Abstract
The mitogen activated protein kinase cascade is a highly conserved signal pathway in plants. The exogenous chemicals, like hormones, can trigger a series of signalling cascades, including MAPK pathway, to modulate the plant physiology. Upon activation, some MAPKs are phosphorylated. It is important to develop methods that can detect changes in the phosphorylation status of MAPKs in plants when they come in contact with external chemicals. This method describes the exogenous treatment of Arabidopsis protoplasts with Kinetin and subsequent detection of the activated MAPKs. This method is useful for studying the effect of exogenously applied chemical compounds on the MAPK signaling cascade in Arabidopsis.
Keywords: MAP kinase, Phosphorylation, In vitro kinase assay, Signal transduction, Immunoprecipitation
Materials and Reagents
Equipment
Procedure
Representative data
Representative data is shown in Figure 1. Figure 1. Western blot detection of MPK3 and MPK6 phosphorylation in Arabidopsis protoplasts after kinetin treatment. Arabidopsis mesophyll protoplasts were isolated and treated overnight with the indicated concentrations of kinetin. Protoplasts were harvested and isolated proteins were analyzed by Western blotting with the anti-p42/44 ERK antibody. The activation of the MAP kinases was observed when kinetin is applied at the lower concentrations. The bottom panel shows the amido black staining of RuBisCo (~55 KD) performed after the Western of the same blot as loading control. Source: Sheikh et al. (2014) Figure 2. General graphical overview of the protoplast isolation. The figure highlights the carefully cut leaves in the enzyme solution and the use of cut tips for handling the protoplast to minimize the damage.
Notes
Recipes
Acknowledgments
Authors thank Prof. Dr. Sabine Rosahl at Leibniz IPB, Halle Germany for providing Kinetin solution and Dr. Lennart Eschen Lippold for kind help with Protoplasts. Authors thank Dhammaprakash Wankhede and Nicole Bauer for technical help. Financial support from Department of Biotechnology, Government of India is acknowledged.
References
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