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Exploring the Biochemical and Phylogenetic Fingerprint of Australian Native Plants for Sustainable Use of Saline Lands

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Exploring the biochemical and phylogenetic fingerprint of Australian native plants for sustainable use of saline lands Shanthi Safrina Maria Monica Joseph (MSc Biotechnology) This thesis is presented for the degree of Doctor of Philosophy March 2014 Department of Chemistry and Biotechnology Faculty of Science, Engineering and Technology Swinburne University of Technology Melbourne, Australia Abstract The remarkably rich Australian native vegetation has developed some unique morphological and genetic mechanisms to adapt to severe drought, salinity and water logging. However, the utilisation and significance of Australian native plant bio- resources has been under-exploited, with relatively few dedicated studies, particularly in comparison to crop plants such as rice, wheat or barley. This project investigated the unique gene pool of certain Australian salinity-tolerant plants (three saltbushes- Atriplex nummularia, A. semibaccata, A. amnicola and four Acacia species- Acacia victoriae, A. salicina, A. pendula and A. stenophylla). The osmoprotectants glycine betaine (GB), proline and trehalose known to impart salt tolerance were investigated in these plants. Genes encoding the enzymes choline monooxygenase (CMO) and betaine aldehyde dehydrogenase (BADH) involved in GB biosynthesis were identified in the three saltbushes. In-silico analyses of their cDNA sequences and predicted proteins revealed valuable predictive data pertaining to their extremely conserved functional and structural motifs, subcellular localisation and physico-chemical properties. Gene expression analyses indicated that the saltbush genes for CMO and BADH were differentially expressed in leaves and roots, with significant up-regulation (>1.5 fold change) of CMO and/or BADH mRNA in the leaf tissues indicating that these genes serve as ideal candidates in transgenic work to enhance salt tolerance in salt sensitive plants. Chemical analyses indicated that Atriplex semibaccata and A. nummularia produced high quantities of the compound GB in their leaves under salt stress relative to reported low levels in cereals such as barley (2.5 to 10.6 fold change differences). The Acacia species, on the other hand, did not produce any detectable levels of GB. Proline production was enhanced by salt in both Atriplex (two fold) and Acacia species (four fold). HPLC analysis for trehalose detection indicated its absence under salt stress, signifying that trehalose accumulation may not be involved in salt tolerance of these native plants. Another dimension of this study was the use of molecular phylogenetics for assisting in identification of further salinity tolerant Acacia species. On the basis of the comparative biology hypothesis that closely related species are most likely to share traits, using DNA markers, a phylogenetic tree of 178 Acacia species including four candidate salt tolerant species, i.e., Acacia pendula, A. salicina, A. victoriae and A. stenophylla used in the Kamarooka land reclamation project (Australia), was i constructed. Based on phylogenetic relatedness, their historically known potential in agroforestry, and seed availability, 15 species were tested for salt tolerance under controlled laboratory conditions. A method was developed for comprehensive analyses of the datasets using three salt tolerance indices to rank the species. Two new highly tolerant and three moderately salt-tolerant Acacia species were thus identified. These will be further used for testing in field conditions (at Kamarooka). In summary, the data on GB analysis, in light of reports from literature on GB-associated health benefits in grazing animals, highlight the potential of saltbushes as sustainable, unseasonal mixed fodder species. Further, the work provides strong rationale for the use of initial phylogenetic screening for large genera, such as Acacia, for identifying candidate species for agroforestry and provides an experimental methodology for this purpose. ii Acknowledgements How great are your works, O Lord, how profound your thoughts! (Psalm 92:5) A journey of a thousand miles begins within a single step. I am truly indebted to my principal supervisor Prof Mrinal Bhave for giving me the opportunity to take that step into this exciting world of plant biotechnology. I am grateful to you for your timely feedback and guidance throughout this project. I have admired your hard work, enthusiasm and ‘eye’ for perfection, and for patiently reading every line of what I wrote (be it good, bad or even worse). Thank you. I am immensely thankful to my co- supervisor Dr Daniel Murphy, I couldn’t have asked for a better supervisor. Thank you for always being no more than just an email away. Your