POSTERS - ComBio · The project’s aim was to see how anatomical traits (namely ... Carotenoids are organic pigments that are naturally synthesized by plants, algae, and some photosynthetic

ComBio2018 s Darling Harbour, Sydney s 23 - 26 September, 2018 Page 103

POSTERS

Tuesday - Wednesday

Page 104 ComBio2018 s Darling Harbour, Sydney s 23 - 26 September, 2018

POSTERS

POS-TUE-001 POS-WED-002


INFLUENCE OF LEAF ANATOMICAL VARIATION BETWEEN SORGHUM BICOLOR GENOTYPES IN RESPONSE TO DIFFERENT GROWTH TEMPERATURES ON WATER USE EFFICIENCY

Al-Salman Y.M.M.1, 2, Cano J.1, 2, Pan L.1, 2, 3, Koller F.1, 2 and Ghannoum O.1, 2 1Hawkesbury Institute for the Environment, Western Sydney University, Locked Bag 1797, Penrith 2751, NSW, Australia. 2ARC Centre of Excellence for Translational Photosynthesis, The Australian National University, Acton 2601, ACT, Australia. 3Sichuan Agricultural University, Sichuan Province, China.

Rising temperatures and world population will impact food security. Sorghum bicolor (great millet) is an important crop that is known for being drought-resistant. Evaluating the characteristics that enable S.bicolor to conserve water use will enable identification of traits to look for in future breeding efforts. C4 leaves of S.bicolor show improved water conservation capacity due to their carbon concentrating mechanism and high intrinsic water use efficiency (iWUE = carbon assimilation/stomatal conductance). Among the main determinants of assimilation and conductance capacities influencing WUE are the leaf’s anatomical characters. The project’s aim was to see how anatomical traits (namely leaf stomatal and vein characters) covariate and reassemble to cope with different growth temperatures in different Sorghum genotypes. Ten Sorghum genotypes were selected based on field-based variation in leaf width and water use efficiency values, and 3 plants per genotype (n=3) were grown at 3 different temperatures (22oC, 28oC, 35oC). Leaf samples from the middle of the leaf blade were cut, fixed in formaldehyde and stored in ethanol and later used for anatomical assays. Confocal microscopy was used to image the leaf surface and obtain stomatal and leaf epidermis characters. Leaves sections were also cleared and stained and scanned to obtain leaf vein characters. Anatomical data was paired with previously measured gas exchange and hydraulic conductance data to establish the relationship between anatomical variation and water use efficiency under different temperatures.

SHEDDING LIGHT ON HOW PHOTOISOMERISATION OF CIS-CAROTENES CAN RAPIDLY REGULATE NUCLEAR GENE EXPRESSION IN PLANTS

Alagoz Y., Anantanawat K., Papanicolaou A. and Cazzonelli C. Western Sydney University, Hawkesbury Institute for the Environment, 2753 Richmond Sydney Australia.

Carotenoids are organic pigments that are naturally synthesized by plants, algae, and some photosynthetic bacteria. The biosynthesis of carotenoids consists of a multistep pathway regulated by key environmental factors such as like light and temperature. Unlike bacteria, plants have evolved four additional enzymes that generate cis-carotene intermediates, mostly in fruits and in dark grown tissues. The light-mediated conversion of cis-carotenes to their cis/trans geometric isomers is an enigmatic phenomenon referred as photoisomerisation. The in-vivo conversion of prolycopene to all-trans-lycopene is a rate-limiting step before the branching of the pathway to ε- and β-carotenoids that drive photosynthetic and photoprotective functions. In-vitro photoisomerisation has been shown to be catalysed by hours of light and is reversible by higher temperatures. Here we have developed a novel in-vivo bioassay to shed real light on the photoisomerisation mechanism by exposing tomato mutant defective in the CAROTENOID ISOMERASE to different intensities of light. We demonstrate that high light can mediate photoisomerisation of z-carotene to neurosporene (orange) and tetra-cis-lycopene to all-trans-lycopene (pinkish-red pigment) rapidly. Our preliminary analysis also reveals rapid changes in nuclear gene expression. We hypothesise that cis-carotenes can act like photo-switches and oxidative cleavage of cis-carotenes can generate apocarotenoid signals (ACS) in the chloroplast to enable the synergistically feedback control over nuclear gene expression. As such the phytochromes, cryptochromes and apocarotenoid signalling molecules generated during/after photoisomerisation of cis-carotenes can mediate the early steps of photomorphogenesis.

DOES THE EXPRESSION OF ARABIDOPSIS THALIANA ACYL-COENZYME A-BINDING PROTEIN 6 MEDIATE COLD/FREEZING STRESS TOLERANCE IN TRANSGENIC CANOLA?

Alahakoon A.Y.1, Tongson E.J.1, Meng W.3, Ye Z.3, Chye M.L.3, Golz J.F.2, Russell D.A.1 and Taylor P.W.J.1 1Faculty of Veterinary and Agricultural Sciences, The University of Melbourne, Parkville, VIC, Australia. 2School of Biosciences, The University of Melbourne, Parkville, VIC, Australia. 3School of Biological Sciences, The University of Hong Kong, Pokfulam, Hong Kong.

Cold and freezing stress heavily affects the performance of canola (Brassica napus L.) crops in temperate countries, reducing seed yield and quality. To test the potential use of a low temperature stress mediating gene acyl-coenzyme A-binding protein 6 (ACBP6) from Arabidopsis thaliana in canola, rapid-cycling B. napus plants overexpressing the Arabidopsis ACBP6 were developed using Agrobacterium-mediated transformation. Four independent To lines confirmed as transgenic by western blot analysis and reverse transcription PCR were maintained to T3. These lines were tested for cold stress at vegetative stage and freezing stress at seed setting stage in non-acclimated and cold-acclimated conditions. The cold stressed ACBP6 vegetative plants recovered well and showed a higher yield potential than the cold stressed wild-type plants. Under both cold-acclimated and non-acclimated conditions the harvest index of three of the transgenic plants was around 0.20 while it was around 0.10 for wild-type plants and a single transgenic line. The percentage of fully viable seeds of freezing treated plants at the seed setting stage was significantly higher in transgenic plants (38-73%) than in wild-type plants (<27%). Cold-acclimation did not improve the cold stress tolerance of rapid-cycling plants, and indeed acted as an additional cold stress during the experiment. However, overexpression of Arabidopsis ACBP6 in canola is potentially useful in generating crops which are more tolerant to cold and freezing stress even in the absence of cold-acclimation.

RESPONSES IN ROOTS ACCOUNTS FOR DIFFERENTIAL SALT TOLERANCE IN QUINOA ACCESSIONS

Bazihizina N.1, 2, Cuin T.A.1, KianiPouya A.1 and Shabala S.1 1Tasmanian Institute for Agriculture, College of Science and Engineering, University of Tasmania. 2Department of Agrifood Production and Environmental Sciences, University of Florence.

Epidermal bladder cells (EBCs) play a key role in the salt tolerance of salt-secreting halophytes by acting as an external salt dump under conditions of excess Na. Nevertheless, after testing the salinity tolerance of more than 100 quinoa accessions, we observed dramatic differences in EBC density. Increase in EBC density were not always correlated with greater salt tolerance. This suggests that some quinoa accessions are capable of enhanced salt tolerance without relying on external Na sequestration. To elucidate possible mechanism(s) underlying this enhanced tolerance in accessions with low EBC density, four accessions with contrasting salt tolerance and EBC density were selected. Different physiological and electrophysiological parameters were evaluated after long and short-term treatment with NaCl. The combined data indicate that additional tolerance mechanisms operate in roots rather than shoots in salt tolerant accessions. In particular, our data indicate a greater reactive oxygen scavenging capacity in tolerant accessions, which improves root functionality following salt stress. This ultimately results in improved leaf stomatal regulation and enhanced salt tolerance.


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REGULATION OF PHOTOSYNTHESIS BY SUGAR SENSING IN C3 AND C4 PLANTS

Benning U.1, Henry C.1, Watson-Lazowski A.1, Koller F.1, Furbank R.2 and Ghannoum O.1 11ARC Centre of Excellence for Translational Photosynthesis, Hawkesbury Institute for the Environment, Western Sydney University, Australia. 22ARC Centre of Excellence for Translational Photosynthesis, Research School of Biology, Australian National University, Australia.

Photosynthesis is important for plant fitness. By producing sugar from light, water and carbon dioxide, photosynthesis provides the plant with energy to grow, produce seeds and survive. When photosynthesis is curtailed, plants remobilise stored resources which may become depleted. When growth is limited, reduced sink strength may feedback on photosynthesis. This dynamic relationship between photosynthesis, growth and sugar production is integral component of plant function. My thesis will focus on two sugar sensors in plants: Target of Rapamycin (TOR), Hexokinase (HXK). I will investigate how these sensors differentially regulate growth and photosynthesis of model C3 and C4 plants.

SILICON APPLICATION : A POTENTIAL SOLUTION TO DROUGHT STRESS IN LENTIL

Biju S., Fuentes S. and Gupta D. School of Food and Agriculture,Faculty of Veterinary and Agricultural Sciences, The University of Melbourne, Parkville, Victoria-3010, Australia.

