A method to detect oxidative stress by monitoring changes in the extracellular antioxidant capacity in plant suspension cells

Detection of H2O2 in the supernatant of plant suspension cells is often used to indicate the time and extent of the oxidative burst during interactions with either bacteria or pathogen-related elicitors. We have found that suspensions of plant cells, depending on conditions, may produce considerable levels of extracellular phenolics that can function as antioxidants and prevent or suppress the detection of H2O2. These compounds can be used as substrates by extracellular peroxidases to scavenge stoichiometric amounts of H2O2. When this occurs during plant/pathogen interactions it can mask both the timing and extent of the oxidative burst if detection of free H2O2 is the only technique used. We have developed a chemiluminescent technique that will account for the H2O2 scavenged by these extracellular metabolites. A known quantity of H2O2 is added to samples and allowed to react with the extracellular antioxidants. The amount of H2O2 that remains is then determined by adding luminol to the sample and measuring luminol-dependent-chemiluminescence. The difference between treated and control samples represents the amount of H2O2 that has been produced by the cells in response to the treatment. We have found that this technique provides a better estimate of both the magnitude and timing of the oxidative burst in bacterial/suspension cell systems

Scavenging of H\u3csub\u3e2\u3c/sub\u3eO\u3csub\u3e2\u3c/sub\u3e and Production of Oxygen by Horseradish Peroxidas

Peroxidases catalyze many reactions, the most common being the utilization of H2O2 to oxidize numerous substrates (peroxidative mode). Peroxidases have also been proposed to produce H2O2 via utilization of NAD(P)H, thus providing oxidant either for the first step of lignification or for the oxidative burst associated with plant-pathogen interactions. The current study with horseradish peroxidase characterizes a third type of peroxidase activity that mimics the action of catalase; molecular oxygen is produced at the expense of H2O2 in the absence of other reactants. The oxygen production and H2O2-scavenging activities had temperature coefficients, Q10, of nearly 3 and 2, which is consistent with enzymatic reactions. Both activities were inhibited by autoclaving the enzyme and both activities had fairly broad pH optima in the neutral-to-alkaline region. The apparent Km values for the oxygen production and H2O2- scavenging reactions were near 1.0 mM H2O2. Irreversible inactivation of horseradish peroxidase by exposure to high concentrations of H2O2 coincided with the formation of an absorbance peak at 670 nm. Addition of superoxide dismutase (SOD) to reaction mixtures accelerated the reaction, suggesting that superoxide intermediates were involved. It appears that horseradish peroxidase is capable of using H2O2 both as an oxidant and as a reductant. A model is proposed and the relevance of the mechanism in plant-bacterial systems is discussed

An Induced Hypersensitive-Like Response Limits Expression of Foreign Peptides via a Recombinant TMV-Based Vector in a Susceptible Tobacco

BACKGROUND: By using tobacco mosaic virus (TMV)-based vectors, foreign epitopes of the VP1 protein from food-and-month disease virus (FMDV) could be fused near to the C-terminus of the TMV coat protein (CP) and expressed at high levels in susceptible tobacco plants. Previously, we have shown that the recombinant TMV vaccines displaying FMDV VP1 epitopes could generate protection in guinea pigs and swine against the FMDV challenge. Recently, some recombinant TMV, such as TMVFN20 that contains an epitope FN20 from the FMDV VP1, were found to induce local necrotic lesions (LNL) on the inoculated leaves of a susceptible tobacco, Nicotiana tabacum Samsun nn. This hypersensitive-like response (HLR) blocked amplification of recombinant TMVFN20 in tobacco and limited the utility of recombinant TMV vaccines against FMDV. METHODOLOGY/PRINCIPAL FINDINGS: Here we investigate the molecular mechanism of the HLR in the susceptible Samsun nn. Histochemical staining analyses show that these LNL are similar to those induced in a resistant tobacco Samsun NN inoculated with wild type (wt) TMV. The recombinant CP subunits are specifically related to the HLR. Interestingly, this HLR in Samsun nn (lacking the N/N'-gene) was able to be induced by the recombinant TMV at both 25°C and 33°C, whereas the hypersensitive response (HR) in the resistant tobacco plants induced by wt TMV through the N/N'-gene pathways only at a permissive temperature (below 30°C). Furthermore, we reported for the first time that some of defense response (DR)-related genes in tobacco were transcriptionally upregulated during HLR. CONCLUSIONS: Unlike HR, HLR is induced in the susceptible tobacco through N/N'-gene independent pathways. Induction of the HLR is associated with the expression of the recombinant CP subunits and upregulation of the DR-related genes

Oxygen metabolism in plantlbacteria interactions: characterization of the oxygen uptake response of plant suspension cells

