Activity Changes in Selected Enzymes from Soybean Leaves Following Ozone Exposure

Soybeans [Glycine max(L.) Merr.] were harvested at various time periods after a 2-h exposure to either 0 or 0.5 μ1/1 ozone to determine the effects of ozone on selected enzymes. Carbohydrate metabolism was modified by a depression of glyceraldehyde 3-phaosphate dehydrogenase and an activation of glucose 6-phosphate dehydrogenase. Ozone did not alter the levels of RNase, protease, acid phosphatase or esterase as might be expected if ozone enhanced leaf senescence. The activities of phenylalanine ammonia lyase, polyphenol oxidase and peroxidase were initially depressed and then stimulated following the ozone exposure. The reactions of soybeans to an acute ozone stress were more nearly akin to those elicited in response to other stresses than to the process of senescence.     

Alteration of Response of Bean Leaves to Compatible and Incompatible Bacteria

Injection of Red Mexican bean leaves with Pseudomonas phascolicolaRace 2 (compatible, 18 h before P. mors-prunorum or P. phaseolicolaRace 1 (incompatible), or simultaneous inoculation with compatibleand incompatible bacteria (3:1) greatly delayed the appearanceof hypersensitive responses. When compatible bacteria were inoculated12 h or less before incompatible bacteria, or when the ratioof these bacteria was 1:1 or 0.3:1 in simultaneous inoculation,hypersensitive responses did develop. Earlier inoculation withincompatible bacteria delayed the appearance and severity ofdisease symptoms following later inoculation with compatiblebacteria. When selected areas of leaves were inoculated with compatiblebacteria, effects on hypersensitive responses were confinedto these areas when the whole leaf was inoculated later withincompatible bacteria. Inoculation through the upper leaf surfacewith incompatible bacteria did not affect susceptible responseswhen compatible bacteria were inoculated 24 h later throughthe lower surface. Treatment of leaves with heat-killed bacteria or live bacteriain numbers insufficient to cause hypersensitive responses didnot prevent development of these responses following later inoculationwith incompatible bacteria. Use of heat-killed bacteria didsuppress hypersensitive responses in tobacco leaves. Injectionof leaves with cycloheximide (20 p.p.m.) 30 min before inoculationwith incompatible bacteria suppressed leakage of electrolytesand browning of tissues associated with hypersensitive responses.Cycloheximide had little effect on leakage of electrolytes fromleaves inoculated with compatible bacteria or with the developmentof susceptible responses. Exposure of leaves to chloroform vapourdelayed hypersensitive responses by 24 h; treatment with peroxidasehad no effect.     

Increased Resistance to the Spread of Tobacco Mosaic Virus in Pinto Bean Leaves Caused by Sugar and Light

Ultraviolet light (UV) irradiation increased expansion of TMV lesions in detached Pinto bean primary leaves incubated in darkness. However, if after UV-irradiation the leaves were incubated in the light, no increase in lesion expansion occurred. The light effect was considered not to be due to photorepair of UV damaged DNA, since non-photorepairing treatments such as incubation in red light, or delayed exposure to white light after UV irradiation also prevented increase in lesion expansion. The effect of visible light in preventing TMV-lesion enlargement was shown to be related to photosynthetic energy supply to the host cell defense mechanism since incubation of infected leaves in the presence of the photosynthesis inhibitor 3-(3,4-dichlorophenyl)-l,l-dimethyl urea (DCMU) in light caused large lesions whether leaves were irradiated by UV or not. Supplying 0.1 M sucrose in the dark also inhibited lesion enlargement in UV-irradiated or nonirradiated leaves. Dinitrophenol (DNP) negated the sucrose effect in the dark. However, in light incubation, DNP did not induce large lesions indicating that DNP did not interfere with energy supply in the light. It is concluded that the Pinto bean leaf cells can use energy derived both from mitochondria and chloroplasts for building the resistance mechanism to virus spread. In this case, cellular resistance to virus spread seems to be correlated with callose deposition on the walls of noninfected cells adjacent to the necrotic cells. Energy supply in various forms will assist host cells in building the resistance mechanism as well as retarding senescence. Detachment, prolonged dark incubation, or exogenous supply of DNP led to accelerated senescence which in turn led to secondary enlargement of lesions. The cause of such secondary enlargement may be explained by starvation of cells and disappearance of callose.     