Lentil (Lens culinaris, Medik.) is the fifth most important grain legume growing in the arid and semi-arid regions of the world. Drought stress is one of the major constraints leading up to 60% production losses in lentil. Application of silicon (Si) has been shown to be a promising technique to improve drought tolerance; however, the physiological mechanisms and interactions involved are not fully understood, especially in legumes. Consequently, in the present study, glasshouse experiments were conducted to evaluate the effect of Si in drought-stressed lentil. Seven lentil genotypes, ILL 6002 and Indianhead (drought-tolerant), Flash, PBA Jumbo 2 and Nipper (moderately drought-tolerant), and PI 468898 and ILL 1796 (drought-sensitive) were subjected to drought stress at the onset of flowering. Results showed that the negative effects of drought stress have been significantly ameliorated and apparently seen by higher concentrations of chlorophyll pigments and improved photosynthetic efficiency in lentil supplied with Si. Si effect on osmoregulation decreased the membrane damage in drought-stressed lentil. Drought stress significantly increased the production of reactive oxygen species and induced oxidative damage, while added Si reversed these effects. Furthermore, the addition of Si significantly stimulated the efficiency of the glutathione-ascorbate cycle by increasing the concentrations of glutathione and ascorbate as well as the activities of antioxidant enzymes like ascorbate peroxidase, guaiacol peroxidase, catalase, superoxide dismutase, glutathione reductase and dehydro-ascorbate reductase. Furthermore, Si treatment enhanced the plant biomass and yield. Thus, Si could ameliorate adverse effects of drought stress in lentil likely by increasing photosynthetic efficiency and biochemical defence responses.

TOWARDS THE DEVELOPMENT OF POINT-OF-CARE ELECTRO-CATALYTIC BIOSENSORS FOR DIAGNOSTICS OF Botrytis spp. OF THE TEMPERATE LEGUMES

Bilkiss M.1, Bar I.1, Brownlie J.1, Shiddiky M.J.A.1, 2 and Ford R.1 1School of Environment and Science, Griffith University, Nathan Campus, QLD 4111, Australia. 2Queensland Micro- and Nanotechnology Centre, Griffith University, Nathan Campus, QLD 4111, Australia.

Botrytis grey mould (BGM), caused by Botrytis cinerea and Botrytis fabae, separately or within a complex, causes serious yield losses in legumes during conducive seasons in Australia and worldwide. A greater success in Integrated Disease Management (IDM) approaches to prevent this loss would result from fast, accurate and cost-effective diagnosis and quantification of the causal pathogen(s). Tools to enable this would provide a large opportunity to save on targeted fungicide chemistries input costs and enable the faster application of other management options. The existing immunogenic and molecular probe type diagnostic methods, based on whole genome sequencing, PCR amplification or antibodies, are time consuming and offer varying levels of specificity and/or sensitivity. As an alternative, we have developed species-specific molecular biosensors for the fast, accurate, sensitive detection and quantification of the mycelium and spore derived nucleic acid of both of the target pathogens. For this, two sets of species-specific primers, Bc-F/Bc-R for Botrytis cinerea and Bfa-F/Bfa-R for Botrytis fabae, were designed. Initially, probe sensitivities were determined (100 fg/μl equivalent to 2 genome copies/μl) using multiplexed quantitative PCR. Simultaneously a specific and sensitive assay for the electro catalytic detection of the target pure fungal DNA using functionalised magnetic nanoparticles has been accessed and showed 100 times more sensitivity than the quantitative PCR towards the development of point-of-care diagnostic biosensors for Botrytis spp. of the temperate legumes.

SEED PRIMING WITH MALIC ACID CONFERS LOW PH STRESS TOLERANCE TO WHEAT (TRITICUM AESTIVUM L.) SEEDLINGS BY UPREGULATING ANTIOXIDANT DEFENSE AND METHYLGLYOXAL DETOXIFICATION SYSTEM

Bhuyan M.H.M.B.1, 2, Hasanuzzaman M.3, Mahmud J.A.4, Hossain M.S.1, Hossen M.S.1, 3, Masud A.A.C.1, 3 and Fujita M.1 1Laboratory of plant stress response, Department of Applied Biological Science, Faculty of Agriculture, Kagawa University, Miki-Cho, Kita-Gun, Kagawa 761-0795, Japan. 2Bangladesh Agricultural Research Institute, Joydebpur, Gazipur-1701, Bangladesh. 3Department of Agronomy, Sher-e-Bangla Agricultural University, Sher-e-Bangla Nagar, Dhaka-1207, Bangladesh. 4Department of Agroforestry and Environmental Sciences, Sher-e-Bangla Agricultural University, Sher-e-Bangla Nagar, Dhaka-1207, Bangladesh.

Low soil pH causes from H+ rhizotoxicity, inhibits plant growth, development and reduces crop yields. Hence, the present study was carried out to reveal the possible mechanisms of acidity stress tolerance in wheat after seed priming with malic acid (MA). Hydroponically grown 8-day-old wheat seedlings (Triticum aestivum L. cv. BARI Gom-25) were exposed to acidic growing media (pH 4.0), alone and combined with 0.5 mM and 1.0 mM MA for 3 and 6 days in order to investigate the effect of acidity stress as well as role of MA counteracting the stress. Acidity (pH 4.0) created oxidative stress by the overproduction of reactive oxygen species (ROS) and methylglyoxal (MG) in wheat leaf tissue, which is clearly supported by higher lipid peroxidation and membrane damage. It also disturbed the water status and chlorophyll biosynthesis in wheat seedlings. The component of antioxidant defense and glyoxalase system also disrupted owing to acidity stress. Acidity induced damages with growth inhibition increased in a day dependent manner. On the other hand, MA upregulated the majority of the antioxidants, which helps to scavenge toxic ROS and MG in studied seedlings; thus finally improve the water status, photosynthetic pigments as well as the growth of the plant. The performance of MA was prominent after 6 days of stress. Therefore, our results suggested that the seed priming with MA rendered wheat more tolerance to acidity-induced oxidative damage at the early seedling stage.


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IDENTIFYING OXIDATIVE STRESS RESPONSIVE GENES IN CHICKPEA (CICER ARIETINUM)

Booth N., Soole K., Jenkins C., Sweetman C. and Day D. Flinders University.

Chickpea (Cicer arietinum) is the second most important food legume globally, with 11.62 million tonnes yielded annually. However, it is anticipated that to meet global food demand, annual production must increase to 17 million by 2020. Chickpea production is currently limited because of its sensitivity to abiotic stresses. Under environmental stress, oxidative stress due to over production of Reactive Oxygen Species (ROS) causes cellular damage, reducing plant productivity through impairing photosynthetic carbon gain and leaf growth rate. This highlights the need to improve crop tolerance under these conditions, through identification of biomarkers for molecular breeding and genetic manipulation. A bioinformatics approach with a focus on potential antioxidant genes was undertaken to identify oxidative stress responsive genes in chickpea, as well as gene orthologs from soybean (Glycine max) and Medicago trancatula. It was found that genes encoding late embryogenesis abundant (LEA) proteins, peroxiredoxins (Prx), uncoupling proteins (UCPs) and a potential UPOX protein, as well as proteins involved in synthesis of proline, galactinol, and glutathione, were highly up-regulated under oxidative stress. To verify these responses, 21-day-old chickpeas (cv. Rupali) were subjected to each of three environmental stressors: salinity, drought and paraquat, with tissue samples collected at various time points for growth rate analysis, malondialdehyde measurements and expression analysis. The gene expression response to oxidative stress was quantified using qRT-PCR for both root and leaf tissue, identifying a cassette of oxidative stress responsive genes for potential use as biomarkers in chickpea.

CONTROLLING SODIUM AND POTASSIUM TRANSPORT ACROSS CHLOROPLAST MEMBRANE IS A KEY DETERMINANT TO MAINTAIN PHOTOSYNTHESIS DURING SALT STRESS

Bose J.1, Jayakannan M.1, Malik S.1, 2, Shabala S.3 and Tyerman S.1 1ARC Centre of Excellence in Plant Energy Biology and School of Agriculture, Food and Wine, University of Adelaide, PMB 1, Glen Osmond SA 5064, Australia. 2Federal University of Maranhão, São Luís, MA, Brazil. 3School of Land and Food, University of Tasmania, Private Bag 54, Hobart, Tas 7001, Australia.

Salt stress affects photosynthesis by causing ionic imbalance within chloroplasts. A comparison between glycophytes and halophytes revealed that halophytes are superior in regulating sodium and potassium concentration inside chloroplasts during salt stress. However, the molecular identity of ion transport systems involved in sodium and potassium homeostasis in chloroplasts during salt stress are not fully understood. A pyruvate (BASS2- bile acid: sodium symporter2) transporter and a sodium-proton exchanger (NHD1) have been suggested to move sodium in and out of chloroplasts. We studied sodium, potassium and proton transport across isolated chloroplasts of Arabidopsis thaliana BASS2 and NHD1 knockout mutants using ion-selective micro-electrode (MIFE) technique under salt stress. Chloroplasts from the bass2 mutant had reduced sodium entry and potassium loss. The opposite was the case for the ndh1 mutant. The implications of altered sodium and potassium transport in chloroplasts of these mutants on photosynthetic performance and growth under salinity will be presented and discussed.

