Enzyme Inhibitor Studies Reveal Complex Control of Methyl-D-Erythritol 4-Phosphate (MEP) Pathway Enzyme Expression in Catharanthus roseus
Figure 8
Time course of in vivo cycloheximide treatment as compared to simultaneous treatment with fosmidomycin or clomazone, respectively, and subsequent turnover of DXS and DXR proteins in isolated chloroplasts.
(A) for each plant, two pairs of mature leaves were injected with 100 µM cycloheximide solution alone, or in combination with 50 µM fosmidomycin or 50 µM clomazone, respectively. For each time point, young leaves were collected from three independent plants and processed for immunoblot analysis of DXS, DXR and HDS, respectively. (B) chloroplasts from young leaves of 6-week-old soil-grown C. rosues plants, treated with 50 µM fosmidomycin, 50 µM clomazone, or water (control) were isolated 78 hours after leaf injection (see (A) this Fig.). 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 and fosmidomycin or clomazone (50 µM). Aliquots were taken at 0, 15, 30 and 60 minutes and used for protein extraction. DXS and DXR proteins were detected by immunoblot. 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 fosmidomycin or clomazone samples (see also Fig. 5C).