Projected Carbon Dioxide to Increase Grass Pollen and Allergen Exposure Despite Higher Ozone Levels

One expected effect of climate change on human health is increasing allergic and asthmatic symptoms through changes in pollen biology. Allergic diseases have a large impact on human health globally, with 10–30% of the population affected by allergic rhinitis and more than 300 million affected by asthma. Pollen from grass species, which are highly allergenic and occur worldwide, elicits allergic responses in 20% of the general population and 40% of atopic individuals. Here we examine the effects of elevated levels of two greenhouse gases, carbon dioxide (CO₂), a growth and reproductive stimulator of plants, and ozone (O₃), a repressor, on pollen and allergen production in Timothy grass (Phleum pratense L.). We conducted a fully factorial experiment in which plants were grown at ambient and/or elevated levels of O₃ and CO₂, to simulate present and projected levels of both gases and their potential interactive effects. We captured and counted pollen from flowers in each treatment and assayed for concentrations of the allergen protein, Phl p 5. We found that elevated levels of CO₂ increased the amount of grass pollen produced by ∼50% per flower, regardless of O₃ levels. Elevated O₃ significantly reduced the Phl p 5 content of the pollen but the net effect of rising pollen numbers with elevated CO₂ indicate increased allergen exposure under elevated levels of both greenhouse gases. Using quantitative estimates of increased pollen production and number of flowering plants per treatment, we estimated that airborne grass pollen concentrations will increase in the future up to ∼200%. Due to the widespread existence of grasses and the particular importance of P. pratense in eliciting allergic responses, our findings provide evidence for significant impacts on human health worldwide as a result of future climate change.     

Daily Fluctuations of Antioxidants in Bean (Phaseolus vulgaris L.) Leaves As Affected by the Presence of Ambient Air Pollutants

During the period 0800–1700 h (GMT) of a summer day, youngleaves were collected every 20 min from Phaseolus vulgaris L.cv. Horticultural plants grown in open-top field chambers locatedat an urban site in northern Italy and exposed either to ambientlevels of gaseous air pollutants or to filtered ambient air.Ascorbic and dehydroascorbic acids, GSH and GSSG, superoxidedismutase, ascorbate peroxidase, dehydroascorbate reductase,GSSG reductase, GSH peroxidase, catalase, guaiacol peroxidase,chlorophylls, carotenoids, soluble protein and dry weight weremeasured in these leaves. The main differences between treatedand control leaves were observed during the period 1100–1530h and concerned superoxide dismutase, catalase, ascorbate peroxidase,ascorbate/dehydroascorbate and GSH/GSSG ratios, chlorophylls,carotenoids and dry weight. On the basis of the pollution climateobserved at the experimental site on the day of leaves sampling,ozone appeared to be the causative agent of the observed divergencesamong the time patterns of antioxidants in treated and controlplants. It was deduced that ozone can induce oxidative stressvia the production of superoxide radical anion and hydrogenperoxide. (Received June 1, 1992; Accepted December 9, 1992)     

Effects of Ozone and Sulfur Dioxide on Phyllosphere Fungi from Three Tree Species