GENERATION OF VITAMIN C ENRICHED RICE TO IMPROVE IRON BIOAVAILABILITY

Broad R.C.1, Bonneau J.P.1, O’Brien M.1, Beasley J.T.1, Roden S.M.2, Eftekhari F.N.2, Hellens R.P.2 and Johnson A.A.T.1 1School of Biosciences, The University of Melbourne, VIC 3010, Australia. 2Centre for Tropical Crops and Biocommodities, Queensland University of Technology, QLD 4001, Australia.

Breeding strategies to produce iron (Fe) biofortified crops typically focus on increasing Fe density and/or reducing inhibitors of Fe absorption, with less attention given to compounds that enhance Fe absorption. Ascorbate, most commonly known as vitamin C, is a potent enhancer of Fe bioavailability in human digestion. Because many staple crops have little to no ascorbate, enriching crops with ascorbate could provide a novel means of improving human Fe nutrition. This study aims to investigate the effects of increased ascorbate biosynthesis in rice on grain Fe bioavailability and plant growth. Rice plants genetically engineered to constitutively overexpress the rice GDP-L-galactose phosphorylase (OsGGP) gene, encoding the key rate-limiting enzyme involved in ascorbate biosynthesis, contained 8.7-fold more ascorbate in germinated grain, 1.8-fold more ascorbate in seedling shoots, and 4.5-fold more ascorbate in seedling roots, relative to wild-type rice. At maturity, however, the OsGGP overexpressing rice plants contained less ascorbate in shoots and had reduced expression of the endogenous OsGGP gene, correlated with significant yield penalties, indicating that OsGGP overexpression has several pleiotropic effects on rice physiology. Therefore, as an alternative strategy to increase ascorbate biosynthesis in rice, the CRISPR/Cas9 gene-editing system has been used to induce mutations in the OsGGP upstream open reading frame (uORF), a negative translational regulator of the OsGGP coding sequence. Here we present findings from several molecular and phenotypic analyses of the OsGGP overexpressing rice plants, including an Fe bioavailability assessment of ascorbate-enriched, germinated grain, and summarize findings of CRISPR/Cas9-induced mutations in the OsGGP uORF sequence.

THE APPLICATION OF FOURIER TRANSFORM MID-INFRARED (FTIR) SPECTROSCOPY TO IDENTIFY VARIATION IN CELL WALL COMPOSITION OF SETARIA ITALICA ECOTYPES

Brown C.W.1, Grof C.P.L.1 and Martin A.P.2 1Centre for Plant Science, School of Environmental and Life Sciences, University of Newcastle, University Drive, Callaghan, NSW 2308, Australia. 2Rapid Phenotyping, 9 Gipps St, Carrington, NSW, 2294, Australia.

Cell wall composition in monocotyledonous grasses has been identified as a key area of research for developing better feedstocks for forage and biofuel production. Setaria viridis and its close domesticated relative Setaria italica, have been chosen as suitable monocotyledonous models for more economically significant plants possessing the C4 pathway of photosynthesis including sorghum, maize, sugarcane, switchgrass and Miscanthus x giganteus. Accurate Partial Least Squares Regression (PLSR) models to predict S. italica cell wall composition in stem tissue have been generated, based upon Fourier transform mid-Infrared (FTIR) spectra and calibrated with wet chemistry determinations of ground S. italica stem material measured using a modified version of the US National Renewable Energy Laboratory (NREL) two stage acid hydrolysis protocol. The models facilitated a high-throughput screening analysis for glucan, xylan, Klason lignin and acid soluble lignin in a collection of 183 natural S. italicavariants and clustered them into classes, some possessing unique cell wall chemotypes. Genes encoding key catalytic enzymes of the lignin biosynthesis pathway were compared from different developmental regions within an elongating stem internode in selected S. italica variants. A high level of conservation with matching expression profiles between selected variants was evident. Expression in S. italica was determined by quantitative reverse transcription polymerase chain reaction (RT-qPCR), and closely mirrored expression profiles of homologous genes in equivalent developmental regions of elongating internodes of S. viridis by RNASeq.


POSTERS


POS-TUE-015 POS-TUE-016

COLI-PLAST: EXPRESSING AND BIOENGINEERING HIGHER PLANT RUBISCO IN E. COLI

Buck S. and Whitney S. ARC Centre of Excellence for Translational Photosynthesis. Research School of Biology. The Australian National University. Canberra, ACT 2601, Australia.

Ribulose bisphosphate carboxylase oxygenase (Rubisco) is a key enzyme in the process of carbon fixation in photosynthesis, and as such it directly or indirectly supports almost all life on earth. It is has some marred properties for such a crucial enzyme; being both slow and non-specific, catalyzing the oxygenation as well as the carboxylation of RuBP resulting in an energy intensive regeneration process. Synthetic biology and bioengineering methods for the improvement of Rubisco have been hindered by its inability to be functionally expressed in the commonly used bacterial expression host Escherichia coli. This is likely because the biogenesis requirements of plant Rubisco cannot be met in E. coli due to incompatibilities in the protein folding and assembly machinery between E. coli and chloroplasts. This study aims to develop expression capabilities in E. coli suitable for improving the catalysis of higher plant Rubisco. This involves the expression of functional tobacco Rubisco in E. coli through its co-expression with the necessary ancillary proteins needed for its biogenesis and metabolic repair. This system will complement existing Rubisco directed evolution E. coli systems that have successfully identified mutations that improve the kinetics of Archaea and cyanobacteria Rubisco - and hopefully translated to selecting improved crop Rubisco mutants useful for subsequent testing in planta.

DIFFERENTIAL EXPRESSION OF LACCASES IN BOTRYTIS CINEREA IN THE PRESENCE OF INDUCERS AND DURING THE INFECTION OF GRAPES

Buddhika U.V.A., Savocchia S., Schmidtke L.M. and Steel C.C. National Wine and Grape Industry Centre, Charles Sturt University, School of Agriculture and Wine Sciences, Wagga Wagga, NSW, 2678.

Botrytis cinerea (grey mould) produces oxidative enzymes, such as laccases, during the infection of plant tissues. Laccases are induced by gallic acid and copper, however, the responsive genes are not known. Although three laccase encoding genes (LAC1, LAC2, LAC3) have been reported, no virulence determinants have been identified in B. cinerea to date. This study characterised the expression patterns of laccases from B. cinerea infected grapes with respect to different inducers. Laccase activity in the culture filtrate of B. cinerea was measured and laccase transcripts were quantified in a laccase-inducing medium supplemented with inducers (gallic acid, CuSO4). Increased laccase activity occurred 2-days post-inoculation along with increased expression of LAC2 mRNA in response to copper. The purified enzyme was confirmed as LAC2, 63kDa in size by ESI/MS/MS analysis. The expression of laccase mRNA transcripts was analysed from B. cinerea infected Vitis vinifera berries (cvs Thompson Seedless and Chardonnay) during disease development and resulted in consistent expression of LAC3 and inconsistent expression of LAC1 and LAC2 suggesting involvement of LAC3 in initiation and the progression of disease in grapes. To confirm the expression of LAC3 during disease development, ESI/MS/MS analysis of the purified protein from infected grapes was performed, following bioinformatics analysis. The resulting protein was 26kDa in size and harbored the functional group ‘Glyco-hydro 12 superfamily’, responsible for the hydrolysis of polysaccharides. The presence of this domain indicates the protein function in plant cell wall and membrane degradation during pathogen penetration and in the disease progression, although ESI/MS/MS analysis did not confirm the protein as LAC3. In conclusion, LAC3 is potentially a determinant of virulence while LAC2 is copper inducible. LAC3 expression is presumably induced by gallic acid. Further experiments are warranted to confirm LAC3 as the determinant of virulence and its induction by gallic acid.

PLANTS INCREASE CO2 UPTAKE BY ASSIMILATING NITROGEN VIA THE PHOTORESPIRATORY PATHWAY

Busch F.A.1, Sage R.F.2 and Farquhar G.D.1 1Research School of Biology and ARC Centre of Excellence for Translational Photosynthesis, Australian National University, Acton, ACT, Australia. 2Department of Ecology and Evolutionary Biology, University of Toronto, Toronto, Ontario, Canada.

Photorespiration is a major bioengineering target for increasing crop yields as it is often considered a wasteful process due to the release of previously fixed CO2. Photorespiratory metabolism is integrated into leaf metabolism and thus may have certain benefits. Using a combination of mathematical modelling and photosynthetic gas exchange measurements, we show that plants can increase their rate of photosynthetic CO2 uptake when assimilating nitrogen de novo via the photorespiratory pathway. Plants achieve this by fixing carbon as amino acids in addition to carbohydrates. When plants were fed nitrate they had higher rates of CO2 assimilation under photorespiratory than low-photorespiratory conditions, while plants lacking nitrate nutrition exhibited lower stimulation of CO2 uptake. We modified the widely used Farquhar, von Caemmerer and Berry photosynthesis model to include the carbon and electron requirements for nitrogen assimilation via the photorespiratory pathway. Our modified model improves predictions of photosynthetic CO2 uptake and of rates of photosynthetic electron transport. The results highlight how photorespiration can improve photosynthetic performance despite reducing the efficiency of Rubisco carboxylation.

