On the role of sugar compartmentation and stachyose synthesis in symplastic phloem loading Dissertation zur Erlangung des Doktorgrades der Mathematisch-Naturwissenschaftlichen Fakultäten der Georg-August-Universität zu Göttingen Vorgelegt von Olga Vladimirovna Voitsekhovskaja aus St.-Petersburg, Rußland Göttingen 2001 D7 Referentin: PD Dr. Gertrud Lohaus Koreferent: Prof. Dr. Hans-Walter Heldt Tag der mündlichen Prüfung: 30.01.2002 Content 1. Introduction ............................................................................................. 1 1.1. Types of companion cells in minor vein phloem....................................... 2 1.2. Symplastic (dis)continuity between mesophyll and phloem ..................... 3 1.3. Apoplastic phloem loading mode............................................................... 4 1.4. Symplastic phloem loading mode .............................................................. 5 1.5. Relationship between companion cell types and the types of translocated sugars ............................................................................................ 7 1.6. The polymerization trap model and the role of stachyose synthesis in symplastic phloem loading................................................................................ 10 1.7. The aim of this thesis and the choice and characterization of model plants. ................................................................................................................ 12 1.7.1. The aim of this thesis.......................................................................................12 1.7.2. Phloem sap composition and characterization of minor vein anatomy in two related species, Alonsoa meridionalis and Asarina barclaiana........................12 1.7.3. Choice of the model plants..............................................................................15 2. Materials and Methods............................................................... 17 2.1. Materials..................................................................................................... 17 2.1.1. Biochemicals.....................................................................................................17 2.1.2. Enzymes used in molecular biology ...............................................................18 2.1.3. Kits....................................................................................................................18 2.2. Growth conditions...................................................................................... 19 2.2.1. Plants ................................................................................................................19 2.2.2. Bacteria.............................................................................................................20 2.2.3. Plasmids............................................................................................................22 2.3. Extraction of metabolites ........................................................................... 23 2.3.1. Chloroform:methanol extraction ...................................................................23 2.3.2. Isolation of galactinol and antirrhinoside from plant material...................24 2.3.2.1. Preparative isolation of galactinol......................................................24 2.3.2.2. Preparative isolation and confirmation of the chemical structure of antirrhinoside...............................................................................................25 2.4. Extraction of apoplastic sap (intercellular washing fluid) ......................... 26 2.5. Determination of enzyme activities ........................................................... 26 2.5.1. Glucose-6-phosphate dehydrogenase (G6PDH)............................................27 2.5.2. NADP-dependent Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) ....................................................................................................................27 2.5.3. Phosphoenolpyruvate carboxylase (PEPCx).................................................28 2.5.4. α-Mannosidase .................................................................................................28 2.5.5. Malate dehydrogenase.....................................................................................28 ii Content 2.5.6 Stachyose synthase activity assays.................................................................. 29 2.5.6.1 Establishment of the AmSTS activity assay using leaves of Alonsoa ........................................................................................................... 29 2.5.6.2 AmSTS activity assay in protein extracts of E. coli BL21 (DE3) (pET*/STS) ..................................................................................................... 30 2.6 Non-aqueous fractionation of leaves...........................................................30 2.6.1. Preparation of leaf material........................................................................... 30 2.6.2. Non-aqueous fractionation of leaf tissue in a density gradient................... 30 2.6.3. Determination of the activities of marker enzymes in the gradient fractions...................................................................................................................... 32 2.6.4. Determination of metabolite concentrations in the gradient fractions ...... 32 2.6.5. Calculation of the subcellular distribution of metabolites .......................... 32 2.6.6. Determination of the chlorophyll:protein ratio............................................ 33 2.7. Protein determination .................................................................................34 2.8. Sugar analysis.............................................................................................34 2.8.1. HPLC................................................................................................................ 34 2.8.2. Enzymatic measurements of glucose, fructose and sucrose ........................ 35 2.9. Determination of the osmolality of the leaf sap.........................................36 2.10. Analyses in single cell sap .......................................................................36 2.10.1. Single cell sampling....................................................................................... 36 2.10.2. Osmolality of single cell sap ......................................................................... 37 2.10.3. Sugar concentrations in single cell sap........................................................ 37 2.11. Inhibition of phloem translocation ...........................................................39 2.12. Study of sugar uptake from the apoplast in vivo......................................39 2.12.1. Feeding experiments ..................................................................................... 39 2.12.2. Gas and H2O exchange mesurements.......................................................... 40 2.12.3. Recordings of pH- and membrane potential changes................................ 40 2.13. Electron microscopy and determination of partial volumina of subcellular compartments..................................................................................41 2.14. Light microscopy......................................................................................43 2.15. Isolation of total RNA and genomic DNA from plant tissues .................45 2.15.1. Isolation of RNA from Asarina and Alonsoa.............................................. 45 2.15.2. Isolation of genomic DNA from Alonsoa .................................................... 46 2.15.3. Isolation of DNA from Arabidopsis thaliana F1 transformants................ 46 2.16. Plasmid DNA isolation from bacteria ......................................................47 2.16.1. Isolation of plasmid DNA from E. coli (miniprep and maxiprep)............ 47 2.16.1.1. Plasmid DNA miniprep isolation using a triton boiling protocol .... 48 2.16.1.2. Plasmid DNA miniprep isolation with the Qiagen Miniprep Kit .... 48 2.16.1.3. Plasmid DNA maxiprep isolation with E.Z.N.A. Plasmid Miniprep Kit.................................................................................................... 48 2.16.2. Isolation of plasmid DNA from Agrobacterium tumefaciens..................... 49 2.17. Determination of RNA and DNA concentration by spectrophotometry .49 2.18. Purification and precipitation of DNA and RNA.....................................50 2.19. Agarose gel electrophoresis of DNA and RNA .......................................50 Content iii 2.19.1. Separation of DNA ........................................................................................50 2.19.2. DNA gel blots (Southern blots).....................................................................51 2.19.3. Separation of RNA ........................................................................................52 2.19.4. RNA gel blot (Northern blot)........................................................................53 2.19.5. Hybridization of DNA and RNA gel blots...................................................54 2.19.5.1. Labelling of probes...........................................................................54 2.19.5.2. Hybridization and washing of blots..................................................54 2.20. Gene cloning ............................................................................................ 55 2.20.1. Elution of DNA fragments