Multicolor pigments in tobacco.
Transient expression of biosynthetic enzymes in the leaves of Nicotiana benthamiana is a very efficient and currently popular way to produce complex secondary metabolites quickly and in sufficient quantities. In recent years, this tobacco expression platform has contributed to the rapid elucidation of several biosynthetic pathways of complex natural products and has greatly accelerated the growth of knowledge in this field. However, this does not include anthocyanins, whose expression in Nicotiana benthamiana works poorly and has so far only been successful in the production of delphinidin 3-O-rutinoside, which is considered a precursor for some but not all complex anthocyanins. The production of other anthocyanins, however, would require the repression of certain endogenous flavonoid biosynthetic genes with simultaneous transient expression of other genes.
IPB scientists have now developed an N. benthamiana expression platform that enables the production of any desired basic anthocyanin by infiltrating a few gene constructs. For the synthesis of more complex anthocyanins, species-specific biosynthetic genes can also be introduced into the tobacco leaves. The Halle scientists’ strategy is based on the overexpression of certain anthocyanin biosynthesis genes that are insufficiently or not at all expressed in N. benthamiana, while at the same time inactivating other genes that could hinder the production of certain anthocyanins. With the help of this platform, the researchers succeeded in obtaining the basic anthocyanins pelargonidin 3-O-glucoside, cyanidin 3-O-glucoside and delphinidin 3-O-glucoside in just a few days and in large quantities.
In general, substance production in Nicotiana benthamiana is based on cloning the required biosynthetic genes into an expression vector controlled by the robust constitutive 35S promoter of the cauliflower mosaic virus. After infiltration of the construct into the tobacco leaves by agrobacteria, the introduced genes are transiently expressed. In this process, several genes can be simultaneously transferred into the plant using different agrobacterial strains and transcribed there. This method enables the elucidation of entire metabolic pathways, even if the exact sequence of the successive enzymatic reactions is not known. The new expression platform now enables the elucidation of biosynthetic pathways of complex anthocyanins and provides a possible strategy for rapid and high-yield production of these compounds and their non-natural derivatives.
Anthocyanins are water-soluble pigments that occur in the majority of higher plants and give many flowers and fruits their beautiful red, purple, or blue colors. They are produced in the cytosol by several enzymatic reactions from the precursor molecule phenylalanine and are stored in the vacuole. The first colored and stable products are the basic compounds pelargonidin 3-O-glucoside, cyanidin 3-O-glucoside, and delphinidin 3-O-glucoside. In many plants, these basic anthocyanins undergo further modifications, such as glycosylation, acylation, and methylation, which affect the color and stability of the pigments. Anthocyanins are used as food coloring for confectionery, jam, canned fruit, and baking products. The colors show antioxidative, anti-inflammatory, and vasoprotective effects.