CARBON PARTITIONING IN NITROGEN-FIXING ROOT NODULES Dissertation zur Erlangung des Doktorgrades der Mathematisch-Naturwissenschaftlichen Fakultäten der Georg-August-Universität zu Göttingen vorgelegt von Maria Schubert, geb. Ramenskaia aus St. Petersburg, Russland Göttingen 2002 D 7 Referent: Prof. Dr. Hans-Walter Heldt Korreferentin: Prof. Dr. Christiane Gatz Tag der mündlichen Prüfung: 30.10.2002 Content 1. Introduction.............................................................................................................1 1.1. Degradation of sucrose in sink organs................................................................ 1 1.1.1. The role of sucrose in the plant cell................................................................. 1 1.1.1.1. Sucrose synthase .................................................................................... 2 1.1.1.2. Invertases ................................................................................................ 2 1.2. Transport of sugars ............................................................................................... 4 1.2.1. Phloem loading................................................................................................ 4 1.2.2. Phloem unloading and post-phloem transport................................................. 5 1.2.2.1. Symplastic sugar transport ...................................................................... 5 1.2.2.2. Apoplastic phloem unloading and post-phloem transport........................ 6 1.2.2.3. Sugar transporters in apoplastic sugar transport..................................... 7 1.3. The regulation of phloem unloading and sink strength in different systems .. 9 1.3.1. Developmental sinks ....................................................................................... 9 1.3.2. Optional sinks................................................................................................ 10 1.4. Nitrogen-fixing root nodule symbioses ............................................................. 10 1.4.1. Nitrogen fixation in nodules ........................................................................... 11 1.4.2. Infection of plants and nodule formation........................................................ 12 1.4.3. Nodule structure ............................................................................................ 13 1.4.4. Phylogenetic relationship of root nodule symbioses...................................... 15 1.4.5. Carbon sources supplied by the host plant to nitrogen-fixing microsymbionts.. ...................................................................................................................... 15 1.4.6. Carbon transport and metabolism in nitrogen-fixing root nodules................. 17 1.5. Aim of this thesis ................................................................................................ 18 2. Materials and methods………………………………………………………………..20 2.1. Materials................................................................................................................ 20 2.1.1. Plant material ................................................................................................ 20 2.1.2. Bacterial and yeast strains ............................................................................ 20 2.1.3. Oligonucleotides (Primers) ............................................................................ 21 2.1.4. Plasmides...................................................................................................... 21 2.1.5. Enzymes........................................................................................................ 22 2.1.5.1. Restriction enzymes................................................................................ 22 2.1.5.2. Other enzymes and kits .......................................................................... 22 2.1.6. Chemicals...................................................................................................... 23 Content ii 2.1.7. Other materials and devices.......................................................................... 24 2.1.8. Culture media ................................................................................................ 24 2.1.8.1. Plant media............................................................................................. 24 2.1.8.2. Bacterial media ....................................................................................... 25 2.1.8.3. Media for Saccharomyces cerevisiae ..................................................... 27 2.1.8.4. Medium additives.................................................................................... 28 2.2. Plant culture methods and growth conditions .................................................. 28 2.3. RNA isolation from plant tissue.......................................................................... 29 2.3.1. RNA isolation from Medicago truncatula and Datisca glomerata (modified after Burgos et al., 1995)............................................................................... 29 2.3.2. RNA isolation from Casuarina glauca............................................................ 30 2.4. Isolation of plasmid DNA from bacteria and yeast ........................................... 31 2.4.1. Plasmid mini-preparation protocol „ Triton Boiling“........................................ 31 2.4.2. Mini preparation of bacterial plasmid DNA for the sequence analysis .......... 31 2.4.3. Maxi preparation of bacterial plasmid DNA ................................................... 32 2.4.4. Preparation of plasmid DNA from yeast ........................................................ 32 2.5. Concentration and purification of DNA or RNA solutions ............................... 32 2.5.1. Precipitation of nucleic acids ......................................................................... 32 2.5.2. Phenol-chloroform extraction and precipitation of DNA................................. 33 2.6. Electrophoretic separation of DNA and RNA .................................................... 33 2.6.1. TEA-Agarose gel electrophoresis.................................................................. 33 2.6.2. Separation of RNA on agarose gel for Northern blots................................... 34 2.7. Northern blot hybridization ................................................................................. 34 2.7.1. RNA transfer to nylon membranes (Northern blotting) .................................. 34 2.7.2. DNA probe labelling with α-[32P]-dATP.......................................................... 35 2.7.3. Hybridization.................................................................................................. 35 2.8. First strand cDNA synthesis (Reverse transcription)....................................... 36 2.9. Amplification of DNA fragments ......................................................................... 36 2.9.1. Polymerase chain reaction (PCR) ................................................................. 37 2.9.2. Design of synthetic oligonucleotide primers .................................................. 37 2.10. Rapid amplification of cDNA ends (RACE): 5´-RACE ....................................... 38 2.11. DNA sequencing................................................................................................... 39 2.12. Cloning methods .................................................................................................. 40 2.12.1. Digestion with restriction enzymes ................................................................ 40 2.12.2. Phosphatase treatment ................................................................................. 40 2.12.3. Filling-in of 5´ overhanging ends with Klenow fragment ................................ 41 Content iii 2.12.4. Isolation of DNA fragments from agarose gels.............................................. 41 2.12.5. Ligation.......................................................................................................... 42 2.13. Transformation of Escherichia coli .................................................................... 42 2.13.1. Preparation of competent E.coli cells ............................................................ 42 2.13.2. Transformation of competent E.coli cells....................................................... 43 2.13.3. Characterisation of transformants ................................................................. 44 2.14. Yeast transformation ........................................................................................... 45 2.15. Bacterial and yeast glycerol cultures................................................................. 46 2.16. Extraction of sugars............................................................................................. 46 2.16.1. Chloroform-methanol extraction .................................................................... 46 2.16.2. Preparative isolation of unknown sugars / sugar derivates from Datisca ...... 47 2.16.3. Ethanol extraction.......................................................................................... 48 2.16.4. Perchlorate extraction.................................................................................... 49 2.16.5. Acetone extraction......................................................................................... 49 2.17. Sugar analysis by high-performance liquid chromatography (HPLC) ............ 49 2.18. Sugar analysis