Recombinant expression and functional characterisation of a putative clathrin assembly protein from arabidopsis thaliana

Abstract

The need to develop agricultural crops with persistent resistance to the vagaries of climate change has led plant biotechnologists to strategically focus on those plant molecules involved in the maintenance and sustenance of homeostasis. One such plant molecule that is typically involved in the various signal transduction and cellular communication systems is the cyclic adenosine 3′,5′-monophosphate (cAMP) which naturally, is generated by the enzyme, adenylate cyclase (AC). Even though ACs have previously been experimentally proven to be centrally involved in numerous stress response systems in animals, prokaryotes and lower eukaryotes, their existence and/or functional properties in higher plants have until recently, been a very serious matter of debate and elusiveness. To date, only four ACs have since been confirmed in higher plants, and specifically in the Arabidopsis thaliana, Zea mays, Nicotiana benthamiana and Hippeastrum hybridum plants. Since it is inconceivable that a single AC per plant can account for all the cAMP-dependent processes in plants, we 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. From our findings, a preliminary bioinformatic analysis of this protein showed that this putative candidate is actually a multi-domain, multi-functional protein with a possible role in AC-dependent stress response and adaptation mechanisms. We then cloned and recombinantly expressed its AC-domain-containing fragment of AtCAP (AtCAP-AC) in the chemically competent E. cloni BL21 (DE3) pLysS DUOs cells and unequivocally demonstrated its ability to induce the generation of endogenous cAMP in this prokaryotic expression system. More so, we also undoubtedly demonstrated a complementation system of the mutant non-lactose fermenting cyaA SP850 Escherichia coli strain by this recombinant AtCAP-AC protein, to eventually ferment lactose and as a result of this protein’s ability to generate the most required cAMP molecule for this process. In addition, we also undertook the purification system of this truncated AtCAP-AC protein followed by the functional characterization of its in vitro AC activities, which in turn revealed that this recombinant is indeed a bona fide soluble adenylate cyclase (sAC), whose physiological and biochemical roles may be mediated by cAMP and possibly, via a calmodulin-dependent signalling system. Lastly and in order to gain more insights into the possible physiological roles of this AtCAP-AC protein in higher plants, we then bioinformatically analysed its expressional profiles using the various computational and web-based bioinformatic tools, and found out that the protein is centrally involved in responses to biotic stress factors, whose systems are both cAMP- and SORLIP1AT core motif-dependent.