Comparison of Indole-3-acetic Acid Oxidation in Peroxidases from Rust-Infected Resistant Wheat Leaves

Seven peroxidase isozyme fractions were isolated from rust-infectedresistant wheat leaves by means of ammonium sulfate fractionation,pH precipitation, ion exchange chromatography and gel filtration.Three isozymes showed a single peroxidative band in the electrophoreticgels. The catalytic activity of the enzymes on non-physiologicalsubstrates was comparable to that of commercial horseradishperoxidase. When compared to isozyme 9, isozyme 10 had twicethe activity on guaiacol and eugenol but only one-fifth theoxidative activity on p-phenylenediamine and o-dianisidine.The IAA oxidation activity was compared among purified enzymefractions. Isozyme 10 was the only enzyme which could destroythe auxin without phosphate and manganese cofactors. All theother enzymes, including isozyme 9, showed the activity onlywhen both cofactors were present. The possible involvement ofthese peroxidases in IAA destruction in the resistant tissueis discussed. (Received June 14, 1984; Accepted October 15, 1984)     

Effect of Paraquat on Tomato Roots Cultured in Vitro Susceptible and Resistant to Nematodes

Roots from two tomato cultivars, one resistant (Rossol) andthe other susceptible (Roma VF) to root-knot nematodes, werecultured in vitro and tested for their response to paraquat,an herbicide which generates superoxide in cells. The effectof paraquat on the permeability of root membranes was testedby the safranine method. Membranes from resistant roots wereinjured more markedly than susceptible ones. A statistical analysisof the variations on a series of enzyme activities from rootsincubated for 2 days with paraquat was carried out. Superoxidedismutase was repressed in treated resistant roots togetherwith ascorbate and glutathione peroxidases. In susceptible rootsthese enzymes were not significantly affected by paraquat treatmentexcept for ascorbate peroxidase which increased slightly. Syringaldazineand polyphenol oxidases, two enzymes active in the hypersensitiveresponse to nematodes, increased in resistant but not in thesusceptible cultivar. p-Phenilendiamine-pyrocathecol oxidasewas not affected while catalase increased markedly by paraquattreatment in both cultivars. (Received January 21, 1991; Accepted August 6, 1991)     

Proline involvement in the common sage antioxidant system in the presence of NaCl and paraquat

To elucidate proline antioxidant properties in common sage (Salvia officinalis L.) plants, they were treated with paraquat (a producer of superoxide radical) and/or NaCl and also with paraquat and proline at the stage of 4–5 true leaves. The paraquat solution (1 ml containing 0.1 μmol of the agent) was applied to the leaf surface; NaCl (200 mM) and proline (the final concentration of 5 mM) were added to nutrient medium. Experimental plants were firstly kept in darkness for 12 h, then illuminated, and in 3, 6, and 12 h, leaves and roots were fixed for biochemical analyses. The results obtained are in agreement with the supposition of proline antioxidant properties. In particular, it was established that paraquat induced a slight increase in the proline level in the leaves during dark period of plant growth and also during subsequent 3 h after light switching on. This transient proline accumulation in the leaves was accompanied by its level decrease in the roots. Proline addition to the nutrient medium of paraquat-treated plants neutralized paraquat damaging action on the leaves. In the presence of paraquat, proline treatment reduced the accumulation in the roots of hydrogen peroxide and malondialdehyde, the product of membrane lipid peroxidation. It also affected indirectly the activities of superoxide dismutase (SOD) and free, covalently bound, and ionically bound peroxidases. Keeping in mind that, in the presence of paraquat, superoxide-induced changes in SOD activity in the roots were negatively correlated with the level of proline, which content was the highest during the last hours of experiments, we can conclude that proline antioxidant effects are manifested only after 12 h of stressor action, whereas antioxidant enzymes are involved in ROS scavenging during the earlier stage of damaging factor action.     

