Figure 2.
Changes in Isoprenoid/Carotenoid Gene Expression Levels and Enzyme Activities Resulting from the Expression of an Additional Psy-1.
The Psy-1 variety PS-1C26R2 (T7) was used for the determination of gene expression and enzyme activity. One fruit (at the mature green stage, 36 to 37 d after anthesis) from three representative Psy-1 and wild-type plants was pooled and pulverized into a homogeneous powder as described in Methods. Total RNA was then extracted from an aliquot of this material. Quantitative real-time RT-PCR was performed with gene-specific primers for (1) Dxs, (3) Ggpps-1, (4) Ggpps-2, (5) Psy-1, (6) Psy-2, (7) Pds, (8) Zds, (9) CrtISO, (10) the Cyc-B gene, and (11) Lcy-B. Enzymes are numbered similarly, with the addition of (2) IPP isomerase. Expression data were normalized to the expression of actin. The data represent se ± (n = 3 to 8). Student's t tests illustrate statistical significance (* P < 0.05, ** P < 0.01, and *** P < 0.001). The black bars indicate wild-type determinations, and the checkered bars are for the Psy-1 transgenic variety. Enzyme activities were performed on extracts determined from the same tissue used for RNA analysis. Specific assays for DXS, GGPPS, PSY, PDS, and lycopene cyclase were performed as described in Methods. The protein levels used were as follows: 440 μg for the Psy-1 and wild-type extracts used in the assays of phytoene desaturation and lycopene cyclization; 200 and 100 μg for the wild type and Psy-1 extracts, respectively, used to assay DXS, IPI, GGPPS, and PSY. Typically, substrate levels were ∼50,000 dpm phytoene and 20,000 dpm lycopene formed from 1 μCi 14C-IPP. In all cases, activity was expressed as dpm incorporated/mg protein/h. Determinations were performed at least in triplicate. Data are shown as se (n = at least 3).