Short-term effects of ozone (O₃) on phyllosphere fungi were studied by examining fungal populations from leaves of giant sequoia (Sequoiadendron giganteum (Lindl.) Buchholz) and California black oak (Quercus kelloggii Newb.). Chronic effects of both O₃ and sulfur dioxide (SO₂) were studied by isolating fungi from leaves of mature Valencia orange (Citrus sinensis L.) trees. In this chronic-exposure experiment, mature orange trees were fumigated in open-top chambers at the University of California, Riverside, for 4 years with filtered air, ambient air plus filtered air (1:1), ambient air, or filtered air plus SO₂ at 9.3 parts per hundred million. Populations of Alternaria alternata (Fr.) Keissler and Cladosporium cladosporioides (Fres.) de Vries, two of the four most common fungi isolated from orange leaves, were significantly reduced by chronic exposure to ambient air. In the short-term experiments, seedlings of giant sequoia or California black oak were fumigated in open-top chambers in Sequoia National Park for 9 to 11 weeks with filtered air, ambient air, or ambient air plus O₃. These short-term fumigations did not significantly affect the numbers of phyllosphere fungi. Exposure of Valencia orange trees to SO₂ at 9.3 parts per hundred million for 4 years reduced the number of phyllosphere fungi isolated by 75% compared with the number from the filtered-air treatment and reduced the Simpson diversity index value from 3.3 to 2.5. A significant chamber effect was evident since leaves of giant sequoia and California black oak located outside of chambers had more phyllosphere fungi than did seedlings within chambers. Results suggest that chronic exposure to ambient ozone or SO₂ in polluted areas can affect phyllosphere fungal communities, while short-term exposures may not significantly disturb phyllosphere fungi.     

The Influence of Plant-Growth Factors on the Initiation and Growth of Crown-Gall Tumours on Primary Pinto Bean Leaves

The number of tumours developing on primary pinto bean leaves(Phaseolus vulgaris L. variety Pinto) inoculated with Agrobacteriumtumefaciens (Smith and Town.) Conn, strain B6, was increasedby as much as 100 per cent through the addition of several plant-growthfactors and antagonists of some of these factors to the freshlyinoculated leaves. Naphthylacetic acid, gibberellic acid, (2-chloroethyl)trimethylammonium chloride, and adenine gave a biphasic responsewith optimal promotions of tumour initiation at 10^-5 to 10^-4mg/ml. Tri-iodobenzoic acid and 4-chlorophenoxyisobutyric acidwere most active at 10^-1 mg/ml. Mean tumour diameter showeda direct correlation with tumour number in these experiments.The results show tumour initiation to be sensitive to growthfactor changes, possibly through a heightened traumatic responseof the wounded leaves. The growth of the tumours was unaffectedby these additions except as they altered tumour number andthis secondarily affected tumour growth.     

Evidence for a Tumor-associated Factor Active in the Promotion of Crown-gall Tumor Growth on Primary Pinto Bean Leaves

The growth of crown-gall tumors on primary pinto bean leaves (Phaseolus vulgaris L. cv. “Pinto”) between day 3 and day 6 after inoculation was found to be proportional to the number of tumors on the leaves. Similar differences observed in the growth of tumors induced by adenine, methionine and asparagine requiring mutants of Agrobacterium tumefaciens (Smith and Town.) Conn appear to be due to the same phenomenon. Tumors induced by these auxotrophs thus show no obvious growth differences from those induced by the prototrophic strain despite the lower specific infectivity and the existence of a mutational lesion in these bacteria. A diffusible growth factor(s) produced by the tumor tissue is proposed to account for the relation between tumor number and early tumor growth.     

Changes in Activities of Enzymes of Carbon Metabolism in Leaves during Exposure of Plants to Low Temperature

The aim of this study was to determine the response of photosynthetic carbon metabolism in spinach and bean to low temperature. (a) Exposure of warm-grown spinach and bean plants to 10°C for 10 days resulted in increases in the total activities of a number of enzymes, including ribulose 1,5-bisphosphate carboxylase (Rubisco), stromal fructose 1,6 bisphosphatase (Fru 1,6-P₂ase), sedoheptulose 1,7-bisphosphatase (Sed 1,7-P₂ase), and the cytosolic Fru 1,6-P₂ase. In spinach, but not bean, there was an increase in the total activity of sucrose-phosphate synthase. (b) The CO₂-saturated rates of photosynthesis for the cold-acclimated spinach plants were 68% greater at 10°C than those for warm-acclimated plants, whereas in bean, rates of photosynthesis at 10°C were very low after exposure to low temperature. (c) When spinach leaf discs were transferred from 27 to 10°C, the stromal Fru 1,6-P₂ase and NADP-malate dehydrogenase were almost fully activated within 8 minutes, and Rubisco reached 90% of full activation within 15 minutes of transfer. An initial restriction of Calvin cycle fluxes was evident as an increase in the amounts of ribulose 1,5-bisphosphate, glycerate-3-phosphate, Fru 1,6-P₂, and Sed 1,7-P₂. In bean, activation of stromal Fru 1,6-P₂ase was weak, whereas the activation state of Rubisco decreased during the first few minutes after transfer to low temperature. However, NADP-malate dehydrogenase became almost fully activated, showing that no loss of the capacity for reductive activation occurred. (d) Temperature compensation in spinach evidently involves increases in the capacities of a range of enzymes, achieved in the short term by an increase in activation state, whereas long-term acclimation is achieved by an increase in the maximum activities of enzymes. The inability of bean to activate fully certain Calvin cycle enzymes and sucrose-phosphate synthase, or to increase nonphotochemical quenching of chlorophyll fluorescence at 10°C, may be factors contributing to its poor performance at low temperature.     

