Biochemical Evidence for the Activation of Distinct Subsets of Mitogen-Activated Protein Kinases by Voltage and Defense-Related Stimuli

Abstract

Activation of mitogen-activated protein (MAP) kinases is a common reaction of plant cells in defense-related signal transduction pathways. To gain insight into the mechanisms that determine specificity in response to a particular stimulus, a biochemical approach has been employed. Photoautotrophic suspension culture cells of tomato (Lycopersicon peruvianum) were used as experimental system to characterize MAP kinase activation by different stress-related stimuli. An elicitor preparation of the tomato-specific pathogen Fusarium oxysporum lycopersici was shown to result in the simultaneous induction of four kinase activities that could be separated by ion-exchange chromatography. The simultaneous activation of multiple MAP kinases was further substantiated by distinct pharmacological and immunological properties: a differential sensitivity toward various protein kinase inhibitors and a differential cross-reaction with isoform-specific MAP kinase antibodies. In contrast to the two fungal elicitors chitosan and the F. oxysporum lycopersici preparation, the plant-derived stimuli polygalacturonic acid and salicylic acid were shown to activate distinctly different subsets of MAP kinases. Application of a voltage pulse was introduced as a transient stress-related stimulus that does not persist in the culture. Voltage application activates a distinct set of MAP kinases, resembling those activated by salicylic acid treatment, and generates a refractory state for the salicylic acid response. The inhibitory effect of nifedipine indicates that current application may directly affect voltage-gated calcium channels, thus, providing a tool to study various calcium-dependent pathways.During their whole life, plants need to cope with a variety of attacking pathogens. They developed appropriate defense responses that protect them against impairment by most of them. These defense responses against several different pathogens have been extensively studied, however, the components that determine the specificity of the defense gene activation remain to be elucidated. After a plant cell is challenged by an elicitor, one or more signal transduction pathways are invoked by a ligand-receptor interaction (Nü rnberger, 1999) that lead to the activation of a set of defense-related genes appropriate for the defense against the attacking pathogen (Jabs et al., 1997).The involvement of mitogen-activated protein (MAP) kinases in biotic and abiotic stress-mediated defense gene activation has been extensively studied and MAP kinases that respond to elicitors (Zhang et al., 1998), wounding (Stratmann and Ryan, 1997), cold and drought stress (Jonak et al., 1996), salinity (Munnik et al., 1999), and endogenous signals (Zhang and Klessig, 1997) have been described. The activities were assigned to MAP kinase genes by the observation of coordinated transcriptional up-regulation and the use of specific antibodies. In-depth analysis of MAP kinase activation in tobacco revealed that the MAP kinase salicylate-induced protein kinase is activated by salicyli...

Trisubstituted Purines Are Useful Tools for Developing Potent Plant Mitogen-Activated Protein Kinase Inhibitors

“…10,15) Different inhibitory potencies of Bohemine, Olomoucine, and Roscovitine versus plant MAPKs have been reported. 12) This indicates that the inhibitors can be useful in the characterization of plant MAPK, and suggest the possibility of inhibitor development mediated by interaction between chemical structure and protein kinase structure using C2, C6, N9-trisubstituted purines.…”

“…To ensure the same starting conditions for all cultures of an experiment, cultures were mixed and divided again 24 h before treatment. Elicitor was prepared by the procedure described previously, 12) and was applied to 100 mg/ml E-FOl, the elicitor preparation of the wilt-inducing fungus F. oxysporum lycopersici. The cells were harvested at different time points by centrifugation at 1000 g, snap freezing in liquid nitrogen, and were stored at À80 C until analysis.…”

“…These compounds have been reported to have inhibitory effects on the activity of CDKs, 16,20,21) and different potencies to inhibit the activity of plant MAPKs. 12) This indicates that a minor modification of the cytokinin structure can have a dramatic impact on protein kinase activity and selectivity.…”

“…11,12) In higher plants, gain-of-function and the loss-offunction mutants are commonly used in the study of the physiological roles of MAPK, which, however, is limited by the availability of such mutants. In addition, functional redundancy can complicate analysis.…”

Stress Response Monitoring of Photoautotrophic Higher Plant Suspension Cultures by Fluorescence Imaging for High-Throughput Toxic Compound Screening

“…The phytotoxicity of DCMU is caused by blocking electron transport flow in PSII at the QB site terminating the photosynthetic light reactions and causing formation of singlet oxygen which results in lipid peroxidation (reviewed in [52]). As plant material we have used PA cultures of the model plant tomato (Solanum peruvianum, [53]) that is also an important vegetable crop plant. In our case study, ten concentrations of DCMU and deio- Figure 1 and the quantitative evaluation of the data in Table 1.…”