Effect of heavy metals and temperature on the oxalate oxidase activity and lignification of metaxylem vessels in barley roots

The effect of Cd on oxalate oxidase (OxO) activity and its localisation were analysed in barley root. In Cd-treated roots OxO activity was strongly induced in the region 2–4 mm behind the root tip and in the area toward the root base. In situ analyses showed that Cd-induced OxO activity was localised to the cell wall (CW) of early metaxylem vascular bundles and surrounding parenchyma cells and was accompanied by lignification of metaxylem vessels. OxO activation was also observed during treatment with other heavy metals (HMs), salt treatment and at elevated non-optimal temperature. In contrast to HM activation of OxO and lignification, high temperature and NaCl indeed activated OxO but did not induce lignification of metaxylem vessels. These results suggest that oxalate oxidase as an H₂O₂-generating enzyme is activated in response to several stresses, however the ectopic lignification of metaxylem vessels is activated specifically by HMs. This HM-induced premature root xylogenesis due to ectopic lignification of metaxylem vessels probably causes shortening of the root elongation zone and therefore a reduction in root growth.     

Elevated oxalate oxidase activity is correlated with Al-induced plasma membrane injury and root growth inhibition in young barley roots

In order to characterise the possible mechanisms involved in Al toxicity some functional characteristics were analysed in young barley (Hordeum vulgare L.) seedlings cultivated between moistened filter paper. Transfer of germinated barley seeds into hydroponic culture system caused significant stress, which was manifested by root-growth inhibition and elevated Evans blue uptake of root tips. Hydroponics caused stress unabled the analysis of Al-induced stress in the young barley roots during the first day of cultivation. Several (3–4) days are required for adaptation of barley seedlings to hydroponics in spite of strong aeration of the medium. Using filter paper compared to cultivation in solution application of much higher Al concentrations were required to inhibit root growth. Al-induced root growth inhibition, Al uptake, damage of plasma-membrane (PM) permeability of root cells, as well as elevated oxalate oxidase - OxO (EC 1.2.3.4) activity were significantly correlated. While 1 mM Al concentration had no effect on barley roots growing on filter paper, 5 to 100 mM Al concentration inhibited root growth, enhanced cell death and induced oxalate oxidase activity with increasing intensity. The time course analysis of OxO gene expression and OxO activity showed that 10 mM Al increased OxO activity as soon as 3 h after exposure of roots to Al reaching its maximum at about 18 h after Al application. These results indicate that expression of OxO is activated very early after exposure of barley to Al, suggesting its role in oxidative stress and subsequent cell death caused by Al toxicity in plants.     

Specific and constitutive expression of oxalate oxidase during the ageing of leaf sheaths of ryegrass stubble

Changes in the activity of oxalate oxidase (OxO) and of the concentrations of oxalate and H₂O₂ were investigated during the ageing of leaf sheaths of ryegrass (Lolium perenne L.) stubble. The accumulation of H₂O₂ during ageing coincides with the increases of both oxalate level and OxO activity. Western and Northern blot analyses using protein and RNA extracts of the different categories of leaf sheaths suggested that OxO gene expression, as well as Ca-oxalate synthesis, are crucial events of ageing for leaf sheaths. Immunocytochemistry experiments have revealed that OxO, which is an extracellular enzyme, is nearly always present in the parenchymatous cells surrounding the vascular bundles and in the cells of the lower epidermis. Overall, results suggest that in ryegrass that synthesizes both Ca-oxalate and OxO, the production of H₂O₂ and Ca²⁺ during ageing of stubble might be involved in the constitutive defences against pathogens, thus allowing the phloem mobilization of nutrient reserves from the leaf sheaths towards elongating leaf bases of ryegrass.     

Oxalate oxidase: a novel reporter gene for monocot and dicot transformations

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.     

Effect of heavy metals on root growth and peroxidase activity in barley root tip

In the present work, we investigated the alteration of oxidative and peroxidative activities of peroxidases (PODs) along the longitudinal root axis of barley seedlings during heavy metal (HM; e.g., Cd, Cu, Hg, Ni, Pb) treatment. Analysis of the individual root segments revealed that all of the analyzed HMs caused an increase of guaiacol-POD activity, however to a different extent and spatial distribution. Cd-induced ferulic acid POD activity was observed along the whole root tip (RT), while Cu and Hg caused its increase in the meristematic zone and Ni mainly at the end of the differentiation zone of RT. The activation of coniferyl alcohol POD by HMs was detected along the whole RT. HM-induced hydrogen peroxide-generating POD activity was localized mainly to the elongation zone of RT. Elevated chlorogenic acid POD activity was observed in the meristematic zone and at the end of the differentiation zone of RTs. The activation of several PODs is probably associated with enhanced H₂O₂ production and lignification as a defense response of roots to several HM, to prevent their uncontrolled flux. On the other hand, this defense response is accompanied by root growth inhibition, due to the enhanced rigidification of cell wall and accelerated differentiation of RTs.     