willingness to help, constructive criticism, words of support and positive attitude encouraged me all the way. To my friends, Abi (Dr Abirami Ramalingam), Rue (Dr Runyararo Hove) and Azadeh joon, I can’t thank you guys enough for always being there. Your understanding, advice and invaluable friendship helped me remain sane. And to sisi, I am glad your theory of ‘fear it but do it anyway’ worked, thank you for your encouragement, support and your other motivational quotes (too many to pen down!). To Kaylass, thank you for being a good listener and a great friend. To all the past and current students of my research group, Ms Saifone Chuaboonmee, Dr Huimei Wu, Dr Shee Ping Ng, Dr Peter Gollan, Dr Rebecca Alfred, Atul, Yen and Guri; my other friends and colleagues, especially, Mr and Mrs Azad, Elisa, Jafar, Chris, Bita, Qudsia, Vanu, Shaku, Suchetana, Rashida, Dhivya, Babu, Jun, Snehal, Avinash, thank you for your friendly interactions and company. I would also like to acknowledge Ms Soula Mougos, Ms Angela McKellar, Ms Savithri Galappathie, Mr Chris Key and Mr Ngan Nguyen for technical assistance, and staff from Swinburne Research, especially, Ms Robyn Watson for her invaluable time and support. I am exceedingly grateful to the Australian Research Council and SUT for my scholarship and project funding, the Royal Botanic Gardens (Melbourne) for providing Acacia herbarium samples, the Northern United Forestry Group for advice on some of the species used in this project, and Dr Joe Miller and Ms Kristy Lam (CSIRO Canberra) for help and support with DNA sequencing. iii I thank my patron saint, St. Anthony, I have knocked the doors of your church far too many times, and you have never let me down. My heartfelt thanks goes to my parents who always gave me the freedom to make my own choices and taught me the value of being human above all else. Thanks to my sisters and friends back home who encouraged me and believed in my efforts. Last but NEVER the least, this whole journey of mine couldn’t have been possible without the unconditional love and companionship of my partner. Richard, your selfless sacrifice, willingness to always help and ‘cooking’, kept me strong and going. Words can’t express my gratitude for all your kindness, and now we can gladly look forward to the ‘big’ day ahead! iv This thesis is lovingly dedicated to my parents and Richard… v Declaration I, Shanthi Safrina Maria Monica Joseph, declare that this PhD thesis entitled ‘Exploring the biochemical and phylogenetic fingerprint of Australian native plants for sustainable farming on saline lands’ is no more than 100,000 words in length, exclusive of tables, figures, appendices, references and footnotes. This thesis contains no material that has been submitted previously, in whole or in part, for the award of any other academic degree or diploma, and has not been previously published by another person. Except where otherwise indicated, this thesis is my own work. Shanthi Safrina Maria Monica Joseph 2014 vi Publications arising from this work Refereed journal articles Joseph S, Murphy DJ, Miller JT and Bhave M (2013) Rapid identification of Acacia species with potential salt tolerance using nuclear ribosomal DNA markers. Sustainable Agriculture Research, 2 (4): 77-86. Joseph S, Murphy DJ and Bhave M (2013) Glycine betaine biosynthesis in saltbushes (Atriplex spp.) under salinity stress. Biologia, 68: 879-895. Joseph S, Murphy DJ, Miller JT and Bhave M (2013). Application of molecular markers for identification of potential salt tolerant plant species for use in agroforestry and saline land reclamation. APCBEE Procedia, 5, 514-519. Manuscript under preparation Joseph S, Murphy DJ and Bhave M (2013) Testing of salt tolerant Acacia species from a phylogenetically pre-selected subset for saline land utilization (Under review) Oral conference presentations Joseph S, Murphy DJ, Miller JT and Bhave M (2013) Unlocking the potential uses of Australian native trees. ‘Ecology and the Environment’ student conference 11 November 2013, Bundoora, Australia. Joseph S, Murphy DJ, Miller JT and Bhave M (2013) Application of molecular markers for identification of potential salt tolerant plant species for use in agroforestry and saline land reclamation. 4th International Conference on Environmental Science and Development, 19-20 January 2013, Dubai, UAE. Joseph S, Murphy DJ and Bhave M (2011) Identification of genes encoding osmoprotectants in Australian native plants. Annual conference of the Australian Society for Biochemistry and Molecular Biology (ComBio 2011) 25-29 September 2011, Cairns, Australia. vii Abbreviations BADH Betaine aldehyde dehydrogenase BLAST Basic Local Alignment Search Tool BDT Big Dye Terminator bp Base pair (s) CDH Choline dehydrogenase cDNA Complementary DNA CDS Coding sequence CMO Choline monooxygenase COX Choline oxidase C-terminal
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