THE PYRENOID OF ZYGNEMA: ISOLATION AND CHARACTERIZATION Item Type text; Dissertation-Reproduction (electronic) Authors Rosowski, James R. Publisher The University of Arizona. Rights Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author. Download date 28/09/2021 02:41:35 Link to Item http://hdl.handle.net/10150/290230 This dissertation has been microfilmed exactly as received 70-4685 ROSOWSKI, James Ray, 1938- THE PYRENOID OF ZYGNEMA: ISOLATION AND CHARACTERIZATION. University of Arizona, Ph.D., 1969 Botany- University Microfilms, Inc., Ann Arbor, Michigan THE PYRENOID OF ZYGNBMA: ISOLATION AND CHARACTERIZATION t>y James Ray Rosowslci A Dissertation Submitted to the Faculty of the DEPARTMENT OF BIOLOGICAL SCIENCES In Partial Fulfillment of the Requirements For the Degree of DOCTOR OF PHILOSOPHY WITH A MAJOR IN BOTANY In the Graduate College THE UNIVERSITY OF ARIZONA 19 6 9 THE UNIVERSITY OF ARIZONA GRADUATE COLLEGE I hereby recommend that this dissertation prepared under my direction by James Ray Rosowski entitled The Pyrenoid of Zvenema: Isolation and Characterization be accepted as fulfilling the dissertation requirement of the degree of Ph. D. • / / C ^' j^'c, /V, /U? Dissertation Director After inspection of the final copy of the dissertation, the following members of the Final Examination Committee concur in its approval and recommend its acceptance:* ,f<ySi / IlL /yy/ ' V 7/t 7 /£ ? 1//7 tt? llnifi /)n • Thi/ approval and acceptance (iJs contingent on the candidate's adequate performance and^-tlefense of this dissertation at the final oral examination. The inclusion of this sheet bound into the library copy of the dissertation is evidence of satisfactory performance at the final examination. STATEMENT BY AUTHOR This dissertation has been submitted in partial fulfillment of requirements for an advanced degree at The University of Arizona and is deposited in the University Library to be made available to borrowers under rules of the Library. Brief quotations from this dissertation are allowable without special permission, provided that accurate acknowledgment of source is made. Requests for permission for extended quotation from o.r reproduction of this manuscript in whole or in part may be granted by the head of the major department or the Dean of the Graduate College when in his judgment the proposed use of the material is in the interests of scholarship. In all other instances, however, permission must be obtained from the author. SIGNED: A CKN OWL ED GMENTS The author is indebted to Dr. Robert W. Hoshaw for his suggestion of the subject of this dissertation and for his guidance during the research and writing stages. In addition, the author wishes to express here his sincere appreciation to Dr. Iloshaw for time and energies spent in discussions related not only to this dissertation but also to past endeavors. The author would like to thank Dr. Paul G. Bartels, Dr. AlJ.ce M. Boyle, Dr. Robert M. Harris, and Dr. Merritt R. Nelson for reading and criticizing this manuscript. A special thanks goes to Dr. Kaoru Matsuda for en­ couragement of the author to attempt the isolation of pyrenoids, for discussions related to this work, and for provision of the research facilities for this activity. The valuable discussions and assistance of Edward James Pegelow, Jr. are also gratefully acknowledged. The very capable technical assistance of Mrs. Kathie Mitchell was appreciated. The author is indebted to Dr. Wayne R. Ferris for the use of the electron microscope, to Dr. Hebcr P. liostetter for several ultrasections, to Dr. Richard G. Jensen for suggesting Nitex filtration, to Mr. and Mrs. William Clark for typing the manuscript, and to Dr. David J. Wolosliin for his German translations. A very iii special thanks to my wife Sue ^for assistance with th manuscript and for her continual support. TABLE OF CONTENTS Page LIST OF ILLUSTRATIONS vii LIST OF TABLES ' . xiv ABSTRACT xv INTRODUCTION 1 GENERAL MATERIALS AND METHODS 6 Experimental Organisms 6 Isolation Technique 6 Medium and Transfer Technique 7 Growth Conditions 10 Light Photomicrography 12 THE PYRENOID AS A MORPHOLOGICAL UNIT 13 Light Microscopy 13 The Cell 13 Growth l'± Nuclear and Cell Division l6 Division of the Pyrenoid 17 Chloroplast Mutant 19 Electron Microscopy 20 Fixation and Dehydration 21 Embedding 21 Sectioning . 22 Observations 23 Discussion of Pyx-enoid U1 tras true tur e . 2h THE PYRENOID AS AFFECTED BY LIGHT AND BY CHEMICAL AGENTS 27 Starch Synthesis 27 On a 12:12 hr Light:Dark Cycle 28 In Destarched Cells 29 In a Micx"obeam of Light 38 Light Intensity 4l Materials and Methods . 42 Results and Discussion ^3 v vi TABLE OF CONTENTS —Continued * Page Division 45 On ci 12:12 hi" Light:Dark Cycle 49 On a .1.2:12 hr Light:Dark Cycle Followed by Continuous Darkness 5^ On a 12:12 hr Light:Dark Cycle Followed !