Molecular and Functional Characterization of a Novel Microtubule Associated Protein from Arabidopsis thaliana

Abstract

The question of whether cyclic 3?,5?-adenosine monophosphate (cAMP) is an authentic second messenger molecule in higher plants has kept plant scientists busy for many decades, this being due to methodological challenges encountered in previous years, when techniques developed for its analysis in mammalian tissues were merely adopted for plant material. cAMP is a product of ATP hydrolysis catalyzed by the enzyme adenylate cyclase (AC). These two groups of molecules (cAMP and ACs) play an important role in mediating environmental stimuli to physiological responses in living cells. In this study, our work focused on an Arabidopsis thaliana microtubule associated protein (AtMTA) encoded by the At3g02930 gene, which was recently bioinformatically annotated as an AC candidate. In order to elucidate its possible functional roles in higher plants, a preliminary bioinformatic analysis of its encoding gene, the At3g02930, was conducted, where it was found that AtMTA is implicated in the photosynthetic processes of plants as well as the reciprocal meiotic recombination functions. Furthermore, to assess for its possible AC activity, total mRNA from 6 weeks old A. thaliana leaf material was extracted and used as a template in a specialized RT-PCR system for amplification of the At3g02930 gene fragment bearing the putative AC catalytic center (AtMTA-AC). The amplified gene fragment was then cloned into a pTrcHis-TOPO expression vector to form a pTrcHis-TOPO:AtMTA-AC recombinant construct, which was then used to transform competent Escherichia coli BL21 (DE3) pLysS cells. Protein expression then proceeded after induction of the transformed cell cultures with 1 mM IPTG. After expression, the resultant recombinant protein was then purified on an Ni-NTA affinity system under non-native denaturation conditions because the expressed recombinant product was found to be mostly expressed in its insoluble state. This was then followed by its refolding into its native and soluble form via a linear gradient on the same purification system. The resultant protein product was then tested for its possible endogenous, in vitro and in vivo AC activities followed by a further characterization of such an activity. The endogenous and in-vitro assays were determined via a cAMP-specific enzyme immunoassay system, whereby it was shown indisputably, that the AtMTA-AC protein has the ability to induce the generation of endogenous cAMP in a prokaryotic expression system while at the same time, capable of generating cAMP as a pure product. On the other hand, the in vivo activity was tested and determined via a complementation testing, where it was strongly confirmed that this recombinant protein is indeed a bona fide functional AC molecule capable of generating cAMP from ATP, and rescuing a non-lactose mutant E. coli strain into a lactose fermenting host. Results from all the three tests showed that this putative protein has some inherent endogenous, in vitro and in vivo AC activities and therefore, confirming it as a higher plant AC with a possible cAMP-mediated signaling function. Lastly, a detailed overview of the bioinformatic expressional profile of the At3g02930 gene was conducted, where the gene was found to be directly linked to three other genes on the microarray network which are the: At5g52280 and At1g64330 genes, which function as myosin heavy chain-related proteins and At3g53350 that functions as a repressor of primer (ROP) interactive partner 4. Thus since co-expressed genes are generally expected to be involved in related and/or similar cellular functions, then the AtMTA-AC gene may potentially be a myosin heavy chain-related protein or a ROP interactive partner 4 with essential roles in growth and development. The gene could also response to various environmental stress factors.