Overexpression of Farnesyl Diphosphate Synthase in Arabidopsis Mitochondria Triggers Light-dependent Lesion Formation and Alters Cytokinin Homeostasis

To investigate the role of mitochondrial farnesyl diphosphate synthase (FPS) in plant isoprenoid biosynthesis we characterized transgenic Arabidopsis thaliana plants overexpressing FPS1L isoform. This overexpressed protein was properly targeted to mitochondria yielding a mature and active form of the enzyme of 40 kDa. Leaves from transgenic plants grown under continuous light exhibited symptoms of chlorosis and cell death correlating to H₂O₂ accumulation, and leaves detached from the same plants displayed accelerated senescence. Overexpression of FPS in mitochondria also led to altered leaf cytokinin profile, with a reduction in the contents of physiologically active trans-zeatin- and isopentenyladenine-type cytokinins and their corresponding riboside monophosphates as well as enhanced levels of cis-zeatin 7-glucoside and storage cytokinin O-glucosides. Overexpression of 3-hydroxy-3-methylglutaryl coenzyme A reductase did not prevent chlorosis in plants overexpressing FPS1L, but did rescue accelerated senescence of detached leaves and restored wild-type levels of cytokinins. We propose that the overexpression of FPS1L leads to an enhanced uptake and metabolism of mevalonic acid-derived isopentenyl diphosphate and/or dimethylallyl diphosphate by mitochondria, thereby altering cytokinin homeostasis and causing a mitochondrial dysfunction that renders plants more sensitive to the oxidative stress induced by continuous light.     

Drought-induced senescence is characterized by a loss of antioxidant defences in chloroplasts

The relationship between drought, oxidative stress and leaf senescence was evaluated in field‐grown sage (Salvia officinalis L.), a drought‐susceptible species that shows symptoms of senescence when exposed to stress. Despite the photoprotection conferred by the xanthophyll cycle, drought‐stressed senescing leaves showed enhanced lipid peroxidation, chlorophyll loss, reduced photosynthetic activity and strong reductions of membrane‐bound chloroplastic antioxidant defences (i.e. β‐carotene and α‐tocopherol), which is indicative of oxidative stress in chloroplasts. H₂O₂ accumulated in drought‐stressed senescing leaves. Subcellular localization studies showed that H₂O₂ accumulated first in xylem vessels and the cell wall and later in the plasma membrane of mesophyll cells, but not in chloroplasts, indicating reactive oxygen species other than H₂O₂ as direct responsible for the oxidative stress observed in the chloroplasts of drought‐stressed senescing leaves. The strong degradation of β‐carotene and α‐tocopherol suggests an enhanced formation of singlet oxygen as the putative reactive oxygen species responsible for oxidative stress to senescing chloroplasts. This study demonstrates that oxidative stress in chloroplasts mediates drought‐induced leaf senescence in sage growing in Mediterranean field conditions.     

Metalliferous and non-metalliferous populations of Viola tricolor represent similar mode of antioxidative response

Heavy metal-contaminated sites are excellent areas to examine the antioxidative machinery responsible for physiological adaptations of many plant species.Superoxide dismutase (SOD), guaiacol peroxide (GPX), ascorbate peroxide (APX), catalase (CAT) activity and hydrogen peroxide (H₂O₂) content were analyzed in leaves and roots of Viola tricolor (Viola) from contaminated soils (‘Bukowno’, ‘Saturn’, ‘Warpie’ heaps), and non-contaminated soil (‘Zakopane meadow’) to examine the level of oxidative stress and antioxidative response.In leaves, six isoforms of SOD were recognized. Roots possessed two additional bands, named manganese superoxide dismutase (MnSOD)-like form (MnSODI) and Cu/ZnSOD-like form (Cu/ZnSODIV). The H₂O₂ content in leaves ranged from 554 to 5 098 μmol H₂O₂/g f.w. and was negatively correlated with CAT activity. The non-contaminated population was characterized by the lowest CAT activity combined with the highest H₂O₂ concentration. Two isoforms of CAT, CAT-1 and CAT-2, were recognized in leaves of plants from non-contaminated and contaminated sites, respectively. In roots of individuals from two heaps (‘Warpie’ and ‘Saturn’), two distinct bands for each CAT isoform were observed. A slower migrating band may be an aggregate, exhibiting CAT and MnSODs activities. Both peroxidases (APX and GPX) presented the same pattern of activity, depending on the organ, indicating that in leaves and roots APX and GPX were regulated in parallel.Differences in enzyme activities and H₂O₂ content between plants from different contaminated sites were statistically significant, but were tightly maintained at a very similar level. Prolonged and permanent heavy metal stress evoked a very similar mode of antioxidative response in specimens of analyzed metalliferous populations not causing measurable oxidative stress. Thus, our results clearly indicate that V. tricolor is a taxon well adapted to heavy metal-contaminated soils, and that differences in enzyme activities and H₂O₂ content result from adjustment of plants to a variety of conditions.     