LOW-INPUT TRANSCRIPTOMICS OF DEVELOPING POLLEN ISOLATED FROM A SINGLE ANTHER

Chakkatu S.P., Day R., Harrop T. and Brownfield L. University of Otago, Dunedin, New Zealand.

Pollen are the male gametophytes of plants that deliver the sperm cells to the female gametes. Development of pollen from a unicellular microspore to a mature triploid pollen grain is a highly complex process involving precise and co-ordinated regulation of cell cycle, specification and differentiation of two different cell types. A key challenge in plant biology is to understand the genes that participate in this complex regulation. However, transcriptomic analysis of developing pollen has been a challenge due to their relative inaccessibility within an anther and also because of a tough outer pollen wall. Due to these challenges, previous studies of pollen transcriptomes have used either whole anthers (comprising parental tissue) or pollen from multiple plants encompassing a broad developmental window. To enable transcriptomic analysis on pollen at a particular developmental stage and from a small number of plants, we have developed a protocol to analyze pollen from a single anther in Arabidopsis thaliana. This protocol involves removal of an anther from a bud. The anther is ruptured and the released pollen is washed before being frozen for storage. The remaining anther material with pollen is fixed and later stained to be viewed under a microscope to determine its developmental stage. The frozen pollen is chemically lysed and used in direct cDNA synthesis. We will report on our development and testing of the method for RT-qPCR, digital droplet PCR and RNA sequencing and its use to study gene expression in pollen.


POSTERS



EXPLORING FLOWERING AND PHOTOPERIOD RESPONSE MECHANISMS IN LEGUMES

Contreras B., Van der Schoor J., Hecht V. and Weller J.L. School of Natural Sciences, University of Tasmania, Hobart, Tasmania, Australia.

Flowering time and its responsiveness to photoperiod is a key trait in the adaptation of plants to different climates and locations. The genetic control of flowering is well described in the model plant Arabidopsis but not as well understood in other species including many crops. Legumes are an important crop group that includes two clades showing opposite response to photoperiod: warm season species such as soybean and bean, which are short-day plants, and temperate species such as chickpea and pea which are long-day plants. Pea (Pisum sativum) has been an important model system for investigating the legume long-day mechanism and exploring similarities and differences with the main Arabidopsis model. Previous studies have characterized an expanded family of FT (florigen) genes pea that collectively play a conserved role as environmentally-regulated mobile flowering signals. Certain upstream regulators of FT genes are also conserved with Arabidopsis, but a key difference is the lack of a clear role for homologs of CONSTANS (CO), which in Arabidopsis is a major FT activator that integrates light and circadian clock signalling for photoperiod measurement. The absence of this central point of regulation raises questions about the nature of the molecular mechanism for photoperiod response in pea and how other conserved components may be acting. This study investigates two specific aspects of this system in order to further assess the conservation of photoperiod-response pathways; 1) the role of the pea ortholog of CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1), a major regulator of CO protein stability in Arabidopsis and 2) the mechanism of action of LATE BLOOMER 1 (LATE1), an ortholog of Arabidopsis GIGANTEA which has an important role in regulating CO transcription.

DEVELOPMENT OF A HIGH THROUGHPUT, SEMI-QUANTITATIVE DOT BLOT TO DETECT HME PROTEIN IN RYEGRASS

Cookson R., Roberts N., Crowther T., Beechey-Gradwell Z., Cooney L., Bryan G. and Winichayakul S. AgResearch Grasslands, Tennent Drive, Private Bag 11008, Palmerston North 4442, New Zealand.

High Metabolizable Energy (HME) technology utilizes two recombinant proteins to increase the energy content, change the fatty acid profile, and increase the biomass, of transformed plants. Over-expression of optimised Tropaeolum majus diacylglycerol acyltransferase (DGAT) and a modified oleosin from Sesamum indicum seeds allows excess lipid to be stored in cross-linked oil bodies in leaf tissue. Our HME ryegrass (Lolium perenne) breeding programme has resulted in large populations of T2 segregating lines, which require a fast, accurate assessment of HME protein presence in order to distinguish between null and HME seedlings. Here we describe the development of an immuno-dot blot system to detect sesame oleosin via a simple plant extraction, which can be configured for high throughput or formulated as a semi-quantitative assay for smaller numbers of samples. Due to the robustness of detection after various tissue treatments (including freeze drying), the assay offers an improved approach for detecting sesame oleosin in ryegrass blades, compared to SDS-PAGE immunoblotting. In addition, cost and speed of result per sample extracted are greatly improved. Validation of the protocol involved demonstration that the sesame oleosin protein level positively correlated with the HME traits and with Fatty Acid Methyl Esterification (FAMES) Gas Chromatography-Mass Spectrometry (GC-MS) data. Using generic laboratory equipment, this method provides a valuable tool for the rapid identification of plants expressing HME-based technologies.

SODIUM FLUXES ALONG THE ARABIDOPSIS ROOT

Cuin T.A., Bazihizina N. and Shabala S. Tasmanian Institute of Agriculture, University of Tasmania, Hobart, Tasmania.

Despite decades of research into plant salinity tolerance, the patterns of root sodium fluxes remain unclear. The current consensus is that large quantities of sodium go passively into root cells and are subsequently expelled in a futile cycle that consumes large amounts of metabolic energy. However, no transport system for cellular sodium influx has been definitively identified, and only one transporter, SOS1, is recognised as mediating sodium efflux. However, this antiporter is localised only to the root tip. What happens along the rest of the root? Perhaps sodium does not enter the cell at all, but instead emanates from sodium cycling in the apoplast [1]. We need to assess how much and where sodium enters the root to discern whether cellular cycling is actually significant. There is no point developing crops with increased sodium export if its efflux is from the apoplast, not the cell. NaCl induces membrane depolarisation of at least 60 mV in Arabidopsis roots [2]. This implies large sodium influxes into cells. Also in [2], we demonstrated that sos mutations affect the ion transport patterns in the entire root, not just the tip. We now revisit this work with an improved sodium LIX [3], measuring net sodium fluxes along Arabidopsis roots. Using transport mutants and pharmacology, we are deciphering the extent of sodium cycling in the root and starting to answer questions as to the nature and location of root sodium transport. [1] Britto and Kronzucker, 2015. J. Plant Physiol 186-187: 1-12 [2] Shabala et al., 2005. Planta 222: 1041-1050 [3] Jayakannan et al., 2011. J. Plant Physiol 168: 1045-1051.

CHARACTERIZATION OF TOBACCO AQUAPORINS: THE SEARCH FOR CO2 PORES

De Rosa A., Groszmann M. and Evans J.R. Division of Plant Sciences, Research School of Biology, The Australian National University, Canberra, ACT, 2601, Australia.

Photosynthesis is a vital process that sustains nearly all life on earth and its efficiency can be limited by the diffusion of carbon dioxide (CO2) from the atmosphere to the chloroplast. A strategy to increase photosynthetic efficiency could be to facilitate the journey of CO2 to the chloroplasts by increasing mesophyll conductance. Lipid bilayers are highly permeable to CO2, however biological membranes -such as plasma membrane and chloroplast envelope- are heavily populated by proteins that reduce the area available for CO2 diffusion. Aquaporins are pore forming proteins that facilitate the transmembrane transport of not only water, but also a range of small molecules and gases. Aquaporins in the plasma membrane intrinsic proteins (PIP) sub-family have been shown to enhance permeability of the membrane to CO2, making them ideal study targets in attempts to facilitate the diffusion of CO2 to the chloroplast. Our research aims to characterize PIP aquaporins in tobacco, a model species for photosynthesis. To date one PIP gene has been described in tobacco to be a CO2 pore, NtAQP1, however beyond this there is little characterization of tobacco aquaporins in the literature. We used tomato and potato gene homologs to identify all aquaporins in the tobacco genome, resulting in the discovery of 76 aquaporin genes and 29 genes in the PIP sub-family. From these finding we will be able to identify PIPs that may facilitate CO2 diffusion across biological membranes and assess their physiological importance.


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DROUGHT-INDUCED SENESCENCE OF MEDICAGO TRUNCATULA NODULES INVOLVES SERPIN AND FERRITIN TO CONTROL PROTEOLYTIC ACTIVITY AND IRON LEVELS

Dhanushkodi R.1, Matthew C.2, McManus M.T.1 and Dijkwel P.P.1 1Institute of Fundamental Sciences, Massey University, Palmerston North, New Zealand. 2Institute of Agriculture and Environment, Massey University, Palmerston North, New Zealand.

