Enzyme inhibitor studies reveal complex control of methyl-D-erythritol 4-phosphate (MEP) pathway enzyme expression in Catharanthus roseus

PLoS One. 2013 May 1;8(5):e62467. doi: 10.1371/journal.pone.0062467. Print 2013.

Abstract

In Catharanthus roseus, the monoterpene moiety exerts a strong flux control for monoterpene indole alkaloid (MIA) formation. Monoterpene synthesis depends on the methyl-D-erythritol 4-phosphate (MEP) pathway. Here, we have explored the regulation of this pathway in response to developmental and environmental cues and in response to specific enzyme inhibitors. For the MEP pathway entry enzyme 1-deoxy-D-xylulose 5-phosphate synthase (DXS), a new (type I) DXS isoform, CrDXS1, has been cloned, which, in contrast to previous reports on type II CrDXS, was not transcriptionally activated by the transcription factor ORCA3. Regulation of the MEP pathway in response to metabolic perturbations has been explored using the enzyme inhibitors clomazone (precursor of 5-ketochlomazone, inhibitor of DXS) and fosmidomycin (inhibitor of deoxyxylulose 5-phosphate reductoisomerase (DXR)), respectively. Young leaves of non-flowering plants were exposed to both inhibitors, adopting a non-invasive in vivo technique. Transcripts and proteins of DXS (3 isoforms), DXR, and hydroxymethylbutenyl diphosphate synthase (HDS) were monitored, and protein stability was followed in isolated chloroplasts. Transcripts for DXS1 were repressed by both inhibitors, whereas transcripts for DXS2A&B, DXR and HDS increased after clomazone treatment but were barely affected by fosmidomycin treatment. DXS protein accumulated in response to both inhibitors, whereas DXR and HDS proteins were less affected. Fosmidomycin-induced accumulation of DXS protein indicated substantial posttranscriptional regulation. Furthermore, fosmidomycin effectively protected DXR against degradation in planta and in isolated chloroplasts. Thus our results suggest that DXR protein stability may be affected by substrate binding. In summary, the present results provide novel insight into the regulation of DXS expression in C. roseus in response to MEP-pathway perturbation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Catharanthus / drug effects
  • Catharanthus / enzymology*
  • Chloroplasts / drug effects
  • Chloroplasts / enzymology
  • Cloning, Molecular
  • Cycloheximide / pharmacology
  • DNA, Complementary / genetics
  • Enzyme Inhibitors / pharmacology*
  • Erythritol / analogs & derivatives*
  • Erythritol / metabolism
  • Fosfomycin / analogs & derivatives
  • Fosfomycin / pharmacology
  • Gene Expression
  • Gene Expression Regulation, Plant
  • Herbicides / pharmacology
  • Isoenzymes / genetics
  • Isoenzymes / metabolism
  • Isoxazoles / pharmacology
  • Metabolic Networks and Pathways
  • Organ Specificity
  • Oxazolidinones / pharmacology
  • Paraquat / pharmacology
  • Plant Leaves / drug effects
  • Plant Leaves / enzymology
  • Plant Proteins / genetics
  • Plant Proteins / metabolism*
  • Plant Roots / drug effects
  • Plant Roots / enzymology
  • Promoter Regions, Genetic
  • Proteolysis
  • Sugar Phosphates / metabolism*
  • Transferases / antagonists & inhibitors
  • Transferases / genetics
  • Transferases / metabolism*

Substances

  • 2-C-methylerythritol 4-phosphate
  • DNA, Complementary
  • Enzyme Inhibitors
  • Herbicides
  • Isoenzymes
  • Isoxazoles
  • Oxazolidinones
  • Plant Proteins
  • Sugar Phosphates
  • Fosfomycin
  • clomazone
  • fosmidomycin
  • Cycloheximide
  • Transferases
  • deoxyxylulose-5-phosphate synthase
  • Paraquat
  • Erythritol

Grant support

Funding came from Heidelberg University Core Funding and a DAAD stipend to Mei Han. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.