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Physiological and Biochemical Responses of Bald Cypress to Salt Stress Nathan T UNF Digital Commons UNF Graduate Theses and Dissertations Student Scholarship 2013 Physiological and Biochemical Responses of Bald Cypress to Salt Stress Nathan T. Lauer University of North Florida Suggested Citation Lauer, Nathan T., "Physiological and Biochemical Responses of Bald Cypress to Salt Stress" (2013). UNF Graduate Theses and Dissertations. 454. https://digitalcommons.unf.edu/etd/454 This Master's Thesis is brought to you for free and open access by the Student Scholarship at UNF Digital Commons. It has been accepted for inclusion in UNF Graduate Theses and Dissertations by an authorized administrator of UNF Digital Commons. For more information, please contact Digital Projects. © 2013 All Rights Reserved Physiological and Biochemical Responses of Bald Cypress to Salt Stress by Nathan Lauer A thesis submitted to the department of Biology in partial fulfillment of the requirements for the degree of Master of Science in Biology UNIVERSITY OF NORTH FLORIDA COLLEGE OF ARTS AND SCIENCES July, 2013 The thesis of Nathan Lauer is approved: (Date) ________________________ ____________ Dr. Cliff Ross ________________________ ____________ Dr. Courtney Hackney ________________________ ____________ Dr. Jim Gelsleichter ________________________ ____________ Dr. Tim Sherman Accepted for the Department of Biology: ________________________ ____________ Dr. Dan Moon Chair Accepted for the College of Arts and Sciences: ________________________ ____________ Dr. Barbara Hetrick Dean Accepted for the University: ________________________ ____________ Dr. Len Roberson Dean of Graduate Studies Table of Contents: Title page i Certificate of approval ii Table of contents iii List of figures iv Abstract vii Chapter 1: General Introduction 1 Chapter 2: The development and utilization of biomarkers to detect sub-lethal 14 salinity stress within Bald Cypress (Taxodium distichum (L.) Rich.). Chapter 3: Preliminary investigation into the cellular mechanism of salinity 63 tolerance in Bald Cypress (Taxodium distichum (L.) Rich.). Chapter 4: Conclusions and future directions 100 References 103 Vita 113 iii List of Tables and Figures Figure 1.1 Ten year historical salinity data within the lower St. John’s River. Figure 1.2 “Ghost Swamp” associated with the lower St. Johns River. Figure 2.1 Location of field sampling sites. Figure 2.2 Cypress root sodium content from the greenhouse study. Figure 2.3 Cypress root hydrogen peroxide content from the greenhouse study. Figure 2.4 Cypress root respiration from the greenhouse study. Figure 2.5 Cypress leaf sodium content from the greenhouse study. Figure 2.6 Maximum quantum yield of Cypress from the greenhouse study. Figure 2.7 Non-photochemical quenching of Cypress from the greenhouse study. Figure 2.8 Cypress leaf hydrogen peroxide content from the greenhouse study. Figure 2.9 Cypress leaf free proline content from the greenhouse study. Figure 2.10 Localization of ROS in Cypress leaf tissue from the greenhouse study. Figure 2.11 Relationship between leaf hydrogen peroxide content and maximum quantum yield of Cypress from the greenhouse study. Figure 2.12 Cypress leaf superoxide dismutase activity from the greenhouse study. Figure 2.13 Cypress leaf peroxidase activity from the greenhouse study. Figure 2.14 In-gel analysis of Cypress leaf peroxidase isozymes from the greenhouse study. Figure 2.15 Cypress leaf sodium content from the field study. Figure 2.16 Maximum quantum yield of Cypress from the field study. Figure 2.17 Non-photochemical quenching of Cypress from the field study. Figure 2.18 Cypress leaf lipid peroxidation from the field study. iv Figure 2.19 Cypress leaf free proline content from the field study. Figure 2.20 Cypress leaf superoxide dismutase activity from the field study. Figure 2.21 Cypress leaf peroxidase activity from the field study. Figure 2.22 Relationship between leaf superoxide dismutase and leaf peroxidase in Cypress from the field study. Figure 3.1 Maximum quantum yield from the detached Cypress leaf experiment. Figure 3.2 Relationship between Cypress leaf sodium content and maximum quantum yield from the detached leaf experiment. Figure 3.3 Relationship between Cypress leaf sodium content and maximum fluorescence from the detached leaf experiment. Figure 3.4 Relationship between Cypress leaf sodium content and initial fluorescence from the detached leaf experiment. Figure 3.5 Peroxidase activity from the detached Cypress leaf experiment. Figure 3.6 In-gel analysis of a ~37 kDa peroxidase isozyme from the detached Cypress leaf experiment. Figure 3.7 Transpiration from the detached Cypress leaf experiment. Figure 3.8 Peroxidase activity from detached Cypress leaves treated with clofibrate. Figure 3.9 In-gel analysis of a ~37 