Recombinant Expression and Molecular Elucidation of the Biochemical Functions of a Novel Linker Histone-like Protein from Arabidopsis thaliana

Abstract

Plants are sessile organisms and have evolved to live in environments exposed to various stress factors that may occur singly and/or in combination and as such, plants have to respond and adapt accordingly for survival. The threat of global climatic change has caused serious concerns among scientists as crop growth could be severely affected by changes in key climatic variables. In this regard therefore, food security thus has to heavily depend on the development of crop plants with increased resistance to environmental stress. As a result of this reason, plant biotechnologists have strategically began focusing on those plant molecules involved in maintaining and sustaining functional homeostasis. Cyclic 3',5'-adenosine monophosphate (cAMP) and its generating enzyme, adenylate cyclase (AC), are one group of such plant molecules that plant biotechnologists are currently focusing on. ACs have previously been experimentally proven to be centrally involved in a number of stress response systems in prokaryotes, higher eukaryotes and animals, while their existence and/or functional properties in higher plants have until recently, been a very serious matter of debate and elusiveness. To date, only eight A. thaliana protein ACs have since been confirmed in higher plants while only one has been confirmed in lower plants. Those in higher plants are the Zea mays pollen signaling protein, the Arabidopsis pentatricopeptide repeat-containing protein, the Nicotiana benthamiana adenylyl cyclase protein, a Hippeastrum hybridum adenylyl cyclase protein, two A. thaliana K+-uptake permeases, an Arabidopsis clathrin assembly protein and an A. thaliana leucine-rich repeat protein. Apparently, with the knowledge that a single AC per plant cannot account for all the cAMP-dependent processes in plants, this study was thereof set out to enzymatically and functionally characterize a novel probable AC candidate from A. thaliana in the form of a putative LHL protein (AtLHL: At3g18035), with a view of elucidating the elusiveness of its exact biological, functional and physiological roles in higher plants. Findings from our preliminary analysis of this protein using various bioinformatic programs and tools showed that this putative candidate is actually a binding signalling candidate with a possible role in cAMP-dependent adaptation and stress response mechanisms. More so, when this putative protein was cloned and recombinantly expressed in some chemically competent E. cloni BL21 (DE3) pLysS DUOs cells, the protein demonstrated its ability to generate endogenous cAMP in this prokaryotic expression system thereby raising the cAMP levels to over 2 folds. Again, the recombinant protein when expressed in the SP850 Escherichia coli mutant, it demonstrated its catalytic ability to generate cAMP from ATP whereby it functionally rescued this AC-deficient E. coli mutant from a non-lactose fermenter into a lactose fermenter – data that unequivocally provided evidence that the protein is indeed a functional AC. Additionally and when purified, the recombinant demonstrated to generate significant cAMP in vitro AC activity that was Mn²⁺-, Ca²⁺ and CO₃²⁻-dependent, reminiscent of all known soluble ACs (sACs). In summary, we therefore, concluded that the LHL protein is an essential plant protein candidate that may play an essential role in the possible management of plant stress-related factors for the enhancement of cultivar status and ultimately, crop productivity.