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Determination of cAMP in plant cells by a modified enzyme immunoassay method

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Abstract

Presently, there is no doubt about the functioning of the adenylate cyclase signaling system in plants, but the role of this system in various physiological–biochemical processes has been investigated insufficiently. Cyclic adenosine monophosphate (cAMP), the key component produced by adenylate cyclase, whose concentrations in plant cells vary rather widely, is the indicator of functional activity for this signaling way. In the latter case, in the process of determination of concentrations of this messenger, one encounters difficulties related to insufficient sensitivity of the methods most frequently applied. In this connection, the proposed mechanism is a modification of the method of the enzyme immunoassay (EIA), which is based on immediate measurement of cAMP concentrations in the sample with the use of antibodies. This modification allows us to determine the concentrations of cAMP with the precision of 5 pM, which exceeds the sensitivity of other methods by approximately 10 times. The specificity of the assay has been confirmed by other two independent tests––the capillary electrophoresis and the nuclear magnetic resonance (NMR). It has also been compared to the data obtained with the use of the commercial kit from Sigma–Aldrich. The modification has been tested on such plant objects as in vitro potato plants, and suspension cells of potato and Arabidopsis.

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References

  • Brown EG, Newton RP, Smith CI (1980) A cyclic AMP-binding protein from barley seedlings. Phytochemistry 19:2263–2267. doi:10.1002/ps.805

    Article  CAS  Google Scholar 

  • Chin K, Moeder W, Yoshioka K (2009) Biological roles of cyclic-nucleotide-gated ion channels in plants: What we know and don’t know about this 20 member ion channel family. Botany 87:668–677. doi:10.1139/B08-147

    Article  CAS  Google Scholar 

  • Chin K, Moeder W, Abbel-Hamid H, Shahinas D, Gupta D, Yoshioka K (2010) Importance of the alphaC-helix in the cyclic nucleotide binding domain for the stable channel regulation and function of cyclic nucleotide gated ion channels in Arabidopsis. J Exp Bot 61:2383–2393. doi:10.1093/jxb/erq072

    Article  CAS  PubMed  Google Scholar 

  • Cooke CJ, Smith CJ, Walton TJ, Newton RP (1994) Evidence that cyclic AMP is involved in the hypersensitive response of Medicago sativa to a fungal elicitor. Phytochem 35:889–894. doi:10.1002/ps.805

    Article  CAS  Google Scholar 

  • Gehring C (2010) Adenyl cyclases and cAMP in plant signaling––past and present. Cell Commun Signal 8:15. doi:10.1186./1478-811x-8-15

    Article  PubMed  Google Scholar 

  • Gilman AG (1970) A protein binding assay for adenosine 3′,5′-cyclic monophosphate. Proc Natl Acad Sci 67:305–312. doi:10.1073/pnas.0700609104

    Article  CAS  PubMed  Google Scholar 

  • Ichikawa T, Suzuki Y, Czaja I (1997) Identification and role of adenylyl cyclase in auxin signaling in higher plants. Nature 390:698–701

    CAS  PubMed  Google Scholar 

  • Kalinowsky H-O, Berger S, Braun S (1988) Carbon-13 NMR spectroscopy. John Wiley & Sons, New York, p 776

    Google Scholar 

  • Karimova FG, Tyrikina EV, Zaharova OY (2005) cAMP-dependent phosphorylation of pea proteins, induced by forskolin. Russ J Plant Physiol 52:21–28

    Article  CAS  Google Scholar 

  • Lomovatskaya LA, Romanenko AS, Krivolapova NV, Kopytchuk VN, Salyaev RK (2006) Activities of transmembrane and “soluble” adenylate cyclases in potato cell organelles during bacterial pathogenesis. Doklady Biol Sci 409:1–3. doi:10.1134/S001249660604020x

    Article  Google Scholar 

  • Lomovatskaya LA, Romanenko AS, Filinova NV (2008) Plant adenylate cyclases. J Recept Signal Transduct 28:531–542. doi:10.1080/10799890802602308

    Article  CAS  Google Scholar 

  • Ma W, Smigel A, Verma R, Berkowitz G (2009a) Cyclic nucleotide gated channels and related signaling components in plant innate immunity. Plant Signal Behav 4:277–282. doi:10.4161/psb.4.4.8103

