Enzyme Inhibitor Studies Reveal Complex Control of Methyl-D-Erythritol 4-Phosphate (MEP) Pathway Enzyme Expression in Catharanthus roseus
Figure 5
Time course of in vivo clomazone treatment on the expression of MEP pathway genes, and subsequent degradation of DXS and DXR proteins in isolated chloroplasts.
For each plant, two pairs of mature leaves were injected with a 50 µM clomazone solution (or water for control) until the entire leaf blades were fully soaked, using a 1 ml needleless syringe applied to the lower epidermis. For each time point, young leaves were pooled from three independent plants and processed for MEP pathway protein and transcript analysis, respectively. (A) DXS, DXR and HDS proteins were detected by immunoblot. Equal sample loading was confirmed by Coomassie staining. The arrow marks the position of mature CrDXS2A protein. (B) transcript amounts for DXS isoforms (1, 2A & 2B), DXR and HDS were determined by qPCR relative to the geometric mean of multiple reference genes according to Vandesompele et al.[60]. The experiment was performed 3 times, values from a representative experiment are presented ± SD. (C) chloroplasts were isolated from young leaves of 6-week-old soil-grown C. rosues control and 50 µM clomazone-treated plants (see (A) this Figure) 78 hours after treatment. Chloroplasts were incubated for 1 h in the light (100 µmol m−2 s−1) at 25°C in the presence of 5 mM ATP. Aliquots were taken at 0, 15, 30 and 60 minutes and used for protein extraction. DXS and DXR proteins were detected by immunoblot. Note that to obtain similar signal intensity at time point 0, the loading amount of protein from control samples (chloroplast isolated from water infiltrated plants) was twice that of clomazone samples.