The Presence of an Enzyme that Converts IndoIe-3-acetamide into IAA in Wild and Cultivated Rice

The enzymatic conversion of indole-3-acetamide (IAM) to IAA,which is the second step in the IAM pathway (tryptophan IAM IAA) was investigated in calluses derived from various dicotyledonousand monocotyledonous plants. A simple method, using analysisby HPLC to measure the conversion of naphthaleneacetamide (NAM)to naphthaleneacetic acid (NAA) was employed for the detectionof IAM hydrolase activity. Among calluses from 27 plants tested,only callus from a cultivated strain of rice (Oryza sativa C5924)had high conversion activity similar to that of crown gall cells,and very weak activity was found in calluses from lucern andorange. In addition to the presence of the conversion activity,we confirmed that radioactivity from ³H-IAM was incorporatedinto IAA in a cell-free system from O. sativa C5924. An extractof roots of rice seedlings exhibited twice the activity of thatin an extract of shoots. IAM hydrolase activity was observedin calluses from all lines of rice callus examined, irrespectiveof whether they were wild or cultivated lines, with the exceptionof O. grandiglumis W1194 and O. branchyantha W656, while otherspecies of Gramineae exhibited no activity. These results suggestthe possibility that this enzyme may play a specific role inrice. (Received August 23, 1990; Accepted November 29, 1990)     

Influence of Glyphosate on Nitrate Reductase Activity in Soybean (Glycine max)

Experiments were conducted to determine the influence of glyphosate[N-(phosphonomethyl)glycine] on extractable nitrate reductaseactivity during light and dark growth of soybean (Glycine max)seedlings. Glyphosate (5?10^–4 M), applied via root-feedingto three-day-old etiolated seedling, significantly reduced enzymeactivity in roots (48 to 96 h) and leaves (96 h) of seedlingsplaced in the light, but had little effect on enzyme activityin cotyledons compared to enzyme levels in tissues of untreatedseedlings. During dark-growth, nitrate reductase activity increasedwith time in cotyledons of untreated seedlings (activity about85-fold less than in cotyledons of light-grown plants) but muchlower enzyme levels were found in cotyledons of glyphosate-treatedseedlings after 72 and 96 h. In leaves of dark-grown seedlings,glyphosate reduced nitrate reductase levels by 95%. Most inhibitionof extractable enzyme activity occurred in newly developingorgans (leaves and roots) which correlates well with reportsthat glyphosate is rapidly translocated to these sites. However,the fact that glyphosate inhibits growth prior to lowering enzymeactivity levels indicates a secondary effect on nitrate reductase. (Received May 18, 1984; Accepted February 12, 1985)     

Changes in Activity of Malate Dehydrogenase, Catalase, Peroxidase and Superoxide Dismutase in Leaves of Halimione portulacoides (L.) Aellen Exposed to High Sodium Chloride Concentrations

Plants of Halimione portulacoides were grown in nutrient solutionscontaining NaCl at concentrations ranging from 0–2.0 MNaCl. They survived in this environment at least for 20 days.Malate dehydrogenase (MDH), catalase, peroxidase and superoxidedismutase (SOD) were extracted from the leaves of such plantsand enzyme activity was assayed in the absence of salt. Sodium chloride at low concentration (0–0.5 M) stimulatedthe activities of MDH and catalase but inhibited them at concentrationshigher than 0.5 M. Peroxidase and SOD were hardly affected byexposure to salinity in vivo. Salinity in vivo also affectedthe K_m and the V_max of the enzymes. The possibility that thethree enzymes (catalase, peroxidase and SOD) have a role inprotecting the leaf cells against oxygen toxicity caused byfree radicals, that may be formed in cells when growing undersaline and extreme climatic conditions, is discussed. Halimione portulacoides (L.) Aellen, salinity, catalase, peroxidase, superoxide dismutase     

Isolation and Characterization of Photosynthetically Active Cells from Submersed and Floating Leaves of the Aquatic Macrophyte Potamogeton nodosus Poir

