Tissue-Specific Expression of Germin-Like Oxalate Oxidase during Development and Fungal Infection of Barley Seedlings

Oxalate oxidase activity was detected in situ during the development of barley seedlings. The presence of germin-like oxalate oxidase was confirmed by immunoblotting using an antibody directed against wheat germin produced in Escherichia coli, which is shown to cross-react with barley (Hordeum vulgare) oxalate oxidase and by enzymatic assay after electrophoresis of the protein extracts on polyacrylamide gels. In 3-d-old barley seedlings, oxalate oxidase is localized in the epidermal cells of the mature region of primary roots and in the coleorhiza. After 10 d of growth, the activity is detectable only in the coleorhiza. Moreover, we show that oxalate oxidase is induced in barley leaves during infection by the fungus Erysiphe graminis f. sp. hordei but not by wounding. Thus, oxalate oxidase is a new class of proteins that responds to pathogen attack. We propose that oxalate oxidase could have a role in plant defense through the production of H2O2.     

Overexpression of a gene encoding hydrogen peroxide-generating oxalate oxidase evokes defense responses in sunflower

Oxalate oxidase (OXO) converts oxalic acid (OA) and O(2) to CO(2) and hydrogen peroxide (H(2)O(2)), and acts as a source of H(2)O(2) in certain plant-pathogen interactions. To determine if the H(2)O(2) produced by OXO can function as a messenger for activation of defense genes and if OXO can confer resistance against an OA-producing pathogen, we analyzed transgenic sunflower (Helianthus annuus cv SMF3) plants constitutively expressing a wheat (Triticum aestivum) OXO gene. The transgenic leaf tissues could degrade exogenous OA and generate H(2)O(2). Hypersensitive response-like lesion mimicry was observed in the transgenic leaves expressing a high level of OXO, and lesion development was closely associated with elevated levels of H(2)O(2), salicylic acid, and defense gene expression. Activation of defense genes was also observed in the transgenic leaves that had a low level of OXO expression and had no visible lesions, indicating that defense gene activation may not be dependent on hypersensitive response-like cell death. To further understand the pathways that were associated with defense activation, we used GeneCalling, an RNA-profiling technology, to analyze the alteration of gene expression in the transgenic plants. Among the differentially expressed genes, full-length cDNAs encoding homologs of a PR5, a sunflower carbohydrate oxidase, and a defensin were isolated. RNA-blot analysis confirmed that expression of these three genes was significantly induced in the OXO transgenic sunflower leaves. Furthermore, treatment of untransformed sunflower leaves with jasmonic acid, salicylic acid, or H(2)O(2) increased the steady-state levels of these mRNAs. Notably, the transgenic sunflower plants exhibited enhanced resistance against the OA-generating fungus Sclerotinia sclerotiorum.     

Cell wall oxalate oxidase modifies the ferulate metabolism in cell walls of wheat shoots

Oxalate oxidase (OXO) utilizes oxalate to generate hydrogen peroxide, and thereby acts as a source of hydrogen peroxide. The present study was carried out to investigate whether apoplastic OXO modifies the metabolism of cell wall-bound ferulates in wheat seedlings. Histochemical staining of OXO showed that cell walls were strongly stained, indicating the presence of OXO activity in shoot walls. When native cell walls prepared from shoots were incubated with oxalate or hydrogen peroxide, the levels of ester-linked diferulic acid (DFA) isomers were significantly increased. On the other hand, the level of ester-linked ferulic acid (FA) was substantially decreased. The decrease in FA level was accounted neither by the increases in DFA levels nor by the release of FA from cell walls during the incubation. After the extraction of ester-linked ferulates, considerable ultraviolet absorption remained in the hemicellulosic and cellulose fractions, which was increased by the treatment with oxalate or hydrogen peroxide. Therefore, a part of FA esters may form tight linkages within cell wall architecture. These results suggest that cell wall OXO is capable of modifying the metabolism of ester-linked ferulates in cell walls of wheat shoots by promoting the peroxidase action via supply of hydrogen peroxide.     

