55c1348b591a96e54240d5bc0300b73c Pla_a2020v9n4p462iENG.pdf 33148917af5d9360b0e2c8f0578590ae1918594f Pla_a2020v9n4p462iENG.pdf 9578951612d7de0e465c5562d9114596816cf2554a718e204b1553cdc23957c6 Pla_a2020v9n4p462iENG.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 Mar 15 13:02:01 2022 CET 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: 10612914 bytes Optimized: yes PDF version: 1.5 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- VVBZKI+Pxsy Type 1 Builtin yes yes no 918 0 OROQVZ+VnURWPalladioL Type 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yes no 399 0 OICKOF+PalatinoLinotype TrueType WinAnsi yes yes no 408 0 OICLEF+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 410 0 OICLFG+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 412 0 OICMNA+TimesNewRoman CID TrueType Identity-H yes yes yes 415 0 OICKOF+PalatinoLinotype TrueType WinAnsi yes yes no 433 0 OICLEF+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 435 0 OICLFG+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 437 0 OICMNA+TimesNewRoman CID TrueType Identity-H yes yes yes 440 0 OICKNE+PalatinoLinotype CID TrueType Identity-H yes yes yes 473 0 OICKOF+PalatinoLinotype TrueType WinAnsi yes yes no 475 0 OICLEF+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 477 0 OICLFG+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 479 0 OICMNA+TimesNewRoman CID TrueType Identity-H yes yes yes 482 0 TGMEOR+Rpcxr Type 1 Builtin yes yes no 619 0 LGZKLX+Rpxi Type 1 Builtin yes yes no 622 0 Jhove (Rel. 1.22.1, 2019-04-17) Date: 2022-03-16 02:29:06 CET RepresentationInformation: Pla_a2020v9n4p462iENG.pdf ReportingModule: PDF-hul, Rel. 1.12.1 (2019-04-17) LastModified: 2022-03-15 13:07:35 CET Size: 10612914 Format: PDF Version: 1.5 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf Profile: Linearized PDF PDFMetadata: Objects: 0 FreeObjects: 1 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: Introduction Item: Title: Results Children: Item: Title: Cloning and Functional Validation of cDNAs Encoding a Second DXS Isoform, CMS, CMK and HDR Item: Title: MEP Pathway Genes Are Highly Coexpressed in IPAP Cells of Young Leaves Item: Title: MEP Pathway Enzymes Are Targeted to Plastids Item: Title: Punctuated Pattern of Localization of DXS and DXR Requires the Whole Protein Sequence Item: Title: Punctuated Localization of DXS and DXR is Likely Caused by Protein Overexpression Item: Title: Discussion Item: Title: Material and Methods Children: Item: Title: Plant Material and Cell Culture Conditions Item: Title: RNA Extraction and cDNA Synthesis Item: Title: Cloning of the cDNA Sequences from the C. Roseus MEP Pathway Enzymes Item: Title: Protein Production and E. coli Complementation Item: Title: Gene Expression Measurements (Real-Time PCR Analysis) Item: Title: Tissue Fixation, Embedding in Paraffin and Microtomy Item: Title: RNA In Situ Hybridization Item: Title: Bioinformatic Sequence Analysis Item: Title: Coexpression Network Analysis Item: Title: FP-Fusion Constructs for MEP Pathway Enzymes Localization Studies Item: Title: Organelle Markers Item: Title: Cell Transformations and Epifluorescence Microscopy Item: Title: Transmission Electron Microscopy Immunogold Labeling Item: Title: References Filters: FilterPipeline: FlateDecode Fonts: Type1: Font: BaseFont: QTLELQ+Pxbsy