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Thesis Template MOLECULAR AND FUNCTIONAL CHARACTERISATION OF AN OSMOTIN GENE FROM THE RESURRECTION PLANT TRIPOGON LOLIIFORMIS By Thi Thuy Trang LE BSc of Agronomy (Hons.) MSc of Plant Science Submitted in fulfilment of the requirements for the degree of Doctor of Philosophy at the Centre for Tropical Crops and Biocommodities Science and Engineering Faculty Queensland University of Technology 2018 Molecular and Functional Characterisation of an Osmotin Gene from the Resurrection Plant Tripogon loliiformis 1 Intentionally blank 2 Molecular and Functional Characterisation of an Osmotin Gene from the Resurrection Plant Tripogon loliiformis Keywords Abiotic stress, Agrobacterium-mediated transformation, Agro-infiltration, BiFC, cell membrane integrity, co-localisation, cold stress, cold tolerance, confocal microscopic analysis, Coomassie stain, drought stress, drought tolerance, dry biomass, electrolyte leakage, embryogenic callus, EYFP, genetic engineering, GO term enrichment, Gus-reporter gene, Gus stain, His tag, hygromycin resistant marker, infiltration, KDEL, leaf damage, morphology, membrane protein tracker, Myc, NaCl, Nicotiana benthamiana, Nicotiana tabacum, Oryza sativa, osmotin, panicle length, plasmid cloning, photosynthesis, protein extraction, protein microarray, protein- protein interaction, protein purification, protein structure, qRT-PCR, recombinant proteins, resurrection plant, rice, ROS, RWC, salinity stress, salinity stress tolerance, transcriptional expression, transgenic plant, transient expression, Tripogon loliiformis, Ubi promoter, yield component. Molecular and Functional Characterisation of an Osmotin Gene from the Resurrection Plant Tripogon loliiformis i ii Molecular and Functional Characterisation of an Osmotin Gene from the Resurrection Plant Tripogon loliiformis Abstract Abiotic stresses such as drought, salinity, and extreme temperature are key factors that limit crop productivity and are major constraints to meeting global food demands. The development of stress-tolerant crops that mitigate the effects of abiotic stresses on crop productivity is crucially needed to sustain agricultural production. The narrow genetic variation of stress-tolerant traits among crops and sexual barrier between species have restricted the success of developing stress-tolerant crops by conventional breeding. Genetic engineering of crops with stress-tolerant traits is a promising approach for improving stress tolerance in crops. Prior to the development of stress tolerant crops by genetic engineering, it is essential that key genes regulating stress-tolerant traits are characterised. Stress-tolerant traits in plants are genetically coded. Naturally tolerant species, such as the resurrection plant Tripogon loliiformis, represent an ideal starting point for the identification of genes encoding stress-tolerant traits. T. loliiformis has evolved mechanisms to tolerate extreme dehydration down to 4% relative water content (RWC), facilitate cellular protection and survival during desiccation, and to rapidly recover within 48-72 h of rehydration to full metabolic activity. These mechanisms hold great potential for the introduction into crop species. It is important to identify the key regulators of these stress-tolerance mechanisms for effective transfer of stress- tolerant traits into crop species. This PhD study describes the molecular characterisation of an osmotin gene (TlOsm) from the desiccation tolerant plant T. loliiformis. The conserved and novel characteristics of TlOsm were identified in comparison with two osmotins, OsOlp1_A from a drought-tolerant cultivar and OsOlp1_I from a drought-sensitive cultivar, of the stress sensitive species Oryza sativa (rice). Protein structural-to-functional predictions, the response of transgenic rice plants constitutively expressing each of the osmotins to cold, drought, and salinity stress, and the profiles of plant proteins interacting with three osmotins on the Arabidopsis protein microarrays (chip) and in living Nicotiana benthamiana plants were analysed. The results revealed the common and unique characteristics of TlOsm, the profiles of TlOsm protein interactors, possible Molecular and Functional Characterisation of an Osmotin Gene from the Resurrection Plant Tripogon loliiformis iii pathways involving TlOsm, and functional roles of TlOsm in enhanced rice tolerance to cold, drought, and salinity stresses. The results highlight the higher functional efficacy of TlOsm, compared to rice osmotins, in regulating plant response to stress, indicate the potential use of TlOsm for developing crops tolerant to multiple abiotic stresses, and provide useful information for elucidating molecular mechanisms underlying osmotin functions in regulating plant response to stress. Transcriptional profiling of TlOsm during