Nitric oxide acts as an antioxidant and delays methyl jasmonate-induced senescence of rice leaves

In the present study, we evaluate the protective effect of nitric oxide (NO) against senescence of rice leaves promoted by methyl jasmonate (MJ). Senescence of rice leaves was determined by the decrease of protein content. MJ treatment resulted in (1) induction of leaf senescence, (2) increase in H₂O₂ and malondialdehyde (MDA) contents, (3) decrease in reduced form glutathione (GSH) and ascorbic acid (AsA) contents, and (4) increase in antioxidative enzyme activities (ascorbate peroxidase, glutathione reductase, peroxidase and catalase). All these MJ effects were reduced by free radical scavengers such as sodium benzoate and GSH. NO donors [N-tert-butyl-α-phenylnitrone (PBN), sodium nitroprusside, 3-morpholinosydonimine, and AsA+NaNO₂] were effective in reducing MJ-induced leaf senescence. PBN prevented MJ-induced increase in the contents of H₂O₂ and MDA, decrease in the contents of GSH and AsA, and increase in the activities of antioxidative enzymes. The protective effect of PBN on MJ-promoted senescence, MJ-increased H₂O₂ content and lipid peroxidation, MJ-decreased GSH and AsA, and MJ-increased antioxidative enzyme activities was reversed by 2-(4-carboxy-2-phenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide, a NO-specific scavenger, suggesting that the protective effect of PBN is attributable to NO released. Reduction of MJ-induced senescence by NO in rice leaves is most likely mediated through its ability to scavenge active oxygen species including H₂O₂     

Role of the Ascorbate-Glutathione Cycle of Mitochondria and Peroxisomes in the Senescence of Pea Leaves

We investigated the relationship between H₂O₂ metabolism and the senescence process using soluble fractions, mitochondria, and peroxisomes from senescent pea (Pisum sativum L.) leaves. After 11 d of senescence the activities of Mn-superoxide dismutase, dehydroascorbate reductase (DHAR), and glutathione reductase (GR) present in the matrix, and ascorbate peroxidase (APX) and monodehydroascorbate reductase (MDHAR) activities localized in the mitochondrial membrane, were all substantially decreased in mitochondria. The mitochondrial ascorbate and dehydroascorbate pools were reduced, whereas the oxidized glutathione levels were maintained. In senescent leaves the H₂O₂ content in isolated mitochondria and the NADH- and succinate-dependent production of superoxide (O₂^·−) radicals by submitochondrial particles increased significantly. However, in peroxisomes from senescent leaves both membrane-bound APX and MDHAR activities were reduced. In the matrix the DHAR activity was enhanced and the GR activity remained unchanged. As a result of senescence, the reduced and the oxidized glutathione pools were considerably increased in peroxisomes. A large increase in the glutathione pool and DHAR activity were also found in soluble fractions of senescent pea leaves, together with a decrease in GR, APX, and MDHAR activities. The differential response to senescence of the mitochondrial and peroxisomal ascorbate-glutathione cycle suggests that mitochondria could be affected by oxidative damage earlier than peroxisomes, which may participate in the cellular oxidative mechanism of leaf senescence longer than mitochondria.     

Effects of cytokinin and senescence-inducing factors on expression of P ARR5 -GUS gene construct during leaf senescence in transgenic Arabidopsis thaliana plants