    Article  CAS  PubMed  Google Scholar 

  • Ma W, Qi Z, Smigel A, Walker RK, Verma R, Berkowitz GA (2009b) Ca2+, cAMP, and transduction of non-self perception during plant immune responses. Proc Natl Acad Sci 106:20995–21000. doi:10.1073/pnas.0905831106

    Article  CAS  PubMed  Google Scholar 

  • Martinez-Atienza J, Van Ingelgem C, Roef L, Maathuis FJM (2007) Plant cyclic nucleotide signaling. Plant Signal Behav 2:540–543. doi:10.4161/psb

    PubMed  Google Scholar 

  • Moutinho A, Hussey PJ, Trevawas AJ, Malho R (2001) Cyclic AMP acts as a second messenger in pollen tube growth and reorientation. Proc Natl Acad Sci 98:10481–10486. doi:10.1073/pnas.0700609104

    Article  CAS  PubMed  Google Scholar 

  • Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497. doi:10.1104/pp.109.900312

    Article  CAS  Google Scholar 

  • Newton RP, Gibbs N, Moyse CD, Wiebers J, Brown EG (1980) Mass spectrometric identification of adenosine 3′:5′-cyclic monophosphate isolated from a higher plant tissue. Phytochem 19:1909–1911. doi:10.1002/ps.805

    Article  CAS  Google Scholar 

  • Newton R, Roef L, Witters E, VanOnckenen H (1999) Cyclic nucleotides in higher plants: the enduring paradox. New Phytol 143:427–455. doi:10.1111/j.1469-8137.2007.02063.x

    Article  CAS  Google Scholar 

  • Nicolaev VO, Lohse MJ (2006) Monitoring of cAMP synthesis and degradation in living cells. Physiology 21:86–92. doi:10.1152/physiol.00057.2005

    Article  Google Scholar 

  • Polya GM, Bowman JA (1981) Resolution and properties of two high affinity cyclic adenosine 3′,5′-monophosphate-binding proteins from wheat germ. Plant Physiol 68:577–584. doi:10.1104/pp.108.118935

    Article  CAS  PubMed  Google Scholar 

  • Richards H, Das S, Smith C et al (2002) Cyclic nucleotidate content of tobacco BY-2 cells. Phytochem 61:531–537. doi:10.1002/ps.805

    Article  CAS  Google Scholar 

  • Roef L, Witters E, Gadeyne J, Marcssen K, Newton RP, VanOncenen H (1996) Analysis of 3′:5′-cAMP and adenylyl cyclase activity in higher plants using polyclonal chicken egg yolk antibodies adenylate cyclase. Analit Biochem 233:188–196. doi:10.1016/j.ab.2008.08.016

    Article  CAS  Google Scholar 

  • Rosenberg N, Pines M, Sela J (1982) Adenosin 3, 5-cyclic monophosphate–its release in a higher plant by an exogeneous stimulus as detected by radioimmunoassay. FEBS Letters 137:105–107. doi:10.1016/j.febslet.2009.12.058

    Article  CAS  Google Scholar 

  • Tijssen P (1985) Practice and theory of enzyme immunoassays. Elsevier. Amsterdam/New York, pp xxvi + 549

  • White AD, Zenser TV (1971) Separation of cyclic 3:5-nucleoside monophosphates from other nucleotides on aluminium oxide columns. Application to the assay of adenyl cyclase and guanyl cyclase. Analit Biochem 41:372–396. doi:10.1016/j.ab.2008.08.016

    CAS  Google Scholar 

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Authors and Affiliations

  1. Laboratory of Phytoimmunology, Siberian Institute of Plant Physiology and Biochemistry, Siberian Branch of the Russian Academy of Sciences, Irkutsk, Russia

    L. A. Lomovatskaya, A. S. Romanenko & N. V. Filinova

  2. Laboratory of Physics Methods Science, Siberian Institute of Plant Physiology and Biochemistry, Siberian Branch of the Russian Academy of Sciences, Irkutsk, Russia

    L. V. Dudareva

Authors
  1. L. A. Lomovatskaya
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  2. A. S. Romanenko
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  3. N. V. Filinova
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  4. L. V. Dudareva
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Corresponding author

Correspondence to L. A. Lomovatskaya.

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Communicated by B. Li.

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Lomovatskaya, L.A., Romanenko, A.S., Filinova, N.V. et al. Determination of cAMP in plant cells by a modified enzyme immunoassay method. Plant Cell Rep 30, 125–132 (2011). https://doi.org/10.1007/s00299-010-0950-5

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  • DOI: https://doi.org/10.1007/s00299-010-0950-5

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