A Noninvasive Technique for Monitoring Peroxidative and H2O2-Scavenging Activities during Interactions between Bacterial Plant Pathogens and Suspension Cells

Abstract

Stimulation of active oxygen metabolism occurs during the early stages of interactions involving bacteria and plant cell suspensions. Although many cellular processes are known to affect active oxygen metabolism in plants, it is not known which of these factors affect active oxygen levels during plant-bacteria interactions. Extracellular peroxidases have been shown to participate in both the production and utilization of active oxygen species such as H,O, and superoxide. Catalase and other scavenging mechanisms also affect the overall level of active oxygen. In this study the luminol-dependent chemiluminescent reaction previously used to measure H,O, levels in suspension cells was modified to allow the assay of both peroxidase and H,O,-scavenging activity. The early stages of the interactions between tobacco (Nicofiana fabacum) and Pseudomonas syringae pv syringae, as well as between soybean (Glycine max) and P. syringae pv glycinea, were investigated. This method of monitoring peroxidase and H,O,-scavenging activity proved to be rapid, sensitive, and nonintrusive, allowing the processing of multiple samples using intact cells or cell-free preparations. The results from the study demonstrate that the scavenging activities can be significant and must be considered when studying active oxygen production in biological interactions.As the study of plant-microbe interactions has become more molecular, there has been an increased use of specialized model systems to allow the study of specific phenomena. Studies of the bacteria-induced HR found that inoculation with incompatible species produces rapid plant cell death in cell suspensions similar to that found during the HR in whole plant tissues (Baker et al., 1993a;Baker and Mock, 1994). The use of suspension cells in addition to whole plants has allowed identification of early plant responses, which precede hypersensitive cell death (Atkinson et al., 1985b;Keppler et al., 1989;Baker et al., 1991Baker et al., , 1993a. One of these responses is the production of active oxygen. The response consists of a rapid, transient production a few minutes after addition of the incompatible bac-

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

“…The concentration of H 2 0 2 in the beakers over a 2 h period was estimated by LDC [5]. The scavenging rate constant for this concentration of potato cells was determined from this data with the aid of SAAM I1 software (SAAM Institute Inc., Seattle, WA, U.S.A.) as previously mentioned [8].…”

Hydrodynamic stress induces monoterpenoid oxindole alkaloid accumulation by Uncaria tomentosa (Willd) D. C. cell suspension cultures via oxidative burst

“…To 325 mL of the supernatant were added with 1,000 mL phosphate buffer (sodium phosphate 0.1 M, pH 7.0), later 150 mL luminol (2.5 mM in sodium phosphate buffer 1.0 M, pH 7.0) and finally 25 mL peroxidase (6 mU mL À1 ). Luminescence was recorded immediately for 3 min at 30 s intervals in a luminometer (Betascout 2007, Perkin Erlmer Life Sciences, Turku, Finland) and the maximum level was registered (Baker et al, 1995). Phosphate buffer (1325 mL) was used as a blank and its luminescence value was subtracted from that of the…”

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

“…Cell suspensions, 25 ml, contained in 50 ml beakers were equilibrated on a rotary shaker at 180 rpm and 25°C for 0.5 h. Bacterial cultures of P. syringae pv. syringae 61 were maintained and prepared as previously described [1]. The wild-type (WT) isolate caused an HR reaction on tobacco while the Tn5 insertion mutant (B7) did not.…”