The Inhibition of Photosynthesis after Exposure of Bean Leaves to Various Low Levels of CO2

Mature first leaves of Phaseolus vulgaris L. were exposed tolow partial pressures of CO₂ (7, 6 and 0 Pa CO₂) for 24 h. Afterexposure of leaves to 6 Pa CO₂ for 24 h, there was a reductionin the carbon exchange rate (CER) at all partial pressures ofCO₂ at which measurements were made. After exposure to 7 PaCO₂, the CER decreased only at high partial pressures of CO₂.The rates of electron transport from water to methyl viologen,through the whole chain, decreased in parallel with the decreasein CER measured at 90 Pa CO₂. One site of inhibition in leavesexposed for 24 h to 6 Pa CO₂ appeared to be the intersystemelectron-transport chain since there were no significant changesin the activities of PSI and PSII, as determined from the levelof P-700 and measurement of fluorescence, respectively. Anotherinhibitory phenomenon appeared to be a negative change in theactivation state of Rubisco, while the level of Rubisco wasunaffected by the exposure to 6 Pa CO₂. These decreases in photosyntheticactivity caused by depletion of CO₂ explains at least in part,the inhibition of photosynthesis that is caused by rain treatment[Ishibashi and Terashima (1995) Plant Cell Environ. 18: 431]. (Received September 19, 1996; Accepted March 10, 1997)     

Ozone,Sulfur Dioxide,and Ultraviolet B Have Similar Effects on mRNA Accumulation of Antioxidant Genes in Nicotiana plumbaginifolia L

We have studied the expression of antioxidant genes in response to near ambient conditions of O3, SO2, and ultraviolet B (UV-B) in Nicotiana plumbaginifolia L. The genes analyzed encode four different superoxide dismutases (SODs), three catalases (Cat1, Cat2, and Cat3), the cytosolic ascorbate peroxidase (cyt APx), and glutathione peroxidase (GPx). The experimental setup for each treatment was essentially the same and caused no visible damage, thus allowing direct comparison of the different stress responses. Our data showed that the effects of O3, SO2, and UV-B on the antioxidant genes are very similar, although the response to SO2 is generally less pronounced and delayed. The effects of the different stresses are characterized by a decline in Cat1, a moderate increase in Cat3, and a strong increase in Cat2 and GPx. Remarkably, SODs and cyt APx were not affected. Analysis of SOD and APx expression in the ozone-sensitive Nicotiana tabacum L. cv PBD6 revealed that induction of the cytosolic copper/zinc SOD and cyt APx occurs only with the onset of visible damage. It is proposed that alterations in mRNA levels of catalases and GPx, but not of SODs and cyt APx, form part of the initial antioxidant response to O3, SO2, and UV-B in Nicotiana.     

Growth of Lesions Caused by Botrytis fabae on Field Bean Leaves in Relation to Foliar Bacteria, Non-enzymic Phytotoxins, Pectic Enzymes and Osmotica

