Roles of Ros Scavenging Enzymes and Aba in Desiccation Tolerance in Ferns
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ROLES OF ROS SCAVENGING ENZYMES AND ABA IN DESICCATION TOLERANCE IN FERNS By Kwanele Goodman Wandile Mkhize Submitted in fulfilment of the academic requirements of Master of Science in the Discipline of Biological Sciences School of Life Sciences College of Agriculture, Engineering and Science University of KwaZulu-Natal Pietermaritzburg Campus South Africa December 2018 Preface The experimental work described in this thesis was carried out by the candidate while based in the Discipline of Biological Sciences School of Life Sciences, of the College of Agriculture, Engineering and Science, University of KwaZulu-Natal, Pietermaritzburg campus, under the supervision of Professor Richard P. Beckett, from January 2017 to December 2018. The research was financially supported by the National Research Foundation (South Africa). The contents of this work have not been submitted in any form to another university and, except where the work of others is acknowledged in the text, the results reported are due to investigations by the candidate. __________________________ Signed: Mkhize K.G.W __________________________ Signed: Beckett R.P Date: 30/11/2018 i Declaration 1: Plagiarism I, Kwanele Goodman Wandile Mkhize, student number: 213501630 declare that: (i) The research reported in this dissertation, except where otherwise indicated or acknowledged, is my original work; (ii) This dissertation has not been submitted in full or in part for any degree or examination to any other university; (iii) This dissertation does not contain other persons’ data, pictures, graphs or other information, unless specifically acknowledged as being sourced from other persons; (iv) This dissertation does not contain other persons’ writing, unless specifically acknowledged as being sourced from other researchers. Where other written sources have been quoted, then: a) Their words have been re-written but the general information attributed to them has been referenced; b) Where their exact words have been used, their writing has been placed inside quotation marks, and referenced; (v) Where I have used material for which publications followed, I have indicated in detail my role in the work; (vi) This dissertation is primarily a collection of material, prepared by myself, published as journal articles or presented as a poster and oral presentations at conferences. In some cases, additional material has been included; (vii) This dissertation does not contain text, graphics or tables copied and pasted from the Internet, unless specifically acknowledged, and the source being detailed in the dissertation and in the References sections. _____________________________________ Signed: Mkhize K.G.W Date: 30/11/2018 ii Declaration 2: Publications CONFERENCE CONTRIBUTION 1. South African Association of Botanists (SAAB) annual congress, January 2018, University of Pretoria. Oral presentation on the Roles of ROS scavenging enzymes in desiccation tolerance in ferns. Authors: Mkhize, K.G.W., Beckett, R.P. 2. South African Association of Botanists (SAAB) annual congress, January 2019, University of Johannesburg. Oral presentation on the The effect of long-term dehydration on desiccation tolerance mechanism of Loxogramme abyssinica and Crepidomanes inopinatum. Authors: Mkhize, K.G.W., Beckett, R.P. __________________________ Signed: Mkhize K.G.W __________________________ Signed: Beckett R.P Date: 30/11/2018 iii Abstract Roles of ROS scavenging enzymes and ABA in desiccation tolerance in ferns The fern species Loxogramme abyssinica and Crepidomanes inopinatum occur widely in KwaZulu Natal Afromontane forests. Although both species are suspected to be desiccation tolerant (DT), little is known about their mechanisms of tolerance. The present study was undertaken to characterize DT in both ferns, specifically the roles of the reactive oxygen species (ROS) scavenging enzymes superoxide dismutase (SOD) and peroxidase (POX), sugar afccumulation, and abscisic acid. Broadly speaking, DT mechanisms are either constitutive (always present) or induced in response to stress. C. inopinatum dries rapidly because it is a “filmy” fern, lacking a cuticle; in contrast L. abyssinica possesses a cuticle, and therefore dries slowly. It was predicted that the fast-drying C. inopinatum would rely mainly on constitutive mechanisms, while the slow-drying L. abyssinica would depend on inducible mechanisms. Plant were collected from the field, transported to the laboratory and then subjected to desiccation. Two methods of desiccation were used. The first one was a relatively mild, slow and short term (48 h) desiccation over calcium acetate, and the second was a harsher, rapid and long term (1 week) desiccation over silica gel. Measurement of chlorophyll fluorescence parameters showed that both species displayed rapid recovery during rehydration after slow or fast desiccation, confirming that both species are genuinely poikilohydric. POX activity remained constant in both species during slow desiccation and subsequent rehydration, suggesting that ROS scavenging by POX is a constitutive DT mechanism. However, the absolute POX activity of C. inopinatum was much higher than in L. abyssinica. Rapid long term desiccation reduced POX activity in both species, but the activity recovered during rehydration. In both species, slow desiccation increased SOD activity, and activity declined to original values during rehydration. Slow and fast drying increased the concentrations of soluble sugars in both species, and concentrations rapidly declined to initial values during rehydration. ABA pretreatment had little effect on DT in either species, although tolerance was slightly increased in L. abyssinica. Results of this study suggested that both species depend largely on inducible DT mechanisms. Counter to the original hypothesis, inducible mechanisms occur even in filmy ferns that desiccate rapidly. The results from this project will contribute to our understanding of how ferns can survive in stressful environments in South Africa, and potentially could help improve abiotic stress tolerance in crop plants. iv Keywords: Desiccation tolerance, Loxogramme abyssinica and Crepidomanes inopinatum Reactive oxygen species (ROS), Peroxidase (POX), Superoxide dismutase (SOD), Soluble sugars, Abscisic acid (ABA), Rapid and Slow desiccation. v Acknowledgements I would like to deeply express my gratitude to my supervisor Prof R.P Beckett. From the start of my postgraduate studies, he showed me extreme support, guidance in all my work and valuable feedback he gave me that enabled me to grow and be where I am today as a scientist. Most of all, I’m greatly gratified for an extra mile of financial support he provided through difficult times. He really played a positive role in my life. I also thank Dr Farida Minibayeva for positive inputs towards experiments conducted here. Secondly, I would like to thank NRF (South Africa) for funding me on this project and also the University of KwaZulu-Natal for providing me with all the necessary facilities for the research, I’m truly grateful. Finally yet importantly, I am grateful for the support, words of encouragement and prayers from my family and friends. Particularly, my friends from UKZN have played a huge role towards the success of this project. All the motivation, help with proof reading and stats and the love they showed me, gave me hope and strength to perform at my best. vi Table of Contents Page Preface......................................................................................................................................... i Declaration 1: Plagiarism ........................................................................................................... ii Declaration 2: Publication......................................................................................................... iii Abstract .................................................................................................................................... iv Acknowledgments .................................................................................................................... vi Table of content ...................................................................................................................... vii List of tables ............................................................................................................................. xi List of figures .......................................................................................................................... xii List of abbreviations ............................................................................................................... xv CHAPTER 1: Literature review ................................................................................................ 1 1.1 The pteridophytes ................................................................................................................ 1 1.2 Occurrence of DT in the pteridophytes ............................................................................... 1 1.3 Morphological and anatomical mechanisms of DT in the pteridophytes ......................... 11 1.4 Physiological and biochemical mechanisms of DT in the pteridophytes ......................... 11 1.4.1 DT mechanisms based on avoiding ROS formation ...................................................... 12 1.4.2 DT mechanisms based on antioxidant enzymes ............................................................ 12 1.4.3 DT mechanisms based on non-enzymatic antioxidants