Kirsi-Marja Oksman-Caldentey

Regulation of Secondary Metabolism in Tobacco Cell Cultures

In addition to primary metabolites, which are essential for life and development, plants also synthesize a number of low molecular weight compounds, so-called secondary metabolites. These compounds have important functions for plants in survival and competing in the environment, in protection against UV light as well as in various defence-related reactions. Up to date, about 100,000 plant secondary metabolites have been isolated (Verpoorte 2000). Most plant constituents that are used medicinally are secondary metabolites, and up to 25% of the contemporary drugs contain an active compound originating from plants. In addition, secondary metabolites are of interest for people as flavours, fragrances, pesticides and dyes. An important group of pharmacologically active compounds consists of alkaloids. Up to 15,000 alkaloids have been characterised since the identification of the first alkaloid morphine from the opium poppy in 1806 (Kutchan 1995). One of the most studied plants is tobacco, belonging to the genus Nicotiana, which was named after the French diplomat Jean Nicot who, in the middle of the sixteenth century, started to popularise tobacco in Europe. Tobacco secondary metabolites have been extensively studied and more than 2500 compounds have been identified. However, the biosynthetic pathways and metabolism of these compounds need further elucidation (Nugroho and Verpoorte 2002). A lot of work concerning the biosynthetic studies has been done using plant cell cultures in order to overcome the problems caused by cultivation of the whole plants. The aim of this chapter is to give an overview of the nicotine alkaloid biosynthesis in tobacco callus and cell suspension cultures including tobacco BY-2 cell culture, and offer an insight into the variables affecting the alkaloid production in these systems. In addition, metabolism of other secondary compounds in tobacco cell cultures is discussed. Today, ample possibilities to study secondary metabolism are allowed by novel techniques, such as genome-wide gene identification, which is demonstrated here by using tobacco BY-2 cell culture.

Functional characterisation of genes alkaloid biosynthesis in involved in pyridine tobacco

Although secondary metabolism in Nicotiana tabacum (L.) (tobacco) is rather well studied, many molecular aspects of the biosynthetic pathways and their regulation remain to be disclosed, even for prominent compounds such as nicotine and other pyridine alkaloids. To identify players in tobacco pyridine alkaloid biosynthesis a functional screen was performed, starting from a tobacco gene collection established previously by means of combined transcript profiling and metabolite analysis. First, full-length cDNA clones were isolated for 34 genes, corresponding to tobacco transcript tag sequences putatively associated with pyridine alkaloid metabolism. Full-length open reading frames were transferred to pCaMV35S-steered overexpression vectors. The effects of plant transformation with these expression cassettes on the accumulation of nicotine and other pyridine alkaloids were assessed in transgenic tobacco Bright-Yellow 2 (BY-2) cell suspensions and hairy root cultures. This screen identified potential catalysers of tobacco pyridine metabolism, amongst which a lysine decarboxylase-like gene and a GH3-like enzyme. Overexpression of the GH3-like enzyme, presumably involved in auxin homeostasis and designated NtNEG1 (Nicotiana tabacum Nicotine-Enhancing GH3 enzyme 1), increased nicotine levels in BY-2 hairy roots significantly. This study shows how functional genomics-based identification of genes potentially involved in biosynthetic pathways followed by systematic functional assays in plant cells can be used at large-scale to decipher plant metabolic networks at the molecular level.