Abstract
Extracellular ATP functions as an important signaling molecule in both plants and animals. In plants, ATP is released in the extracellular region of cells in response to environmental perturbations, such as herbivory, cellular damage, or other abiotic and biotic stimuli, which is then perceived by the purinoceptor P2K1 as a damaged-self signal for activation of defense responses. Given its involvement in various physiological processes, quantification of extracellular ATP is important for further understanding of its molecular function. In this chapter, we describe a method for the accurate and reliable determination of extracellular ATP concentrations in plant cell culture media based on the luciferase-luciferin reaction, using either end-point or real-time detection assays. The protocol can be easily performed with any luminometer within 1 h after sample collection. Although we use Arabidopsis suspension cells, the protocol described can be optimized for any cell type.
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References
Drury AN, Szent-Györgyi A (1929) The physiological activity of adenine compounds with special reference to their action upon the mammalian heart. J Physiol 68:213–237
Burnstock G (1972) Purinergic nerves. Pharmacol Rev 24:509–581
Burnstock G, Williams M (2000) P2 purinergic receptors: modulation of cell function and therapeutic potential. J Pharmacol Exp Ther 295:862–869
Cekic C, Linden J (2016) Purinergic regulation of the immune system. Nat Rev Immunol 16:177–192
Khakh BS, Burnstock G (2009) The double life of ATP. Sci Am 301:84–92
Pavenstadt H, Gloy J, Leipziger J et al (1993) Br J Pharmacol 109:953–959
Jaffe MJ (1973) The role of ATP in mechanically stimulated rapid closure of the Venus flytrap. Plant Physiol 51:17–18
Lew RR, Dearnaley JDW (2000) Extracellular nucleotide effects on the electrical properties of growing Arabidopsis thaliana root hairs. Plant Sci 153:1–6
Reichler SA, Torres J, Rivera AL et al (2009) Intersection of two signalling pathways: extracellular nucleotides regulate pollen germination and pollen tube growth via nitric oxide. J Exp Bot 60:2129–2138
Steinebrunner I, Wu J, Sun Y et al (2003) Disruption of apyrases inhibits pollen germination in Arabidopsis. Plant Physiol 131:638–1647
Tanaka K, Gilroy S, Jones AM et al (2010) Extracellular nucleotide signaling in plants. Trends Cell Biol 20:601–608
Tanaka K, Choi J, Cao Y et al (2014) Extracellular ATP acts as a damage associated molecular pattern (DAMP) signal in plants. Front Plant Sci 5:466
Tripathi D, Zhang T, Koo AJ et al (2018) Extracellular ATP acts on jasmonate signaling to reinforce plant defense. Plant Physiol 176:511–523
Tripathi D, Tanaka K (2018) A crosstalk between extracellular ATP and JA signaling pathways. Plant Signal Behav 13:e1432229
Thomas C, Rajagopal A, Windsor B et al (2000) A role for ectophosphatase in xenobiotic resistance. Plant Cell 12:519–533
Kim SY, Sivaguru M, Stacey G (2006) Extracellular ATP in plants. Visualization, localization, and analysis of physiological significance in growth and signaling. Plant Physiol 142:984–992
Rieder B, Neuhaus HE (2011) Identification of an Arabidopsis plasma membrane-located ATP transporter important for anther development. Plant Cell 23:1932–1944
Dark A, Demidchik V, Richards SL et al (2011) Release of extracellular purines from plant roots and effect on ion fluxes. Plant Signal Behav 6:1855–1857
Wu SJ, Liu YS, Wu JY (2008) The signaling role of extracellular ATP and its dependence on Ca2+ flux in elicitation of Salvia miltiorrhiza hairy root cultures. Plant Cell Physiol 49:617–624
Weerasinghe RR, Swanson SJ, Okada SF et al (2009) Touch induces ATP release in Arabidopsis roots that is modulated by the heterotrimeric G-protein complex. FEBS Lett 583:2521–2526
Webb TE, Simon J, Krishek BJ et al (1993) Cloning and functional expression of a brain G-protein-coupled ATP receptor. FEBS Lett 324:219–225
Lustig KD, Shiau AK, Brake AJ et al (1993) Expression cloning of an ATP receptor from mouse neuroblastoma cells. Proc Natl Acad Sci U S A 90:5113–5117
Ralevic V, Burnstock G (1998) Receptors for purines and pyrimidines. Pharmacol Rev 50:413–492
Abbracchio MP, Burnstock G, Boeynaems JM et al (2006) International Union of Pharmacology LVIII: update on the P2Y G protein-coupled nucleotide receptors: from molecular mechanisms and pathophysiology to therapy. Pharmacol Rev 58:281–341
Choi J, Tanaka K, Cao Y et al (2014a) Identification of a plant receptor for extracellular ATP. Science 343:290–294
Choi J, Tanaka K, Liang Y et al (2014b) Extracellular ATP, a danger signal, is recognized by DORN1 in Arabidopsis. Biochem J 463:429–437
Miyatake K, Sakuraba H, Kitaoka S (1987) Separation and determination of adenine nucleotides by high-performance liquid chromatography. Agric Biol Chem 51:253–255
Khlyntseva SV, Bazel’ YR, Vishnikin AB et al (2009) Methods for determination of adenosine triphosphate and other adenine. J Anal Chem 64:657
Bhatt DP, Chen X, Geiger JD et al (2012) A sensitive HPLC-based method to quantify adenine nucleotides in primary astrocyte cell cultures. J Chromatogr B Analyt Technol Biomed Life Sci 889-890:110–115
Fitzgerald RS, Shriahata M, Chang I et al (2009) The impact of hypoxia and low glucose on the release of acetylcholine and ATP from the incubated cat carotid body. Brain Res 1270:39–44
Acknowledgments
This project was supported by the National Science Foundation (grant no. IOS-1557813) and USDA NIFA (Hatch project 1015621). PPNS No. 0758, Department of Plant Pathology, College of Agriculture, Human and Natural Resource Sciences, Agricultural Research Center, Hatch Project No. WNP00008 and WNP00833, Washington State University, Pullman, WA, 99164-6430, USA.
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Ramachandran, S.R., Kumar, S., Tanaka, K. (2019). Quantification of Extracellular ATP in Plant Suspension Cell Cultures. In: Gassmann, W. (eds) Plant Innate Immunity. Methods in Molecular Biology, vol 1991. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9458-8_5
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DOI: https://doi.org/10.1007/978-1-4939-9458-8_5
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