草酸氧化酶在水稻胚芽鞘衰老中的作用

草酸氧化酶（OxO）催化草酸氧化产生CO2和H2O2,其在植物发育及防御过程中可能具有重要作用。本文以水稻品种‘湘糯1号’（‘Xiangnuo 1’）为材料,对胚芽鞘中的OxO及其生理功能进行了研究。结果表明,胚芽鞘中的H2O2含量在其衰老时增加;OxO活性在浸种后96h时较低,之后也迅速增加,在240h达到最高;而可溶性蛋白、O2-·和草酸含量以及过氧化氢酶（CAT）活性则随着胚芽鞘的衰老迅速降低。由于H2O2能够诱导细胞死亡,推测OxO可能通过降解草酸产生H2O2参与胚芽鞘的衰老。     

Purification and characterization of oxalate oxidase from wheat seedlings

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.     

Determination of oxalate in plant tissues with oxalate oxidase prepared from wheat

A method for determination of oxalate with oxalate oxidase (OxO, EC 1.2.3.4) prepared from wheat bran, is based on specific oxidation of oxalate to produce H₂O₂. The H₂O₂ formed was colorimetrically determined using horseradish peroxidase-catalyzed oxidation of 4-aminoantipyrine and N,N-dimethylaniline by H₂O₂. The new method was tested on rice, buckwheat, soybean and oxalis leaves, showing it is precise, sensitive, inexpensive, highly reproducible and simple to perform. Good agreement could be obtained between this method and the HPLC.     

光照强度对小麦苗期草酸氧化酶活性的影响

以小麦品种‘烟优361’(Triticum aestivum L.cv.Yanyou 361)萌发4 d幼苗为试验材料,分析了草酸氧化酶(OxO)在幼苗中的定位和表达,以及光照强度处理对小麦幼苗OxO活性的影响。实验结果显示,萌发后小麦幼苗的OxO分布在子叶与根的连接处和成熟的根中,其活性随光照强度的增加而下降;200μmol.m-2.s-1的强光显著抑制了OxO活性,该处理培养4 d幼苗的OxO活性仅为40μmol.m-2.s-1光照培养条件下的18.7%;强光还缩短OxO在苗期的表达时间,抑制了OxO的mRNA表达量。同时,光照强度还能影响小麦幼苗中H2O2的含量,200μmol.m-2.s-1处理幼苗的H2O2的含量显著下降,其培养4 d的幼苗H2O2含量仅为40μmol.m-2.s-1光照强度培养条件下的18.0%。研究发现,光照强度可通过调节OxO的活性和表达量来控制H2O2的产量,从而影响幼苗的生长发育。     

小麦种子成熟和萌发过程中的假萌发素活性

用SDS-PAGE方法研究了假萌发素(ψG)在小麦种子成熟和萌发过程中活性的变化.结果表明:在种子成熟过程中只有ψG表达,扬花后10 d,在颖壳、内外桴、种皮和果皮中皆可检测到ψG的草酸氧化酶活性,随着发育进程的推进,ψG的活性增大.在种子萌发过程中,在小麦品种中育5号的维管束过渡区中除了萌发素G和G'外,还可检测到ψG的草酸氧化酶活性.由于ψG在种子成熟过程中主要存在于颖壳、内外桴、果皮及种皮这些保护组织中,且开始大量表达的时间正是生长接近停止时,于是推测ψG很可能通过降解草酸产生H2O2而推动这些组织细胞壁的木质化.     

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.     

Effects of salt and alkali stress on growth,accumulation of oxalic acid,and activity of oxalic acid-metabolizing enzymes in <Emphasis Type="Italic">Kochia sieversiana</Emphasis>

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 Na₂SO₄ 1:1), and alkali stress (200 mM, a molar ratio of NaHCO₃ and Na₂CO₃ 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 phosphoenolpyruvate (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.     

Cadmium induces premature xylogenesis in barley roots

The effect of Cd on H₂O₂ production, peroxidase (POD) activity and root hair formation were analyzed in barley root. Cd causes a strong H₂O₂ burst in the root region 0–6 mm behind the root tip. POD activity was activated in root tip and raised toward the root base in Cd treated roots. In situ analyses showed that both elevated H₂O₂ production and POD activity are localized in the early metaxylem vascular bundles. Cd induces root hair formation in the region 2 to 4 mm behind the root tip that was not detected in control roots. These results suggest that Cd-induced root growth inhibition is at least partially the consequence of Cd-stimulated premature root development involving xylogenesis and root hair formation, which is correlated with shortening of root elongation zone and therefore with root growth reduction.     

