Kathleen Helmstedt 15th PSSC  19 June 2019 - PowerPoint PPT Presentation

using tobacco glandular trichomes to understand n.
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Kathleen Helmstedt 15th PSSC  19 June 2019 PowerPoint Presentation
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Kathleen Helmstedt 15th PSSC  19 June 2019
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Kathleen Helmstedt 15th PSSC  19 June 2019

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  1. Using tobacco glandular trichomes to understand the regulation of the isoprenoid biosynthesis in plants Leibniz Institute of Plant Biochemistry Halle (Saale), Germany Kathleen Helmstedt 15th PSSC  19 June 2019

  2. The MEP and the MEV pathway supply the precursors for the isoprenoids MEP – Methyl-erythritol-phosphate , MEV – Mevalonate, IPP – Isopentenylpyrophosphate, DMAPP – Dimethylallylpyrophosphate MEP and MEV: Synthesis of IPP and DMAPP DMAPP IPP IPP and DMAPP: universal precursormoleculesfortheclass of isoprenoids • Wide rangeofmetabolites • Over 55 000 knownmolecules • Differa lot in structureandfunction

  3. Diversity of isoprenoids Primary metabolism Secondarymetabolism Plant hormones Chlorophyll Monoterpenes Abscisic acid Limonene Carotenoids Sesquiterpenes Brassinosteroids Artemisinine Ubiquinones Diterpenes Gibberellins

  4. The role of the MEP and MEV pathway in the cell Mevalonate pathway MEP Pyruvate + GA-3P Acetyl-CoA DXS DXP AACT DXR Acetoacetyl-CoA MEP HMGS Cytosol MCT HMG-CoA CDP-ME HMGR CMK Mevalonate CDP-MEP Peroxisome MVK MDS MecPP MVAP HDS pMVK HMBPP MVAPP HDR MDC IDI IPP DMAPP DMAPP IPP IDI FPS GPS FPP GPP GGPPS GGPP Phytosterols Dolichols Protein prenylation Monoterpenes Diterpenes Gibberellins, ABA Strigolactones Brassinosteroids Plastid Sesquiterpenes Triterpenes Chlorophyll Carotenoids Plastoquinones

  5. Glandulartrichomesproduce large quantitiesofisoprenoids • Enhanced expression of MEP genes • Hair-like structures of epidermis cells on leaf surface • Specialized secondary metabolites with defense-related activity MEP Model organism: Nicotianatabacum Diterpenes High production of diterpenes in the glandular trichomes(labdanoids, cembranoids) Z-Abienol α-CBTdiol

  6. Research aims Howistheisoprenoidbiosynthesisvia MEV and MEP pathway regulated in tobaccoglandulartrichomes? • Are their feedback mechanisms which control the isoprenoid flux in tobacco trichomes? • How are the MEP pathway enzymes organized within the plastid?

  7. N. tabacumvarieties affected in diterpene synthesis Ambalema 3H02 (Ambalema x Colorado) Pyr + GA-3P Pyr + GA-3P Colorado Pyr + GA-3P MEP IPP + DMAPP IPP + DMAPP IPP + DMAPP Downstream pathways Diterpenes GGPP GGPP GGPP Labdanes Labdanes Labdanes Cembrenes Cembrenes Cembrenes Transcriptomicanalysis: RNA-Seq  Blockedditerpenesynthesis

  8. Gene knock-out of CPS2 in N. tabacum cv. Ambalema via CRISPR/Cas Ambalema 3H02 (Ambalema x Colorado) CRISPR Ambalema Pyr + GA-3P Pyr + GA-3P Pyr + GA-3P MEP IPP + DMAPP IPP + DMAPP IPP + DMAPP Downstream pathways CPS2 Diterpenes GGPP GGPP GGPP Labdanes Labdanes Labdanes Cembrenes Cembrenes Cembrenes Metabolomicanalysis  Same phenotype?

  9. Changed MEP gene expression in the no diTPs line (3H02) RNA-Seqdataforglandulartrichomesof different N. tabacumlines: MEP Pyruvate + GA-3P DXS DXP DXR ** ** MEP MCT CDP-ME * CMK CDP-MEP MDS MecPP * HDS *** HMBPP * *** HDR * * IPP DMAPP * * GPS GPP GGPPS GGPP n = 3; *p < 0,05, **p < 0,01, ***p < 0,001  Blockedditerpenesynthesisleadstoreducedexpressionof MEP branchpoint genes Diterpenes

  10. Reduced abundance of MEP pathway metabolites (3H02, cps2) MEP Pyruvate + GA-3P DXS DXP DXR MEP * MCT CDP-ME CMK CDP-MEP MDS MecPP HDS HMBPP HDR IPP DMAPP GPS GPP GGPPS GGPP ** ** ** ** Diterpenes

  11. Effects on MEP downstream processes GGPPS2 ** Diterpenes GGPP Ambalema trc Colorado trc PSY 3H02 trc Chlorophyll Carotenoids Chlorophyll content: Carotenoidbiosynthesis:

  12. Enhanced expression of HMGR2 gene (3H02) MEV Acetyl-CoA HMGR2t AACT Ambalema trc Acetoacetyl-CoA ** Colorado trc HMGS 3H02 trc HMG-CoA HMGR Mevalonate MVK MVAP pMVK MVAPP MDC IDI DMAPP IPP FPS FPP

