A University of Sussex DPhil thesis Available online via Sussex Research Online: http://sro.sussex.ac.uk/ This thesis is protected by copyright which belongs to the author. This thesis cannot be reproduced or quoted extensively from without first obtaining permission in writing from the Author The content must not be changed in any way or sold commercially in any format or medium without the formal permission of the Author When referring to this work, full bibliographic details including the author, title, awarding institution and date of the thesis must be given Please visit Sussex Research Online for more information and further details Responses of Suaeda maritima to flooding and salinity By Gazala M. Alhdad Presented for the degree of Doctor of Philosophy in the School of Life Sciences at the University of Sussex Submitted October 2012 ii Declaration The contents of this thesis are the original work by the author, except where otherwise stated. I hereby declare that this has not and will not be submitted in whole or in part to any other university for the award of any other degree. Signature……………………………………………………….Gazala Alhdad iii UNIVERSITY OF SUSSEX GAZALA M. ALHDAD RESPONSES OF SUAEDA MARITIMA TO FLOODING AND SALINITY ABSTRACT Suaeda maritima is an annual halophyte commonly found in salt-marshes. Its salt tolerance has been well studied, though there is little published on the effect of simultaneous waterlogging. The effects of saline waterlogging on growth, antioxidants (glutathione and total polyphenolic compounds, antioxidant activity) and oxidative damage were investigated with simulated tides in a controlled glasshouse and on plants collected from the field. Flooded shoots possessed higher levels of antioxidants than those from plants growing in well-drained situations, in the glasshouse and the field. The effects of hypoxia, (simulated in nutrient solution by flushing with nitrogen in a solution containing a low concentration of agar, which limits convection within the solution and so the transport of oxygen from the air) were determined on growth and trace metal concentrations, in plants grown in different concentrations of artificial seawater (100 and 350 mM Na + at low pH, > pH 5.5), in sand/mud irrigated with half- strength fresh seawater (at high pH, ca 7-8) and in different concentrations of manganese and iron in solution culture. High salt concentration reduced accumulation of trace metals in plants. Optimal growth occurred in 14 µM Fe and 1 mM Mn. Accumulation of trace metals was reduced at high pH, with more accumulating in the roots than the shoots. Hypoxia increased soluble sugars in shoots and roots, and this was affected by the salt concentration. Hypoxia also caused adventitious root development in hydroponic experiments, while in sand, adventitious root development was greater in drained than flooded conditions. Hypoxia significantly reduced shoot sodium concentration, sodium flux and bypass flow, at low and high salt concentrations. In high salt conditions, S. maritima reduced its transpiration rate and improved its water use efficiency. It was also shown that the roots contained high lactate concentrations under aerated and hypoxic conditions. S. maritima demonstrated many adaptations for tolerating extreme hypoxia. iv Acknowledgements Above all, I thank God for having given me the strength and patience to carry on this project and for blessing me with many great people, who have been my greatest support in both my personal and professional life. There are many people to whom I owe a great debt of gratitude and without whose help I could not have completed this work: to all those people I am extremely grateful. Firstly, I would like to express my deep gratitude to Prof. Timothy J. Flowers and Dr. Mohammed J. Al-Azzawi, my supervisors, for the opportunity they have given me to study at the University of Sussex, for their invaluable supervision and guidance during my DPhil study, for their unconditional advice and support, for the valuable discussions, especially in the later stages of my work, and for their encouragement during my D Phil. I appreciate all members of the School of Life Sciences, particularly Diane Ellis, Carol Jones, Dr Bualuang Faiyue, Dr Ulya Bashtanova, Dr Ruth Ibemesin, Dr Anne Wetson, Dr Carla Cassaniti, Dr Ruth and Dr Hanaa Kamal, Dr Riadh Ksouri, Dr Alaa Abdul- Sada, Dr Raghad Al-salhi and Prof. Christian Zörb for all their assistance. I am also grateful to Chris Dadswell for helping me use ICP-MS and GC-MS, and to Dr Naomi Ewald for the teaching of statistics. I wish to thank Dr. Charlotte for teaching me to analyse glutathione at Royal Botanic Gardens, Kew, and thanks also go to Dr Martyn Stenning and Dan Young for their help in the collection and transport of vast quantities of seawater, and to the Brighton Sea Life Centre for supplying the seawater. My thanks to Michael Schofield for training me in the preparation of stained microtome-cut sections and light-microscopy techniques. I would also like to thank the people of Libya and the Ministry of Higher Education of Libya for the financial support in Libya and the UK. Last but not least. I would like to express my sincere thanks to my husband Al-Zanati, for his untold support and encouragement throughout this time, especially regarding my studies. I also thank my daughters Lamar and Meyer for their patience and for creating a very nice environment. Finally, I am extremelygrateful to my parents, sisters and v brothers for their encouragement and support during my studies. Without the help of Allah and then the help of all the people mentioned above, this work could not have been completed. This thesis is dedicated to those most dear to me - my beloved family. Many thanks to you all..... Gazala, October 2012 vi List of publications Gazala M. Alhdad, Charlotte E. Seal, Mohammed J. Al-Azzawi, Timothy J. Flowers (2012) The effect of combined salinity and waterlogging on the halophyte Suaeda maritima : The role of antioxidants. Environmental and Experimental Botany 87, 120– 125 vii Contents Declaration ....................................................................................................................... ii ABSTRACT .................................................................................................................... iii Acknowledgements ......................................................................................................... iv List of publications ......................................................................................................... vi List of Figures ................................................................................................................ xii List of Tables .............................................................................................................. xviii Abbreviations ............................................................................................................... xxi Chapter 1 : General introduction .................................................................................. 1 1.1.2 The effects of salinity on plants with an emphasis on halophytes 2 1.1.2.1 Growth .................................................................................................................. 2 1.1.2.2 Osmotic effects...................................................................................................... 4 1.1.2.2.1 Osmotic adjustment, ion compartmentation ...................................................... 5 1.1.2.2.2. Compatible solutes ............................................................................................ 5 1.1.2.3 Effects on mineral nutrition .................................................................................. 6 1.1.2.4 Antioxidants .......................................................................................................... 8 1.1.2.5. The pathways of Na + uptake in halophytes .......................................................... 9 1.1.2.5.1 Apoplastic pathway (Bypass flow) .................................................................. 10 1.1.2.5.2 Symplastic pathway ......................................................................................... 11 1.2 Tolerance to flooding ................................................................................................ 13 1.2.1. Adventitious root 13 1.2.2 Aerenchyma and internal O 2 movement 14 1.2.3 Anoxia tolerance 17 1.3 The effects of combination of waterlogging and salinity on plants with an emphasis on halophytes .................................................................................................................. 18 1.3.1 The halophyte Suaeda maritima 18 1.4 Research objectives ................................................................................................... 20 Chapter 2 : The effect of combined salinity and waterlogging on the halophyte Suaeda maritima : the role of antioxidants ................................................................... 23 2.1. Introduction .............................................................................................................. 23 2.2 Materials and Methods .............................................................................................. 25 2.2.1. Plants materials and culture conditions 25 2.2.2 The tidal flow tank system 26 2.2.3 Glutathione Analysis 30 viii 2.2.3.1. Glutathione half-cell reduction potential (EGSSG/2GSH ) ....................................... 31 2.2.4. Lipid