Abstract
Heterotrimeric GTP-binding proteins (G-proteins) and G-protein-coupled receptors are important signaling components in eukaryotes. In plants, the G-proteins are involved in diverse physiological processes, some of which are exerted via changes in the level of cytosolic free calcium concentration ([Ca2+]cyt). Various techniques have been developed to measure the change of [Ca2+]cyt, e.g., calcium-sensitive microelectrodes, chemical fluorescent dyes, and biosensors based on luminescent or fluorescent indicators. In this chapter, we describe a protocol for in vivo [Ca2+]cyt measurement in G-protein mutants expressing aequorin, a luminescent-based calcium biosensor, to extend our knowledge about G-protein mediated Ca2+ signaling. This method is also applicable to other early signaling events that are mediated by changes in [Ca2+]cyt levels.
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References
Gilman AG (1987) G proteins: transducers of receptor-generated signals. Annu Rev Biochem 56:615
Sprang SR (1997) G protein mechanisms: insights from structural analysis. Annu Rev Biochem 66:639
Jones AM, Assmann SM (2004) Plants: the latest model system for G-protein research. EMBO Rep 5:572
Gelli A, Higgins VJ, Blumwald E (1997) Activation of plant plasma membrane Ca2+-permeable channels by race-specific fungal elicitors. Plant Physiol 113:269
Ueguchi-Tanaka M et al (2000) Rice dwarf mutant d1, which is defective in the alpha subunit of the heterotrimeric G protein, affects gibberellin signal transduction. Proc Natl Acad Sci U S A 97:11638
Wu Y et al (2007) Heterotrimeric G-protein participation in Arabidopsis pollen germination through modulation of a plasmamembrane hyperpolarization-activated Ca2+-permeable channel. New Phytol 176:550
Zhang W, Jeon BW, Assmann SM (2011) Heterotrimeric G-protein regulation of ROS signalling and calcium currents in Arabidopsis guard cells. J Exp Bot 62:2371
Shimomura O, Johnson FH, Saiga Y (1962) Extraction, purification and properties of aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea. J Cell Comp Physiol 59:223
Brini M et al (1995) Transfected aequorin in the measurement of cytosolic Ca2+ concentration ([Ca2+]c). A critical evaluation. J Biol Chem 270:9896
Knight MR, Campbell AK, Smith SM, Trewavas AJ (1991) Transgenic plant aequorin reports the effects of touch and cold-shock and elicitors on cytoplasmic calcium. Nature 352:524
Tanaka K, Swanson SJ, Gilroy S, Stacey G (2010) Extracellular nucleotides elicit cytosolic free calcium oscillations in Arabidopsis. Plant Physiol 154:705
Tanaka K, Gilroy S, Jones AM, Stacey G (2010) Extracellular ATP signaling in plants. Trends Cell Biol 20:601
Qi Z et al (2010) Ca2+ signaling by plant Arabidopsis thaliana Pep peptides depends on AtPepR1, a receptor with guanylyl cyclase activity, and cGMP-activated Ca2+ channels. Proc Natl Acad Sci U S A 107:21193
Ranf S et al (2012) Defense-related calcium signaling mutants uncovered via a quantitative high-throughput screen in Arabidopsis thaliana. Mol Plant 5:115
Wan J et al (2012) LYK4, a LysM receptor-like kinase, is important for chitin signaling and plant innate immunity in Arabidopsis. Plant Physiol 160:396
Baum G, Long JC, Jenkins GI, Trewavas AJ (1999) Stimulation of the blue light phototropic receptor NPH1 causes a transient increase in cytosolic Ca2+. Proc Natl Acad Sci U S A 96:13554
Harada A, Sakai T, Okada K (2003) Phot1 and phot2 mediate blue light-induced transient increases in cytosolic Ca2+ differently in Arabidopsis leaves. Proc Natl Acad Sci U S A 100:8583
Nomura H et al (2012) Chloroplast-mediated activation of plant immune signalling in Arabidopsis. Nat Commun 3:926