Characterization of recombinant barley oxalate oxidase expressed by Pichia pastoris

Oxalate oxidase catalyzes the oxidation of oxalate to carbon dioxide and hydrogen peroxide, making it useful for clinical analysis of oxalate in biological fluids. An artificial gene for barley oxalate oxidase has been used to produce functional recombinant enzyme in a Pichia pastoris heterologous expression system, yielding 250 mg of purified oxalate oxidase from 5 L of fermentation medium. The recombinant oxalate oxidase was expressed as a soluble, hexameric 140 kDa glycoprotein containing 0.2 g-atom Mn/monomer with a specific activity of 10 U/mg, similar to the properties reported for enzyme isolated from barley. No superoxide dismutase activity was detected in the recombinant oxalate oxidase. EPR spectra indicate that the majority of the manganese in the protein is present as Mn(II), and are consistent with the six-coordinate metal center reported in the recent X-ray crystal structure for barley oxalate oxidase. The EPR spectra change when bulky anions such as iodide bind, indicating conversion to a five-coordinate complex. Addition of oxalate perturbs the EPR spectrum of the Mn(II) sites, providing the first characterization of the substrate complex. The optical absorption spectrum of the concentrated protein contains features associated with a minor six-coordinate Mn(III) species, which disappears on addition of oxalate. EPR spin-trapping experiments indicate that carboxylate free radicals (CO2*-) are transiently produced by the enzyme in the presence of oxalate, most likely during reduction of the Mn(III) sites. These features are incorporated into a turnover mechanism for oxalate oxidase.     

Increase of ascorbic acid content and nutritional quality in spinach leaves during physiological acclimation to low temperature

The effect of acclimation to 10 °C on the leaf content of ascorbic and oxalic acids, was investigated in spinach (Spinacia oleracea L.). At 10 °C the content of ascorbic acid in leaves increased and after 7 days it was about 41% higher than in plants remaining under a 25 °C/20 °C day/night temperature regime. In contrast, the content of oxalate, remained unchanged. Transfer to 10 °C increased the ascorbic but not the oxalic acid content of the leaf intercellular washing fluid (IWF). Oxalate oxidase (OXO EC 1.2.3.4) activity was not detected in extracts of leaf blades. Therefore, oxalic acid degradation via OXO was not involved in the control of its content. Our results show that low temperature acclimation increases nutritional quality of spinach leaves via a physiological rise of ascorbic acid that does not feed-forward on the content of oxalic acid.     

Functional characterization of seed coat-specific members of the barley germin gene family

The present project aimed to isolate testa-, pericarp- and epicarp-specific gene promoters for the developing caryopsis of barley (Hordeum vulgare L.). These might be applied in transgenic plants to express antifungal agents or modify metabolic pathways. A testa-specific 379-nucleotide fragment was cloned by differential amplification and used to screen a bacterial artificial chromosome (BAC) library of 6.3 haploid genome equivalents. Fifty-three clones containing genes encoding for proteins of the germin family were found. Characterization of the clones identified a minimum of six seed coat- and eight leaf-specific germin genes. Four seed coat- and one leaf-specific genes were sequenced. The deduced primary structure of the proteins revealed a remarkable conservation of the manganese(II) binding His and Glu residues and β-barrel secondary structure of oxalate oxidase – also in barley, wheat, rice and Arabidopsis germins, for which an enzymatic activity has not yet been identified. The oxalate oxidase and germins of barley and other species are synthesized with a conserved pre-sequence of 23 or 24 amino acids for targeting into the cell wall. β-Glucuronidase expression with the barley germin F gene promoter occurs specifically in the testa and epicarp of the developing barley caryopsis, while expression with the B gene promoter is restricted to the testa. Oxalate oxidase activity is prominent in the epicarp and the root tips of the developing embryo. A family tree based on primary structure homologies of germins distinguishes three groups: oxalate oxidases, leaf-specific germins and seed coat-specific germins.     

Increased Septoria musiva resistance in transgenic hybrid poplar leaves expressing a wheat oxalate oxidase gene

A cDNA clone of a wheat germin-like oxalate oxidase (OxO) gene regulated by the constitutive CaMV 35S promoter was expressed in a hybrid poplar clone, Populus × euramericana (`Ogy'). Previous studies showed that OxO is likely to play an important role in several aspects of plant development, stress response, and defense against pathogens. In order to study this wheat oxalate oxidase gene in woody plants, the expression of this gene and the functions of the encoded enzyme were examined in vitro and in vivo in transgenic `Ogy'. The enzyme activity in the transformed `Ogy' was visualized by histochemical assays and in SDS-polyacrylamide gels. It was found that the wheat OxO gene is expressed in leaves, stems, and roots of the transgenic `Ogy' plants and the encoded enzyme is able to break down oxalic acid. Transgenic `Ogy' leaves were more tolerant to oxalic acid as well as more effective in increasing the pH in an oxalic acid solution when compared to untransformed controls. In addition, when leaf disks from `Ogy' plants were inoculated with conidia of the poplar pathogenic fungus Septoria musiva, which produces oxalic acid, the OxO-transformed plants were more resistant than the untransformed controls.     

