A Putative Protein with No Known Function in Arabidopsis thaliana Harbors a Domain with Adenylyl Cyclase Activity

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 (AtAC261-388) 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 AtAC261-388 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 AtAC261-388 can complement AC-deficiency (cyaA mutation) in SP850 cells when expressed in this mutant Escherichia coli strain.

Elaborating the Functional Roles of a Leucine-Rich Repeat Protein from Arabidopsis thaliana

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 trans-membrane 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, stimulus-specific 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. Findings from anatomical and developmental expression analyses showed that AtLRR is mostly expressed in pollen grains and flowers, senescing leaves as well as during the development of seeds, shoots, roots, seedlings, leaves, flowers, and siliques, linking it to the three key plant physiological processes of reproduction, defense and development respectively. Lastly, findings from co-expression, functional enrichment, stimulus-specific expression and promoter analyses, showed that AtLRR is mostly co-expressed with several other proteins linked to disease resistance, plant reproduction and plant development. Activities and functions of such protein are also commonly regulated by cAMP via a common W-box promoter. So, all in all, our study managed to establish that besides being strongly involved in disease resistance against biotrophic and hemi-biotrophic pathogens, AtLRR also plays key roles in plant development (seed, shoot, root, seedling, leaf, and silique development) and reproduction (flowering, and pollen tube growth and re-orientation), whereby it effects its functions via a W-box or WRKY transcription factor, TTGACY, mediated by cAMP.