Tripogon loliiformis elicits a rapid physiological and structural response to dehydration for desiccation tolerance

Resurrection plants can withstand extreme dehydration to an air-dry state and then recover upon receiving water. Tripogon loliiformis (F.Muell.) C.E.Hubb. is a largely uncharacterised native Australian desiccation-tolerant grass that resurrects from the desiccated state within 72 h. Using a combination of structural and physiological techniques the structural and physiological features that enable T. loliiformis to tolerate desiccation were investigated. These features include: - (i) a myriad of structural changes such as leaf folding, cell wall folding and vacuole fragmentation that mitigate desiccation stress; - (ii) potential role of sclerenchymatous tissue within leaf folding and radiation protection; - (iii) retention of ~70% chlorophyll in the desiccated state; - (iv) early response of photosynthesis to dehydration by 50% reduction and ceasing completely at 80 and 70% relative water content, respectively; - (v) a sharp increase in electrolyte leakage during dehydration, and; - (vi) confirmation of membrane integrity throughout desiccation and rehydration. Taken together, these results demonstrate that T. loliiformis implements a range of structural and physiological mechanisms that minimise mechanical, oxidative and irradiation stress. These results provide powerful insights into tolerance mechanisms for potential utilisation in the enhancement of stress-tolerance in crop plants.

Formation of specialized propagules resistant to desiccation and cryopreservation in the threatened moss Ditrichum plumbicola (ditrichales, bryopsida)

Background and Aims Successful cryopreservation of bryophytes is linked to intrinsic desiccation tolerance and survival can be enhanced by pre-treatment with abscisic acid (ABA) and sucrose. The pioneer moss Ditrichum plumbicola is naturally subjected to desiccation in the field but showed unexpectedly low survival of cryopreservation, as well as a poor response to pre-treatment. The effects of the cryopreservation protocol on protonemata of D. plumbicola were investigated in order to explore possible relationships between the production in vitro of cryopreservation-tolerant asexual propagules and the reproductive biology of D. plumbicola in nature. Methods Protonemata were prepared for cryopreservation using a four-step protocol involving encapsulation in sodium alginate, pre-treatment for 2 weeks with ABA and sucrose, desiccation for 6 h and rapid freezing in liquid nitrogen. After each stage, protonemata were prepared for light and electron microscopy and growth on standard medium was monitored. Further samples were prepared for light and electron microscopy at intervals over a 24-h period following removal from liquid nitrogen and re-hydration. Key Results Pre-treatment with ABA and sucrose caused dramatic changes to the protonemata. Growth was arrested and propagules induced with pronounced morphological and cytological changes. Most cells died, but those that survived were characterized by thick, deeply pigmented walls, numerous small vacuoles and lipid droplets in their cytoplasm. Desiccation and cryopreservation elicited no dramatic cytological changes. Cells returned to their pre-dehydration and cryopreservation state within 2 h of re-hydration and/or removal from liquid nitrogen. Regeneration was normal once the ABA/sucrose stimulus was removed. Conclusions The ABA/sucrose pre-treatment induced the formation of highly desiccation- and cryopreservation-tolerant propagules from the protonemata of D. plumbicola. This parallels behaviour in the wild, where highly desiccation-tolerant rhizoids function as perennating organs allowing the moss to endure extreme environmental conditions. An involvement of endogenous ABA in the desiccation tolerance of D. plumbicola is suggested.

Trehalose accumulation triggers autophagy during plant desiccation

Global climate change, increasingly erratic weather and a burgeoning global population are significant threats to the sustainability of future crop production. There is an urgent need for the development of robust measures that enable crops to withstand the uncertainty of climate change whilst still producing maximum yields. Resurrection plants possess the unique ability to withstand desiccation for prolonged periods, can be restored upon watering and represent great potential for the development of stress tolerant crops. Here, we describe the remarkable stress characteristics of Tripogon loliiformis, an uncharacterised resurrection grass and close relative of the economically important cereals, rice, sorghum, and maize. We show that T. loliiformis survives extreme environmental stress by implementing autophagy to prevent Programmed Cell Death. Notably, we identified a novel role for trehalose in the regulation of autophagy in T.loliiformis. Transcriptome, Gas Chromatography Mass Spectrometry, immunoblotting and confocal microscopy analyses directly linked the accumulation of trehalose with the onset of autophagy in dehydrating and desiccated T. loliiformis shoots. These results were supported in vitro with the observation of autophagosomes in trehalose treated T. loliiformis leaves; autophagosomes were not detected in untreated samples. Presumably, once induced, autophagy promotes desiccation tolerance in T.loliiformis , by removal of cellular toxins to suppress programmed cell death and the recycling of nutrients to delay the onset of senescence. These findings illustrate how resurrection plants manipulate sugar metabolism to promote desiccation tolerance and may provide candidate genes that are potentially useful for the development of stress tolerant crops.

