Ultrastructure, transport and the state of phosphorus in Pisolithus tinctorius David Allan Orlovich Doctor of Philosophy The University of New South Wales 1994 UNIVERSITY Or N.S.VV. 1 6 J'.'-j \TA LI BR/' TIES Contents Abstract............................................................................................................................. i Acknowledgements........................................................................................................ii Certificate of originality....................................................................................... iii 1 Phosphorus and mycorrhizas Introduction..................................................................................................................... 1 Phosphorus nutrition and mycorrhizas.........................................................................1 Phosphorus uptake......................................................................................................... 2 Phosphorus storage........................................................................................................2 Phosphorus transport.....................................................................................................4 Aim..................................................................................................................................5 2 Polyphosphate granules are an artefact of specimen preparation in Pisolithus tinctorius Introduction.....................................................................................................................6 Materials and methods....................................................................................................7 Treatment of hyphae to precipitate metachromatic granules......................... 7 Structure, cytochemistry and X-ray microanalysis of granules in chemically fixed hyphae......................................................................7 Freeze-substitution, ion localisation and X-ray microanalysis..................... 8 Results..............................................................................................................................9 Precipitation of metachromatic granules during conventional specimen preparation....................................................................................9 Evidence that metachromatic granules contain polyphosphate....................10 Comparison of vacuole content after conventional procedures and freeze-substitution...................................................................................... 10 Discussion...................................................................................................................11 Polyphosphate granules and specimen preparation...................................... 11 Cation exchange with polyphosphate in vivo............................................... 12 Significance in transport.................................................................................. 13 3 Ultrastructure of the vacuole system in Pisolithus tinctorius Introduction...................................................................................................................23 Materials and Methods................................................................................................. 24 Rapid freezing and freeze-substitution.......................................................... 24 Measurements and statistics............................................................................24 Results........................................................................................................................... 25 General ultrastructure of the tip region and vacuoles and tubules in cells close to the hyphal tip................................................................... 25 Ultrastructure of cells behind the growing tip...............................................27 Discussion.................................................................................................................. 28 Clusters and tubules.........................................................................................28 Fine filaments...................................................................................................30 Golgi bodies and smooth membrane cistemae............................................. 31 Mature hyphae................................................................................................. 34 4 Structure and development of the dolipore septum in Pisolithus tinctorius Introduction...................................................................................................................45 Materials and Methods................................................................................................. 46 Results........................................................................................................................... 47 Clamp connections and dolipore septa.......................................................... 47 Early development of the dolipore septum.......................................... 47 The pore complex of mature septa................................................................ 48 Discussion..................................................................................................................50 General considerations.................................................................................... 50 Membrane furrowing and the filamentous ring...........................................51 Structure of the septal pore complex at maturity.......................................... 53 Transport and communication through the dolipore.....................................54 5 Observations on ion redistribution within a specimen during freeze-substitution, embedding and X-ray microanalysis Introduction...................................................................................................................62 The application of freeze-substitution............................................................62 General procedure for freeze-substitution.....................................................62 Theoretical aspects of freeze-substitution for ion localisation.....................63 Limitations of X-ray microanalysis......................................................64 Experimental rationale............................................................................. 65 Materials and Methods................................................................................................. 65 Sandwiched droplets........................................................................................65 Solutions frozen in contact with each other......................................... 66 Freeze-dried droplets....................................................................................... 66 Freeze-substitution..........................................................................................66 X-ray microanalysis.........................................................................................67 Results........................................................................................................................... 68 Ultrastructure of freeze-substituted solutions...............................................68 Elemental distribution in solutions sandwiched together after freezing..................................................................................................... 68 Elemental distribution in solutions frozen in contact with each other.............................................................................................................69 Elemental distribution in freeze-dried droplets............................................. 70 Discussion..................................................................................................................70 Retention of water soluble ions by freeze-substitution................................ 70 Interpretation of X-ray microanalysis results....................................... 71 Beam damage...................................................................................................71 Contamination of sections with atmospheric water......................................72 6 Symplastic transport in Pisolithus tinctorius Introduction...................................................................................................................88 Symplastic translocation in fungi................................................................................88 Compartmentation of P, into vacuoles in hyphal tips...............................................89 Storage and translocation in large vacuoles...............................................................89 Translocation in mycorrhizas......................................................................................90 Mechanism of translocation to the Hartig net............................................................91 7 References......................................................................................................................93 8 Appendix........................................................................................................................105 Abstract The ultrastructure of hyphae was studied in the ectomycorrhizal fungus Pisolithus tinctorius (Pers.) Coker & Couch. Polyphosphate granules were found to be artefacts of conventional specimen preparation. Granules were not produced by glutaraldehyde fixation but appeared