Studies into the molecular genetics of alkaloid synthesis in the genus Nicotiana

The presence of pyridine alkaloids (mainly nicotine, nor-nicotine, anabasine and anatabine) is a characteristic feature of species within the Nicotiana genus. Most species contain predominately nicotine or nor-nicotine, with a few species, including Nicotiana glauca (tree tobacco), accumulating anabasine as a major component of their alkaloid fraction. Previous studies in this laboratory have indicated that within 24 hours of aerial damage (apex removal/leaf damage), the transcript levels of several alkaloid biosynthetic genes increase in the leaves ofN. glauca plants, but not the roots. This precedes an increase in leaf anabasine concentrations 5-7 days after wounding (Sinclair et ai, 2004). A more detailed investigation of this wound response in N. glauca was undertaken in the present study. It was determined that increased levels of anabasine biosynthesis occurs predominately in the upper leaves ofN. glauca plants, in response to apex removal, with very little stimulation in alkaloid synthesis occurring in response to leaf wounding alone. This response was shown to be phytohormone dependent, mediated via a reduction in auxin concentration or the application of methyl jasmonate. To further investigate the molecular mechanisms governing the leaf based induction of alkaloid biosynthesis in N. glauca, the genomic DNA sequences of three genes involved in alkaloid biosynthesis, quinolinate phosphoribosyltransferase (QPT), ornithine decarboxylase (ODC) and A622, were isolated, following the creation and screening of a genomic library. Two versions of the QPT gene were isolated (NgQPTl and NgQPT2) from the genomic library of N. glauca. Both genes are highly homologous throughout their coding regions, and appear capable of encoding full length QPT proteins. However, they contain markedly divergent 5’ regulatory regions and intronic regions. NgQPT2 seems to be the predominant transcript in both leaves and roots of N. glauca plants, with the proportion of the NgQPT2 transcript in the total QPT transcript pool, appearing to increase in both tissues in response to apex removal. Fusion of 2.4 kb from the NgQPT2 5’ regulatory region to uidA, and transfer into hairy roots and transgenic plants, showed the promoter to be responsive to jasmonate. However, it appears to lack the necessary elements that confer responsiveness to apex removal and leaf based expression in transgenic N. glauca plants. The AP-2 transcription factors, NtORCl and NtJAPl, which have previously been shown to activate the Nicotiana sylvestris putrescine N-methyltransferase (PMT) promoter (DeSutter et al., 2005), were tested for their ability to activate the N. glauca QPT2 promoter. Using a tobacco cell protoplast transient assay system, NtORCl, but not NtJAPl, was shown to activate the NgQPT2 promoter. The vi endogenous QPT transcript was also shown to accumulate in N. glauca hairy root lines overexpressing the NtORCl gene. Previous studies have indicated that the ODC enzyme from Nicotiana glutinosa can decarboxylate both ornithine and lysine (Lee and Cho, 2001a). To determine whether this enzyme is involved in the synthesis of anabasine in N. glauca, ODC transcript levels were down-regulated by RNAi technology in N. glauca hairy root cultures. ODC transcript downregulation had very little effect upon anabasine levels, although a decrease in nicotine concentration was observed in many lines. This suggests that lysine decarboxylation, leading to cadaverine and anabasine synthesis, occurs via a different enzyme in this species. As a point of comparison, ODC transcript levels were also down-regulated in N. tabacum hairy root cultures, with a marked decrease in nicotine, and a concurrent increase in anatabine concentrations being observed in a number of lines. One copy of the A622 gene was isolated from the genome of N. glauca. At the onset of this study, a functional role for A622 in alkaloid biosynthesis had not been established. To examine the role of A622 in anabasine biosynthesis, transcript levels were down-regulated in N. glauca plants, via RNAi technology. A substantial reduction in anabasine levels was observed in transgenic plants and their offspring containing the RNAi construct. These results confirm an important role for the A622 enzyme in the synthesis of anabasine in N. glauca. This aspect of the current study has been published recently (DeBoer et al., 2009)