MOLECULAR AND PHYSIOLOGICAL STUDIES OF SALT TOLERANCE IN THE SALT-SECRETOR MANGROVE AVICENNIA OFFICINALIS Pavithra Amruthur Jyothi-Prakash (M. Sc. Biochemistry, University of Mysore, India) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY (Ph. D.) DEPARTMENT OF BIOLOGICAL SCIENCES FACULTY OF SCIENCE NATIONAL UNIVERSITY OF SINGAPORE 2015 Declaration I hereby declare that this thesis is my original work and it has been written by me in its entirety. I have duly acknowledged all the sources of information which have been used in the thesis. This thesis has also not been submitted for any degree in any university previously. Pavithra Amruthur Jyothi-Prakash January 2015 ii Acknowledgements The journey started with a simple curiosity towards natural processes and all it took for this thesis to happen was a lot of courage, hard work, enthusiasm, endurance, persistence and sacrifices. But it could not have been completed without support from others. I would like to extend my gratitude for everyone who contributed to my success. I would like to express my sincere gratitude to my supervisors, Prof. Prakash Kumar and A/P Loh Chiang Shiong for their continuous support, patience and guidance, which has shaped me to become a better person and a budding scientist. I would like to thank both my Plant Biology and Plant Morphogenesis lab members for timely guidance and support whenever required. I would like to thank Dr. Tan Wee Kee for helping me initiate the project. Special thanks to Dr. Pannaga, for her involvement in discussions, suggestions and comments on the experimental design and setup. This has significantly contributed to the quality of my work. My thesis would not have been complete without her valuable inputs. I would like to thank Dr. Ram, Dr. Petra, Dr. Vijay and Dr. Vivek for sharing their expertise and for the technical advice related to my work. It was a pleasure working closely with Dr. Pratibha for a few experiments which gave me a good experience in troubleshooting. I would like to extend my thanks to Bhushan, Amrit and Anindita for a fun-filled environment in the lab and immense support during tough times. My sincere gratitude to Mrs. Ang, our lab officer who has been supportive in catering the laboratory needs without delay. I would like to thank Dr. iii Bijayalakshmi Mohanty for her constant support and immense help with bioinformatic analyses in my work. I would like to thank Prof. Mathew M. K. from NCBS, Bangalore, India who supported a part of aquaporin work by providing his resources. I am thankful for all his lab members who helped me during my work. My special thanks to Savita Bhagat and Shishupal Singh who were instrumental in completing my work at NCBS. I would like to extend my thanks to Department of Biological Sciences, NUS Environmental Research Enterprise and SPORE, NUS for the providing me the Research Scholarship. Consumables and traveling grant for overseas research work was supported by the Singapore National Research Foundation under its Environmental & Water Technologies Strategic Research Programme and administered by the Environment & Water Industry Programme Office (EWI) of the PUB, Singapore, NRF-EWI-IRIS (2P 10004/81) (R-706-000-010-272). My sincere thanks go to NParks Board, Singapore for extending permission to collect mangrove samples in Singapore. Many thanks to my friends Pradeep, Bidhan, Pramila, Narayani, Madhuri, Manali, Varuna and Pavithra Singaravelu who were a wonderful company during my Ph. D. My special thanks to Shruti, Ananya and Kameshwari for the quality time we had together at UTown residence, which added incredible memories to my Ph. D. journey. I acknowledge the support of my entire family for believing in my potential and special thanks to my parents, my brother and my husband for their continuous support and understanding, without them this journey would not have been iv possible. Finally, thanks to all my teachers, for their support, teachings and blessings. Lastly, I am grateful to all the helping hands, which were extended to me at the right time, which would be a long list to mention here. Thank you one and all. v Publications (Parts of the contents of the thesis are described in these articles) 1. Jyothi-Prakash PA, Mohanty B, Wijaya E, Lim TM, Lin Q, Loh C-S and Kumar PP (2014) Identification of salt gland-associated genes and characterization of a dehydrin from the salt secretor mangrove Avicennia officinalis. BMC Plant Biology 14-291 2. Krishnamurthy P, Jyothi-Prakash PA, Qin Lin, He Jie, Lin Q, Loh C-S, Kumar PP (2014) Role of root hydrophobic barriers in salt exclusion of a mangrove plant Avicennia officinalis. Plant, Cell & Environment, 37(7):1656-1671 Other Publications: (Content from this article is not related to the thesis) El-Sharkawy, S. Sherif, W. El Kayal, A. Mahboob, K. Abubaker, P. Ravindran, P. A. Jyothi-Prakash, P. P. Kumar and S. Jayasankar (2013). Characterization of gibberellin-signalling elements during plum fruit ontogeny defines the essentiality of gibberellin in fruit development. Plant Molecular Biology: 1-15 Conference Contributions: 1. 