d58cf2597ceef8faae30cf2215294dfa pmc_32272573.pdf 18771f34cc24b147600d3a6e32d8bb95d84007f8 pmc_32272573.pdf dcebed57385765077c356d83ce2ad46a4fe88d2398f98ce17240a6ffb60b9889 pmc_32272573.pdf Title: Cellular and Subcellular Compartmentation of the 2C-Methyl-D-Erythritol 4-Phosphate Pathway in the Madagascar Periwinkle Subject: The Madagascar periwinkle (Catharanthus roseus) synthesizes the highly valuable monoterpene indole alkaloids (MIAs) through a long metabolic route initiated by the 2C-methyl-D-erythritol 4-phosphate (MEP) pathway. In leaves, a complex compartmentation of the MIA biosynthetic pathway occurs at both the cellular and subcellular levels, notably for some gene products of the MEP pathway. To get a complete overview of the pathway organization, we cloned four genes encoding missing enzymes involved in the MEP pathway before conducting a systematic analysis of transcript distribution and protein subcellular localization. RNA in situ hybridization revealed that all MEP pathway genes were coordinately and mainly expressed in internal phloem-associated parenchyma of young leaves, reinforcing the role of this tissue in MIA biosynthesis. At the subcellular level, transient cell transformation and expression of fluorescent protein fusions showed that all MEP pathway enzymes were targeted to plastids. Surprisingly, two isoforms of 1-deoxy-D-xylulose 5-phosphate synthase and 1-deoxy-D-xylulose 5-phosphate reductoisomerase initially exhibited an artifactual aggregated pattern of localization due to high protein accumulation. Immunogold combined with transmission electron microscopy, transient transformations performed with a low amount of transforming DNA and fusion/deletion experiments established that both enzymes were rather diffuse in stroma and stromules of plastids as also observed for the last six enzymes of the pathway. Taken together, these results provide new insights into a potential role of stromules in enhancing MIA precursor exchange with other cell compartments to favor metabolic fluxes towards the MIA biosynthesis. Keywords: 2C-methyl-D-erythritol 4-phosphate pathway; compartmentation; Catharanthus roseus; stromules; GFP imaging; localization artifact; alkaloids Author: Grégory Guirimand, Anthony Guihur, Catalina Perello, Michael Phillips, Samira Mahroug, Audrey Oudin, Thomas Dugé de Bernonville, Sébastien Besseau, Arnaud Lanoue, Nathalie Giglioli-Guivarc’h, Nicolas Papon, Benoit St-Pierre, Manuel Rodríguez-Concepcíon, Vincent Burlat and Vincent Courdavault Creator: LaTeX with hyperref package Producer: pdfTeX-1.40.18 CreationDate: Tue Apr 7 09:03:08 2020 CEST ModDate: Tue Apr 7 09:03:08 2020 CEST Custom Metadata: yes Metadata Stream: no Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 25 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 10602986 bytes Optimized: no PDF version: 1.5 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- CHFALQ+URWPalladioL-Ital Type 1 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2024-10-02 04:12:20 CEST RepresentationInformation: pmc_32272573.pdf ReportingModule: PDF-hul, Rel. 1.12.4 (2023-03-16) LastModified: 2023-03-31 11:44:28 CEST Size: 10602986 Format: PDF Version: 1.5 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 0 FreeObjects: 7 IncrementalUpdates: 0 DocumentCatalog: ViewerPreferences: HideToolbar: false HideMenubar: false HideWindowUI: false FitWindow: true CenterWindow: false DisplayDocTitle: false NonFullScreenPageMode: UseNone Direction: L2R ViewArea: CropBox ViewClip: CropBox PrintArea: CropBox PageClip: CropBox PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: ぉのぴひはつふっぴどはの〠 Item: Title: げづびふぬぴび〠 Children: Item: Title: ぃぬはのどので〠ちのつ〠うふのっぴどはのちぬ〠ざちぬどつちぴどはの〠はて〠っいぎぁび〠ぅのっはつどので〠ち〠こづっはのつ〠いじこ〠ぉびはてはひね〬〠ぃきこ〬〠ぃきか〠ちのつ〠えいげ〠 Item: Title: きぅぐ〠ぐちぴとぷちべ〠ぇづのづび〠ぁひづ〠えどでとぬべ〠ぃはづへばひづびびづつ〠どの〠ぉぐぁぐ〠ぃづぬぬび〠はて〠すはふので〠がづちぶづび〠 Item: Title: きぅぐ〠ぐちぴとぷちべ〠ぅのぺべねづび〠ぁひづ〠ごちひでづぴづつ〠ぴは〠ぐぬちびぴどつび〠 Item: Title: ぐふのっぴふちぴづつ〠ぐちぴぴづひの〠はて〠がはっちぬどぺちぴどはの〠はて〠いじこ〠ちのつ〠いじげ〠げづぱふどひづび〠ぴとづ〠しとはぬづ〠ぐひはぴづどの〠こづぱふづのっづ〠 Item: Title: ぐふのっぴふちぴづつ〠がはっちぬどぺちぴどはの〠はて〠いじこ〠ちのつ〠いじげ〠どび〠がどにづぬべ〠ぃちふびづつ〠ぢべ〠ぐひはぴづどの〠くぶづひづへばひづびびどはの〠 Item: Title: いどびっふびびどはの〠 Item: Title: きちぴづひどちぬ〠ちのつ〠きづぴとはつび〠 Children: Item: Title: ぐぬちのぴ〠きちぴづひどちぬ〠ちのつ〠ぃづぬぬ〠ぃふぬぴふひづ〠ぃはのつどぴどはのび〠 Item: Title: げぎぁ〠ぅへぴひちっぴどはの〠ちのつ〠っいぎぁ〠こべのぴとづびどび〠 Item: Title: ぃぬはのどので〠はて〠ぴとづ〠っいぎぁ〠こづぱふづのっづび〠てひはね〠ぴとづ〠ぃ〮〠げはびづふび〠きぅぐ〠ぐちぴとぷちべ〠ぅのぺべねづび〠 Item: Title: ぐひはぴづどの〠ぐひはつふっぴどはの〠ちのつ〠ぅ〮〠っはぬど〠ぃはねばぬづねづのぴちぴどはの〠 Item: Title: ぇづのづ〠ぅへばひづびびどはの〠きづちびふひづねづのぴび〠〨げづちぬ〭ごどねづ〠ぐぃげ〠ぁのちぬべびどび〩〠 Item: Title: ごどびびふづ〠うどへちぴどはの〬〠ぅねぢづつつどので〠どの〠ぐちひちててどの〠ちのつ〠きどっひはぴはねべ〠 Item: Title: げぎぁ〠ぉの〠こどぴふ〠えべぢひどつどぺちぴどはの〠 Item: Title: あどはどのてはひねちぴどっ〠こづぱふづのっづ〠ぁのちぬべびどび〠 Item: Title: ぃはづへばひづびびどはの〠ぎづぴぷはひに〠ぁのちぬべびどび〠 Item: Title: うぐ〭うふびどはの〠ぃはのびぴひふっぴび〠てはひ〠きぅぐ〠ぐちぴとぷちべ〠ぅのぺべねづび〠がはっちぬどぺちぴどはの〠こぴふつどづび〠 Item: Title: くひでちのづぬぬづ〠きちひにづひび〠 Item: Title: ぃづぬぬ〠ごひちのびてはひねちぴどはのび〠ちのつ〠ぅばどてぬふはひづびっづのっづ〠きどっひはびっはばべ〠 Item: Title: 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