title: Enzyme inhibitor studies reveal complex control of methyl-D-erythritol 4-phosphate (MEP) pathway enzyme expression in Catharanthus roseus.
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.
authors: Centre for Organismal Studies (COS) Heidelberg, Heidelberg University, Heidelberg, Germany.
authors: Mei
authors: M
authors: Han
authors: Simon C
authors: SC
authors: Heppel
authors: Tao
authors: T
authors: Su
authors: Jochen
authors: J
authors: Bogs
authors: Yuangang
authors: Y
authors: Zu
authors: Zhigang
authors: Z
authors: An
authors: Thomas
authors: T
authors: Rausch
mesh_terms: D030008:Catharanthus / Q000187:drug effects / Q000201:enzymology*; D002736:Chloroplasts / Q000187:drug effects / Q000201:enzymology; D003001:Cloning, Molecular; D003513:Cycloheximide / Q000494:pharmacology; D018076:DNA, Complementary / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004896:Erythritol / Q000031:analogs & derivatives* / Q000378:metabolism; D005578:Fosfomycin / Q000031:analogs & derivatives / Q000494:pharmacology; D015870:Gene Expression; D018506:Gene Expression Regulation, Plant; D006540:Herbicides / Q000494:pharmacology; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D007555:Isoxazoles / Q000494:pharmacology; D053858:Metabolic Networks and Pathways; D009928:Organ Specificity; D023303:Oxazolidinones / Q000494:pharmacology; D010269:Paraquat / Q000494:pharmacology; D018515:Plant Leaves / Q000187:drug effects / Q000201:enzymology; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D018517:Plant Roots / Q000187:drug effects / Q000201:enzymology; D011401:Promoter Regions, Genetic; D059748:Proteolysis; D013403:Sugar Phosphates / Q000378:metabolism*; D014166:Transferases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
chemical_list: C114232:2-C-methylerythritol 4-phosphate; D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D006540:Herbicides; D007527:Isoenzymes; D007555:Isoxazoles; D023303:Oxazolidinones; D010940:Plant Proteins; D013403:Sugar Phosphates; D005578:Fosfomycin; C095255:clomazone; C024640:fosmidomycin; D003513:Cycloheximide; D014166:Transferases; C109461:deoxyxylulose-5-phosphate synthase; D010269:Paraquat; D004896:Erythritol
references: Sci Rep. 2011;1:9
references: 22355528
references: Plant J. 2009 Nov;60(3):424-35
references: 19594711
references: Phytochemistry. 2005 Aug;66(15):1797-803
references: 16054176
references: Curr Pharm Des. 2004;10(19):2391-400
references: 15279616
references: Plant J. 2005 Oct;44(1):155-66
references: 16167903
references: Plant Mol Biol. 2007 Sep;65(1-2):13-30
references: 17611800
references: Plant Cell Physiol. 2007 Feb;48(2):322-31
references: 17213228
references: Plant Physiol. 2006 May;141(1):75-84
references: 16531478
references: Metab Eng. 2011 Mar;13(2):234-40
references: 21144909
references: Plant Cell Environ. 2009 Jan;32(1):82-92
references: 19021881
references: Biochim Biophys Acta. 2000 Dec 15;1517(1):159-63
references: 11118631
references: Genome Biol. 2002 Jun 18;3(7):RESEARCH0034
references: 12184808
references: Annu Rev Plant Physiol Plant Mol Biol. 1999 Jun;50:47-65
references: 15012203
references: Biochem Soc Trans. 2000 Dec;28(6):792-3
references: 11171210
references: Plant J. 2002 Aug;31(3):243-54
references: 12164805
references: Mol Plant. 2010 Sep;3(5):904-16
references: 20591838
references: Plant Cell. 2005 Feb;17(2):628-43
references: 15659625
references: Biotechnol Prog. 2003 May-Jun;19(3):1105-8
references: 12790690
references: Proc Natl Acad Sci U S A. 2006 Apr 4;103(14):5614-9
references: 16565214
references: Curr Med Chem. 2004 Mar;11(5):607-28
references: 15032608
references: Proc Natl Acad Sci U S A. 1991 Mar 15;88(6):2042-5
references: 11607160
references: J Biol Chem. 2001 Jun 22;276(25):22901-9
references: 11264287
references: Proc Natl Acad Sci U S A. 2003 May 27;100(11):6866-71
references: 12748386
references: Trends Plant Sci. 2008 Dec;13(12):619-23
references: 18948055
references: Plant Methods. 2005 Dec 18;1:13
references: 16359558
references: FEBS Lett. 1998 Sep 4;434(3):413-6
references: 9742965
references: Mol Cell Proteomics. 2009 Aug;8(8):1789-1810
references: 19423572
references: Cell. 2012 Jun 22;149(7):1525-35
references: 22726439
references: J Exp Bot. 2009;60(10):2933-43
references: 19584121
references: Plant Mol Biol. 2006 Nov;62(4-5):683-95
references: 16941216
references: J Antibiot (Tokyo). 2010 Oct;63(10):583-8
references: 20808315
references: Prog Lipid Res. 2012 Apr;51(2):95-148
references: 22197147
references: Int J Mol Sci. 2009 Jul 10;10(7):3168-85
references: 19742130
references: Plant Cell. 2008 May;20(5):1303-15
references: 18469163
references: Plant Physiol. 2002 Nov;130(3):1079-89
references: 12427975
references: Plant Cell Rep. 2007 Jul;26(7):907-15
references: 17333024
references: Nat Prod Rep. 2006 Aug;23(4):532-47
references: 16874388
references: Science. 2000 Jul 14;289(5477):295-7
references: 10894776
references: J Biosci. 2003 Sep;28(5):637-46
references: 14517367
references: Plant Physiol. 1990 Mar;92(3):777-84
references: 16667349
references: Mol Biol Evol. 2007 Aug;24(8):1596-9
references: 17488738
references: Plant J. 2008 Oct;56(1):86-100
references: 18557838
references: J Exp Bot. 2011 Mar;62(6):2023-38
references: 21199890
references: Plant Mol Biol. 2007 Sep;65(1-2):177-87
references: 17634747
references: Plant Cell. 1999 May;11(5):887-900
references: 10330473
references: Plant J. 2004 Apr;38(1):131-41
references: 15053766
references: Plant J. 2000 Mar;21(6):571-8
references: 10758508
references: Plant J. 2008 May;54(4):763-84
references: 18476877
references: Plant Mol Biol. 2007 Oct;65(3):243-57
references: 17687625
references: PLoS One. 2012;7(8):e43038
references: 22916202
references: Proc Natl Acad Sci U S A. 2001 Jul 17;98(15):8915-20
references: 11427737
references: Planta. 1990 Mar;180(4):603-12
references: 24202107
references: J Biol Chem. 2007 Jul 27;282(30):21573-7
references: 17442674