Abstract
Adenylate cyclases (ACs) are enzymes capable of converting adenosine-5′-triphosphate to cyclic 3′, 5′-adenosine monophosphate (cAMP). In animals and lower eukaryotes, ACs and their product cAMP have firmly been established as important signalling molecules with important roles in several cellular signal transduction pathways. However, in higher plants, the only annotated and experimentally confirmed AC is a Zea mays pollen protein capable of generating cAMP. Recently a number of candidate AC-encoding genes in Arabidopsis thaliana have been proposed based on functionally assigned amino acids in the catalytic center of annotated and/or experimentally tested nucleotide cyclases in lower and higher eukaryotes. Here we detail the cloning and recombinant expression of functional candidate AC domains using, as an example, the A. thaliana pentatricopeptide repeat-containing protein (AtPPR-AC; At1g62590). Through a complementation test, in vivo adenylate cyclase activity of candidate recombinant molecules can be prescreened and promising candidates can subsequently be further evaluated in an in vitro AC immunoassay.
Key words
- Arabidopsis thaliana
- Pentatricopeptide (PPR)
- Adenylate cyclase (AC)
- Adenosine-5′-triphosphate (ATP)
- Cyclic 3′,5′-adenosine monophosphate (cAMP)
- Lactose fermenters
- Enzyme immunoassay
This is a preview of subscription content, access via your institution.
Buying options
Springer Nature is developing a new tool to find and evaluate Protocols. Learn more
References
Robison GA, Butcher RW, Sutherland EW (1968) Cyclic AMP. Annu Rev Biochem 37:149–174
Goodman DB, Rasmussen H, DiBella F et al (1970) Cyclic adenosine 3′:5′-monophosphate-stimulated phosphorylation of isolated neurotubule subunits. Proc Natl Acad Sci USA 67:652–659
Gerisch G, Hülser D, Malchow D et al (1975) Cell communication by periodic cyclic-AMP pulses. Philos Trans R Soc Lond B Biol Sci 272:181–192
Ashton AR, Polya GM (1978) Cyclic adenosine 3′:5′-monophosphate in axenic rye grass endosperm cell cultures. Plant Physiol 61:718–722
Butcher RW, Baird CE, Sutherland EW (1968) Effects of lipolytic and antilipolytic substances on adenosine 3′,5′-monophosphate levels in isolated fat cells. J Biol Chem 243:1705–1712
Amrhein N (1977) The current status of cyclic AMP in higher plants. Annu Rev Plant Physiol 28:123–132
Meier S, Ruzvidzo O, Morse M et al (2010) The Arabidopsis wall-associated kinase-like 10 gene encodes a functional guanylyl cyclase and is co-expressed with pathogen defense related genes. PLoS One 5:e8904
Kurosaki F, Nishi A (1993) Stimulation of calcium influx and calcium cascade by cyclic AMP in cultured carrot cells. Arch Biochem Biophys 302:144–151
Carricarte VC, Bianchini GM, Muschietti JP et al (1988) Adenylate cyclase activity in a higher plant, alfalfa (Medicago sativa). Biochem J 249:807–811
Li W, Luan S, Schreiber SL et al (1994) Cyclic AMP stimulates K+ channel activity in mesophyll cells of Vicia faba L. Plant Physiol 106:957–961
Moutinho A, Hussey PJ, Trewavas AJ et al (2001) cAMP acts as a second messenger in pollen tube growth and reorientation. Proc Natl Acad Sci USA 98:10481–10486
DeYoung BJ, Innes RW (2006) Plant NBS-LRR proteins in pathogen sensing and host defense. Nat Immun 7:1243–1249
Newton RP, Smith CJ (2004) Cyclic nucleotides. Phytochemistry 65:2423–2437
Kwezi L, Meier S, Mungur L et al (2007) The Arabidopsis thaliana brassinosteroid receptor (AtBRI1) contains a domain that functions as a guanylyl cyclase in vitro. PLoS One 2:7
Mulaudzi T, Ludidi N, Ruzvidzo O et al (2011) Identification of a novel Arabidopsis thaliana nitric oxide-binding molecule with guanylate cyclase activity in vitro. FEBS lett 585:2693–2697
Gehring C (2010) Adenyl cyclases and cAMP in plant signaling—past and present. Cell Commun Signal 8:15
Nakamura T, Schuster G, Sugiura G et al (2004) Chloroplast RNA-binding and pentatricopeptide repeat proteins. Biochem Soc Trans 32:571–574
Kotera E, Tasaka M, Shikanai T (2005) A pentatricopeptide repeat protein is essential for RNA editing in chloroplasts. Nature 433:326–330
Schmitz-Linneweber C, Williams-Carrier R, Barkan A (2005) RNA immunoprecipitation and microarray analysis show a chloroplast pentatricopeptide repeat protein to be associated with the 5′ region of mRNAs whose translation it activates. Plant Cell 17:2791–2804
Kurien BT, Scofield RH (2009) Nonelectrophoretic bidirectional transfer of a single SDS-PAGE gel with multiple antigens to obtain 12 immunoblots. Methods Mol Biol 536:55–65
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254
Tang WJ, Stanzel M, Gilman AG (1995) Truncation and alanine-scanning mutants of type I adenylyl cyclase. Biochemistry 34:14563–14572
Tesmer JJ, Dessauer CW, Sunahara RK et al (2000) Molecular basis for P-site inhibition of adenylyl cyclase. Biochemistry 39:14464–14471
Geng W, Wang Z, Zhang J et al (2005) Cloning and characterization of the human soluble adenylyl cyclase. Am J Physiol 288:C1305–C1316
Oh M-H, Kim HS, Wu X et al (2012) Calcium/calmodulin inhibition of the Arabidopsis BRASSINOSTEROID-INSENSITIVE 1 receptor kinase provides a possible link between calcium and brassinosteroid signalling. Biochem J 443:515–523
Chen Y, Cann MJ, Litvin TN et al (2000) Soluble adenylyl cyclase as an evolutionarily conserved bicarbonate sensor. Science 289:625–628
Acknowledgement
This material is based upon work supported financially by the National Research Foundation but any opinion, findings and conclusions or recommendations expressed in this material are those of the author(s) and therefore the NRF does not accept any liability in regard thereto.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2013 Springer Science+Business Media New York
About this protocol
Cite this protocol
Ruzvidzo, O., Dikobe, B.T., Kawadza, D.T., Mabadahanye, G.H., Chatukuta, P., Kwezi, L. (2013). Recombinant Expression and Functional Testing of Candidate Adenylate Cyclase Domains. In: Gehring, C. (eds) Cyclic Nucleotide Signaling in Plants. Methods in Molecular Biology, vol 1016. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-441-8_2
Download citation
DOI: https://doi.org/10.1007/978-1-62703-441-8_2
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-440-1
Online ISBN: 978-1-62703-441-8
eBook Packages: Springer Protocols