Enzyme Inhibitor Studies Reveal Complex Control of Methyl-D-Erythritol 4-Phosphate (MEP) Pathway Enzyme Expression in Catharanthus roseus
Figure 4
Activation of DXS isoforms' promoters (CrDXS1p, CrDXS2Ap and CrDXS2Bp) by the transcription factor ORCA3, analyzed via transient expression of promoter-luciferase fusions in leaves of 6-week old C. roseus plants.
Results are expressed as fold change of promoter activation, based on four independent assays. The promoter activity was determined by the ratio of luciferase (LUC) activity to renilla expression. Dual CaMV 35S promoter was employed as a positive control showing high LUC activity, while promoterless LUC control (pGreenII 0800-LUC vector) reflected the LUC background value. DXS promoter sequences were fused to the firefly luciferase and transiently co-transformed with ORCA3 or without (mock control). Agrobacteria-mediated transient transformation was carried via leaf infiltration of 6-week-old soil-grown C. roseus plants. Two days after transformation, infiltrated leaves were harvested and subjected to dual-luciferase reporter assay. Error bars represent the standard deviation of four independent experiments for promoter activity analysis. The asterisks represent significant difference (** p<0.001, * p<0.1 by student's t-test).