Photosynthetically active cells were isolated by enzymic digestionof floating and submersed leaves of the heterophyllous aquaticmacrophyte Potamogeton nodosus Poir. The yields of cells isolatedfrom floating leaves represented approximately 25% of the leafprotein or chlorophyll, while cell yields from submersed leaveswere only 3%. Photosynthetic activity was maximal in cells isolatedfrom submersed leaves 10 to 14 days after germination of thewinterbuds. Floating leaves were induced by treatment of theplants with abscisic acid. Cells from induced floating leavesshowed maximum photo synthetic rates between 9 and 21 days posttreatment.Phosphoglycerate, fructose 1,6-bisphosphate, sulfate and phosphatewere without significant effect on photosynthesis in eithercell type indicating that the cells were substantially intact.Half-saturation of photosynthesis for bicarbonate was at 0.6mM (pH 7.6) for cells from both leaf types, and the maximumrate was greater for cells from floating leaves. The light intensityfor half-saturation of photosynthesis was approximately 95 µEm^–2s^–1 for cells from both leaf types, and the maximumrate was greater for cells from floating leaves. (Received September 19, 1984; Accepted December 6, 1984)     

Proline antioxidant role in the common ice plant subjected to salinity and paraquat treatment inducing oxidative stress

Leaves of 4-week-old (juvenile) and 9-week-old (adult) plants of the halophyte Mesembryanthemum crystallinum L. (the common ice plant), cultured under controlled conditions in the phytotron, were treated with paraquat (0.1 μM), which produces superoxide radical, and (or) paraquat combined with introduction of NaCl (100 mM) or proline (5 mM) into nutrient medium. After a 20-h dark period (23°C), plants were transferred into light (4 h at 54.1 W/m² of photosynthetically active radiation) for stimulation of O°₂⁻ formation in plastids. Activities of antioxidant enzymes, the contents of MDA, H₂O₂, chlorophyll, and free proline were measured in leaves. Plant responses in two age groups, which differed in the type of photosynthesis (juvenile plants had C₃ type of photosynthesis, whereas adult plants were at the transition stage to Crassulacean Acid Metabolism (CAM) photosynthesis), differed in the levels of constitutive proline and proline, induced by NaCl and paraquat, as well as in activities of superoxide dismutase (SOD) and catalase. Changes in SOD activity and proline accumulation in response to paraquat treatment combined with NaCl revealed opposite dependence to accumulation of proline: the more proline accumulated in leaves, the lower activity of the enzyme. In response to paraquat treatment, the content of chlorophylls a and b most drastically declined in juvenile plants. Negative effect of salinity on the content of chlorophylls was lower than that of paraquat and was almost the same in plants of both age groups. Protective effect of exogenous proline was most profound in the case of paraquat treatment. Exogenous proline decreased the rate of lipid peroxidation, the content of superoxide radical and, consequently, SOD activity (almost fivefold), and increased the content of chlorophylls (a and b) in leaves of adult plants. The obtained data suggest that stress-induced accumulation of proline in the common ice plant has both osmoprotectory and antioxidant functions.     

Effect of Light and Benzyladenine on Dark-Treated Growing Rice (Oryza sativa) Leaves II. Changes in Peroxidase Activity

Changes in peroxidase activity were studied in the attachedfirst leaf of dark-treated Oryza sativa L. cv. Bala seedlingsin response to benzyladenine and light treatments during laterperiods of leaf growth, prior to maturation. Darkness causeda mild decrease in peroxidase activity; but in illuminated leaves,the enzyme activity was stable at all times. There was a sharprise in peroxidase activity in dark-treated leaves upon lightor benzyladenine application, irrespective of the time of treatment.Benzyladenine treatment to illuminated leaves also caused arise in peroxidase activity. Exogenous hydrogen peroxide, glycolateand amizol resulted in a rise in peroxidase activity, whichwas further enhanced by benzyladenine treatment in both lightand dark incubated leaves. Proline maintained chlorophyll levels,whereas hydroxyproline caused chlorophyll degradation. Benzyladenineenhanced the proline effect and counteracted the hydroxyprolineeffect on chlorophyll. Both proline and hydroxyproline increasedperoxidase activity in the leaves of light and dark incubatedseedlings, and the enzyme activity further increased after benzyladeninetreatment. (Received December 7, 1984; Accepted May 8, 1985)     