草酸脱羧酶及其应用

草酸脱羧酶是一种含锰的酶,在白腐菌中广泛存在,少数低等真菌和细菌中也能产生。目前,至少10多种草酸脱羧酶得到了分离和纯化。该酶是一种氨基酸残基在379个左右的单体酶,一般都为酸性糖蛋白,含有2个锰离子,形成2个活性区域;表面一些氨基酸被不同程度地糖基化。晶体结构和其它一些波谱学研究解释了其空间结构和可能的电子传递机制。运用PCR技术和cDNA文库技术,越来越多的草酸脱羧酶基因被克隆。已研究的该酶基因中都含有17个左右的内含子,这些内含子在活性域位置上有比较高的保守性。一些特殊氨基酸序列的存在决定了该酶的表达形式为诱导型,菌株的基因调控序列中含有一段受草酸化合物作用的序列。该酶在一些酵母和植物等异源表达系统中有成功表达的报道。该酶的应用主要集中在以下几方面：造纸废水中的草酸盐降解;食品中的草酸降解;草酸生物检测（如,临床诊断）等。     

Ethanol increases sensitivity of oxalate oxidase assays and facilitates direct activity staining in SDS gels

Oxalate oxidase, and H₂O₂-generating enzyme, has been characterized from several plants, and is widely used for clinical detection of oxalate. Using a germin-like oxalate oxidase from barley leaves, we have developed and optimized novel methods for measuring oxalate oxidase activity. As oxalate oxidase is SDS-tolerant, its activity can be detected directly in SDS-PAGE gels in the presence of ethanol. This ethanol-dependent method is a hundred times more sensitive than the current methods. Furthermore, ethanol also improves the sensitivity of oxalate oxidase assays performed in solution. We found at least a 10-fold increase in sensitivity in comparison to a current method. The assay in solution is, in addition, useful for detection of oxalate. This elevation in sensitivity may be due to the immobilization of the enzyme in protein precipitates as a result of the treatment with ethanol.     

A root-specific O-methyltransferase gene expressed in salt-tolerant barley

A cDNA encoding an O-methyltransferase (OMT) was isolated from salt-tolerant barley roots by subtraction hybridization with cDNAs of salt-tolerant barley roots as a tester cDNA and cDNAs of the salt-sensitive barley roots as a driver cDNA. The deduced amino acid sequence showed significant identity with plant caffeic acid/5-hydroxyferulic acid OMTs. Southern blot analysis showed that the OMT gene was a single copy in both salt-tolerant and -sensitive barley. The cloned gene was expressed in a wheat germ cell-free system to produce the OMT, which had methylating activity for caffeic acid. Northern blot analysis showed that the OMT gene was expressed constitutively in the salt-tolerant barley roots and the expression level was increased 1.5 times by salt stress, but the salt-sensitive barley showed no expression of the gene in roots and leaves.     

植物源草酸氧化酶筛选及应用潜力评价

以44科88种植物叶片为酶源,检测出具有OXO活性的植物34科60种,其中OXO比活力较高(>0.02U/mg protein且>0.2U/g·fw)的有30种。为评估酶法清除动物体内草酸的可能性,研究了松果菊、白花败酱、草珊瑚、白鹤灵芝和甘草的OXO性质。结果表明,5种植物OXO都具有极强的酸、热稳定性;松果菊、白鹤灵芝和白花败酱OXO活性不受胃蛋白酶、NaCl和CaCl₂抑制,具有较好的应用潜力;甘草OXO活性不受NaCl和CaCl₂抑制,但受胃蛋白酶降解;而草珊瑚OXO受NaCl、CaCl₂及胃蛋白酶抑制。     

Effects of ionic substances in bleaching filtrates and of lignosulfonates on the activity of oxalate oxidase from barley