18th Biological Sciences Graduate Congress, December 2014, Malaysia Oral presentation: Molecular and physiological studies of salt secretion in mangrove plant. Pavithra A. J, Loh C-S, Kumar PP 2. 17th Biological Sciences Graduate Congress, December 2012, Thailand Oral presentation: Molecular and physiological studies of salt secretion in mangrove plant. Pavithra A. J, Loh C-S, Kumar PP vi Contents Declaration . ii Acknowledgements iii Publications . vi Summary . ix List of Tables . xii List of Figures xii List of abbreviations xiv Chapter 1: Introduction . 1.1 Salt and soil salinity 1.2 Status of mangrove forest in Singapore . 1.3 Salt balance in mangrove plants . 1.4 Effect of salinity on growth and development of plants . 1.5 Mechanisms to minimize damage from high salinity . 10 1.6 Objectives of the study and approach 17 Chapter : Materials and methods .21 2.1 Plant materials and growth conditions 21 2.2 Plasmid construction 30 2.3 Plant transformation . 35 2.4 Seed sterilization and germination assay in Arabidopsis 39 2.5 Southern blotting 40 2.6 Isolation and transfection of Arabidopsis mesophyll protoplasts . 41 2.7 Physiological methods 42 2.8 Quantification of hormones . 46 2.9 Non-radioactive RNA In Situ Hybridization . 48 2.10 Functional assay of AoDHN1 in E. coli . 54 2.11 Swelling assay in Xenopus laevis oocyte system . 55 2.12 Tissue preparation for subtractive hybridization . 56 2.13 Tissue preparation for transcriptome analysis 57 Chapter : Physiological and morphological studies in Avicennia officinalis 60 3.1 Background 60 3.1 Results 63 3.3 Discussion 78 vii Chapter : Subtractive Hybridization study 86 4.1 Background 86 4.2 Results 89 4.3 Discussion 107 Chapter : Transcriptome study .116 5.1 Background 116 5.2 Results 119 6.3 Discussion 129 Chapter : Aquaporin study .135 6.1 Background 135 6.2 Results 143 5.3 Discussion 172 Chapter : Limitations and recommendations 181 Chapter : General conclusions .185 References: 189 viii Summary Salt secretion is a specialized salt tolerance mechanism observed in mangrove plants. Several studies on mangroves had focussed on the structure of salt glands and salt secretion pattern, while, very few studies described the functional aspects of salt secretion. The present study focussed on preliminary analysis of salt filtration at the roots, salt secretion at the leaves and the effect of salt concentration on secretion using Avicennia officinalis seedlings that were not exposed to salt previously. Furthermore, to understand the molecular mechanisms underlying the secretion at the salt glands, differential gene expression in response to salt treatment was examined in the salt gland-rich tissues using subtractive hybridization and transcriptomics. The present study showed that the amount of salt in the external medium plays an important role in triggering salt secretion. Higher concentration of salt in the external medium leads to increases in xylem salt content and secretion rate. Increased levels of the stress hormones, namely, abscisic acid (ABA) and jasmonic acid (JA) were observed in salt-treated seedling tissues, but the other hormones such as gibberellins (GAs) and salicylic acid (SA) did not show significant variation in relation to salt treatment. Using subtractive hybridization method, an attempt was made to identify key genes that are differentially regulated in salt gland-rich adaxial epidermal tissues of leaves. Among the 34 genes that were enriched in the salt gland-rich tissue, a Dehydrin gene (AoDHN1) showed nearly 6fold increase in expression. Dehydrins are known to be involved in stressix remediation in other plants, and hence, AoDHN1 was chosen for further analysis. AoDHN1 expression was high in A. officinalis leaves and specifically in salt glands as indicated by quantitative RT-PCR and in situ hybridization. To check its stress-remediation effect, AoDHN1 was expressed in E. coli cells that were subjected to salinity and drought stress conditions. The growth of E. coli cells expressing AoDHN1 was significantly higher compared to control cells without AoDHN1, suggesting a significant role for AoDHN1 in mediating salt stress. Aquaporins are known to play an important role during drought and salt stress conditions and are also known to be involved in salt secretion in mangroves. Therefore, three aquaporin genes from A. officinalis, namely, AoPIP1.1, AoPIP1.2 and AoPIP2.2 were cloned and sequenced. These aquaporins showed significant increase in transcript levels within 90min of drought stress, but not in response to ABA and salinity treatments. From a functional assay in Xenopus laevis oocytes, AoPIP1.1 and AoPIP1.2 were found to exhibit water transport activity. Also, expression of AoPIP1.2 was high in salt gland-rich tissues compared to the transcript levels of AoPIP1.1 and AoPIP2.2. 