FontSubset: true FirstChar: 121 LastChar: 121 FontDescriptor: FontName: QTLELQ+Pxbsy Flags: Symbolic FontBBox: 5, -978, 1382, 913 FontFile: true Font: BaseFont: RKUPQK+URWPalladioL-Bold FontSubset: true FirstChar: 3 LastChar: 122 FontDescriptor: FontName: RKUPQK+URWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: OOWXJH+URWPalladioL-BoldItal FontSubset: true FirstChar: 67 LastChar: 67 FontDescriptor: FontName: OOWXJH+URWPalladioL-BoldItal Flags: Symbolic FontBBox: -170, -300, 1073, 935 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: VVBZKI+Pxsy FontSubset: true FirstChar: 0 LastChar: 121 FontDescriptor: FontName: VVBZKI+Pxsy Flags: Symbolic FontBBox: -21, -994, 1292, 912 FontFile: true Font: BaseFont: WSKZKL+URWPalladioL-Roma FontSubset: true FirstChar: 2 LastChar: 252 FontDescriptor: FontName: WSKZKL+URWPalladioL-Roma Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: OVNEXW+EURM10 FontSubset: true FirstChar: 22 LastChar: 22 FontDescriptor: FontName: OVNEXW+EURM10 Flags: Symbolic FontBBox: -32, -243, 1060, 720 FontFile: true Font: BaseFont: OROQVZ+VnURWPalladioL FontSubset: true FirstChar: 161 LastChar: 243 FontDescriptor: FontName: OROQVZ+VnURWPalladioL Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: QANVCD+Rpxr FontSubset: true FirstChar: 11 LastChar: 62 FontDescriptor: FontName: QANVCD+Rpxr Flags: Symbolic FontBBox: -40, -283, 904, 728 FontFile: true Font: BaseFont: TGMEOR+Rpcxr FontSubset: true FirstChar: 169 LastChar: 174 FontDescriptor: FontName: TGMEOR+Rpcxr Flags: Symbolic FontBBox: -212, -286, 1344, 973 FontFile: true Font: BaseFont: YSPQJI+VnURWPalladioL-Bold FontSubset: true FirstChar: 178 LastChar: 189 FontDescriptor: FontName: YSPQJI+VnURWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: CHFALQ+URWPalladioL-Ital FontSubset: true FirstChar: 3 LastChar: 122 FontDescriptor: FontName: CHFALQ+URWPalladioL-Ital Flags: Symbolic FontBBox: -170, -305, 1010, 941 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: LGZKLX+Rpxi FontSubset: true FirstChar: 14 LastChar: 14 FontDescriptor: FontName: LGZKLX+Rpxi Flags: Symbolic FontBBox: -162, -280, 856, 733 FontFile: true XMP: application/pdf 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 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. Cellular and Subcellular Compartmentation of the 2C-Methyl-D-Erythritol 4-Phosphate Pathway in the Madagascar Periwinkle 2020-04-07T15:03:08+08:00 LaTeX with hyperref package 2022-03-15T13:02:01+01:00 2022-03-15T13:02:01+01:00 2C-methyl-D-erythritol 4-phosphate pathway; compartmentation; Catharanthus roseus; stromules; GFP imaging; localization artifact; alkaloids pdfTeX-1.40.18 False This is pdfTeX, Version 3.14159265-2.6-1.40.18 (TeX Live 2017/W32TeX) kpathsea version 6.2.3 uuid:77b7feeb-efb3-4098-ab02-e2c53fc91096 uuid:96e95e3a-8447-4763-abfe-9ec5d4a60e6b Pages: Page: Sequence: 1 Annotations: Annotation: Subtype: Link Rect: 75, 757, 183, 793 Annotation: Subtype: Link Rect: 474, 757, 519, 793 Annotation: Subtype: Link Rect: 186, 640, 197, 654 Annotation: Subtype: Link Rect: 290, 640, 301, 654 Annotation: Subtype: Link Rect: 471, 640, 482, 654 Annotation: Subtype: Link Rect: 389, 626, 400, 640 Annotation: Subtype: Link Rect: 493, 626, 504, 640 Annotation: Subtype: Link Rect: 368, 613, 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