kDa peroxidase isozyme from detached Cypress leaves treated with clofibrate. Figure 3.10 Transpiration from detached Cypress leaves treated with clofibrate. Figure 3.11 Peroxidase activity from detached Cypress leaves pre-treated with BFA and salt stressed. v Figure 3.12 In-gel analysis of a ~37 kDa peroxidase isozyme from detached Cypress leaves pre-treated with BFA and salt stressed. Figure 3.13 Transpiration from detached Cypress leaves pre-treated with BFA and salt stressed. Figure 3.14 Peroxidase activity from detached Cypress leaves pre-treated with DPI and salt stressed. Figure 3.15 In-gel analysis of a ~37 kDa peroxidase isozyme from detached Cypress leaves pre-treated with DPI and salt stressed. Figure 3.16 Transpiration from detached Cypress leaves pre-treated with DPI and salt stressed. vi Abstract Bald Cypress (Taxodium distichum (L.) Rich.) is native to freshwater wetlands of Florida. The vitality of cypress within coastal freshwater wetlands is threatened by saltwater intrusion. Biomarkers to detect sub-lethal salinity stress were developed using a controlled greenhouse study. Cypress saplings maintained at elevated salinities of 4 and 8‰ exhibited a decrease in maximum quantum yield (MQY) and an increase in non- + photochemical quenching (NPQ). Cypress leaves exhibited an increase in Na , H2O2, and free proline content compared to plants maintained in freshwater. These biomarkers were used to detect salinity stress within a population of cypress associated with the lower St. Johns River where saltwater intrusion is occurring. Cypress in a basin swamp exhibited signs of salinity stress with low MQY and elevated NPQ values compared to Cypress at other sites. Cypress leaves at the basin swamp also had the highest Na+, lipid peroxidation, and proline content compared to plants at other sites. Detached Cypress leaf experiments were conducted to explore the mechanisms of salt tolerance. Detached cypress leaves were first exposed to elevated NaCl concentrations for 24, 48, or 72 hours. Elevated salinity caused a decrease in leaf transpiration for all times tested. Total peroxidase activity exhibited an increase in response to salt stress after 48 hours. Enhanced peroxidase activity was found to be associated with the induction of a ~37 kDa peroxidase isoform. Treatment of leaves with clofibrate caused an increase in activity of the ~37 kDa peroxidase. Pre-treatment of leaves with brefeldin A (BFA) blocked the induction of the ~37 kDa peroxidase associated with salt stress. Pre-treatment of Cypress leaves with diphenyliodonium (DPI) blocked the decrease in transpiration associated with vii salt stress, suggesting that H2O2 is enzymatically produced within the stomata in response to salt stress. viii Chapter 1: General Introduction 1.1 Bald Cypress and Sea Level Rise in the St. Johns River Bald Cypress (Taxodium distichum (L.) Rich.) is a large deciduous softwood tree native to the Southeastern United States and are often dominant in coastal freshwater wetlands where they create important structural habitats for numerous organisms (Bonner, 1974; Harris & Mulholland, 1983; Brightman, 1984; McMahan & Davis, 1984; Harris & Vickers 1984; Wilhite & Toliver, 1990). They provide a number of important ecosystem services which make them assets to coastal freshwater habitats. For example, cypress swamps are capable of retaining large quantities of water which aids in flood control (Ewel, 1985). In addition, cypress are incredibly windfirm and are able to protect surrounding areas from winds up to hurricane force (Wilhite & Toliver, 1990). Finally, Cypress have been documented to improve water quality by serving as biological filters removing excess nutrients and pollutants from porewater (Wharton, 1977; Dierberg & Brezonik, 1983). These aforementioned characteristics make Bald Cypress an important member of coastal freshwater wetland communities and an important candidate for conservation and protection. The vitality of coastal freshwater wetlands is currently being threatened by saltwater intrusion associated with both global and local occurrences. Globally, sea level is predicted to rise by 30 cm within the next 100 years (IPCC, 2007). Locally, 1 anthropogenic activities such as dredging and surface water withdrawal are predicted to contribute to saltwater intrusion within the lower St. Johns River. As a result, the freshwater/saltwater boundary in estuaries is beginning to shift upstream. This phenomenon places environmental stress on coastal freshwater wetlands and ultimately causes a shift in ecosystems from freshwater wetlands into oligohaline marshes. The St. Johns River is located in Northeast Florida and enters the Atlantic Ocean near Jacksonville, FL. The lower reach of the river is characterized by a limited elevational gradient and slow freshwater
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