Extracts from spreading chocolate spot lesions contained a heat-stable,water-soluble, ether- or ethanol-insoluble phytotoxic fraction.Elution from Sephadex G75 indicated that the molecular weightsof the toxic compounds were between 10000 and 30000 daltons.The toxins were adsorbed on DEAE Sephadex and eluted from itwith 0.1–0.2 M NaCl. Toxic activity was enhanced in solutionswith low osmotic potentials similar to those found in lesions.Controlling the growth of contaminating bacteria in lesionswith antibiotics appeared to reduce the levels of heat-stabletoxins extracted and to reduce the rate of increase in lesionsize. There were high levels of polygalacturonase (PG) activity inlesion extracts; large quantitites of PG, but little or no trans-eliminases,were produced by Botrytis fabae in liquid culture. A pectolyticstrain of Bacillus lentus was associated with trans-eliminasesin lesion extracts, and produced transeliminases, but not PG,in liquid culture. Activities of both heat-stable phytotoxins and of pectic enzymesmay depend on fungal isolate and types and populations of contaminatingbacteria. Botrytis fabae L., Bacillus, Vicia faba L., phytotoxins, pectic enzymes, chocolate spot     

广东园林绿化植物对大气污染的反应及污染物在叶片的积累

根据盆栽试验植物生长参数(株高、基径、冠幅、叶面积、新叶增长率和叶片脱落率),生理特性(包括净光合速率、叶绿素荧光特性,叶细胞膜渗漏率和叶绿素含量),植物伤害状况,以及长期生长在野外污染环境中的植物生长及受害状况,评价了125种木本植物对空气污染敏感性的反应。被评价的植物其生长环境为酸雨、硫化物、氟化物和粉尘复合污染的工业点,交通繁忙点和居民点。被评价植物对大气污染的敏感性反应分为3类：1．抗性强有35种;2．抗性中等有41种;3.最敏感(抗性弱)有49种。同时还分析了48种植物叶片中S、F、Pb的累积量。研究结果可为中国热带南亚热带相似大气环境地区建立城市植被和生态公益林提供植物种选择的依据,     

Some Features of NADP-Dependent Isocitrate Dehydrogenase Functioning in Pea Leaves upon Exposure to Salt Stress

In pea seedlings transferred to a medium containing 3% NaCl, NADP-dependent isocitrate dehydrogenase (NADP-IDH) activity in leaves increased more than twofold during the first hour, decreased, and increased again (approximately 1.5 times) by the 14th hour of exposure. Some catalytic and regulatory properties of NADP-IDH were studied using enzyme preparations purified from normal plants and plants exposed to salt stress. The results showed that K _m for NADP-IDH in reactions with isocitrate and NADP decreased under stress by factors of 2 and 3, respectively. Specific features of enzyme activity regulation by citrate and trans-aconitate under these conditions were revealed. The inhibitory effects of -aminobutyric acid (GABA) and 2-oxoglutarate on NADP-IDH from plants exposed to salt stress was stronger than that on the enzyme from normal plants. Glutamine and glutamate slightly activated the enzyme in both cases.     

Plant Resistance Mechanisms to Air Pollutants: Rhythms in Ascorbic Acid Production During Growth Under Ozone Stress

Relationships between ozone (O₃) tolerance and leaf ascorbic acid concentrations in 0₃-susceptible (O₃S) 'Hark' and O₃-resistant (O₃R) 'Hood'soybean, Glycine max (L.) Merr., cultivars were examined with high-performance liquid chromatography (HPLC). Leaf samples were analyzed at 4 h intervals during a 24 h period. Soybean cultivars grown in the greenhouse with charcoal filtered (CF) and nonfiltered (NF) air showed daily oscillations in ascorbic acid production. Highest ascorbic acid levels in leaves during light coincided with highest concentrations of photochemical oxidants in the atmosphere at 2:00 p.m. The resistant genotype produced more ascorbic acid in its trifoliate leaves than did the corresponding susceptible genotype. Under CF air (an O₃-reduced environment) O₃-S and O₃-R cultivars showed rhythms in ascorbic acid production. In NF air (an O₃ stress environment) the O₃-R cultivar alone showed rhythms in ascorbic acid production. Results indicated that superior O₃ tolerance in the Hood soybean cultivar (compared with Hark) was associated with a greater increase in endogenous levels of ascorbic acid. Ascorbic acid may scavenge free radicals and thereby protect cells from injury by O₃ or other oxyradical products. Plants defend themselves against photochemical oxidant stress, such as O₃, by several mechanisms. Experimental evidence indicates that antioxidant defense systems existing in plant tissues may function to protect cellular components from deleterious effects of photochemical oxidants through endogenous and exogenous controls.     

