e10ed16a479cafa0d4ceb9fcb7db3987 pmc_9965820.pdf 771969f8f23995c17e28db945ca0b3a4c46a7355 pmc_9965820.pdf 34bc61011df916481c9fc9e7b1d59904a97e1555190869418d958b06cd689235 pmc_9965820.pdf Title: Nutritional Enrichment of Plant Leaves by Combining Genes Promoting Tocopherol Biosynthesis and Storage Subject: The enrichment of plant tissues in tocochromanols (tocopherols and tocotrienols) is an important biotechnological goal due to their vitamin E and antioxidant properties. Improvements based on stimulating tocochromanol biosynthesis have repeatedly been achieved, however, enhancing sequestering and storage in plant plastids remains virtually unexplored. We previously showed that leaf chloroplasts can be converted into artificial chromoplasts with a proliferation of plastoglobules by overexpression of the bacterial crtB gene. Here we combined coexpression of crtB with genes involved in tocopherol biosynthesis to investigate the potential of artificial leaf chromoplasts for vitamin E accumulation in Nicotiana benthamiana leaves. We show that this combination improves tocopherol levels compared to controls without crtB and confirm that VTE1, VTE5, VTE6 and tyrA genes are useful to increase the total tocopherol levels, while VTE4 further leads to enrichment in -tocopherol (the tocochromanol showing highest vitamin E activity). Additionally, we show that treatments that further promote plastoglobule formation (e.g., exposure to intense light or dark-induced senescence) result in even higher improvements in the tocopherol content of the leaves. An added advantage of our strategy is that it also results in increased levels of other related plastidial isoprenoids such as carotenoids (provitamin A) and phylloquinones (vitamin K1). Keywords: tocopherols; vitamins; biofortification; N. benthamiana; transient expression; chromoplasts Author: Luca Morelli, Laura García Romañach, Gaetan Glauser, Venkatasalam Shanmugabalaji, Felix Kessler and Manuel Rodriguez-Concepcion Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Sat Jan 28 09:05:38 2023 CET ModDate: Sat Jan 28 09:19:47 2023 CET Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 15 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 2226680 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- WLENKM+VnURWPalladioL-Bold Type 1 Custom yes yes no 10 0 HZCUWH+VnURWPalladioL Type 1 Custom yes yes no 15 0 KNPFEX+EURM10 Type 1 Builtin yes yes no 19 0 XFTARB+URWPalladioL-Bold Type 1 Custom yes yes no 23 0 JJDQCR+URWPalladioL-Roma Type 1 Custom yes yes no 28 0 LLXNRS+URWPalladioL-Ital Type 1 Custom yes yes no 32 0 ZZPUEX+ArialMT Type 1C WinAnsi yes yes no 51 0 TWGUDY+TimesNewRomanPS-BoldItalicMT Type 1C WinAnsi yes yes no 54 0 BFGHDH+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 82 0 BFGHDI+PalatinoLinotype CID TrueType Identity-H yes yes yes 85 0 BFGHDJ+PalatinoLinotype TrueType WinAnsi yes yes no 91 0 BFGHDL+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 94 0 BFGHDH+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 105 0 BFGHDI+PalatinoLinotype CID TrueType Identity-H yes yes yes 108 0 BFGHDJ+PalatinoLinotype TrueType WinAnsi yes yes no 114 0 BFGHDL+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 117 0 WXNNPN+CMSY10 Type 1 Builtin yes yes no 127 0 BFGHDH+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 137 0 BFGHDI+PalatinoLinotype CID TrueType Identity-H yes yes yes 140 0 BFGHDJ+PalatinoLinotype TrueType WinAnsi yes yes no 146 0 BFGHDL+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 149 0 BFGHDH+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 159 0 BFGHDI+PalatinoLinotype CID TrueType Identity-H yes yes yes 162 0 BFGHDJ+PalatinoLinotype TrueType WinAnsi yes yes no 168 0 BFGHDL+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 171 0 BFGHDH+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 181 0 BFGHDI+PalatinoLinotype CID TrueType Identity-H yes yes yes 184 0 BFGHDJ+PalatinoLinotype TrueType WinAnsi yes yes no 190 0 BFGHDL+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 193 0 Jhove (Rel. 1.28.0, 2023-05-18) Date: 2023-12-15 02:34:43 CET RepresentationInformation: pmc_9965820.pdf ReportingModule: PDF-hul, Rel. 1.12.4 (2023-03-16) LastModified: 2023-03-02 02:16:51 CET Size: 2226680 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 453 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: section.1 Item: Title: Results and Discussion Destination: section.2 Children: Item: Title: VitE Content in Leaves Can Be Maximised by Enhancing the Biosynthetic Pathway in Combination with the Formation of Artificial Chromoplasts for Storage Destination: subsection.2.1 Item: Title: Physical Treatments Associated with PG Proliferation Can Be Combined with Metabolic Engineering for Additional Tocopherol Enhancement Destination: subsection.2.2 Item: Title: The Levels of Plastidial Isoprenoids Other than Tocopherols and Carotenoids Are Also Impacted by Tocopherol Pathway Engineering and Chromoplast Differentiation Destination: subsection.2.3 Item: Title: Conclusions Destination: section.3 Item: Title: Materials and Methods Destination: section.4 Children: Item: Title: Plant Material and Growth Conditions Destination: subsection.4.1 Item: Title: Gene Constructs Destination: subsection.4.2 Item: Title: Metabolite Analyses Destination: subsection.4.3 Item: Title: Antioxidant Capacity Destination: subsection.4.4 Item: Title: Appendix A Destination: section.A. Item: Title: References Destination: table.caption.6 Info: Title: Nutritional Enrichment of Plant Leaves by Combining Genes Promoting Tocopherol Biosynthesis and Storage Author: Luca Morelli, Laura García Romañach, Gaetan Glauser, Venkatasalam Shanmugabalaji, Felix Kessler and Manuel Rodriguez-Concepcion Subject: The enrichment of plant tissues in tocochromanols (tocopherols and tocotrienols) is an important biotechnological goal due to their vitamin E and antioxidant properties. Improvements based on stimulating tocochromanol biosynthesis have repeatedly been achieved, however, enhancing sequestering and storage in plant plastids remains virtually unexplored. We previously showed that leaf chloroplasts can be converted into artificial chromoplasts with a proliferation of plastoglobules by overexpression of the bacterial crtB gene. Here we combined coexpression of crtB with genes involved in tocopherol biosynthesis to investigate the potential of artificial leaf chromoplasts for vitamin E accumulation in Nicotiana benthamiana leaves. We show that this combination improves tocopherol levels compared to controls without crtB and confirm that VTE1, VTE5, VTE6 and tyrA genes are useful to increase the total tocopherol levels, while VTE4 further leads to enrichment in -tocopherol (the tocochromanol showing highest vitamin E activity). Additionally, we show that treatments that further promote plastoglobule formation (e.g., exposure to intense light or dark-induced senescence) result in even higher improvements in the tocopherol content of the leaves. An added advantage of our strategy is that it also results in increased levels of other related plastidial isoprenoids such as carotenoids (provitamin A) and phylloquinones (vitamin K1). 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