>, Continuous Illumination . 55 On a 6:6:6:6 hr Light:Dark:Light: Dark Cycle 55 Discussion of the Light Cycle Studies . 56 Technique for Staining Pyrenoids 6l Ami do-Schwartz 67 Pronase 68 Triton X-100 70 Sodium Deoxycholate 71 Streptomycin Sulfate 73 B Medium Minus Nitrogen 76 Discussion on the Effects of Chemical Agents 7o ISOLATION OF THE CHLOROPLAST 79 Materials and Methods 79 Observations and Results 82 Discussion 83 ISOLATION OF THE PYRENOID 85 Materials 85 Isolation Procedure 89 Characterization of the Isolated Pyrenoid . 96 Evidence for Loss or Alteration of a Soluble Component. 97 GENERAL DISCUSSION 101 SUMMARY 108 LITERATURE CITED 130 LIST OF ILLUSTRATIONS Figure Page 1. Diagram of inid-prophase. Disintegration of the nucleolus ar.d of the nuclear membrane is shown. Cell wall formation has begun 112 2. Diagram of metaphase 112 3« Diagr"ani of anaphase 112 k. Diagram of telophase 112 5. Diagram of interphase and stage one of pyrenoid division 112 6. Diagram of stage two of pyrenoid division 112 7. Diagram of stage three of pyrenoid division 112 8. Diagram of newly formed daughter cells .... 112 9. Diagram of a normal interphase cell at the end of the light period on a 12:12 hr light: dark cycle 3.12 10. Interphase nuclei of strain 922 after being fixed in Jackson's fixative and stained with propiocannine 113 11. Mid-prophasc nucleus of strain 922 after being fixed in Jackson's fixative and stained with propiocannine 113 12. Late prophase nucleus (11) of strain 922 after being fixed in Jackson's fixative and stained with propiocarmine . 113 13• Anaphase nucleus (a) of strain 922 after being fixed in Jackson's fixative and stained with propiocarmine 113 vii viii LIST OF ILLUSTRATIONS--Continued Figure Page l4. Mid-telophase nuclei (t) of strain 922 after being fixed in Jackson's fixative and stained with propio- carmine 113 15- Interphase nucleus (i) of strain 922 after being fixed in Jackson's fixative and stained with propio- carmine 113 16. Interphase nucleus of strain 922 after being fixed in Jackson's fixative. Phase two of pyrenoid division 113 17* Interphase nucleus of strain 922 after being fijced in Jackson's fixative. Phase three of pyrenoid division is illustrated in one daughter cell (d); the pyrenoid has divided 113 18. Diagram of the interphase nucleus of Gauch's chloroplast mutant strain 1559> top view Il4 19* Diagram of interphase nuclei of daughter cells of Gauch's chloroplast mutant strain 1559? after nuclear division has taken place. Stage one of pyrenoid division, top view Il4 20. Diagram of stage two of pyrenoid division in daughter cells of Gauch's chloro­ plast mutant strain 1559? top view Il4 21. Same as in Figure l8 but a side view ll^t 22. Same as in Figure 19 but a side view. The nuclei have completed their first stage of migration ll't 23. Same as in Figure 20 but a side view. The nuclei are passing between the pair of chloroplasts in each daughter cell ll'l ix LIST OF ILLUSTRATIONS--Continued Figure Page 2k. El ectron micrograph of the pyrenoid area of Zygnenia circumcar.1 natum strain 42 fixed in 1% potassium permanganate. A section through starch grains (S) tangential to the pyrenoid 115 25- Electron micrograph of the pyrenoid of Zy gnenia circ um carinat um strain 'l2 fixed in1%potassium permanganate ..... 115 26. Elec tron micrograph of the pyrenoid of Zygnema circum carin a t um strain k2 fixed in1%potassium permanganate. Tubular branched thylakoids are in the upper half of the pyrenoid while disc-thylakoids are in the lower half . Il6 27• A diagramatic interpretation of a membrane structure found in Figure 26 116 28. Demonstration of propiocarmine stain on pyrenoids of a cell of strain 922 fixed in Jackson1 s fixative 117 29* Cell same as in Figure 28 but treated directly with IKI after fixation in Jackson's fixative. The protoplasts including the pyrenoids show severe shrinkage 117 30. Cell of strain 922 in the dark for six days, unfixed. Treatment with IKI gives no conclusive indication of starch 117 31• Cell same as in Figure 30 but fixed in Jackson's fixative and treated graduall y with IKI to avoid plasinolysis . No conclusive evidence for starch 117 32. Cell same as in Figure 30 but placed under 2,l60 lux intensity for" 1/2 hr, fixed in Jackson's fixative, and trecited gradually with IKI to avoid plasinolysis 117 X LIST OF ILLUSTRATIONS--Continned 0 Figure Page 33 • Cell same as in. Figure 30 but placed under 2,l6o lux intensity for 1 hr, fixed in Jackson's fixative, and treated gradually with IKI to avoid plasmolysis 117 Cell as in Figure 30 but placed under 2 ,l60 1ux intensity for 2 hr, fixed in Jackson's fixative, and treated gradually with IKI to avoid plasinolysis 117 35• Cell as in Figure 30 but placed under 2, 160 lux intensity for 3 hr •> fixed in Jackson's fixative, and t.ree\ted gradually with IKI to avoid V plasmolysis 117 36.