ARR5-gene expression was studied in the course of natural leaf senescence and detached leaf senescence in the dark using Arabidopsis thaliana plants transformed with the P _ARR5 -GUS gene construct. GUS-activity was measured as a marker of ARR5-gene expression. Chlorophyll and total protein amounts were also estimated to evaluate leaf senescence. Natural leaf senescence was accompanied by the progressive decline in the GUS-activity in leaves of the 2nd and 3rd nodes studied, and this shift of GUS-activity was more pronounced than the loss of chlorophyll content. The ability of the ARR5-gene promoter to respond to cytokinin was not eliminated during natural leaf senescence, as was demonstrated by a cytokinin-induced increase in GUS activity in leaves after their detachment and incubation on benzyladenine (BA, 5 × 10⁻⁶ M) in the dark. Leaf senescence in the dark was associated with the further decrease in the GUS-activity. The ARR5-gene promoter response to cytokinin was enhanced with the increase of the age of plants, taken as a source of leaves for cytokinin treatments. Hence, although the expression of the ARR5 gene reduces during natural and dark/detached leaf senescence, the ARR5-gene sensitivity to cytokinin was maintained in both cases and even increased with the leaf age. This data suggest that the ARR5 gene, which belongs to the type-A negative regulators of plant response to cytokinin, could be a feedback regulator able to prevent retardation by cytokinin of leaf senescence when it is important for plant life. Growth regulators either reduced ARR5 gene response to cytokinin during senescence of mature detached leaves in the dark (SA, meJA, ABA, SP) or increased it (IAA), thus modifying the resulting rate of its expression.     

Selenoprotein H Suppresses Cellular Senescence through Genome Maintenance and Redox Regulation

Oxidative stress and persistent DNA damage response contribute to cellular senescence, a degeneration process critically involving ataxia telangiectasia-mutated (ATM) and p53. Selenoprotein H (SelH), a nuclear selenoprotein, is proposed to carry redox and transactivation domains. To determine the role of SelH in genome maintenance, shRNA knockdown was employed in human normal and immortalized cell lines. SelH shRNA MRC-5 diploid fibroblasts under ambient O₂ displayed a distinct profile of senescence including β-galactosidase expression, autofluorescence, growth inhibition, and ATM pathway activation. Such senescence phenotypes were alleviated in the presence of ATM kinase inhibitors, by p53 shRNA knockdown, or by maintaining the cells under 3% O₂. During the course of 5-day recovery, the induction of phospho-ATM on Ser-1981 and γH2AX by H₂O₂ treatment (20 μm) subsided in scrambled shRNA but exacerbated in SelH shRNA MRC-5 cells. Results from clonogenic assays demonstrated hypersensitivity of SelH shRNA HeLa cells to paraquat and H₂O₂, but not to hydroxyurea, neocarzinostatin, or camptothecin. While SelH mRNA expression was induced by H₂O₂ treatment, SelH-GFP did not mobilize to sites of oxidative DNA damage. The glutathione level was lower in SelH shRNA than scrambled shRNA HeLa cells, and the H₂O₂-induced cell death was rescued in the presence of N-acetylcysteine, a glutathione precursor. Altogether, SelH protects against cellular senescence to oxidative stress through a genome maintenance pathway involving ATM and p53.     

Photosynthetic electron flow affects H2O2 signaling by inactivation of catalase in Chlamydomonas reinhardtii

A specific signaling role for H₂O₂ in Chlamydomonas reinhardtii was demonstrated by the definition of a promoter that specifically responded to this ROS. Expression of a nuclear-encoded reporter gene driven by this promoter was shown to depend not only on the level of exogenously added H₂O₂ but also on light. In the dark, the induction of the reporter gene by H₂O₂ was much lower than in the light. This lower induction was correlated with an accelerated disappearance of H₂O₂ from the culture medium in the dark. Due to a light-induced reduction in catalase activity, H₂O₂ levels in the light remained higher. Photosynthetic electron transport mediated the light-controlled down-regulation of the catalase activity since it was prevented by 3-(3′4′-dichlorophenyl)-1,1-dimethylurea (DCMU), an inhibitor of photosystem II. In the presence of light and DCMU, expression of the reporter gene was low while the addition of aminotriazole, a catalase inhibitor, led to a higher induction of the reporter gene by H₂O₂ in the dark. The role of photosynthetic electron transport and thioredoxin in this regulation was investigated by using mutants deficient in photosynthetic electron flow and by studying the correlation between NADP-malate dehydrogenase and catalase activities. It is proposed that, contrary to expectations, a controlled down-regulation of catalase activity occurs upon a shift of cells from dark to light. This down-regulation apparently is necessary to maintain a certain level of H₂O₂ required to activate H₂O₂-dependent signaling pathways.     