Drought is a major constraint for legume growth and yield. Senescence of nitrogen-fixing nodules is one of the early drought responses which may cause nutrient stress in addition to water stress in legumes. For nodule senescence to function as part of a drought-survival strategy, we propose that the intrinsically destructive senescence process must be tightly regulated. M. truncatulaprotease inhibitor and iron scavenger-encoding genes, possibly involved in controlling nodule senescence, were identified. RNAi lines were constructed in which expression of a serpin or ferritins was knocked down. Both wild-type and RNAi lines were subjected to drought stress and nodule activity and plant physiological responses were measured. Drought caused M. truncatulato initiate nodule senescence before plant growth was affected and before an increase in papain-like proteolytic activity and free iron levels was apparent. Knock-down expression of serpin6 and ferritins caused increased protease activity, free iron levels, early nodule senescence and reduced plant growth. The results suggest that M. truncatula nodule-expressed serpin6 and ferritins mediate ordered drought-induced senescence by regulating papain-like cysteine protease activity and free iron levels. This strategy may allow the drought-stressed plants to benefit maximally from residual nitrogen fixation and nutrient recovery resulting from break-down of macromolecules.

CHARACTERISING DEVELOPMENTAL MUTATIONS IN ARABIDOPSIS THALIANA

Downs J. and Jones B. The University of Sydney.

Arabidopsis thaliana is a small, flowering eudicot. This unassuming plant is ideal for genetic research due its small genome and relatively rapid life cycle. These traits make A. thaliana one of the most well-studied plants and yet there is still much that can be gleaned from this plant in terms of understanding general plant function. Reproduction is one of the most important parts of plant life. Understanding how plant reproduction works on a molecular genetic scale can potentially lead to improved crop yields. In this study, two separate mutations were studied in T-DNA insertional mutants. The mutations are associated with fertilisation and embryogenesis, respectively. The mutations and their phenotypes are being characterised to improve the understanding of plant reproductive processes. Embryo termination occurs in 25% of seeds in plants hemizygous for the embryogenesis mutation. The seeds can be distinguished early in development as they appear white, as opposed to the other (75%) green seeds in the siliques. The embryogenesis mutation appears to be monogenic, recessive and homozygous lethal. Embryos within the white seeds are halted at early stages of development and bear aberrant cell division. This causal mutation is being located by whole-genome re-sequencing using an Illumina platform. The second reproductive mutation in this study, the fertilisation mutation was first observed in an arf1 homozygous arf2 heterozygous mutant line. In this line, progeny from self-crossing had a non-Mendelian ratio of genotypes. Novel techniques and confocal microscopy are currently in development to observe the movement of fluorescently labelled pollen tubes within the styles of the flowers. This will help to determine if the phenotype is due to pollen growth, parental influence or aberrant embryogenesis creating the biases in the genotypes. Ultimately, characterising these mutations will help improve knowledge of plant reproductive biology.

OVEREXPRESSION OF PIP1;2 INCREASES MESOPHYLL CONDUCTANCE

Groszmann M., Skinner S., Von Caemmerer S. and Evans J.R. ARC Centre of Excellence for Translational Photosynthesis, Australian National University.

Aquaporins in plants are encoded by multigene families. Plasma membrane intrinsic proteins (PIPs) have been categorised into two groups, but this is not clearly associated with their function. Due to easy methodology, many have been functionally assayed for their ability to alter membrane permeability to water. However, determining CO2 permeability is difficult and while some aquaporins have been shown to increase membrane permeability to CO2 in yeast or Xenopus oocytes, in planta demonstrations are limited. We have been using Arabidopsis to investigate AQP function. We generated Arabidopsis lines overexpressing each plasma membrane intrinsic protein to facilitate in planta characterisation of CO2 permeability. Following initial screening for enhanced growth, several lines of PIP1;2, PIP1;3 and PIP 1;4 were chosen for more detailed characterisation using combined gas exchange and carbon isotope measurements to determine mesophyll conductance. Overexpression of PIP1;2 increased mesophyll conductance and was associated with a decrease in the draw-down in CO2 between intercellular airspaces and sites of carboxylation within chloroplasts. Overexpression of PIP1;4 also increased mesophyll conductance, but as this was associated with increased photosynthetic capacity, further anatomical characterisation is needed to identify the reason. There was no significant effect from overexpressing PIP1;3.

WHITE CLOVER GENETICS DRIVE ROOT SOIL RHIZOBIUM INTERACTIONSWeith S.K.1, 2, Griffiths A.G.2, Ballard R.A.3, Ganesh S.2, Jones E.E.1, Ridgway H.J.4 and Hofmann R.W.1 1Faculty of Agriculture and Life Sciences, Lincoln University, PO Box 85084, Lincoln 7647, New Zealand. 2AgResearch, Grasslands Research Centre, Private Bag 11008, Palmerston North 4442. 3South Australian Research and Development Institute, GPO Box 397, Adelaide, SA 5001, Australia. 4Plant & Food Research Ltd, Private Bag 4704, Lincoln 7608, New Zealand.The legume white clover (Trifolium repens L.) is an important source of biologically fixed nitrogen (N) for temperate agronomy in mixed swards. To date, there has been little focus on identifying and exploiting variation in plant genetic factors underpinning effective symbioses with rhizobia. Combining plant and Rhizobium genetics for improved N fixation, and concomitant reductions in fertiliser use, provides new avenues towards sustainable agriculture. However, in order to breed new white clover cultivars with improved interactions with rhizobia, the genetic factors of phenotypic variation in symbiotic performance need to be examined. In this study, we quantified the variation in symbiotic performance across a large number (n=360) of individuals from 17 white clover cultivars released between 1920 and 2003, in addition to a wild ecotype and a T. repens x T. uniflorum hybrid. Symbiotic responses were assessed using an effective and partially effective Rhizobium strain. There were significant interactions between white clover populations and the two Rhizobium strains. Productivity of the white clover ecotype was low with the partially effective Rhizobium strain, and high with the effective strain. In contrast, some cultivars performed similarly well with both strains. Shoot dry matter production was positively correlated with the number of pink (effective) nodules on the roots of individuals inoculated with the partially effective strain. In contrast, shoot and root symbiotic potentials were positively correlated with the number of pink nodules when inoculated with the effective strain. Canonical discriminant analysis (CDA) separated the population response to N availability. This was reflected by an increased root:shoot ratio (RSR) under the low-nitrogen and partially effective Rhizobium strain treatments. Dry matter production and symbiotic potential increased with effective Rhizobium and N supplemented treatments. Further, the RSR of individuals inoculated with the partially effective Rhizobium strain was positively correlated with the decade of cultivar release: when inoculated with a partially effective N-fixing strain of Rhizobium, older cultivars produced more root biomass relative to shoot biomass than newer cultivars. This study showed that the N-fixing abilities of white clover cultivars play a key role in determining symbiotic performance with Rhizobium strains. The results highlight the potential for manipulating clover genetics to improve nodulation and symbiotic performance of white clover cultivars in the future.


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COMPOUND-SPECIFIC CARBON ISOTOPE ANALYSIS OF POST-PHOTOSYNTHETIC METABOLITES IN C3 AND C4 GRASSESIsrael W.K.1, Koller F.1, Hocart C.2, Tcherkez G.2, Stuart-Williams H.2 and Ghannoum O.1 1ARC Centre of Excellence for Translational Photosynthesis, Hawkesbury Institute for the Environment, Western Sydney University. 2ARC Centre of Excellence for Translational Photosynthesis, The Australian National University.

Breeding crops with improved water-use efficiency (WUEi) has been successful through examining the integrated ratio of the naturally occurring stable carbon isotopes (13C /12C or δ13 C) incorporated during photosynthesis. It is intensively studied among C3 species most notably that of wheat varieties with improved WUE. The carbon concentrating mechanism (CCM) in C4 allows for an enriched δ13C signature relative to C3. Since C4 species are known to have better WUE, attempts have been undertaken to correlate δ13 C with WUE i. In C3 species, WUE is directly correlated to Ci/Ca which can be measured by analysing δ13C. However, δ13C and thus WUE in C4 plants are not consistently correlated because of the CCM and other leaf metabolic reactions including post-photosynthetic fractionation. Thus, the study seeks to examine δ13C signatures of post-photosynthetic metabolites among C4 grasses to ultimately understand the mechanisms of intrinsic water use efficiency; hence, providing a breeding tool for crops with improved WUE. Grasses representing C3 and C4 photosynthetic types and C4 biochemical subtypes (NADP-ME, NAD-ME, and PCK) were grown in glasshouse conditions and whole leaves were assayed for simple sugar, organic acids, and bulk metabolic pool extraction and purification. δ13C signatures were assayed using HPLC coupled with wet oxidiser that directly converts eluted compounds to CO2 and measured using IRMS. Results show that fructose is 13C-enriched by 2.8 ‰ in NADP-ME relative to NAD-ME while sucrose and glucose are not significantly different. PCK metabolites tend to have an intermediate 13C signature when compared to NADP-ME and NAD-ME while the greatest depletion in 13C was observed among C3. These results indicate initial evidence that could potentially explain the variations in 13C signatures among C4 grasses and ultimately develop screening tools for improved WUE.

IDENTIFICATION OF THE ROLE OF BLH GENES IN PLANT LEAF MORPHOGENESIS

Jeon H.W. and Byrne M. School of Biological Sciences, The University of Sydney, Sydney, NSW, Australia.