T. loliiformis plant development and on exposure to cold, heat, drought, and salinity stresses indicated that TlOsm was induced up to a thousand fold upon cold, drought, and salinity stresses. The plasma membrane localisation of TlOsm was observed by confocal microscopy of transgenic Nicotiana tabacum expressing Enhanced Yellow Fluorescent Protein (EYFP)-tagged TlOsm. Sequence analysis of TlOsm revealed its conserved characteristics of an osmotin, the close genetic relationship with monocotyledonous osmotins, and a sequence of 50 AA at its C-terminus that is not homologous to other osmotins. Analyses based on structural-to-functional predictions, compared to two rice osmotins, revealed that TlOsm had more glucan-binding and phosphorylation sites than rice osmotins and four binding sites with enzymatic functions in sugar metabolism, which did not exist in either of rice osmotins. The effects of TlOsm, OsOlp1_A, and OsOlp1_I on enhancing plant tolerance to cold, drought, and salinity stress were compared in transgenic rice expressing each of the osmotin gene. Agrobacterium-mediated transformation was used to generate 42 transgenic rice lines expressing TlOsm, OsOlp1_A, OsOlp1_I, or the Gus reporter gene. Transgenic plants of two successive generations were assessed for enhanced tolerance to cold, drought, and salinity stresses. The results demonstrate the capacity of TlOsm and OsOlp1_A to enhance rice tolerance to cold, drought, and salinity stresses and emphasise the additional efficacy of TlOsm. The tolerant traits passed on to the next generation. The results suggest a positive correlation between functional binding sites of the osmotins and the levels of enhanced stress tolerance in transgenic rice. Interactive protein partners and possible pathways involving the osmotins were revealed by Arabidopsis protein chip assays. The results were validated in N. benthamiana. On the protein chips, 267, 239, and 237 proteins interacted with TlOsm, OsOlp1_A, and OsOlp1_I respectively. Osmotin interactors were found involved iv Molecular and Functional Characterisation of an Osmotin Gene from the Resurrection Plant Tripogon loliiformis in the diverse biological processes and pathways fundamental in plant development and adaptation. The three dominant pathways were fructose and mannose metabolism, glycolysis, and pentose phosphate pathways. Interestingly, 21 proteins interacted only with TlOsm and were involved in plant responses to stress, chemical, and endogenous stimuli. Nine of these 21 proteins are involved in nine pathways that do not contain any interactors of the rice osmotins. Fifteen interactions were validated in planta by Bimolecular Fluorescence Complementation (BiFC) analysis and 14 of those confirmed the results on the chip assays. Protein-protein interaction assays revealed that TlOsm is capable of interacting with more stress-responsive proteins than osmotins from sensitive species, O. sativa. The study contributes significantly to understanding the proteomic evolution of osmotins from tolerant and sensitive species as well as from tolerant and sensitive cultivars within species. The results provide evidence highlighting the potential of T. loliiformis genetic resource for identification of stress-tolerant traits to introduce into crop species. The study also deepens our understanding of the molecular mechanisms underlying osmotin functions in regulating plant stress response. Molecular and Functional Characterisation of an Osmotin Gene from the Resurrection Plant Tripogon loliiformis v List of Publications and Conferences Thi Thuy Trang LE, Brett Williams, and Sagadevan Mundree (2017). An osmotin from the resurrection plant Tripogon loliiformis (TlOsm) confers tolerance to multiple abiotic stresses in transgenic rice. Physiologia Plantarum, doi: 10.1111/ppl.12585. Thi Thuy Trang LE, Brett Williams, and Sagadevan Mundree (2016). Comparative analysis of osmotins from Tripogon loliiformis and Oryza sativa revealed role in abiotic stress tolerance through signalling pathways. Poster in the Combio2016 Workshop, 03-07 Oct 2016, Brisbane, Australia. Thi Thuy Trang LE, Brett Williams, and Sagadevan Mundree (2016). Characterization of an osmotin gene from resurrection plant Tripogon loliiformis. Oral presentation in the 7th International Workshop on Desiccation Sensitivity and Tolerance across Life Forms, 11-15 Jan 2016, Cape Town, South Africa. vi Molecular and Functional Characterisation of an Osmotin Gene from the Resurrection Plant Tripogon loliiformis Table of Contents Keywords .................................................................................................................................. i Abstract ..................................................................................................................................
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