Protection of Chinese hamster ovary cells from paraquat-mediated cytotoxicity by a low molecular weight mimic of superoxide dismutase (DF-Mn)

Paraquat exerts a cytotoxic effect of Chinese hamster ovary cells in culture via the superoxide radical (O₂⁻. We have described a superoxide dismutase (SOD) mimic based on manganese (DF-Mn) which consists of a one-to-one complex between desferrioxamine B (Desferal) and MnO₂. It is a small molecular weight molecule, easy to prepare and possesses considerable stability. It is now shown to protect mammalian cells from paraquat toxicity. Thus, 20 μM DF-Mn affords up to complete protection against the cytotoxicity of 200 μM paraquat in Chinese hamster ovary cells. Desferrioxamine B or MnO₂ alone gave no protection. MnCl₂ or catalase provided little or no protection against the paraquat, respectively. Equivalent amounts of human Cu-Zn SOD in terms of activity, also provided no protection. Copper diisopropylsalicylate (CuDIPS) provided limited, yet significant, protection, but this is explained in terms other than SOD activity. Finally, at higher concentrations, purified human SOD, exerts a limited toxicity as well as a protective ability against paraquat (similar to DF-Mn) both of which are eliminated upon heat denaturation of the enzyme. It appears that the SOD mimic, DF-Mn, can enter mammalian cells and can protect against the cytotoxic effects of O₂⁻.     

NADP+-Dependent Sorbitol Dehydrogenase Found in Apple Leaves

An NADP⁺-dependent sorbitol dehydrogenase that catalyzes sorbitoland glucose was found in apple leaves. The partially purifiedenzyme had optimum activity at pH 9.6 and a K_m value of 128mM for sorbitol. Among the polyols studied, this enzyme showedthe most activity for sorbitol. ¹This paper is contribution A-173 of the Fruit Tree ResearchStation. (Received June 4, 1984; Accepted July 31, 1984)     

Effect of the free radical-generating herbicide paraquat on the expression of the superoxide dismutase (Sod) genes in maize

10-day-old maize leaves were treated with the oxygen free radical-generating herbicide paraquat for 12 h. Paraquat treatments (10(-5) M) resulted in a 40% increase in superoxide dismutase activity and a smaller increase in catalase activity. The increase in total superoxide dismutase (SOD) activity correlates with higher levels of specific isozymes. The chloroplast (SOD-1) and cytosolic (SOD-2 and SOD-4) forms were increased significantly; however, the mitochondrial form (SOD-3) was increased only slightly. Higher levels of SOD-4 and SOD-3 after paraquat exposure were the result of increased synthesis of these proteins, as determined by labeling in vivo with [35S]methionine. Isolation and in vitro translation of polysomes from 10(-5) M paraquat-treated leaves indicated that paraquat increased the amount of polysomal mRNA which codes for SOD-4 and SOD-3. Superoxide dismutase induction does not appear to be a response that is specific to paraquat, since another superoxide-generating compound, juglone, caused a similar increase in total superoxide dismutase activity. Therefore, the effect of these compounds on the expression of the maize Sod genes is exerted via their ability to generate superoxide.     