NADPH oxidase inhibitor diphenyleneiodonium and reduced glutathione mitigate ethephon-mediated leaf senescence,H2O2 elevation and senescence-associated gene expression in sweet potato (Ipomoea batatas)

Ethephon, an ethylene releasing compound, promoted leaf senescence, H₂O₂ elevation, and senescence-associated gene expression in sweet potato. It also affected the glutathione and ascorbate levels, which in turn perturbed H₂O₂ homeostasis. The decrease of reduced glutathione and the accumulation of dehydroascorbate correlated with leaf senescence and H₂O₂ elevation at 72 h in ethephon-treated leaves. Exogenous application of reduced glutathione caused quicker and significant increase of its intracellular level and resulted in the attenuation of leaf senescence and H₂O₂ elevation. A small H₂O₂ peak produced within the first 4 h after ethephon application was also eliminated by reduced glutathione. Diphenyleneiodonium (DPI), an NADPH oxidase inhibitor, delayed leaf senescence and H₂O₂ elevation at 72 h, and its influence was effective only within the first 4 h after ethephon treatment. Ethephon-induced senescence-associated gene expression was repressed by DPI and reduced glutathione at 72 h in pretreated leaves. Leaves treated with l-buthionine sulfoximine, an endogenous glutathione synthetase inhibitor, did enhance senescence-associated gene expression, and the activation was strongly repressed by reduced glutathione. In conclusion, ethephon-mediated leaf senescence, H₂O₂ elevation and senescence-associated gene expression are all alleviated by reduced glutathione and NADPH oxidase inhibitor DPI. The speed and the amount of intracellular reduced glutathione accumulation influence its effectiveness of protection against ethephon-mediated effects. Reactive oxygen species generated from NADPH oxidase likely serves as an oxidative stress signal and participates in ethephon signaling. The possible roles of NADPH oxidase and reduced glutathione in the regulation of oxidative stress signal in ethephon are discussed.     

Phosphatidylinositol 3-phosphate is required for abscisic acid-induced hydrogen peroxide production in rice leaves

Rice leaves produce H₂O₂ in response to abscisic acid (ABA), which results in induction of senescence and accumulation of NH₄⁺. The upstream steps of the ABA-induced H₂O₂ production pathway in rice leaves remain largely unclear. In animal cells, H₂O₂ production in neutrophils is activated by phosphatidylinositol 3-phosphate (PI3P), a product of phosphatidylinositol 3-knase (PI3K). In the present study, we examined whether PI3P plays a role in H₂O₂ production in rice leaves exposed to ABA. We found that PI3K inhibitors LY 294002 (LY) or wortmannin (WM) inhibited ABA-induced H₂O₂ production, senescence and NH₄⁺ accumulation. Hydrogen peroxide almost completely rescued the inhibitory effect of LY or WM. It appears that PI3P plays a role in ABA-induced H₂O₂ production, senescence, and NH₄⁺ accumulation in rice leaves.     

Induction of Betalain Pigmentation in Hairy Roots of Red Beet under Different Radiation Sources

The effects of aluminum on lipid peroxidation and activities of antioxidative enzymes were investigated in detached rice leaves treated with 0 to 5 mM AlCl₃ at pH 4.0 in the light. AlCl₃ enhanced the content of malondialdehyde but not the content of H₂O₂. Superoxide dismutase activity was reduced by AlCl₃, while catalase and glutathione reductase activities were increased. Peroxidase and ascorbate peroxidase activities were increased only after prolonged treatment, when toxicity occurred. The results give evidence that Al treatment caused oxidative stress and in turn, it caused lipid peroxidation.     

Exogenous proline alleviates the effects of H2O2-induced oxidative stress in wild almond species

The effect of proline on the antioxidant system in the leaves of eight species of wild almond (Prunus spp.) exposed to H₂O₂-mediated oxidative stress was studied. The levels of endogenous proline (Pro) and hydrogen peroxide, and the activities of total superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), glutathione reductase (GR), and guaiacol peroxidase (POD) were measured. The degradation of chlorophyll but not carotenoids occurred in leaves in the solution of 5 mM H₂O₂. An increase in membrane lipid peroxidation was observed in H₂O₂ treatment, as assessed by MDA level and percentage of membrane electrolyte leakage (EL). Significant increases in total SOD and CAT activities, as well as decreases in APX and POD activities, were detected in H₂O₂-treated leaves. The three SOD isoforms showed different behavior, as Mn-SOD activity was enhanced by H₂O₂, whereas Fe-SOD and Cu/Zn-SOD activities were inhibited. In addition, Pro accumulation up to 0.1 ??mol/g fr wt, accompanied by significant decreases in ascorbate and glutathione levels, was observed in H₂O₂-treated leaves. After two different treatments with 10 mM Pro + 5 mM H₂O₂, total SOD and CAT activities were similar to the levels in control plants, while POD and APX activities were higher if compared to the leaves exposed only to H₂O₂. Pro + H₂O₂ treatments also caused a strong reduction in the cellular H₂O₂ and MDA contents and EL. The results showed that Pro could have a key role in protecting against oxidative stress injury of wild almond species by decreasing membrane oxidative damage.     