Leaf and inflorescence structure and phylogenetics of Tripogon and affiliated genera (Poaceae: Chloridoideae)

This thesis provides new knowledge on an understudied group of grasses, some of which are resurrection grasses (i.e. able to withstand extreme drought). The sole Australian species (Tripogon loliiformis) is morphologically diverse and could be more than one species. This study sought to determine how many species of Tripogon occur in Australia, their relationships to other species in the genus and to two other genera of resurrection grasses (Eragrostiella and Oropetium). Results of the research indicate there is not enough evidence, from DNA sequence data, to warrant splitting up T. loliiformis into multiple species. The extensive morphological diversity seems to be influenced by environmental conditions. The three genera are so closely related that they could be grouped into a single genus. This new knowledge opens up pathways for future investigations, including studying genes responsible for desiccation tolerance and the conservation of native grasses that occur in rocky habitats.

Structural, physiological and molecular characterisation of the Australian native resurrection grass Tripogon loliiformis (F.Meull) C.E.Hubb during dehydration and rehydration

There is an urgent need to develop crops that can withstand future climates. Results from this thesis demonstrated that a native Australian resurrection grass exhibits structural, physiological and metabolic strategies to tolerate drying. These strategies may be utilized for the generation of stress tolerant crops.

The biology and natural history of Tripogon loliiformis (Poaceae, Chloridoideae), an Australian resurrection grass

Tripogon loliiformis is a desiccation-tolerant grass that occurs throughout mainland Australia. There has been recent interest in this species as a model system for understanding desiccation tolerance in a native grass at the structural, molecular and physiological levels. However, not much is known about the biology and natural history of this species, despite its widespread geographic distribution and remarkable capability of withstanding prolonged drying. We provide an overview of the genus by consolidating information from a wide variety of sources. We report a variety of new and interesting observations on the general biology, ecology and desiccation response of T. loliiformis and conclude by highlighting areas for future research.

The role of MicroRNAs in stress response in the resurrection plant Tripogon loliiformis

Research in this thesis focussed on the improvement of agricultural crops in increasing water use efficiency that impacts global crop productivity. The study identified key genetic regulatory mechanisms that the resurrection plant Tripogon loliiformis utilises to tolerate desiccation. Due to the conserved nature of the pathways involved, this information can be transferred for the enhancement of drought tolerance and water use efficiency in agricultural crops. Specifically this study used high throughput sequencing, microscopy and plant transformation to further the understanding of post-transcriptional regulatory mechanisms. It was shown that T. loliiformis uses microRNAs to regulate pro-survival autophagy pathways to tolerate desiccation.

Genetic Analysis of Tolerance to Rice Tungro Bacilliform Virus in Rice (Oryza sativa L.) Through Agroinoculation

Balimau Putih [an Indonesian cultivar tolerant to rice tungro bacilliform virus (RTBV)] was crossed with IR64 (RTBV, susceptible variety) to produce the three filial generations F1, F2 and F3. Agroinoculation was used to introduce RTBV into the test plants. RTBV tolerance was based on the RTBV level in plants by analysis of coat protein using enzyme-linked immunosorbent assay. The level of RTBV in cv. Balimau Putih was significantly lower than that of IR64 and the susceptible control, Taichung Native 1. Mean RTBV levels of the F1, F2 and F3 populations were comparable with one another and with the average of the parents. Results indicate that there was no dominance and an additive gene action may control the expression of tolerance to RTBV. Tolerance based on the level of RTBV coat protein was highly heritable (0.67) as estimated using the mean values of F3 lines, suggesting that selection for tolerance to RTBV can be performed in the early selfing generations using the technique employed in this study. The RTBV level had a negative correlation with plant height, but positive relationship with disease index value

Morphine-induced receptor endocytosis in a novel knockin mouse reduces tolerance and dependence

Abstract Opioid drugs, such as morphine, are among the most effective analgesics available. However, their utility for the treatment of chronic pain is limited by side effects including tolerance and dependence. Morphine acts primarily through the mu-opioid receptor (MOP-R) , which is also a target of endogenous opioids. However, unlike endogenous ligands, morphine fails to promote substantial receptor endocytosis both in vitro, and in vivo. Receptor endocytosis serves at least two important functions in signal transduction. First, desensitization and endocytosis act as an "off" switch by uncoupling receptors from G protein. Second, endocytosis functions as an "on" switch, resensitizing receptors by recycling them to the plasma membrane. Thus, both the off and on function of the MOP-R are altered in response to morphine compared to endogenous ligands. To examine whether the low degree of endocytosis induced by morphine contributes to tolerance and dependence, we generated a knockin mouse that expresses a mutant MOP-R that undergoes morphine-induced endocytosis. Morphine remains an excellent antinociceptive agent in these mice. Importantly, these mice display substantially reduced antinociceptive tolerance and physical dependence. These data suggest that opioid drugs with a pharmacological profile similar to morphine but the ability to promote endocytosis could provide analgesia while having a reduced liability for promoting tolerance and dependence