Organ Differentiation and Secondary Plant Products

For the second case where metabolites are produced during organ differentiation, organ culture is a suitable alternative even on a large scale (Curtis, 1993; Kusakari et al., 2000). Hairy roots, transformed with Agrobacterium rhizogenes, have also been found to be suitable for the production of secondary metabolites due to their stability and high productivity in hormone-free culture conditions (Oksman- Caldentey, 2002; Shanks and Morgan, 1999), and several medicinal plant species have been successfully transformed in this manner. The selection of high-productivity root lines based on somaclonal variation also offers an interesting option for enhancing productivity. While this type of metabolite-producing culture provides stable material and an interesting field of study, how hairy root formation affects metabolite production is not clear. The hairy root system could have a more complicated level of regulation due to tissue organization and the effect of integrated transgenes (see below).

In higher plants and other complex organisms, certain pathways of secondary metabolism can depend on the general development of the organism, including organ, tissue, and particular specialized cell development (Wiermann, 1981). The biosynthesis and accumulation of several secondary compounds occur during defined developmental stages in an organism. Compounds are not necessarily synthesized in organs and tissues with high levels of accumulation; for example, tropane alkaloids in Atropa and nicotine alkaloids in tobacco are produced in root and transported to aerial parts (Yun et al., 1992).

One of the most well-known examples of cell differentiation regarding secondary metabolites is the glandular trichome for essential oils in mint and related species (Kutchan, 2005a). These glandular cells show a striking differentiation of tubular smooth ER, and the biosynthetic characteristics of glandular trichomes have been examined by EST analysis (Lange et al., 2000). Highly cytotoxic monoterpenoids require this specific structure for biosynthesis and accumulation. While it is not clear whether the process of the formation of glandular trichomes is similar to that of nonglandular trichomes, the successful trichome formation seen on all epidermal surfaces by the constitutive overexpression of transcriptional factor (GL1 and maize R) genes may provide insight into cellular differentiation and metabolite production (Lange and Croteau, 1999).

The extent to which secondary metabolism depends on the development of specific cellular structures is not yet clear. Poppy plants have idioblast cells specialized for the storage of secondary products and laticifers for excretion (Bird et al., 2003; Facchini and St-Pierre, 2005;Kutchan, 2005a;Weid et al., 2004).Terpenoid indole and tropane alkaloids also need similar cellular collaboration (Burlat et al., 2004; Facchini and St-Pierre, 2005; Kutchan,

FIGURE 11.6 Cell-specific gene expression in tropane alkaloids. (A) A model for alkaloid trafficking between different cell types in root. (B) Pericycle cell-specific gene expression of the H6H gene. (C) Cell-specific gene expression of the PMT gene.

2005a; St-Pierre et al., 1999): nonmevalonate pathway enzymes and G10H to produce 10-hydroxygeraniol are localized in internal phloemparenchyma cells, TDC and STR, in the early pathway of terpenoid indole alkaloid biosynthesis, are in epidermal cells, while D4H and DAT, in the late pathway, are in idioblast cells of aerial organs. In tropane alkaloid biosynthesis, the entry enzyme,PMT, is expressed in the cortex and endodermis,whereas the last step of the pathway, H6H, is specifically expressed in pericycle cells in root (Fig. 11.6). These results indicate that intermediates should drive the site of the primary reaction to that of the end reaction. The morphological differentiation of cells would be needed for functional differentiation of each specific reaction in metabolism. However, the molecular basis of the link between function and morphological differentiation is not yet clear. Indeed, this observation can explain why the production of some metabolites requires organ differentiation. For example, the overexpression of a key enzyme (H6H) in tropane alkaloid biosynthesis can improve the production of scopolamine in cultured cells (Yun et al., 1992).