Lt

Figure 7 Utilization of various concentrations of loganin (L) and loganin/tryp-tamine (LT) by lines S1 (transgenic for Sir, (a) and T22 (transgenic for Tdc, (b). Feeding consisted of addition of L or LT in a series of concentrations from 0.4 mM up to 6.4 mM at inoculation. Right-hand y-axis represents percentages of loganin (□) utilized in TIAs. Duplicate cultures were pooled upon harvesting on day 2 and analyzed as single samples (C, control).

Figure 7 Utilization of various concentrations of loganin (L) and loganin/tryp-tamine (LT) by lines S1 (transgenic for Sir, (a) and T22 (transgenic for Tdc, (b). Feeding consisted of addition of L or LT in a series of concentrations from 0.4 mM up to 6.4 mM at inoculation. Right-hand y-axis represents percentages of loganin (□) utilized in TIAs. Duplicate cultures were pooled upon harvesting on day 2 and analyzed as single samples (C, control).

TIA pathway, in this case TDC or STR, was not sufficient to keep elevated TIA levels. Although TDC is known to be a strongly regulated enzyme, which has a transient maximum in activity shortly after subculture on a fresh medium, TDC seems to not be a limiting factor in these cells. Also, STR is not a limiting factor. The following points might be considered in future studies, concerning possible physiological or further biochemical barriers for TIA accumulation in C. roseus cell cultures:

The cellular or subcellular localization of enzymes, precursors, and TIAs

Factors involved in transport of the precursors for TIAs Effect of competitive pathway(s) utilizing TIA precursors Catabolism of TIAs

Factors that control the carbon flux into the pathway

As all the factors are connected with each other, further studies on the regulatory genes and the overexpression of such genes seem a promising approach. Our studies showed that there is a short-term increase in the TIA levels with overexpression of Tdc and Str genes; however, in a complex pathway such as the TIA pathway, overexpression of one or two enzymes seemed to be sufficient only when accompanied by a favorable epigenetic environment. This is in contrast to the overexpression of chalcone isomerase in tomatoes, which resulted in a 70-fold increase of flavanoids (87). An encouraging fact is that expression of the transgenes seems to be stable over long periods despite the fact that in cell suspension cultures there is a strong selection for fast-growing cells.

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