Embryogenic Callus Induction from Coffea arabica Leaf Explants by Benzyladenine

Coffea arabica leaf explants cultured on medium with 5 µM6-benzyladenine (BA) as the sole plant growth regulator producedwhite friable calluses that formed somatic embryos. These calluseshave been subcultured on the same medium for more than 2 yearsand maintain the ability to produce somatic embryos. (Received October 3, 1984; Accepted January 18, 1985)     

Differential reponses to paraquat‐induced oxidative injury in a pea (Pisum sativum) protoplast system

Antioxidant responses to varying degrees of paraquat stress in freshly isolated photosynthesizing pea (Pisum sativum L.) protoplasts from cultivars Progress and Nugget were studied. Leaves of comparable maturity were used for protoplast isolation. Nugget protoplasts were more resistant to paraquat in the micromolar range under our conditions. In Nugget, a non-bleaching paraquat concentration (10 µM) inhibited CO₂-dependent O₂ evolution ca 50% during the first 40 min, remaining at that rate (“coping behavior”) for up to 100 min. In contrast, Progress protoplasts treated with the same concentration of paraquat did not exhibit coping behavior. Antioxidant enzyme activities were unaltered throughout the time course of the experiment in treated protoplasts from Nugget and in chloroplasts isolated from them. Thus, the coping behavior of Nugget protoplasts cannot be attributed to changes in activities of the three antioxidant enzymes tested. Paraquat treatment did not affect antioxidant enzyme activities in Progress protoplasts nor in chloroplasts isolated from them. When higher doses of paraquat were used (12 h, 0.1 mM paraquat), protoplasts from both cultivars were rapidly bleached and total protein decreased to ca 30% of pre-stress levels. Glutathione reductase (GR, EC 1.6.4.2) activity dropped in protoplasts from both cultivars under the severe stress conditions in concert with declines in protein levels. However, superoxide dismutase (SOD, EC 1.15.1.1) activity remained constant over the first 9 h of the time course, increasing to ca 150& of original levels by the final, 12-h time point. The activity of the plastid Cu,Zn isoform, expressed as a percentage of total SOD activity, declined over the time course of the experiment while that of mitochondrial MnSOD appeared to increase. This change in activity of MnSOD correlated with cell decline, therefore, and was not correlated with protection. These data are in agreement with some earlier reports and are compatible with the hypothesis that SOD activity levels increase in response to reactive oxygen species levels, even under conditions leading to cell death.     

Effects of inhibitors on the enzyme system producing C6-aldehydes from C18-unsaturated fatty acids in chloroplasts of Japanese silver (Farfugium japonicum) leaves

When chloroplasts isolated from Farfugium japonicum (Japanesesilver) leaves were used as an enzyme source, the activity ofthe enzyme system producing C₆-aldehydes (cis-3-hexenal andn-hexanal) from C₁₈-unsaturated fatty acids (linolenic and linoleicacids) decreased upon treatment with LAHase from potato. Thisenzyme system could not be separated from chlorophylls and lipidsby detergent treatment and was not affected by light illumination,CCCP or DCMU. The activity of the enzyme system was inhibitedby MB and NTB used as a redox reagent, SKF 525-A as an oxidaseinhibitor and DABCO as a quencher of singlet oxygen, but notby DCIP, PMS and SOD. These data suggest that; i) interactionof the enzyme system with lipids is required for maximal enzymeactivity, ii) this enzyme system may involve electron mediator(s),and iii) singlet oxygen takes part in the enzyme reaction. (Received October 28, 1977; )     

苜蓿抗乙硫氨酸愈伤组织的筛选和特性

用乙硫氨酸为筛选剂,通过幼苗和组织培养筛选得到乙硫氨酸抗性愈伤组织。该愈伤组织在含乙硫氨酸的培养基上表现出较高的半抑制剂量和相对生长量。作为天门冬氨酸途径的产物,甲硫氨酸、异亮氨酸和赖氨酸在所筛选的愈伤组织中分别增加到对照的两倍多,但苏氨酸保持正常水平,另外酪氨酸、半胱氨酸和亮氨酸也有所增加,而在所筛选的愈伤组织中缬氨酸浓度却下降。说明在所筛选愈伤组织中存在一个以上与氨基酸合成相关的酶发生改变。同工酶分析表明,该愈伤组织中出现对照中没有的分子量为44kD的超氧化物歧化酶和分子量为45kD的酯酶谱带。     

Expression of bovine superoxide dismutase in Drosophila melanogaster augments resistance of oxidative stress.