In recent years the accumulation of reactive oxygen species (ROS) has been studied in plant cell suspension systems treated with bacterial pathogens. However, the associated utilization of molecular oxygen has not been well characterized. Using a multi-electrode oxygen analyser, the rates of oxygen consumption by tobacco cells during bacterial interactions were monitored. Heat-killed (HK) bacteria, which initiate an immediate ROS response in plant cells, were used as an elicitor to avoid complications of oxygen consumption by viable bacteria. An increase in oxygen uptake by the tobacco cells occurred within 4 min after addition of HK-bacteria and lasted for about 10 min, returning to a steady state at approximately twice the initial basal rate. The initial burst in oxygen uptake coincided with production of H202. Calculation of the total oxygen consumption by the plant cells indicated that less than 5 % of the increased oxygen uptake was utilized in ROS production. Use of respiratory inhibitors indicated that respiration, especially the cytochrome pathway, played a significant role in this response. Results from the use of K-252, a protein kinase inhibitor, and DPI, an inhibitor of membrane bound NADPH oxidases, indicated that triggering of the oxygen uptake response may involve protein phosphorylation and is at least partially activated by the membrane bound NADPH oxidase activity. The involvement of mitochondrial respiration in the oxygen uptake response described here indicates that early events in plant recognition of pathogens involves more of the cellular machinery than previously hypothesized

Oxidative metabolism in plant/bacteria interactions: characterization of a unique oxygen uptake response of potato suspension cells

Plant suspension cells have been shown to respond to bacteria or microbial elicitors by producing active oxygen as well as increasing oxygen uptake. Here we characterize a unique two stage oxygen uptake response of potato suspension cells to heat-killed bacteria. Stage 1 occurred within minutes after the addition of heat-killed bacteria; the potato suspension cells responded with a rapid increase in oxygen uptake and reached a steady state approximately 50 % greater than the initial basal rate. Stage 2 began 20-30 min after this new steady state was achieved and was characterized by a slow increase in the oxygen uptake rate over the remaining 90 min period. Calculation of the total oxygen consumption by the plant cells indicated that only a small fi-action of the increased oxygen uptake was due to the concomitant production of reactive oxygen species. The protein kinase inhibitor, K-252, inhibited the oxygen uptake response by 80-90 %, suggesting the involvement of protein phosphorylation in the oxygen uptake response. The alternate oxidase inhibitor, SHAM, inhibited the elicited oxygen uptake response by about 25 % while a combination of SHAM and KCN almost completely blocked respiration as well as the elicited response. The data indicate that mitochondrial respiration and, in particular, the alternate oxidase, play a significant role in the elicited oxygen uptake response of potato cells

Continuous production of extracellular antioxidants in suspension cells attenuates the oxidative burst detected in plant microbe interactions

Suspension cells of Solanacearum tuberosum and Nicotiana tabacum placed in fresh buffer rapidly produce and maintain significant pools of extracellular antioxidants. The extracellular antioxidant was detected by first adding a known amount of exogenous H2O2 to samples and then immediately measuring the remaining H2O2. The difference between the amount added and amount remaining was used to determine the antioxidant capacity of the sample. This extracellular antioxidant pool attenuates levels of hydrogen peroxide produced during plant–bacterial interactions. When tobacco cells were inoculated with an isolate Pseudomonas syringae pv. syringae that causes a hypersensitive response much of the antioxidant capacity had been expended neutralizing the oxidative burst characteristic of such plant–microbe interactions. After a brief delay, the levels of extracellular phenolics increased commensurate to antioxidative capacity in freshly transferred cells within 2–4 h. The strong UV absorbance of these extracellular phenolics within 250 and 350 nm was used to follow oxidation upon reaction with H2O2. This extracellular antioxidant pool is an important consideration in cell suspension studies of the plant–microbe oxidative burst. This study demonstrates that the true magnitude and timing of the oxidative burst in cell suspensions is masked by extracellular antioxidants

Oxidative metabolism in plant/bacteria interactions: characterization of the oxygen uptake response of bacteria

An increase in oxygen uptake has been previously described in plant cell suspensions treated with bacteria or bacterial elicitors. These studies, regarding oxygen uptake, have all been undertaken from the perspective of the host plant cell reacting to the invading pathogen. In contrast, here we describe and characterize an increase in oxygen uptake by bacterial cells in response to plant suspensions or autoclaved plant cell filtrates. Autoclaved plant cell filtrates stimulated bacterial oxygen uptake by as much as sevenfold within a few minutes after addition. This oxygen uptake response was proportional to both the concentration of the plant cell filtrate and the concentration of the bacteria. KCN inhibited the bacterial response, suggesting that bacterial respiration may be involved. Unlike the plant oxygen uptake response to bacteria, there was no concurrent H202 accumulation and the NADPH oxidase inhibitor, DPI, had no effect on the bacterial response. Streptomycin, an inhibitor of bacterial protein synthesis, inhibited the bacterial oxygen uptake response to the plant cell filtrate. K-252, a protein kinase inhibitor that strongly inhibits the plant oxygen uptake response to bacteria, had no effect upon the bacterial oxygen uptake response. When potato/bacterial cell suspensions were pretreated with either streptomycin or K-252, the combined plant/bacterial oxygen uptake response was inhibited by 15 or 70 %, respectively. This indicates that as much as 15-30 % of the increased oxygen consumption during plant suspension cell/bacteria interactions may be attributable to bacteria, which comprise less than 1 % of the total cell mass