Florida State University Libraries Electronic Theses, Treatises and Dissertations The Graduate School 2005 Regulation of Guard-Cell Function by the Regulatory Apoplastic Photosynthate Pool Yun Kang Follow this and additional works at the FSU Digital Library. For more information, please contact [email protected] THE FLORIDA STATE UNIVERSITY COLLEGE OF ARTS AND SCIENCES REGULATION OF GUARD-CELL FUNCTION BY THE REGULATORY APOPLASTIC PHOTOSYNTHATE POOL By YUN KANG A Dissertation submitted to the Department of Biological Science in partial fulfillment of the requirements for the degree of Doctor of Philosophy Degree Awarded: Fall Semester, 2005 The members of the committee approve the dissertation of Yun Kang defended on November 4, 2005. ______________________________________ William H. Outlaw Jr. Professor Directing Dissertation ______________________________________ Michael Blaber Outside Committee Member _____________________________________ Hank W. Bass Committee Member _____________________________________ George W. Bates Committee Member _____________________________________ Ross W. Ellington Committee Member _____________________________________ Laura R. Keller Committee Member Approved: ___________________________________________________________ Timothy S. Moerland, Chair, Department of Biological Science The Office of Graduate Studies has verified and approved the above named committee members. ii AKNOWLEDGEMENTS I would like to thank my major advisor, Dr. William H. Outlaw Jr., for his guidance, support and assistance. Special thanks are extended to Drs. Hank W. Bass, George W. Bates, Michael Blaber, and Laura R. Keller for helpful advice. I would like to thank Dr. Karthik Aghoram, Fanxia Meng, Danielle M. Sherdan and Tianran Jiang for assistance in experiments as well as writing. Kara L. Chamberlain and Guorong Zhang are thanked for help in revision of the dissertation. I thank Bruce N. Smith and Giordano B. Fiore for conducting the sugar measurements with HPLC, and Kim A. Riddle and Xixi Jia for microscopy. I also thank Paul Burress and Jason A. Johnson for making the liquid scintillation measurements. Finally, I would like to thank my family for their unconditional love and support. This work was partially supported by a grant from NASA awarded to Dr. William H. Outlaw Jr. iii TABLE OF CONTENTS LIST OF FIGURES ..............................................................................................................vi LIST OF ABBREVIATIONS............................................................................................ viii ABSTRACT..........................................................................................................................ix INTRODUCTION .................................................................................................................1 GUARD-CELL APOPLASTIC PHOTOSYNTHATE ACCUMULATION IS LIMITED OR ABSENT IN THE SYMPLASTIC PHLOEM LOADING PLANT OCIMUM BASILICUM...........................................................................................................................7 Introduction ........................................................................................................................7 Materials and Methods.....................................................................................................10 Plant Material ...............................................................................................................10 Chemicals .....................................................................................................................10 Phloem Exudation ........................................................................................................10 Leaf Conductance.........................................................................................................11 Semiquantitative Histochemical Estimates of Guard-cell Starch Contents and of Guard-cell Potassium Contents ....................................................................................11 Quantitative Histochemical Assay of Guard-cell Sucrose Content..............................11 Bulk-leaf Apoplastic Sap Collection............................................................................12 Bulk-leaf Apoplastic Water Content Measurement .....................................................12 Transmission Electron Microscopy (TEM) and Dwarf Basil Guard-cell-wall Volume and Guard-cell Volume Calculation.............................................................................13 Movement of Extra-foliar Xylem-source Mannitol through the Leaf..........................13 Guard-cell-sugar Extraction for HPLC ........................................................................14 HPLC Analysis of Sugars in Phloem Exudates, Bulk-leaf Apoplastic Sap and Guard-cell Extracts.......................................................................................................14 Results ..............................................................................................................................15 Sugar Analysis of Phloem Exudates Confirmed that Dwarf Basil is a Symplastic Phloem Loader..............................................................................................................15 Diurnal Kinetics of Dwarf-basil Leaf Conductance, Stomatal Aperture Size, and Guard-cell Starch and Potassium Contents Was Typical.............................................16 Dwarf-basil Bulk-leaf Apoplastic Sugar and Galactinol Concentrations were in the Micromolar Range........................................................................................................18 iv Mannitol Fed via the Petiole Accumulated in the Guard-cell Apoplast, Indicating the Continuity of the Bulk-leaf Apoplast in Dwarf Basil...................................................19 Guard-cell Apoplastic Photosynthate Accumulation was Limited or Absent in Dwarf-basil Plants ........................................................................................................23 Discussion ........................................................................................................................25 Dwarf Basil, a Model Symplastic Phloem Loading Species for Guard-cell Studies ...26 Relevance of Sugars in the Bulk-leaf apoplast of Symplastic Phloem loaders to Photosynthesis-dependent Transpiration-linked Sugar Accumulation in the Guard-cell Apoplast......................................................................................................27 Absence of Guard-cell Apoplastic Photosynthate Accumulation in Symplastic Phloem Loading Plant Dwarf Basil..............................................................................28 Physiological and Ecological Implications of Stomatal Control in Symplastic Phloem Loaders and Apoplastic Phloem Loaders.....................................................................29 Summary.......................................................................................................................30 GUARD-CELL APOPLASTIC SUCROSE CONCENTRATION―A LINK BETWEEN LEAF PHOTOSYNTHESIS AND STOMATAL APERTURE SIZE ................................31 Introduction ......................................................................................................................31 Materials and Methods.....................................................................................................32 Plant Material ...............................................................................................................32 Photosynthesis Rate Measurement...............................................................................33 Plant Shading and Growth-cabinet CO2-Concentration Manipulation ........................33 Quantitative Histochemical Assay of Guard-cell Sucrose Content..............................33 Bulk-leaf Apoplastic Sap Collection............................................................................34 Guard-cell-sugar Extraction for HPLC ........................................................................34 HPLC Analysis of Sugars in Bulk-leaf Apoplastic Sap and Guard-cell Extracts ........35 Results ..............................................................................................................................35 Lowered Photosynthesis Rate without Altering Transpiration Rate Was Effected by Shading Plus Decreased Ambient CO2 Concentration.................................................35 Bulk-leaf Apoplastic Sucrose, Glucose and Fructose Concentrations were Lower in Shaded than in Control Plants ......................................................................................37 Lower Leaf Photosynthesis Rate Resulted in Lower Guard-cell Apoplastic Sucrose Content .........................................................................................................................38 Discussion ........................................................................................................................41 OVERALL SUMMARY .....................................................................................................46 REFERENCES ....................................................................................................................48 BIOGRAPHICAL SKETCH ...............................................................................................58 v LIST OF FIGURES Fig. 1. Leaf cross of apoplastic phloem loaders (A) and symplastic phloem loaders (B), showing the accumulation of sucrose around