Plant leaves are one of the most obvious features of plants. After the initiation from the shoot apical meristem, they form diverse leaf shapes determined by leaf morphogenesis. Arabidopsis thaliana, a model of plant biology, has two families of the transcription factor, BEL1-LIKE TALE HOMEODOMAIN (BLH) proteins and KNOTTED1-LIKE TALE HOMEOBOX (KNOX) proteins, that affect various plant aspects as well as leaf development. First, among a total of thirteen BLH proteins, previous studies have shown that BLH2/SAW1 and BLH4/SAW2 are redundantly required for leaf margin development. Second, class II KNOX proteins (KNAT3, 4, 5) have been identified as regulators of the above-ground organ development including leaf formation. In addition to these transcription factors, an auxin-CUC2 feedback loop is a well-known hormonal mechanism that regulates the shape of leaf margins. To understand how plants determine their leaf shape, our study is aiming at the in-depth characterisation of SAW proteins with their functional redundancies and the relationship between BLH and KNOX in terms of the leaf margin development as well as how these proteins interact with the auxin-CUC2 regulation module. We have found that additional knockout of a class II KNOX gene in saw1 saw2 double mutants can enhance the leaf serration phenotype. This observation leads to the possibility of the new BLH-KNOX interactions in leaf shape formation. Consequently, our study will provide an insight into the underlying mechanisms of the leaf development with a better understanding of BLH and KNOX functions.

THE SYNERGISTIC RELATIONSHIP BETWEEN BHLHM1 AND THE AMMONIUM TRANSPORTER AMF1 ACROSS THE RHIZOBIA AND ARBUSCULAR MYCHORIZZAL FUNGI SYMBIOSES IN LEGUMES

Kaiser B.N.1, Ovchinnikova E.1, Wen Z.1, Chiasson D.2 and Wu Y.3 1School of Life and Environmental Sciences, The University of Sydney, 380 Werombi Road, Brownlow Hill, NSW. 2Ludwig-Maximilians-Universitat, Grobhaderner Str. 2-4 82152 Martinsried Germany. 3School of Agriculture Food and Wine, The University of Adelaide, Urrbrae, SA.

The regulation of ammonium transport in plants is traditionally aligned with the activities of AMT1 and AMT2 transport pathways. Previously we characterised the functional relationship between a soybean nodule transcription factor (bHLHm1) and a class of transport protein (AMF1) which permits the transport of ammonium when expressed in yeast and Xenopus laevis oocytes. Both proteins are required for an effective nodule symbiosis, where loss of activity disrupts nodule development and activity. We have further explored the post transcriptional relationship between bHLHm1 and AMF1 homologs in soybean and have confirmed using EMSA analysis and in planta transactivation assays that GmbHLHm1 recognises the promoter sequences of multiple AMF1homologs in soybean. In parallel studies, we have examined the relationship between bHLHm1 and AMF1 orthologs in Medicago truncatula. Both genes are induced upon arbuscular mychorrizal colonisation and the encoded proteins co-located within arbuscular containing root cortical cells. Loss of function of either bhlhm1 or amf1;3 in Medicago roots disrupts AM colonisation and alters the selected expression of other known AMT2 genes linked to AM mediated ammonium transport in arbuscular containing cortical cells.

ACCLIMATION AND ADAPTATION COMPONENTS OF THE TEMPERATURE DEPENDENCE OF PLANT PHOTOSYNTHESIS AT THE GLOBAL SCALE

Kumarathunge D.P.1, Medlyn B.E.1, Drake J.E.2 and Tjoelker M.G.1 1Hawkesbury Institute for the Environment, Hawkesbury Campus, Western Sydney University, Locked Bag 1797, Penrith NSW 2751, Australia. 2Forest and Natural Resource Management, SUNY-ESF, 310B Bray Hall, 1 Forestry Drive, Syracuse, NY 13210, USA.

Temperature dependence of leaf photosynthesis (An-T response) is a key determinant in modelling plant growth. Hence, the way that any Earth System Model (ESM) handles the An-T response is critical. It is known that there are differences in the optimum temperature for net photosynthesis (Topt) across species. However, it is unknown how much each of the underlying component processes (biochemical, stomatal and respiratory) contribute to these differences in optimum. Additionally, it is unknown whether differences across species are largely genetic (adaptation) or plastic (acclimation). In this study, we hypothesise that Topt is more strongly related to climate of origin than growth environment, and that all three component processes contribute to differences in Topt. We quantified and modelled key mechanisms responsible for photosynthetic temperature acclimation and adaptation using a global dataset of photosynthetic CO2 response curves including data from 141 tree species from tropics to Arctic tundra. We separated temperature acclimation and adaptation processes by considering seasonal and common-garden datasets. The observed global variation in the temperature optimum of photosynthesis was primarily explained by changes in biochemistry, rather than stomatal conductance or respiration. We found acclimation to growth temperature to be a stronger driver of this variation, than adaptation to temperature at climate of origin. We developed a summary model to represent photosynthetic temperature responses and adaptation and showed it predicts the observed global variation in optimal temperatures with high accuracy. These novel algorithms should enable improved prediction of the function of global forest ecosystems in a warming climate.


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SILICON-INDUCED ANTIOXIDANT DEFENSE, METHYLGLYOXAL DETOXIFICATION AND METAL CHELATION WORKS COORDINATELY IN ALLEVIATING NICKEL TOXICITY IN Oryza sativa L

Mohsin S.M.1, 2, Hasanuzzaman M.3, Alam M.M.4, Nahar K.5, Bhuyan M.H.M.B.1, 6, Parvin K.1, 7 and Fujita M.1 1Laboratory of Plant Stress Responses, Department of Applied Biological Sciences, Faculty of Agriculture, Kagawa University, Miki-Cho, Kita-Gun, Kagawa 761-0795, Japan. 2Department of Plant Pathology, Faculty of Agriculture, Sher-e-Bangla Agricultural University, Sher-e-Bangla Nagar, Dhaka-1207, Bangladesh. 3Department of Agronomy, Faculty of Agriculture, Sher-e-Bangla Agricultural University, Sher-e-Bangla Nagar, Dhaka-1207, Bangladesh. 4Department of Agriculture, Faculty of Science, Noakhali Science and Technology University, Noakhali-3814, Bangladesh. 5Department of Agricultural Botany, Faculty of Agriculture, Sher-e-Bangla Agricultural University, Sher-e-Bangla Nagar, Dhaka-1207, Bangladesh. 6Bangladesh Agricultural Research Institute, Joydebpur, Gazipur-1701, Bangladesh. 7Department of Horticulture, Faculty of Agriculture, Sher-e-Bangla Agricultural University, Sher-e-Bangla Nagar, Dhaka-1207, Bangladesh.

Nickel (Ni) is an essential plant nutrient but very toxic to plant at supra-optimal concentration and causes inhibition of seed germination, growth, and development of plants. The present study investigates the possible mechanisms of Ni tolerance in rice seedlings by exogenous application of silicon (Si). Hydroponically grown 13-day-old rice (Oryza sativa L. cv. BRRI dhan54) seedlings were exposed to 0.25 mM and 0.5 mM NiSO4 alone and combined with 0.50 mM Na2SiO3 for 3 days to investigate the effect of Ni toxicity as well as the role of Si revoking the stress. Nickel toxicity caused oxidative stress by overproduction of reactive oxygen species (ROS) and methylglyoxal (MG) in seedlings of rice that created higher lipid peroxidation and membrane damage. Nickel stress also reduced growth and biomass, leaf relative water content (RWC), and chlorophyll (chl) content of seedlings. However, non-protein thiol (NPT) content, phytochelatin (PC) and proline (Pro) content increased in Ni-stress. In contrast, the Ni-stressed seedlings supplemented with exogenous Si recovered from water loss, chlorosis, growth inhibition, and oxidative stress. Silicon up-regulated most of the antioxidant defense components as well as glyoxalase systems, which helped to improve ROS and MG detoxification respectively. Hence, these findings suggest that the exogenous application of Si can improve the tolerance of rice seedlings to Ni-toxicity.

TIME COURSES OF LEAF CONTENT OF CYTOCHROME B6F COMPLEX AND PHOTOSYNTHETIC CAPACITY AFTER CHANGES IN GROWTH IRRADIANCE

Murakami K.1, 2, Zhu H.1, 3, Zeng L.D.1, 4, Yi X.P.1, 5, Peng C.L.6, Zhang W.F.5 and Chow W.S.1 1Research School of Biology, The Australian National University, Australia. 2Graduate School of Sciences and Technology for Innovation, Yamaguchi University, Japan. 3School of Life Science and Food Technology, Hanshan Normal University, PR China. 4Department of Life Sciences, Huizhou University, PR China. 5Xinjiang Production and Construction Group, Shihezi University, Shihezi, PR China. 6College of Life Sciences, South China Normal University, PR China.

The content of cytochrome b6f complex, a photosynthetic oxidoreductase between the two photosystems, is a major determinant of the leaf photosynthetic capacity. Although the effect of growth irradiance on the cytochrome content has been investigated intensively, the time course has been poorly characterized. Here, we evaluated the half-life of the cytochrome complex after transfer from high-light to low-light. Pea plants were cultivated in a growth chamber for 18 d under moderately-high light (HL; 360 μmol m-2 s-1) and then subjected to several treatments. When the plants were transferred to low-light (LL; 60 μmol m-2 s-1), the maximum photosynthetic O2 evolution rate (Pmax) of young fully expanded leaves exhibited an exponential decay for 12 d while that of control, leaves kept under HL, was constant. The decrease in Pmax appeared to result from a parallel decrease in the cytochrome content. These decreases were not suppressed by 1.5- and 5-h HL-exposure treatments in LL days. In addition, re-exposure to HL after 4-day LL did not increase the photosynthetic capacity, suggesting that the reduction of the photosynthetic capacity may be irreversible in mature leaves. Some recent results from experiments which attempt to suppress the reduction in leaf photosynthetic capacity will also be discussed.

