## 2 Results and Discussion
### Expression of Isoprenoid Biosynthetic Genes and Isoprenoid Production  
On the basis of the chemical similarity of \(\beta\)-CDs to pectic oligosaccharides are released by the cell wall after a fungal infection, the early induction response after three days of treatment, mainly regarding lutein, Q9 and Q10, thus determining a total increase of intracellular content of isoprenoids in cell cultures of _A. annua_ (Figures 1 and 2), could be explained by the occurrence of a stress response. In _Vitis vinifera_\(\beta\)-CDs have been reported to act as true elicitors by activating different families of transcription factors regulating the expression of genes involved in the trans-resveratrol biosynthetic pathway [4, 5, 18]. In order to analyze the relationship between the transcript profile of biosynthetic genes and the production of isoprenoid compounds, the expression levels of 1-deoxy-d-xylulose-5-phosphate reductio isomerase (_DXR_), farnesyl diphosphate synthase (_FDS_), geranylgeranyl diphosphate synthase (_GGDS_), isopentenyl diphosphate isomerase (_IDI_), lycopene beta-cyclase (_LYC-B_), phytoene synthase (_PS_) and 3-hydroxy-3-methyl-glutaryl-Coenzyme A reductase (_HMGR_) genes were determined in _A. annua_ cell cultures treated for one and three days with DIMEB in comparison with untreated samples. These genes were chosen as key enzymes in the terpenoid biosynthetic routes, the cytosolic MVA and the plastidial MVA independent, as shown in Figure 3.  
The results of Real-Time PCR revealed that in _A. annua_ cell cultures treated for one day with DIMEB, transcript accumulation of all the analyzed genes was lower than in untreated samples (Figure 4A), while in cells treated for three days the expression of _DXR_ was significantly higher (more than 2.5-fold) compared to untreated samples. Slight increases were observed in the expression of _FDS_, _IDI_ and _LYC-B_ genes and no significant changes were detected for the other genes (Figure 4B). _DXR_ encodes for a key enzyme in the plastidial MVA-independent pathway, catalyzing the biosynthesis of methylerythnitol 4-phosphate (MEP), which is the first committed intermediate of the pathway [19]. _DXR_ overexpressing _Arabidopsis thaliana_ plants were reported to have an increased accumulation of plastid isoprenoids including chlorophylls and carotenoids [20]. Thus the up-regulation of _DXR_ gene only observed after three days of DIMEB treatment could account for the increase of carotenoids in our cell system. The considerable increase of chlorophyll levels measured in DIMEB treated cells compared to untreated samples (data not shown) strengthened this hypothesis. On the other hand, a cross talk between the two isoprenoid routes has been demonstrated and an export of isoprenoid C5 units from the plastid to the cytosol can occur in higher plants [21] thus the enhancement of the plastidial route could influence also the formation of cytosolic isoprenoids such as quinones. Figure 3: Isoprenoid biosynthetic pathways in the plant cell. Abbreviations used in **Cytosol**: HMG-CoA, Hydroxyethylglutaryl-coenzyme A; MVA, mevalonic acid; MVP, 5-phosphomevalonate; MVPP, 5-diphosphomevalonate; DMAPP, dimethylallyl diphosphate; IPP, isopenthenyl diphosphate; FPP, farnesyl diphosphate; AACT, acetoacetyl-coenzyme A thiolase; HMGS, 3-hydroxy-3-methyl-glutaryl coenzyme A synthase; _HMGR_, 3-hydroxy-3-methyl-glutaryl coenzyme A reductase; MVK, mevalonate kinase; PMK, phosphomevalonate kinase; PMD, diphosphomevalonate decarboxylase; _IDI_, isopentenyl diphosphate isomerase; _FDS_, famesyl diphosphate synthase.