Agriculture & Horticulture

The need to develop agricultural crops with improved stress tolerance and/or resistance has led plant biotechnologists to strategically focus onto those plant molecules involved in the initiation and sustenance of cellular homeostasis. One such molecule, typically involved in signal transduction, is the cyclic adenosine 3’,5’-monophosphate (cAMP) generated by the enzyme adenylate cyclase (AC). However, even though ACs have previously been experimentally proven to be centrally involved in numerous stress response systems in various organisms (bacteria, fungi and lower eukaryotes), their existence and/or functional roles in higher plants have until recently, been a matter of debate and serious controversy. To date, only four higher plants ACs have been functionally confirmed, specifically in Arabidopsis thaliana, Zea mays, Nicotiana benthamiana and Hippeastrum hybridum. Apparently, since it is inconceivable that a single AC per plant can account for all the currently known and/or reported cAMP-dependent processes in higher plants, we then, in this study, set out to enzymatically and functionally characterize a second probable AC candidate from A. thaliana in the form of a putative clathrin assembly protein (AtCAP: At1g68110), with a view of elucidating its exact physiological and biological roles in higher plants. In this regard, we then firstly conducted a preliminary bioinformatic analysis of this putative protein candidate followed by its molecular cloning, recombinant expression and endogenous activity assaying, then its affinity purification and in vitro functional characterization and finally, its co-expressional and bioinformatic functional analysis. Our findings unequivocally, indicated that this novel protein is indeed a multi-domain, multi-functional bona fide soluble adenylate cyclase (sAC) responsible for biotic stress responses and whose functional activities are essentially mediated by the cAMP via a calmodulin/SORLIP1AT core motifdependent signaling system.