Nitric oxide retards xanthine oxidase-mediated superoxide anion generation in Phalaenopsis flower: an implication of NO in the senescence and oxidative stress regulation

Senescence is a developmentally regulated and highly ordered sequence of events. Senescence leads to abscission of plant organs and eventually leads to death of a plant or part of it. Present study revealed that Phalaenopsis flower undergo senescence due to over activation of O₂ ^·−generating xanthine oxidase (XO), which consequently increases the concentrations of O₂ ^·− leading to enhanced oxidative damage and disturbed cellular redox environment as indicated by increased lipid peroxidation and DHA/AsA + DHA ratio, respectively. While activities of superoxide dismutase (SOD), ascorbate peroxidase (APX), and non-specific peroxidase (POD) were enhanced in sepals and petals of old flower, activities of catalase (CAT) and glutathione reductase (GR) were decreased. Exogenous application of nitric oxide (NO) retarded H₂O₂-induced senescence of Phalaenopsis flower by downregulating activity of XO and concentrations of O₂ ^·−, H₂O₂ and malondialdehyde (MDA, an index of lipid peroxidation). Exogenous application of NO also downregulated SOD activity and upregulated antioxidant enzymes involved in the detoxification of H₂O₂ (CAT and APX), and in the regulation of redox couples viz, monodehydroascorbate reductase (MDHAR) and GR, together with the modulation in non-protein thiol status and DHA/AsA + DHA ratio.     

Antioxidative protection in the leaves of dark-senescing intact barley seedlings

In order to elucidate the possibility of in vivo oxidative modification of Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase, EC 4.1.1.39) as a triggering mechanism for its preferential degradation early in senescence, some antioxidant compounds, protective enzymes, H₂O₂ and protein carbonylation levels were studied in the leaves during dark-induced senescence of barley (Hordeum vulgare L. cv. “Obzor”) seedlings. Analyses were performed in extracts as well as in purified chloroplasts. Some weakening of the antioxidative protection was detected during the treatment: diminution in the ascorbate and non-protein SH (mainly glutathione) pools, lower activities of superoxide dismutase, guaiacol and ascorbate peroxidases. However, no accumulation of H₂O₂ was found, lower level of protein carbonylation in darkness was measured and the percentage of reduced ascorbate was maintained high. Data concerning antioxidant compounds in chloroplasts revealed some impairment of the ascorbate and glutathione pools under induced senescence - the level of non-protein thiols declined during early senescence whereas the ascorbate pool was not significantly changed. The percentage of reduced ascorbate remained high in the chloroplasts and the activities of superoxide dismutase and of ascorbate peroxidase were conserved. Taken together the results are not in accordance with the possibility of in vivo oxidative modification of Rubisco in the case of dark-induced senescence. Our data bring some support to the view about redox regulation of Rubisco turnover in senescence through the pool of the low-molecular chloroplastic thiols.     

H2O2 metabolism during senescence of rice leaves: changes in enzyme activities in light and darkness

The possible role of H₂O₂ metabolism on light-regulated senescence of detached rice leaves was investigated. Light retards senescence but at the same time accumulates more H₂O₂. Light treatment resulted in an increase in malondialdehyde level in detached rice leaves but no membrane leakage was observed in light-treated detached leaves. It seems that there was no direct relationship between lipid peroxidation and deterioration in membrane integrity. The results obtained suggest that retardation of senescence by light is closely related to high activities of superoxide dismutase and ascorbate peroxidase.     

An inhibitor of catalase induced by cold in chilling-sensitive plants

An inhibitor of catalase accumulated when leaves of chilling-sensitive species were stored in the dark at 0°C. The inhibitor could be removed from crude extracts by passing them through a column of Sephadex G-25. After this treatment, the catalase activity of extracts of chilled tissues was found to be equal to that of extracts from unchilled leaves. When chilled tissues were incubated at 20°C, the inhibitor of catalase was lost, unless the tissues had been irreversibly damaged. It specifically inhibited plant catalase, and had no effect on mammalian catalase, plant malic dehydrogenase, or plant superoxide dismutase.