Allen DG, Blinks JR, Prendergast FG (1977) Aequorin luminescence: relation of light emission to calcium concentration – a calcium-independent component. Science 195:996
Campbell AK (1988) Chemiluminescence – principles and applications in biology and medicine. VCH and Ellis Horwood Ltd., New York
Love J, Dodd AN, Webb AA (2004) Circadian and diurnal calcium oscillations encode photoperiodic information in Arabidopsis. Plant Cell 16:956
Torrecilla I, Leganes F, Bonilla I, Fernandez-Pinas F (2000) Use of recombinant aequorin to study calcium homeostasis and monitor calcium transients in response to heat and cold shock in cyanobacteria. Plant Physiol 123:161
Knight H, Trewavas AJ, Knight MR (1996) Cold calcium signaling in Arabidopsis involves two cellular pools and a change in calcium signature after acclimation. Plant Cell 8:489
Logan DC, Knight MR (2003) Mitochondrial and cytosolic calcium dynamics are differentially regulated in plants. Plant Physiol 133:21
Johnson CH et al (1995) Circadian oscillations of cytosolic and chloroplastic free calcium in plants. Science 269:1863
van Der Luit AH, Olivari C, Haley A, Knight MR, Trewavas AJ (1999) Distinct calcium signaling pathways regulate calmodulin gene expression in tobacco. Plant Physiol 121:705
Baubet V et al (2000) Chimeric green fluorescent protein-aequorin as bioluminescent Ca2+ reporters at the single-cell level. Proc Natl Acad Sci U S A 97:7260
Kiegle E, Moore CA, Haseloff J, Tester MA, Knight MR (2000) Cell-type-specific calcium responses to drought, salt and cold in the Arabidopsis root. Plant J 23:267
Jones AM, Ecker JR, Chen JG (2003) A reevaluation of the role of the heterotrimeric G protein in coupling light responses in Arabidopsis. Plant Physiol 131:1623
Ullah H et al (2003) The beta-subunit of the Arabidopsis G protein negatively regulates auxin-induced cell division and affects multiple developmental processes. Plant Cell 15:393
Edwards K, Johnstone C, Thompson C (1991) A simple and rapid method for the preparation of plant genomic DNA for PCR analysis. Nucleic Acids Res 19:1349
Shimomura O (1991) Preparation and handling of aequorin solutions for the measurement of cellular Ca2+. Cell Calcium 12:635
Knight H, Knight MR (1995) Recombinant aequorin methods for intracellular calcium measurement in plants. In Galbraith DW, Bohnert HJ, Bourque DP (eds) Methods in Cell Biology 49:201–216
Dubuisson ML et al (2000) Antioxidative properties of natural coelenterazine and synthetic methyl coelenterazine in rat hepatocytes subjected to tert-butyl hydroperoxide-induced oxidative stress. Biochem Pharmacol 60:471
Shimomura O, Musicki B, Kishi Y, Inouye S (1993) Light-emitting properties of recombinant semi-synthetic aequorins and recombinant fluorescein-conjugated aequorin for measuring cellular calcium. Cell Calcium 14:373
Acknowledgments
We are grateful to Dr. Alan M.Jones (University of North Carolina, USA) for the Arabidopsis heterotrimeric G-protein mutants, and to Dr. Marc R.Knight (University of Oxford, UK) for transgenic Arabidopsis plants expressing aequorin. This work was supported by the US Department of Energy (grant no. DE-FG02-08ER15309) and by the Next-Generation BioGreen 21 Program, Systems and Synthetic Agrobiotech Center, Rural Development Administration, Republic of Korea (grant no. PJ009068).
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Tanaka, K., Choi, J., Stacey, G. (2013). Aequorin Luminescence-Based Functional Calcium Assay for Heterotrimeric G-Proteins in Arabidopsis. In: Running, M. (eds) G Protein-Coupled Receptor Signaling in Plants. Methods in Molecular Biology, vol 1043. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-532-3_5
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DOI: https://doi.org/10.1007/978-1-62703-532-3_5
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