Superoxide dismutases (SOD) play a major role in the intracellular defense against oxygen radical damage to aerobic cells. In eucaryotes, the cytoplasmic form of the enzyme is a 32-kDa dimer containing two copper and two zinc atoms (CuZn SOD) that catalyzes the dismutation of the superoxide anion (O2-) to H2O2 and O2. Superoxide-mediated damage has been implicated in a number of biological processes, including aging and cancer; however, it is not certain whether endogenously elevated levels of SOD will reduce the pathological events resulting from such damage. To understand the in vivo relationship between an efficient dismutation of O2- and oxidative injury to biological structures, we generated transgenic strains of Drosophila melanogaster overproducing CuZn SOD. This was achieved by microinjecting Drosophila embryos with P-elements containing bovine CuZn SOD cDNA under the control of the Drosophila actin 5c gene promoter. Adult flies of the resulting transformed lines which expressed both mammalian and Drosophila CuZn SOD were then used as a novel model for evaluating the role of oxygen radicals in aging. Our data show that expression of enzymatically active bovine SOD in Drosophila flies confers resistance to paraquat, an O2(-)-generating compound. This is consistent with data on adult mortality, because there was a slight but significant increase in the mean lifespan of several of the transgenic lines. The highest level of expression of the active enzyme in adults was 1.60 times the normal value. Higher levels may have led to the formation of toxic levels of H2O2 during development, since flies that died during the process of eclosion showed an unusual accumulation of lipofuscin (age pigment) in some of their cells. In conclusion, our data show that free-radical detoxification has a minor by positive effect on mean longevity for several strains.     

The Relationship of Abscisic Acid to Nitrate Reductase Activity in the Potato Plant

Abscisic acid (ABA) at 3.8 µM suppressed both in vivoand in vitro nitrate reductase activity in roots, stems andleaves of potato plants grown in solution culture. Suppressionwas maximal between 24 and 48 h, followed by recovery of activityat 72 h in roots and leaves and at 96 h in stems. Removal from ABA after 24 h resulted in complete recovery ofnitrate reductase activity in roots by 24 h and partial recoveryin leaves. ABA treatment enhanced nitrate accumulation in roots,decreased that of leaves, but had no effect on stem nitratecontent. ABA enhanced decay of the enzyme following nitrate removal;by 7 h activity in roots was 22.5% of the initial value comparedto 55% in the control. ABA showed a less drastic effect on lossof activity in leaves and stems. These results indicate thatABA suppression of nitrate reductase activity is not dependenton nitrate uptake, and although it reduced leaf nitrate contentthere was no clear relationship between tissue nitrate levelsand the ABA response. (Received September 13, 1984; Accepted July 1, 1985)     

Some Properties of the Arginine Decarboxylase in Vicia faba Leaves

Growth of Vicia faba seedlings is accompanied by a rapid increasein arginine decarboxylase (EC 4.1.1.19 [EC] ) in the leaves and epicotyl.Increased enzyme activity was observed under saline conditionsin the presence of NaCl and with osmotic stress by mannitol.The partially purified enzyme (about 86-fold) readily decarboxylatedL-arginine, while D-arginine, L-homoarginine, L-ornithine andL-lysine were decarboxylated very slowly, and L-citrulline andL-glutamic acid were not decarboxylated. The K_m value was 5.8?10^–4M for L-arginine. The optimal pH and temperature for activitywere 8.5 and 45?C, respectively. p-Chloromercuribenzoate andN-ethylmaleimide were effective inhibitors of the enzyme. Inhibitionby spermidine, putrescine and agmatine suggested a possiblefeed-back mechanism in the pathway of polyamine biosynthesis. (Received October 11, 1983; Accepted February 24, 1984)