Iowa State University Capstones, Theses and Retrospective Theses and Dissertations Dissertations 2000 Carbohydrate regulation of leaf development: Investigating the role of source strength, carbon partitioning and hexokinase signaling in regulating leaf senescence Adam Christopher Miller Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/rtd Part of the Molecular Biology Commons, and the Plant Sciences Commons Recommended Citation Miller, Adam Christopher, "Carbohydrate regulation of leaf development: Investigating the role of source strength, carbon partitioning and hexokinase signaling in regulating leaf senescence " (2000). Retrospective Theses and Dissertations. 12707. https://lib.dr.iastate.edu/rtd/12707 This Dissertation is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. 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Rodermel and Martin Spalding Iowa State University Ames, Iowa 2000 Copyright ® Adam Christopher Miller, 2000. All rights reserved. UMI Number. 9977346 _____ iR UMI UMI Microfomi9977346 Copyright 2000 by Bell & Howell Infomiation and Learning Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. Bell & Howell Information and Learning Company 300 North Zeeb Road P.O. Box 1346 Ann Arbor. Ml 48106-1346 ii Graduate College Iowa State University This is to certify that the Doctoral dissertation of Adam Christopher Miller has met the dissertation requirements of Iowa State University Signature was redacted for privacy. Co-major Professor Signature was redacted for privacy. issor Signature was redacted for privacy. Signature was redacted for privacy. Forthe Gf^cfuate' College iii TABLE OF CONTENTS CHAPTER 1. GENERAL INTRODUCTION 1 Dissertation Organizatioii 1 Background Information and Literature Review 2 References 13 CHAPTER 2. ELEVATED CO, EFFECTS DURING LEAF ONTOGENY: A NEW PERSPECTIVE ON ACCUMATION 24 Abstract 24 Introduction 25 Materials and Methods 27 Results 30 Discussion 32 Acknowledgements 37 References 37 CHAPTER 3. LEAF DEVELOPMENT AND ACCLIMATION TO ELEVATED CO, 44 Introduction 44 Elevated CO, and Tobacco Leaf Development 47 A New Paradigm to Understand Acclimation SO Mechanism of the Temporal Shift 52 References S3 CHAPTER 4. CARBOHYDRATE REGULATION OF LEAF DEVELOPMENT: PROLONGATION OF LEAF SENESCENCE IN RUBISCO ANTISENSE MUTANTS OF TOBACCO 59 Abstract S9 Introduction 60 Materials and Methods 62 Results 64 Discussion 68 Acknowledgements 71 References 71 CHAPTER 5. CHANGE IN CARBON PARTITIONING DURING ARABIDOPSIS LEAF DEVELOPMENT 83 Abstract 83 Introduction 84 Materials and Methods 86 Results 89 Discussion 93 References 97 iv CHAPTER 6. GENERATION OF TRANGENIC ARABIDOPSIS WITH ALTERED LEVELS OF HEXOKINASE (HXKl) ACTIVITY 113 Introduction 113 Materials and Methods 113 Results and Discussion 117 References 120 CHAPTER 7. GENERAL CONCLUSIONS AND FUTURE RESEARCH DIRECTIONS 127 Conclusions and Future Research 127 References 134 ACKNOWLEDGEMENTS 138 1 CHAPTER 1. GENERAL INTRODUCTION Dissertation Oiigaiiization This dissertation is organized into six chapters. Chapter 1 is an introduction to source strength and cart)ohydrate effects on plant systems. Specifically, this background information is divided into three main sections. The first section covers known carbohydrate effects on plant metabolic systems, particularly their effects on gene expression. The second section describes leaf development and factors involved in the senescence phase of development. The third section details the hexokinase sugar-signaling pathway in plants as a potential regulation step in carbohydrate effects on development. Chapters 2,4, and 5 are presented as independent journal papers; chapter 3 is a chapter in a book. Chapter 2 covers the experiments performed on tobacco plants under elevated COj conditions and how this affects the progression of leaf senescence. Chapter 3 summarizes these results and discusses their implications on our understanding of acclimation to high CO; environments. Chapter 4 covers the experiments performed on rbcS antisense mutants of tobacco and how decreased source strength effects on senescence compares to the increased source strength condition. Chapter 5 covers the investigations of wild type Arabidopsis leaf development and how carbon partitioning changes over ontogeny and how these changes relate to hexokinase and invertase activities. Chapter 6 provides a description of the generation of transgenic Arabidopsis plants with altered levels of hexokinase to be used in future analysis. Chapter 7 presents general conclusions and potential future projects. The last section contains the acknowledgements. 2 Background Informatioii and Literature Review Carboiiydrates and Regulation of Gene Expression The ultimate purpose of photosynthesis in higher plants is to fix CO2 and generate carbohydrates. After fixation, there are two main fates for this carbon: production of sucrose for export to other parts of the plant, or as starch for storage (Huber, 1989). These carbohydrates serve as the main source of chemical energy to power all of the cellular processes necessary to sustain life. However, mounting evidence has shown that carbohydrates have a much more complex role in plant metabolism besides simply as an energy supply. Sugars are now implicated as key regulatory molecules in several biochemical pathways. Abundance or insufficient levels of carbohydrates has been seen to either enhance or repress the expression of specific genes (reviewed in Koch, 1996; Graham, 1996). The specificity of action on gene expression suggests that this is a directed effect on sugar-modulated genes only, rather than a more general response to changes in energy supply status (Koch, 1996). Many of the major targets of carbohydrate regulation are genes involved in maintaining their own production and metabolism, consistent with a feedback regulation system (Koch, 1996; Hesse et al., 1995; Ko^mann et al., 1991; Krapp and Stitt, 1994). In general, plentiful amounts of sugars lead to an increase in genes responsible for storage and utilization of carbohydrates and a decrease in production genes. Conversely, sugar depletion enhances production genes and storage mobilization genes (Sheen, 1990; Koch, 1996). Such modifications help the plant maintain an optimum balance between carbohydrate supply and demand. 3 Initially, outside of seed reserves, the "source" of all carbohydrates in the plant arises from photosynthetic activity in the chloroplasts of a "source" leaf. These carbohydrates are eventually converted into the sugar sucrose in the cytosol, and then exported out of the cell. Sucrose is transported via the phloem to tissues that have an energetic requirement for these carbohydrates. Such tissues are also referred to as "sinks" for carbohydrates. Several observations have suggested that photosynthetic production of carbohydrates may be adjusted in response to the demand for these resources by the sinks. When sink demand is low, photosynthesis is down-regulated, and high demand results in increased photosynthesis (Sonnewald and Willmitzer, 1992). The degree of demand by sinks would determine sucrose export out of the source leaf, and ultimately carbohydrate status of the cell. Therefore, low demand for photoassimilates would result in carbohydrate accumulation in source cells, leading to feedback down-regulation of photosynthetic gene expression to reduce production (Stitt, 1991). A series of investigations have contributed support to this hypothesis. Decreased