Molecular bases for signal transduction in plant cells are still largely unknown. Though calcium ion was proposed as a second messenger in plant cells, spatio-temporal changes in cytosolic calcium ion in response to various environmental stimuli have not been well characterized due to the technical difficulties in imaging. We have developed a novel method to load the calcium indicators such as calcium green-1 and indo-1 into the cytoplasm of plant cells including stomatal guard cells. Stomatal pores control gas exchange including H2O loss (transpiration) and CO2 uptake. Understanding the regulation of stomatal pores is important for the future genetic improvement of gas exchange. The hormone absisic acid (ABA) causes stomatal pores to close uder dehydrated conditions. Although much of the ABA signaling pathway is unknown, it has been proposed that an increase in cytoplasmic CA2+ is the earlies known ABA signaling event. Using the newly-developed method to load the calcium indicatiors into the cytoplasm of guard cells as well as epidermal cells of Commelina communis, we monitored the spatio-temporal changes in Ca2+ concentration in response to ABA by using laser fluorescence confocal imaging techniques. Furthermore, we have extended this approach and are pursuing molecular genetic studies to determine how ABA-induced CA2+ increases are affected in ABA signaling mutants of Arabidopsis thaliana. We have also developed ratiometric onfocal imaging technique s for plant cells including Arabidopsis. These studies are allowing a cellular analysis of the function of cloned genetic loci for ABA signaling in Arabidopsis. We are also planning to apply these techniques to other plant cells that recognizes and respons to ligosaccharide elicitors to elucidate the molecular mechanisms of signal transduction for plan-specific denfense responses. We have been using the confocal microscopy and image analysis facilites at the NCMIR, including the program B track developed at the NCMIR.
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