Sequence identity showed Nectarin I is related to wheat germin. Although hydrogen peroxide is readily detectable in tobacco floral nectar, we were unable to demonstrate any oxalate oxidase activity for Nectarin I. A partial cDNA encoding the mature Nectarin I N-terminus was isolated and used to probe a Nicotiana plumbaginifolia genomic library. The Nectarin I gene was isolated and the translated sequence was consistent with both N-terminal and internal cyanogen bromide-derived amino acid sequence. The gene contains a single 386 nt intron and encodes a mature protein of 197 amino acids. [ABSTRACT FROM AUTHOR]

Germins and germin-like proteins (GLPs) constitute a ubiquitous family of plant proteins that seem to be involved in many developmental and stress-related processes. Wheat germin has been extensively studied at the biochemical level: it is found in the apoplast and the cytoplasm of germinating embryo cells and it has oxalate oxidase activity (EC 1.2.3.4). Germin synthesis can also be induced in adult wheat leaves by auxins and by a fungal pathogen but it remains to be determined whether the same gene is involved in developmental, hormonal and stress response. In this work, we have studied the expression of one of the wheat germin genes, named gf-2.8, in wheat as well as in transgenic tobacco plants transformed with either this intact gene or constructs with GUS driven by its promoter. This has allowed us to demonstrate that expression of this single gene is both developmentally and pathogen- regulated. In addition, we show that expression of the wheat gf-2.8 germin gene is also stimulated by some abiotic stresses, especially the heavy metal ions Cd2+, Cu2+ and Co2+. Several chemicals involved in stress signal transduction pathways were also tested: only polyamines were shown to stimulate expression of this gene. Because regulation of the wheat gf-2.8 germin gene is complex and because its product results in developmental and stress-related release of hydrogen peroxide in the apoplast, it is likely that it plays an important role in several aspects of plant growth and defence mechanisms. [ABSTRACT FROM AUTHOR]

Wheat (Triticum aestivum) germin is a homopentameric glycoprotein whose synthesis is allied with seed germination. Germin pentamers show an unusual resistance to dissociation and possess an oxalate oxidase (OxO) activity. In order to increase our knowledge of germin gene expression, the function(s) of germin during development and possible uses in plant genetic engineering, an in vivo expression system is required. To this end, a gene for germin, named gf-2.8, was studied by expressing either promoter-GUS fusions or the intact gene in transgenic tobacco (Nicotiana tabacum) plants. Heterologous gene transcription was monitored in vitro and in vivo by GUS or OxO activity and was found to occur in developing seeds and in seedlings. This transcription was stimulated by auxins, as would be expected because of the presence of putative auxin-responsive elements in the promoter of the gf-2.8 gene. Auxin stimulation also extended to young leaves since OxO activity could be detected in treated but not in untreated leaves. The biochemical characteristics of wheat germin were also conserved in a transgenic host: the OxO activity was present under the form of a doublet co-migrating with germin G and G′ isoforms. Also, germin distributed between a soluble and an apoplastic fractions despite the fact that wheat cell wall substantially differs from tobacco cell wall. Therefore, tobacco constitutes a suitable host for in vivo studies of this monocotyledon gene. [ABSTRACT FROM AUTHOR]

Using differential screening of a root tip cDNA library prepared from an Al-tolerant wheat cultivar ( Triticum aestivum L. cv. Atlas-66) exposed to Al, we have isolated and characterized several wheat aluminum-regulated ( War) cDNAs. Sequence comparison revealed that genes up-regulated by Al correspond to peroxidase ( war4.2), cysteine proteinase ( war5.2), phenylalanine-ammonia lyase ( war7.2), and oxalate oxidase ( war13.2). Two wheat cultivars that differ in their level of tolerance (cv. Atlas-66: tolerant, and cv. Fredrick: sensitive) were used to evaluate the relationship between the accumulation of War mRNAs and Al toxicity, as measured by root growth inhibition (RGI). The mRNA accumulation was modulated to similar levels in both cultivars compared at equivalent RGIs. This indicates that War mRNA accumulation is associated with the toxicity of Al rather than with the cultivar's tolerance. It appears that most of the genes found to be up-regulated by Al share homologies with genes induced by pathogens. This suggests that Al may act as an elicitor of a pathogenesis-related transduction pathway. The potential functions of the up-regulated war genes in cell wall strengthening and Al trapping are discussed. [ABSTRACT FROM AUTHOR]

Wheat germin is a homopentameric 125 kD glycoprotein mainly localized in the cell wall of monocots, and is a specific marker of the onset of growth in germinating seeds. The major objective of this study was to examine the expression and oxalate oxidase activity of two wheat germin isoforms: gf-2.8 and gf-3.8 in transgenic tobacco plants. The transgenic tobacco plants were created with different constructs: 1) one entire excision of gf-2.8 germin promoter and two partially deleted promoter sequences were used to generate 3 independent GUS constructs; 2) the whole gf-2.8 gene construct and the fusion with CaMV 35S promoter; 3) one entire excision of gf-3.8 germin gene and one partially deleted gf-3.8 promoter sequences were used to generate 2 independent GUS constructs; 4) the whole gf-3.8 gene and the fusion with CaMV 35S promoter. Hormonal treatment (auxin and gibberellin), salt treatment, heavy metals (Mn, Fe, Co, Ni, Cu, Zn, Cd, Hg, As) and Al induced high GUS activity in tobacco transformed with entire and one partially deleted of the gf-2.8 gene. The immunoblotting confirmed induction of gf-2.8 gene and its product expressed oxalate oxidase activity in tobacco transformed with the entire gf-2.8 construct. Neither nicotinic acid, salicylic acid, heat shock, cold nor UV-C have enhanced significant GUS activity and germin gf-2.8 synhesis and activity. The germin gf-3.8 constructs with GUS gene and with the entire gf-3.8 sequences gave non-positive response with factors mentioned above. It has been demonstrated that gf-3.8 germin isoform is present as a monomer (Mr 25 kD). The non-active gf-3.8 protein is synthetised in transgenic tobacco plants only under control of the CaMV 35S promoter. Consequently, among two germin isoforms, only the gf-2.8 protein seems to be regulated by hormonal, salt and heavy metal factors. The gf-2.8 oxalate oxidase activity could be then involved in general stress-induced signalling in plant. [ABSTRACT FROM AUTHOR]