CRYOPRESERVATION PROTOCOL DEVELOPMENT FOR SYZIGIUM MAIRE, A RECALCITRANT MYRTACEAE SPECIES

Nadarajan J.1, van der Walt K.2 and Pathirana R.1 1The New Zealand Institute for Plant & Food Research Ltd, Private Bag 11-600, Palmerston North 4442, New Zealand. 2Otari Native Botanic Garden & Wiltons Bush Reserve, 160 Wilton Road, Wilton, Wellington, 6012, New Zealand.

The current threat of myrtle rust to Myrtaceae species including a number indigenous and socio-economically important plants of New Zealand, requires that ex-situ conservation is used to complement in-situ collections. To mitigate the effects of biotic and abiotic threats, ex-situ germplasm conservation is widely applied in many species using protocols for conservation of pollen, seed and clonal germplasm, complemented by in vitro propagation and cryopreservation. The New Zealand Myrtaceae have received little attention in terms of long-term ex-situ conservation. Seed of some Myrtaceae species are considered orthodox and can be stored relatively easily in conventional seed banks. However, some Myrtaceae species exhibit recalcitrant seed storage behaviour, e.g. Syzigium maire, and therefore require cryopreservation of isolated embryos. In this study, desiccation sensitivity profiles of S. maire seeds and excised embryos collected from various natural populations in New Zealand were investigated. S. maire was cryopreserved using excised embryos, encapsulation-dehydration and PVS2 vitrification techniques. Seeds and embryos of S. maire showed extreme sensitivity to desiccation and lost viability completely following desiccation to a moisture content below 20%, confirming its recalcitrant behaviour. No survival was recorded following excised embryo and PVS2 vitrification cryopreservation. Cryopreservation using the encapsulation-dehydration technique resulted in embryo survival and root formation (30%) though complete regeneration was not recorded. Further experiments are underway to refine the cryopreservation protocol for this species including optimisation of the seed harvesting period as seed development stage is a critical factor in the success of a cryopreservation strategy in recalcitrant species.

OVEREXPRESSION OF MICRORNA397 AND ITS EFFECTS ON THE EXPRESSION OF LACCASES INVOLVED IN LIGNIN BIOSYNTHESIS

Nguyen D.Q.1, Furbank R.2, Eamens A.1 and Grof C.1 1Centre for Plant Science, School of Environmental and Life Science, University of Newcastle, Callaghan, NSW, 2308, Australia. 2Research School of Biology, Australian National University, ACT, 2600, Australia.

Lignocellulosic biomass derived from C4 grasses and other energy feedstocks is currently viewed as an attractive, alternative energy source. However, hydrolysis of lignocellulosic biomass to obtain fermentable sugars for the production of bioethanol is an inefficient process due to the presence of a complex network of lignin. Lignin, the third most abundance component in plant cell walls, is a recalcitrant biopolymer that consists of 3 monolignols (p-coumaryl, coniferyl and sinapyl alcohols), the precursors of 3 monomeric lignin subunits, p-hydroxyphenyl (H), guaicyl (G) and syringyl (S), respectively. Monolignols are initially synthesised in cytosol and transported to the apoplasm before they are oxidised and incorporated into the plant cell wall. The incorporation of lignin subunits into the plant cell wall has been reported to be controlled by a class of laccase enzymes. The genetic manipulation of LACCASE (LAC) gene function, or the activity of the post-transcriptional regulator of LAC gene expression, microRNA397 (miR397), is a potentially viable approach to modify plant cell wall composition and structure. Such an approach may maximise the efficiency of hydrolysis, and therefore, enhance the yield of bioethanol, while reducing input costs. The initial goal of this study was to identify putative Setaria viridis LACs (SvLACs), and a targeting small RNA (sRNA), and to profile the expression domains of both the miRNA and its targeted LAC genes across 13 developmentally distinct tissues of S. viridis. Secondly, to transform wild-type Arabidopsis thaliana (Col-0) plants with a miR397 overexpression construct to allow the study of the efficacy of the sRNA in knocking down the expression of Arabidopsis LAC target genes. Finally, the cell walls of the miR397 overexpression plant line will be anatomically and chemically analysed to compare their lignin content to that of non-transformed, wild-type Arabidopsis cell walls.


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MYRTLE MYTH? MANUKA MAKES MORE NECTAR ON IMPOVERISHED SOILS?

Noe S.1, Clearwater M.1, Manley-Harris M.1, Richardson S.2 and Whitehead D.2 1University of Waikato, NZ. 2Landcare Research Manaaki Whenua, Lincoln, NZ.

It has been proposed that the extreme soil nutrient poverty and intense fire regime of Australia results in plants having excess carbon resources. They are still able to photosynthesise, but do not have the soil nutrients with which to combine the produced carbohydrates for nutrient rich tissue growth. This is hypothesised to have resulted in many Australian plants evolving unique, anomalous sinks for this extra carbon, as carbon rich compounds such as wood, oils, and nectar. The manuka plant (Leptospermum scoparium, Myrtaceae) occurs both in Australia and New Zealand, the nectar from its flower being the raw material for the production of the world-renowned manuka honey. Beekeepers have recounted that the best mānuka honey yields are from manuka growing on impoverished soils, suggesting manuka exhibits anomalously high nectar production on poor soils, possibly because of excess carbon. As yet there has been no thorough testing of this theory. This research investigates the effects of soil fertility on manuka growth, flowering, and nectar production, aiming to answer the question – does manuka make more nectar on impoverished soils? Potted manuka plants of two NZ provenances growing in an open field will be supplied with nutrient treatments testing the effects of soil nitrogen and phosphorus. Vegetative growth and photosynthesis; flowering timing, duration, and intensity; and nectar production will be monitored over two successive seasons. Examining the role of soil nutrient status in determining nectar flow for honey production purposes will improve our understanding of the highly variable nature of nectar production. The data generated from this research will contribute to a landscape scale model predicting nectar resources available through time for honey production.

MAGNESIUM DEFICIENCY-INDUCED MAGNESIUM UPTAKE REVEALED BY RADIOTRACER STUDIES

Ogura T.1, Kobayashi N.I.1, Suzuki H.2, Iwata R.3, Nakanishi T.M.1 and Tanoi K.1, 4 1Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan. 2National Institute of Radiological Sciences, National Institutes for Quantum and Radiological Science and Technology, 4-9-1 Anagawa, Inage-ku, Chiba-city, Chiba 263-8555, Japan. 3Cyclotron and Radioisotope Center, Tohoku University, 6-3 Aoba, Aramaki, Aoba-ku, Sendai, Miyagi 980-8578, Japan. 4PRESTO, Japan Science and Technology Agency (JST), 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan.

It has been hypothesized that plants regulate the membrane transport of Mg2+ to maintain the optimum concentration in cells for growth. However, some studies suggest that gene expression of Mg2+ transporters does not increase in response to low Mg2+condition. To investigate the mechanism of Mg2+ transport, we tried to elucidate whether and how Mg2+ uptake responds to environmental changes. To investigate Mg2+ uptake rate in root, we prepared 1-week-old Arabidopsis plants and performed Mg2+uptake experiments using agarose plates with 28Mg as a radiotracer. The Mg2+ uptake rate in root increased in response to low Mg2+ condition for 24 h, however, this increase was not observed in the Mg2+ transporter mutants atmrs2-4 and atmrs2-7. In addition, AtMRS2-4 and AtMRS2-7 gene expression in wild-type Arabidopsis did not increase in low Mg2+ condition. In wild-type Arabidopsis, Mg2+ uptake rate increased within 30 minutes at earliest. The low-Mg2+-induced increase was repressed within 5 minutes when Mg2+ was resupplied. An additional experiment to investigate Mg2+ uptake inhibition with other cations under Mg2+sufficient condition showed that the Mg2+ uptake system was sensitive to a number of divalent and trivalent cations, but not to K+.

EXPLORING THE PHYSIOLOGICAL AND BIOCHEMICAL CHANGES IN TOMATO (Lycopersicon esculentum Mill.) UNDER SALT STRESS AND RECOVERY: ROLE OF ANTIOXIDANT DEFENSE AND GLYOXALASE SYSTEM FOR ATTUNING SALINITY TOLERANCE

Parvin K.1, 2, Bhuyan M.H.M.B.1, 3, Hasanuzzaman M.4, Nahar K.5, Mohsin S.M.1, 6, Hossain M.S.1 and Fujita M.1 1Laboratory of plant stress response, Department of Applied Biological Science, Faculty of Agriculture, Kagawa University, MikiCho, Kita-Gun, Kagawa 761-0795, Japan. 2Department of Horticulture, Sher-e-Bangla Agricultural University, Sher-e-Bangla Nagar, Dhaka-1207, Bangladesh. 3Bangladesh Agricultural Research Institute, Joydebpur, Gazipur-1701, Bangladesh. 4Department of Agronomy, Sher-e-Bangla Agricultural University, Sher-e-Bangla Nagar, Dhaka-1207, Bangladesh. 5Department of Agricultural Botany, Sher-e-Bangla Agricultural University, Sher-e-Bangla Nagar, Dhaka-1207, Bangladesh. 6Department of Plant Pathology, Sher-e-Bangla Agricultural University, Sher-e-Bangla Nagar, Dhaka-1207, Bangladesh.