The Development of Activities of Some Enzymes Concerned with Glycollate Metabolism in Greening Bean Leaves

The activities of phosphoglycollate phosphatase (EC 3.1.3.18 [EC] ),glycollate oxidase (EC 1.1.3.1 [EC] .). catalase (EC 1.11.1.6 [EC] ), theperoxisomal NADH-glyoxylate reductase (EC 1.1.1.26 [EC] ) which isconsidered to function as a hydroxypyruvate reductase in theperoxisomes, and the chloro-plastic NADPH-dependent glyoxylatereductaae, have been measured in extracts prepared from 14-d-olddark-grown bean leaves during the course of their greening inresponse to exposure to continuous illumination. All of theenzymes were found in the dark-grown leaves and on a per-leafbasis the activities increased from 6- to 12-fold with the exceptionof a 2–3-fold increase of NADPH-dependent glyoxylate reductaseduring 96-h greening, while the activities either remained constantor declined during similar periods in darkness. Initial lagperiods were evident before the illumination-induced increasesin enzyme activities. As D-threo-chloramphenicol did not affectthe increase in activity of any of these enzymes it would appearthat the increases were in no way dependent on protein synthesisby 70S ribosomes, or on the development of photosynthetic activity.     

Activities of Carbonic Anhydrase and Carboxylating Enzymes in Chlamydomonas segnis Adapted and Adapting to Air Levels of Carbon Dioxide

Activities of carbonic anhydrase (CA), phosphoenolpyruvate carboxylase(PEPCase) and ribulose 1,5-bisphosphate carboxylase (RuBPCase)were compared in air- and 5% CO₂adapted and adapting cells ofChlamydomonas segnis during the cell cycle in continuously illuminatedsynchronous cultures. Air-adapted cells exhibited considerablylower PEPCase activities, but higher RuBPCase and CA activitiesthan 5% CO²-adapted cells. Most (75 to 88%) of the CA activityin air-adapted cells appeared to be located in the periplasmicspace. Transferring 5% CO₂-adapted cells to air scarcely influencedRuBPCase activity, but led to 85% decrease in the activity ofPEPCase and to 400% increase in that of CA. In such air-adaptingcells, more than half (54 to 70%) of the CA activity was intracellular.The proposal that PEPCase and RuBPCase in addition to CA maybe involved in the regulation of inorganic carbon uptake byair-adapted and adapting cells under CO₂-limiting conditionsis discussed. (Received June 11, 1987; Accepted September 5, 1987)     

Cell Wall Accumulation of Cu Ions and Modulation of Lignifying Enzymes in Primary Leaves of Bean Seedlings Exposed to Excess Copper

Copper is both a nutrient and an environmental toxin that is taken up by plants. In order to determine the subcellular localization of copper and to assess the resulting metabolic changes, we exposed 14-day-old bean seedlings to nutrient solutions containing varying concentrations of Cu²⁺ ions for 3 days. Biochemical analyses revealed that the cell wall was the major site of Cu²⁺ accumulation in the leaves of treated plants. Excess copper modified the activity of lignifying peroxidases in both soluble and ionic cell wall-bound fraction. The activity of ionic GPX (guaiacol peroxidase, EC 1.11.1.7) was increased by 50 and 75 μM CuSO_4. The activities of both ionic CAPX (coniferyl alcohol peroxidase, EC 1.11.1.4) and NADH oxidase were increased by both copper concentrations tested. While soluble CAPX activity decreased in leaves treated by all copper concentrations tested, the activity of soluble NADH oxidase remained unchanged at 50 μM and was enhanced at 75 μM. Treatment with CuSO₄ also increased the abundance of total phenol compounds and induced stimulation in the activity of PAL (phenylalanine ammonia lyase, EC. 4.3.1.5). Using histochemistry in combination with fluorescence microscopy we show that bean leaves from copper-exposed plants displayed biochemical and structural modifications reinforcing the cell walls of their xylem tissues. On the other hand, the perivascular fiber sclerenchyma appeared to be less developed in treated leaves.