Alkaloid Formation and Secondary Metabolism

The most abundant of the alkaloids produced in wild and cultivated tobacco (Nicotiana) are nicotine, nornicotine, anabasine, and anatabine. In most commercial tobacco varieties, nicotine represents 90-95% of the total alkaloid content of the leaf, with the remainder of the alkaloid pool accounted for by minor alkaloids present in varied amounts depending on cultivar. We now understand that the biosynthesis of nicotine and its derivatives is controlled by a variety of factors. Among the major endogenous factors, changes in levels and/or ratios of various phytohormones (e.g., auxin, ethylene) as a consequence of plant development stimulate nicotine formation in the roots and its translocation to the leaves. Removal of the flowering stalk during commercial tobacco growing is well know to induce alkaloid formation and nicotine accumulation, either through a wound response or via auxin derepression.  Biotic stresses (such as insect herbivory) have also been shown to stimulate the synthesis and accumulation of nicotine and its derivatives. This process is tied into a jasmonic acid (JA) response cascade.
 
Using an integrated approach that includes biochemical analysis, genome sequencing and bioinformatics, reverse genetics, and microarray-based gene expression profiling we are trying to identify the genes involved and the transcription factors and basic signal transduction components that control major and minor alkaloid biosynthesis pathways in tobacco. In our work we utilize a combination of approaches to define genes of importance in alkaloid biosynthesis, BY-2 tobacco suspension cultures and whole plant studies along with various gene transformation procedures to identify regulatory motifs and elements in the promoters of genes encoding key enzymes in nicotine and minor alkaloid formation, and various in vivo and in vitro approaches to define the specific transcription factors (TF) and TF complexes that bind these elements to promote or repress gene expression. 

Among the current focuses of the laboratory at present is understanding the role of jasmonic acid (JA) and JA signaling in the regulation of alkaloid formation during development and in response to wounding and resistance to herbivore attack. To this end we are defining the TFs and promoter elements that mediate JA-responsiveness in several key alkaloid biosynthesis genes including putrescine N-methyltransferase, quinolinic acid phosphoribosyltransferase, and nicotine demethylase. To accomplish this we are using BY-2 cell cultures as a model in combination with targeted gene alteration and gene expression profiling with Nimblegen microarrays.