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Current Opinion in Plant Biology 6, 441-445. 203 [...]... secretion and therefore the salt tolerance mechanism in the mangrove Avicennia officinalis These results validate the previous findings that aquaporins play a critical role in salt secretion and water reabsorption in this species While we recognise the need for additional work, these findings help to identify avenues for further research aimed at elucidating the underlying mechanisms of salt secretion and tolerance. .. A officinalis young and mature leaves 65 Figure 3.2: Adaxial and abaxial surfaces of A officinalis fresh and dried leaves 66 Figure 3.3: Determination of salt gland density in A officinalis leaves 67 Figure 3.4: Salt gland structure from A officinalis leaves 69 Figure 3.5: Estimation of ions in xylem sap of A officinalis 71 Figure 3.6: Estimation of ions in leaf secretions of. .. Ultrastructural studies of these salt glands showed sunken corn shaped structure of secretory cells, collecting cells at the bottom and stalk cells are placed between them These secretory cells of the salt glands are covered with a layer of cuticle which provides interstitial space for the movement of water and ions from salt glands to surface of the leaves Under salt stress in secretory cells, an increase in endoplasmic... accumulation of Na+ in leaf tissues, which leads to necrosis of older leaves, starting at the leaf-tips, continues to the margins and later to the petiole of the leaves Causing ionic stress, Na+ and Clalso inhibit metabolic processes including protein synthesis This mainly reduces the growth and yield of the plants by shortening the lifetime of the individual leaves, thus affecting net productivity and crop... responses are some of them Understanding the mechanisms of primary salt tolerance in mangroves and identification of salt tolerant genes from mangrove may lead to effective means to breed and genetically engineer salt tolerant crops 16 1.6 Objectives of the study and approach CHAPTER1 The mangrove Avicennia officinalis is known to exhibit salt tolerance property Among the small number of studies that report... water due to the scarcity of replenishable fresh water Some mangroves possess a characteristic structure in their leaves called salt glands Their role is to remove the excess salt from the plant through leaves Once removed, the salt can either crystallize in the sunlight or get washed off in the rain and wind Salt glands secrete salt in the form of a solution, which includes toxic ions and water However,... both physiological and molecular aspects of salt tolerance in A officinalis The notable contributions of this study include confirmation of the role of AoDHN1 in stress remediation and identification of a water transporting aquaporin AoPIP1.2 Furthermore, the observation that their expression is highly enriched in salt gland cells, suggests that both these genes may play a significant role in salt. .. to be extinct and later they have been rediscovered in Singapore (YANG et al., 2011) Avicennia species are among the most commonly seen species in our mangroves of which Avicennia alba, Avicennia marina, Avicennia rumphiana and Avicennia officinalis are found in Singapore (Ng and Sivasothi, 1999) Avicennia marina is listed as 'Critically Endangered' in the Red List of threatened plants of Singapore... AoPIP1.2 and AoPIP2.2) Furthermore, early responsive genes to salt treatment in the salt gland-rich tissues were identified using transcriptomic approach Various physiological and molecular approaches were taken to study the salt tolerance aspects in Avicennia officinalis The specific objectives of the study were: I Physiological and morphological studies including both fieldgrown and greenhouse-grown Avicennia. .. Another efficient way of eliminating excess salt from the leaves is secretion through salt glands, which is commonly seen in mangrove plants (Kathiresan and Bingham, 2001) Although the natural process of salt secretion is known for decades, only a few studies have attempted to decipher the mechanism of salt gland function (Ding et al., 2010) In addition, tolerance of single cell to high salinity, involving . secretion and therefore the salt tolerance mechanism in the mangrove Avicennia officinalis. These results validate the previous findings that aquaporins play a critical role in salt secretion and. physiological and molecular aspects of salt tolerance in A. officinalis. The notable contributions of this study include confirmation of the role of AoDHN1 in stress remediation and identification of. Sections of A. officinalis young and mature leaves 65 Figure 3.2: Adaxial and abaxial surfaces of A. officinalis fresh and dried leaves 66 Figure 3.3: Determination of salt gland density in A. officinalis