Despite the presence of catalase inhibitor in extracts of chilled plants, no increase in the level of H₂O₂ in chilled tissues was found, suggesting either that the inhibitor is compartmentalized and not in contact with catalase in vivo, or that the level of H₂O₂ is controlled by means other than through catalase activity. Plant tissues normally contain H₂O₂ which is destroyed by catalase when they are damaged. After chilling, H₂O₂ leaking from already injured cells would not be so readily removed by the inhibited catalase, and could contribute to further injury by acting as a source of free radical oxidants.

     

Canopy Light Gradient Perception by Cytokinin

We have recently identified cytokinin as an important xylem-carried signal involved in the photosynthetic acclimation of plants to light gradients in dense canopies. Lower leaves become shaded in a dense canopy and consequently have reduced transpiration rates. our measurements have shown that this results in a reduced delivery of cytokinins carried in the transpiration stream to shaded leaves, as compared to light-exposed leaves. Cytokinins are involved in the regulation of photosynthetic acclimation to the light gradient by stimulating the expression of photosynthetic enzymes in light-exposed leaves. In shaded leaves, the low delivery rate of cytokinin leads to reduced photosynthetic capacity and ultimately senescence. We show evidence for this role of cytokinin, as part of a complex of signaling pathways where other regulatory mechanisms are also involved. A model is presented depicting the regulation of photosynthetic acclimation by cytokinin delivery to leaves dependent on the irradiance they receive.Key Words: canopy light gradient, transpiration, photosynthetic acclimation, cytokinin, nitrate, systemic signaling     

Delay of Senescence of Detached Cucumber Cotyledons by Triadimefon

Changes in contents of reactive oxygen species (O₂ ⁻ and H₂O₂) and non-enzymatic antioxidants, activities of antioxidant enzymes and lipid peroxidation were investigated during senescence of detached cucumber cotyledons dipped in water (control) and 20 mg dm⁻³ triadimefon (TDM). O₂ ⁻ and H₂O₂ accumulation and lipid peroxidation were observed during senescence of cucumber cotyledons, which coincided with a drop in the contents of carotenoids (Car) and ascorbic acid (AsA), and the activities of superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX), and an increase in the activity of peroxidase (POD). However, TDM could significantly inhibit the accumulation of O₂ ⁻ and H₂O₂, and lipid peroxidation by preventing the decrease of CAT, APX, Car and AsA and the increase of POD, while TDM had little effect on SOD activity during the senescence. Therefore we can draw a conclusion that TDM protects the membrane system and retards the senescence of detached cucumber cotyledons. This revised version was published online in July 2006 with corrections to the Cover Date.     

Involvement of the oxidative burst in phytoalexin accumulation and the hypersensitive reaction

The role of the oxidative burst, transient production of activated oxygen species such as H₂O₂ and superoxide (O₂⁻) in elicitation of phytoalexins and the hypersensitive reaction (HR) was investigated in white clover (Trifolium repens L.) and tobacco (Nicotiana tabacum L.). H₂O₂ and O₂⁻ production was measured as chemiluminescence (CL) mediated by luminol, which was added to suspension-cultured white clover just before measurement in an out-of-coincidence mode scintillation counter. Maximum CL occurred between 10 and 20 min after addition of 0.4 × 10⁸ colony-forming units/mL of incompatible Pseudomonas corrugata or 158 μm HgCl₂. Autoclaved P. corrugata produced a slightly higher response. Elicitation of cells with 25 μm HgCl₂ did not produce CL. Preincubation of plant cells in superoxide dismutase, which converts O₂⁻ to H₂O₂, for 2 min before addition of bacteria did not significantly increase maximum CL levels (P ≥ 0.05). Preincubation of plant cells with catalase for 2 min before addition of bacteria prevented the increase in CL, confirming that H₂O₂ is the substrate for the luminol reaction. Addition of live bacteria or HgCl₂ (25 and 158 μm) to white clover increased levels of the phytoalexin medicarpin during a 24-h period, but addition of autoclaved bacteria did not elicit formation of medicarpin. Preincubation of plant cells with catalase, which quenched the bacteria-induced oxidative burst, did not decrease phytoalexin accumulation. Live bacteria infiltrated into Havana 44 tobacco leaf panels induced development of the HR, but autoclaved bacteria did not. Incubation of live bacteria with superoxide dismutase and catalase before infiltration into tobacco leaves did not interfere with development of the HR. Tobacco leaf panels infiltrated with up to 158 μm HgCl₂ did not develop an HR. These results suggest that an oxidative burst consisting of H₂O₂ and O₂⁻ does occur during these two plant defense responses, but it may not be a necessary element of the signaling system for HR and phytoalexin formation.