Adenylyl cyclases (ACs) are a special group of enzymes that catalyze formation of the second messenger molecule, 3',5'-cyclic adenosine monophosphate (cAMP) from 5'-adenosine triphosphate (ATP). Apparently, even though cAMP is increasingly becoming an important signaling molecule in higher plants, the identification of plant ACs has somewhat remained slow. Here we report the recombinant cloning, partial expression and affinity purification of the truncated version (AtAC 261-388 ) of a putative Arabidopsis thaliana protein (AtAC: At3g21465) followed by a demonstration of its inherent enzymatic activity as an AC. Currently, AtAC is not assigned any particular function in A. thaliana but simply annotated as an AC-like protein and, therefore, we targeted it for our study to establish if it is indeed a bona fide AC molecule.From our work, we firstly, show through enzyme immunoassaying and mass spectrometry that the recombinant AtAC 261-388 can generate cAMP from ATP in vitro in a manganese-dependent manner that is activated by calcium and hydrogen carbonate. Secondly, we reveal through computational analysis that the AC center of AtAC is solvent-exposed, and amenable to the unhindered access of ATP as a substrate for catalysis. Lastly, we show that the recombinant AtAC 261-388 can complement AC-deficiency (cyaA mutation) in SP850 cells when expressed in this mutant Escherichia coli strain.

Plants, just like any other living organism, naturally get attacked by various pathogenic microorganisms such as bacteria, fungi and viruses. However, unlike animals that utilize their specialized circulatory macrophage system to protect themselves, plants instead use a multi-layered complex system termed the plant innate immunity, which recognizes pathogens and transducing downstream defense responses. They have developed a unique type of transmembrane receptors or R proteins, which extracellularly, are capable of recognizing pathogen-associated molecular patterns (PAMP) such as flagellin and chitin, while intracellularly, they activate their harbored nucleotide cyclases (NCs) such as adenylyl cyclases (ACs), to generate second messenger molecules such as 3',5'-cyclic adenosine monophosphate (cAMP), which then propagates and magnifies the defense response. To date, only a single R protein from Arabidopsis thaliana (AtLRR) has been shown to possess AC activity as well as having the ability to defend plants against infection by biotrophic and hemi-biotrophic pathogens. Therefore, in order to further broaden information around the functional roles of this protein (AtLRR), we explored it further, using an array of web-based tools or bioinformatics. These included structural analysis, anatomical expression analysis, developmental expression analysis, co-expression analysis, functional enrichment analysis, stimulusspecific expression analysis and promoter analysis. Findings from structural analysis showed that AtLRR is a multi-domain, trans-membrane molecule that is multi-functional, and thus consistent with all known R-proteins.