Functional analysis of dehydration responsive genes in the resurrection grass Sporobolus stapfianus

Crop plant productivity in Australia and worldwide is facing increasing environmental constraints brought upon by drought, cold and salinity. The ability of a plant to survive drought, as well as other abiotic stressors is a trait of particular interest in crop improvement studies. Current research focuses of the use of genes from drought-sensitive plants as a means to increase drought tolerance through genetic modification, however the use of genes from plants that are extremely drought tolerant is a growing area of interest in genetic modification studies. Resurrection plants, such as Sporobolus stapfianus, utilised in this study, are anhydrobiotic organisms that have molecular mechanisms that allow them to dehydrate and go into a state of suspended animation for several months during a period of drought. These plants then restore metabolism 24 hours after a rainfall event. This study presents evidence of molecular mechanisms associated with genes that are transcribed during the dehydration of S. stapfianus. Genes of interest in this process had been isolated in previous work, and a selection of these genes, namely, SDG3i, SDG4i and SDG8i have been chosen for further work in this study. This study provided further support for the transcription of these genes during the dehydration phase of S. stapfianus, and interestingly also in the rehydration phase. Bioinformatic analysis suggests that SDG3i encodes a protein containing at TIM17-like domain, and may be localized to either the mitochondrial inner membrane or peroxisome (Chapter 3). Intriguingly, the protein product of SDG4i was predicted to be involved in chromosome remodeling through its potential for interaction with histone deacetyalase 19 (Chapter 3). Developmental phenotype analysis of transgenic Arabidopsis thaliana (Columbia) over-expressing SDG4i showed that these plants produced had changes in petiole size, shoot branching and height, as well as delayed senescence, increased seed yield, however a reduced seed longevity in long and short day conditions (Chapter 4). Furthermore, the abiotic stress tolerance, particularly to osmotic, salt, cold and endoplasmic reticulum stress was increased (Chapter 5). With these traits of transgenic plants observed, and the potential of SDG4i to be involved in a protein-protein interaction, a yeast 2-hybrid screen was performed to ascertain if these changes could be a result of an interaction between SDG4i and a protein product. It was shown that SDG4i interacts with an early response to dehydration 15- like protein (ERD15). This ERD15 protein acts to up-regulate asparagine-rich proteins (NRPs) to facilitate cell death during dehydration and endoplasmic reticulum stress. The role of SDG4i in S. stapfianus may potentially be to prevent cell death during dehydration, allowing for the induction and/or facilitation of the complete desiccation of S. stapfianus. In addition to revealing protein-protein interactions in desiccation toleranceassociated genes in S. stapfianus, this study also uncovered the first known UDPglycosyltransferase (SDG8i) (chapter 3) to glycosylate a strigolactone-like compound. This was completed using an in vitro coupled enzyme assay with a variety of plant hormones, where SDG8i has the highest affinity for the synthetic strigolactone GR24 (Chapter 7). This supports work completed in prior to this study that A. thaliana overexpressing SDG8i produce plants that have an increased branching phenotype that is generally associated with strigolactone mutant plants. This study provides novel evidence that S. stapfianus transcribes genes that may be affecting hormone homeostasis as well as drought-related senescence processes to allow the plant successfully negotiate a dehydration/rehydration cycle.