CRTI expression in BY2 cells
The tobacco BY2 cells were used as a non-green model system to investigate the effect of CRTI expression on carotenoid content and expression of endogenous carotenoid biosynthesis genes. Analogous to the A. thaliana transformation experiments (see Results), the vector pSToff was used for transformation to mediate the constitutive expression of CRTI driven by the CaMV35S promoter (see Materials and Methods for tissue culture regime and vector construction).
Eight transgenic lines were recovered and investigated for CrtI expression levels and carotenoid content using TaqMan Real-Time-PCR and HPLC analysis, respectively. As shown in Fig. S1 the lines exhibited comparable expression levels for the transgene except line o3, where three to five times higher levels were observed. The carotenoid content in these transgenic cultures showed some variability relative to wild-type cells, ranging from a reduction of about 30% (lines o1, o2) to an increase of 20% to 40% (lines o3, o6-o10). In contrast to A. thaliana and rice leaves, with BY2 transformants it was not possible to find a change in the carotenoid pattern i.e. a decrease in lutein abundance. BY2 cells do not contain this xanthophyll and, therefore, the effect did not appear.
The carotenoid pattern in wild-type and transgenic BY2 tissue cultures did not show any lycopene accumulation, just as with rice and A. thaliana. Small amounts of -carotene (2%) were detected upon HPLC analysis, while neoxanthin and violoaxanthin represented 95% of the total carotenoids.
The lines o1 and o2 with their lowered carotenoid content and line o3 with its high CrtI expression level showing increased carotenoid content relative to the wild-type were chosen for gene expression analysis. Selected endogenous carotenoid biosynthesis genes (coding for PSY, PDS, ZDS, ISO and LCY) were analyzed for their expression levels using TaqMan Real-Time-PCR with tobacco-specific primers (given in Table S2). The results are summarized in Fig. S2.
Considerable variation in the expression of these genes was observed between the transgenic and wild-type cultures. No consistent up- or down-regulation of the expression levels of the investigated genes was found in response to CRTI expression. Given the good reproducibility of TaqMan Real-Time-PCR data obtained from biological replicates (independently harvested materials), this variability is probably due to growth variations of the transgenic cultures. Thus, there is no consistent CRTI-dependent effect on the expression of the investigated genes, as found with A. thaliana and rice, and a regulatory feedback loop originating from all-trans lycopene is not detectable.