Targeting Tryptophan Decarboxylase to Selected Subcellular Compartments of Tobacco Plants Affects Enzyme Stability and in Vivo Function and Leads to a Lesion-Mimic Phenotype

Abstract

Tryptophan decarboxylase (TDC) is a cytosolic enzyme that catalyzes an early step of the terpenoid indole alkaloid biosynthetic pathway by decarboxylation of l-tryptophan to produce the protoalkaloid tryptamine. In the present study, recombinant TDC was targeted to the chloroplast, cytosol, and endoplasmic reticulum (ER) of tobacco (Nicotiana tabacum) plants to evaluate the effects of subcellular compartmentation on the accumulation of functional enzyme and its corresponding enzymatic product. TDC accumulation and in vivo function was significantly affected by the subcellular localization. Immunoblot analysis demonstrated that chloroplast-targeted TDC had improved accumulation and/or stability when compared with the cytosolic enzyme. Because ER-targeted TDC was not detectable by immunoblot analysis and tryptamine levels found in transient expression studies and in transgenic plants were low, it was concluded that the recombinant TDC was most likely unstable if ER retained. Targeting TDC to the chloroplast stroma resulted in the highest accumulation level of tryptamine so far reported in the literature for studies on heterologous TDC expression in tobacco. However, plants accumulating high levels of functional TDC in the chloroplast developed a lesion-mimic phenotype that was probably triggered by the relatively high accumulation of tryptamine in this compartment. We demonstrate that subcellular targeting may provide a useful strategy for enhancing accumulation and/or stability of enzymes involved in secondary metabolism and to divert metabolic flux toward desired end products. However, metabolic engineering of plants is a very demanding task because unexpected, and possibly unwanted, effects may be observed on plant metabolism and/or phenotype.Plants produce large arrays of chemicals, many of which are referred to as secondary metabolites. Despite the minor role initially assigned to these molecules, secondary metabolites are now considered crucial for the interaction of plants with their environment (Verpoorte, 1998). Many secondary metabolites are also important therapeutic agents or pharmaceuticals, and the generally low abundance to which they accumulate has prompted extensive research into their biosynthetic pathways. To date, few plant secondary metabolic pathways have been fully characterized, and some have been partially characterized, although investigation into many is at an early stage. Nevertheless, it is clear that the biosynthesis of secondary metabolites is under strict developmental, temporal, and spatial control in plants (St. Pierre et al., 1999; De Luca and St. Pierre, 2000). As a consequence of the strictly regulated biosynthesis, natural products accumulate to only trace amounts in plants, and their extraction and purification is often difficult and cost intensive. Attempts to use plant cell cultures as an alternative source to natural products have also been problematic, often due to the lack of fully functional pathways required for the production of the target molecules. With the exceptio...

Production of Coumaroylserotonin and Feruloylserotonin in Transgenic Rice Expressing Pepper Hydroxycinnamoyl-Coenzyme A:Serotonin N-(Hydroxycinnamoyl)transferase

“…TDC activity was about 2-fold higher in the roots than in the shoots and ranged from 0.36 to 1.83 pkat mg 21 protein, which was consistent with the values reported by Lopez-Meyer and Nessler (1997) and Ueno et al (2003). As for a negative control of TDC assay, we employed tobacco, which was reported to lack TDC gene (Fiore et al, 2002). TDC activities from leaves and suspension-cell culture of tobacco were not detected at all.…”

Novel Roles for the Polyphenol Oxidase Enzyme in Secondary Metabolism and the Regulation of Cell Death in Walnut

“…Based on extensive pathogen testing, we have concluded that these lesions are not associated with disease. Similar lesion mimic phenotypes have been observed in a variety of transgenic plants in which antioxidant levels (Takahashi et al, 1997), carbohydrate metabolism (Mittler and Rizhsky, 2000), defenseassociated genes (Shirano et al, 2002), or phenolic metabolism (Di Fiore et al, 2002) have been manipulated. The absence of elevated reactive oxygen species levels, SA, and oxidative damage in PPO-silenced lines points to a subtle, potentially metabolic, connection between cell death and PPO in walnut.…”

Modulation of Polyamine Biosynthesis in Transformed Tobacco Plants by Targeting Ornithine Decarboxylase to an Atypical Subcellular Compartment

“…Therefore, targeting enzymes to an atypical subcellular compartment provides a novel strategy for creating additional capacities for rate-limiting steps of a pathway. The feasibility of such a strategy has been illustrated for the biosynthesis of amino acids [14,15] and terpenoid indole alkaloids in plants [16]. In the presented study, we demonstrated that targeting of recombinant ODC to a different and atypical subcellular compartment resulted in significant increased enzyme activity and putrescine levels.…”