Late Maturation of Large Metaxylem Vessels in Soybean Roots: Significance for Water and Nutrient Supply to the Shoot

The large, late metaxylem (LMX) in the roots of soybean beginsdevelopment in the centre of the stele after lignification ofthe early metaxylem poles. Subsequent maturation of the firstappearing LMX elements is gradual. They were never mature inthe 8-d-old seedlings examined. In 10 to 15-d-old plants thefirst LMX matured to open vessels at a mean of 17 cm proximalto the root tip. Additional LMX vessels developed in more proximalregions of the roots and these also matured gradually. Based on calculations from relative vessel diameters, the potentialflow of xylem sap in a single central LMX vessel is 50 timesthat in the total of all the early metaxylem (EMX) vessels ofa typical primary root of soybean. There was a marked dependence of relative leaf area on the lengthof primary root with open LMX vessels. This may result fromthe predicted increased water and nutrient flow to the shoot,facilitated by the opening of the large vessels. It is suggestedthat, as in maize, the living LMX elements may function in ionaccumulation. Dicotyledonous roots, soybean, Glycine max, xylem vessels, xylem maturation, water conduction     

Purification and Properties of Oxalate Oxidase from NaCl Stressed Grain Sorghum Seedlings

The effect of NaCl stress on molecular and biochemical properties of oxalate oxidase (OXO) was studied in leaves of grain sorghum hybrid (var CSH-14) seedlings. There was no effect on molecular weight and number of subunits of the enzyme but it showed some important changes in its kinetic parameters such as K_m for oxalate and V_max. Optimum pH (5.8), activation energy (5.084 kcal mole^?1), time of incubation (6 min) and K_m for oxalate (1.21×10^t-4M) were increased, while V_max (0.182 mmole min^?1) decreased and no change in optimum temperature was observed. This showed that substrate affinity and maximum activity of the enzyme was adversely affected. The specific activity of oxalate oxidase was increased in seedlings grown in a NaCl containing medium compared to normal, which reveals the increased de novo synthesis of the enzyme to sustain oxalate degradation.     

Aluminum-induced cell death of barley-root border cells is correlated with peroxidase- and oxalate oxidase-mediated hydrogen peroxide production

The function of root border cells (RBC) during aluminum (Al) stress and the involvement of oxalate oxidase, peroxidase and H₂O₂ generation in Al toxicity were studied in barley roots. Our results suggest that RBC effectively protect the barley root tip from Al relative to the situation in roots cultivated in hydroponics where RBC are not sustained in the area surrounding the root tip. The removal of RBC from Al-treated roots increased root growth inhibition, Al and Evans blue uptake, inhibition of RBC production, the level of dead RBC, peroxidase and oxalate oxidase activity and the production of H₂O₂. Our results suggest that even though RBC actively produce active oxygen species during Al stress, their role in the protection of root tips against Al toxicity is to chelate Al in their dead cell body.     

Cotton-fiber germin-like protein. II: Immunolocalization,purification, and functional analysis

Cotton (Gossypium hirsutum L.) contains a germin-like protein (GLP), GhGLP1, that shows tissue-specific accumulation in fiber. The fiber GLP is an oligomeric, glycosylated protein with a subunit size of approximately 25.5 kDa. Accumulation of GhGLP1 occurs during the period of fiber elongation [4–14 days post-anthesis (DPA)]. During early phases of fiber development (2–4 DPA), GhGLP1 localizes to cytoplasmic vesicles as shown by confocal immunofluorescent microscopy. In slightly older fibers (7–10 DPA), GhGLP1 localizes to the apoplast. In other plants, germins and GLPs have been reported to have enzymatic activities including oxalate oxidase (OxO), superoxide dismutase, and ADP-glucose pyrophosphatase. Cotton fiber extracts did not contain OxO activity, nor did intact fibers stain for OxO activity. A four-step purification protocol involving ammonium sulfate precipitation of a 1.0 M NaCl extract, ion-exchange chromatography on DEAE-Trisacryl M, lectin-affinity chromatography, and gel filtration chromatography resulted in electrophoretically pure GhGLP1. While 1.0 M NaCl extracts from 10–14 DPA fiber contained superoxide dismutase and phosphodiesterase activities, GhGLP1 could be separated from both enzyme activities by the purification protocol. Although a GLP accumulates in the cotton fiber apoplast during cell elongation, the function of this protein in fiber growth and development remains unknown.Abbreviations ABP Auxin binding protein - AGPPase ADP-Glucose pyrophosphatase/phosphodiesterase - bis-PNPP Bis-p-nitrophenol phosphate - ConA Concanavalin A - DOA Day of anthesis - DPA Days post-anthesis - GLP Germin-like protein - Mn-SOD Manganese superoxide dismutase - OxO Oxalate oxidase - PBS Phosphate-buffered saline