  13. Increase of MEV downstream products (3H02, cps2) Phytosterols MEV Acetyl-CoA AACT Acetoacetyl-CoA HMGS HMG-CoA HMGR Mevalonate MVK MVAP pMVK MVAPP MDC IDI DMAPP IPP FPS FPP Cholesterol Stigmasterol Campesterol Beta-Sitosterol

  14. Transcriptomic profile of trichomes in 3H02 MEP MEV Pyruvate + GA-3P Acetyl-CoA DXS AACT DXP Acetoacetyl-CoA DXR HMGS MEP HMG-CoA MCT HMGR CDP-ME Mevalonate CMK MVK CDP-MEP MVAP MDS MecPP pMVK MVAPP HDS HMBPP MDC IDI HDR DMAPP IPP IPP DMAPP FPS IDI FPP GPS GPP GGPS GGPP PSY Carotenoids Diterpenes

  15. Metabolic and transcriptomic profile of trichomes in 3H02/cps2 MEP MEV Pyruvate + GA-3P Acetyl-CoA DXS AACT DXP Acetoacetyl-CoA DXR HMGS MEP HMG-CoA MCT HMGR CDP-ME Mevalonate CMK MVK CDP-MEP MVAP MDS MecPP pMVK MVAPP HDS HMBPP MDC IDI HDR DMAPP IPP IPP DMAPP FPS IDI FPP GPS GPP GGPS GGPP PSY Phytosterols Carotenoids Diterpenes Chlorophyll

  16. Summary & Outlook CharacterizationofditerpeneaffectedNtlines • The N. tabacumline 3H02 is a unique tool to investigate the feedback mechanisms of blocking the diterpenesynthesis • Generation of genetically defined tobacco line cps2 with help of CRISPR/Cas • Blocking diterpene synthesis leads tofeedback regulation of MEP and MEV pathways • qPCR for cps2 for selected genes • Further metabolomics analysis of the lines (carotenoids, GGPP…) • Screening for transcription factors involved in this feedback regulation • (Promotor-GUS-Assay, Y1H)

  17. Research aims Howistheisoprenoidbiosynthesisvia MEV and MEP pathway regulated in tobaccoglandulartrichomes? • Are their feedback mechanisms which control the isoprenoid flux in tobacco trichomes? • Howarethe MEP pathwayenzymesorganizedwithintheplastid?

  18. MEP enzymes are mainly localized in the stroma of chloroplasts • Co-localization with mCherry fusion constructs of marker proteins • Agrobacterium mediated transient transformation of N. benthamianaleaves Plastidstromamarker:mCherry Merge Chlorophyll NtDXR:GFP Pyruvate + GA-3P DXS DXP 10 µm DXR MEP Plastidstromamarker:mCherry MCT Merge Chlorophyll NtMCT:GFP CDP-ME CMK CDP-MEP MDS MecPP HDS HMBPP 5 µm HDR IPP DMAPP

  19. Plastoglobule localization of the MEP first step enzyme DXS • Co-localization of DXS1 with plastoglobule marker protein AtVTE1 in N. benthamiana Agrobacterium mediated transient transformation: Protoplast transfection: Merge SlDXS1:YFP AtVTE1:mCherry Merge SlDXS1:YFP AtVTE1:mCherry 10 µm 5 µm Merge NtDXS1:GFP AtVTE1:mCherry Merge NtDXS1:GFP AtVTE1:mCherry 10 µm 10 µm Nt – NicotianatabacumSl – Solanum lycopersicumAtVTE1 – ArabidopsisthalianaTocopherolcyclase

  20. Preparation of plastoglobules by organelle fractionation • Lipoprotein particle attached to thylakoids via the outer half of the lipid bilayer • Hydrophobic environment • Function not clear • Proteomic studies in Arabidopsis thaliana: •  No DXS detected plastoglobuli • Fraction 1-3 outer/innerenvelopemembranes • Fraction 4-22 • Fraction 23-37 thylakoidmembranes Rottet, 2015 Besagniet al. 2011 Separation of plastid membrane fractions in N. benthamiana

  21. Analysis of DXS distribution in the plastoglobules via Protein-MS plastoglobuli outer/innerenvelopemembranes (OM/IM) thylakoidmembranes  DXS was detected in plastoglobulefraction Plastoglobulemarkerprotein Thylakoidmarkerprotein

  22. How is DXS associated to the plastoglobule membrane? • DXS: notransmembranedomain • MEP pathwayoccursonly in thestroma DXS ? Rottet, 2015 - DXS islikelyassociated at the outside oftheplastoglobulemembraneandsticks out intothestroma Post-translationalmodifications Protein-Protein Interaction/ Protein complex

  23. Summary & Outlook DXS sub-cellularlocalization • MEP enzymes are mainly localized in the stroma of chloroplasts • Tobacco DXS shows plastoglobule localization • Protein-protein interaction analysis of DXS1 • (BioID system, cross linking)

  24. Acknowledgements Prof. Alain Tissier Dr. Tom Schreiber Dr. Wolfgang Höhenwarter Dr. Karin Gozolka Dr. Gerd Balcke Dr. Stephanie Werner Dr. Benedikt Ahtmer Anja Prange Ramona Schubert All membersofthe SZB-department Thanks for your attention!