The effects of ionic substances in seven industrial filtrates from kraft pulping, mechanical pulping, and sulfite pulping on the activity of oxalate oxidase from barley were investigated by pre‐treatment of the filtrates with ion‐exchange resins prior to enzymatic degradation of the oxalic acid in the filtrates. The pre‐treatment resulted in increased oxalic acid degradation rates in all filtrates, except for one that was obtained from sulfite pulping. The possibility that lignosulfonates, which were present in the filtrate from sulfite pulping, could affect oxalate oxidase was investigated in a separate set of experiments involving four different preparations of lignosulfonates. At a lignosulfonate concentration of 50 mg/mL and a pH of 3.8, only 2–16% of the activity of oxalate oxidase remained. The results show the effects of anionic and cationic substances in bleaching filtrates on oxalate oxidase and indicate that there is an interaction between the enzyme, which has a positive net charge at pH 3.8, and the polymeric anionic lignosulfonates.     

A proposed role of oxalic acid in wood decay systems of wood-rotting basidiomycetes

Abstract: The possible roles of oxalic acid, veratryl alcohol, and manganese were investigated in relation to lignin biodegradation by white-rot basidiomycetes. Oxalate inhibited both lignin peroxidase (LiP) and manganese-peroxidase (MnP). and was decarboxylated by the mediation of veratryl alcohol and Mn. Oxalate was shown to regulate the mineralization of lignin in the in vivo system of Phanerochaete chrysosporium . In the brown-rot wood decay process, oxalic acid may serve as an acid catalyst as well as an electron donor for the Fenton reaction, to breakdown cellulose and hemicellulose. Oxaloacetase and glyoxylate oxidase may play a key role in production of oxalic acid by white-rot and brown-rot basidiomycetes such as Phanerochaete chrysosporium, Coriolus versicolor and Tyromyces palustris . A possible role of oxalate metabolism is discussed in relation to the physiology of wood-rotting fungi.     

Characterization of wheat germin (oxalate oxidase) expressed by Pichia pastoris

High-level secretory expression of wheat (Triticum aestivum) germin/oxalate oxidase was achieved in Pichia pastoris fermentation cultures as an alpha-mating factor signal peptide fusion, based on the native wheat cDNA coding sequence. The oxalate oxidase activity of the recombinant enzyme is substantially increased (7-fold) by treatment with sodium periodate, followed by ascorbate reduction. Using these methods, approximately 1 g (4x10(4) U) of purified, activated enzyme was obtained following eight days of induction of a high density Pichia fermentation culture, demonstrating suitability for large-scale production of oxalate oxidase for biotechnological applications. Characterization of the recombinant protein shows that it is glycosylated, with N-linked glycan attached at Asn47. For potential biomedical applications, a nonglycosylated (S49A) variant was also prepared which retains essentially full enzyme activity, but exhibits altered protein-protein interactions.     

Oxalic acid and oxalate oxidase enzyme in <Emphasis Type="Italic">Costus pictus</Emphasis> D. Don

The leaves of Costus pictus are sour in taste due to the presence of oxalic acid in the leaves. Different stages of leaves were collected and the samples were designated as stage one, stage two and stage three. It was found that oxalate content and oxalate oxidase activity were maximum in second leaf stage followed by first leaf stage and third leaf stage. Drying causes substantial loss of oxalate content and complete loss of oxalate oxidase activity. With various solvents water recovered more oxalate followed by methanol and ethanol while oxalate oxidase activity was maximum in ethanol followed by methanol and water. The ethanol or methanol extract of second leaf stage of C. pictus can be used for isolating active principles. The oxalate oxidase from C. pictus can be used as a cheap source of oxalate oxidase enzyme which is used in oxalate determination in biological fluids. Moreover, the sensitivity of oxalate determination employing oxalate oxidase from C. pictus will be more as oxalate oxidase in C. pictus has K _m 20 times lesser than the oxalate oxidase enzyme from barley seedling.     

Fungal physiology and the formation of calcium oxalate films on stone monuments

Summary Extensive, uniform, yellow-brown films are observed on many monuments. The origin of these films, composed predominantly of calcium oxalate, has been investigated by several authors. Oxalate film formation may be related, in some cases, to the activity of such microorganisms as fungi, which presumably form oxalic acid via the metabolic transformation of organic substances already present on the stone. The present work provides an overview of the physiological factors affecting oxalate synthesis by fungi and of oxalic acid in fungi metabolism.