Both stress and consequent episodes are highly sensitive to plant growth, physiological and biochemical processes. Hereafter, we investigated the plant responses to salinity and recovery process to find out the salt tolerance mechanism in tomato focusing the antioxidant defense mechanism and glyoxalase system. Hydroponically grown 16-d-old tomato seedlings (Lycopersicon esculentum Mill. cv. Pusa ruby) were treated with 150 and 250 mM NaCl for 4 days, and subsequently grown 2 days in salt free condition in order to observe post stress revival. Under saline condition, plants suffered from acute osmotic stress associated with lower relative water content (RWC) of leaf tissues, destruction of photosynthetic pigments, and higher proline content in a dose dependent manner. Henceforth, salinity induced oxidative stress through overproduction of reactive oxygen species (H2O2 and O2

•–), and methylglyoxal (MG); eventually damage the membranes (higher electrolyte leakage), and lipids (elevated malondialdehyde). Salinity also impaired the non enzymatic and enzymatic components of antioxidant defense system and disrupted redox balance. As a result the growth and development of tomato seedlings stagnated. But when the seedlings were allowed to grow in the saline free condition; a dramatic change was visible in the seedlings, and within 2-day of recovery, improved plant growth, water balance and chlorophyll synthesis was observed followed by restoration of the activity of antioxidant defense as well as glyoxalase systems, which scavenge the toxic ROS and lowered the lipid peroxidation and membrane damage. Thus, our result suggested that tomato plant can tolerate salinity, and can undergo a quick post stress recovery by enhancing antioxidant defense mechanism and glyoxalase pathway.

PROFILING THE ABIOTIC STRESS RESPONSIVE MICRORNA LANDSCAPE OF ARABIDOPSIS THALIANA

Pegler J.L., Grof C.P.L. and Eamens A.L. Centre for Plant Science, School of Environmental and Life Sciences, Faculty of Science, University of Newcastle, Callaghan 2308, New South Wales, Australia.

It is well established among interdisciplinary academics contemporary society is challenged with addressing and mitigating the negative consequences that accompany anthropogenic driven climate change. One such negative impact is the increased prevalence (duration and severity) of increasingly unfavourable environmental conditions such as (but not limited to) elevated temperature, drought and salinity: environmental conditions that are well documented to significantly contribute to a reduction in global agricultural crop yield. Confronted with the challenge of providing food security for 9.5 billion people by the year 2050, modern molecular agricultural biology aims to address this issue with the development of superior crop varieties, that is, genetically modifying key global food crops to harbour molecular modifications that result in the ability of the modified cropping species to maintain yield during times abiotic stress. In plants, microRNAs are central regulators of gene expression to; (1) influence all aspects of development; (2) mount a defence response against invading pathogens, and; (3) direct many of the physiological and/or phenotypical changes required by a plant to respond to abiotic stress. Via a high-throughput sequencing approach, the microRNA landscape of wild-type Arabidopsis thaliana exposed to salt, drought or heat stress, was profiled. This explorative analysis identified large microRNA cohorts responsive to each assessed stress. microRNA profiles are currently being experimentally validated via a modified reverse transcriptase quantitative PCR (RT-qPCR) approach. Concurrently, a standard RT-qPCR approach is being applied to quantify the expression of target genes of microRNAs experimentally validated to be abiotic stress responsive.


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SOURCE-SINK METABOLIC RELATIONSHIPS SHAPE β-AMINOBUTYRIC ACID-INDUCED RESPONSE ON FLAX GROWTH (LINUM USITATISSIMUM)

Pontarin N.1, 2, Tchoumtchoua J.1, Gredelj M.1, Quero A.1, Molinie R.1, Mathiron D.3, Van Bohemen A.I.1, Fontaine J.X.1, Sarazin V.2 and Mesnard F.1 1Biologie des Plantes & Innovation (BIOPI), UPJV, Amiens, France. 2Laboulet Semences, Airaines, France. 3PFA, Amiens, France.

At every moment of their lives, plants are constituted of organs at different stages of development. Therefore, plant response to any environmental stimuli could be considered the sum of different interrelated responses, each typical to the various physiological stages coexisting in the same plant. For leaves, the stage of development is generally assigned on the basis of their photosynthetic and metabolic independence, so we can distinguish three populations of leaves: source, transition and sink leaves. β-aminobutyric acid (BABA) is a non-protein amino acid synthesised by plants in a hormone-like response to stress. When exogenously applied, BABA is a powerful priming agent of plant natural defences, active against 80 different pathogens and pests, and a variety of abiotic stresses. Though, at the treatment doses, BABA engenders a growth delay. Within this context, this study aims to characterise BABA-induced response on flax growth in a spatio-temporal fashion. Firstly, flax source, transition and sink leaves were differentiated based on their relative growth rate and their metabolic composition via multivariate analysis. Secondarily, BABA-induced response on the growth of those leaf populations was evaluated during a time frame of ten days. The contribution of expansive and structural growth to the overall leaf growth was assessed by measuring fresh mass, dry mass, leaf surface, water content and transpiration. For further understanding, metabolic changes were investigated by metabolic profiling through GC-MS and LC-MS, and osmotic potential was measured. We observed that BABA-induced response was characterised by an early expansive growth retardation, followed by the recovery of a new homeostatic state, characterised by a lower deposition of biomass. The metabolic reorganisation played a central role in growth regulation, revealing how the various phases of the response were driven by the leaf populations, in different but coordinated fashions.

A TAQMAN-BASED ASSAY TO DETERMINE T-DNA ZYGOSITY IN GM FORAGE

Reid M., Crowther T., Winichayakul S., Roberts N.J., Bryan G. and Richardson K. AgResearch Ltd., Grasslands Research Centre, Private Bag 11008, Tennent Drive, Palmerston North 4442, New Zealand.

The identification of plants with a homozygous transgene is essential to the breeding of genetically modified forage grasses. Using rice as a model, we have developed a zygosity assay based on real-time qPCR and TaqMan hydrolysis probes, which allows us to readily distinguish between plants which are either hemizygous or homozygous for the transgene. Plants transformed with a single T-DNA (containing a synthetic DGAT/Oleosin construct designed to accumulate fatty acids in foliar tissue) were identified by Southern blot hybridisation and used to generate T1 families for these experiments. The segregation ratios of T1 progeny, using standard PCR, were as expected for an inbred species and the qPCR assay reliably differentiated hemizygous and homozygous individuals in each family. These results positively correlate with fatty acid methyl ester (FAMES) analyses, where a significant increase in the C18:1/C18:3 and C18:2/C18:3 fatty acid ratios was observed in homozygous progeny. Quantitative immunoblots performed using a sesame-oleosin antibody also positively correlated with the homozygous progeny identified via the qPCR assay results. This T-DNA zygosity assay is easily modified for use with other plant species and has been adapted for use in our GM ryegrass seed production program, where populations containing a homozygous DGAT/Oleosin construct are currently being developed for field trialling in the United States over the next five years. Development of the assay in rice will be presented.

EVOLUTIONARY DIVERSITY OF LIGHT PARTITIONING BETWEEN PHOTOSYSTEMS (FI) IN LEAVES OF HIGHER PLANTSSagun J.V.1, Badger M.R.2, Chow W.S.2 and Ghannoum O.1 1ARC Centre of Excellence for Translational Photosynthesis, Hawkesbury Institute for the Environment, Western Sydney University, Hawkesbury Campus, Locked Bag 1797, Penrith, NSW 2751, Australia. 2ARC Centre of Excellence for Translational Photosynthesis, Research School of Biology, Australian National University, Canberra ACT, 2601.The cyclic electron flux (CEF) around PSI is essential for efficient photosynthesis because it generates ATP thus balancing ATP/NADPH energy budget, and also protects PSI and PSII against photoinhibition. However, it is difficult to quantify CEF due to the absence of net product of cyclic electron flux. ΔFlux is an upper estimate of CEF and can be calculated as the difference between the total electron flux through PSI (ETR1) and the linear electron flux (LEFO2) through both photosystems. In this study, a method was developed to concurrently measure ETR1 and LEFO2 in leaf discs in CO2-enriched air by combining membrane inlet mass spectrometry with a Dual-PAM/F. Most importantly, the method also allowed for the calculation of fI which represents the fraction of absorbed light that goes to either PSI. A correct value of fI is important for estimation of ETR1 which can be calculated by multiplying fI by the absorbed irradiance (I) and leaf absorptance (~0.85) and PSI photosynthetic efficiency (YI). Value of fI is usually assumed to be 0.5 which means that 50% of the absorbed light is portioned to PSI. But it was hypothesized that this value is higher in C4 plants compared to C3 plants because C4 plants have two photosynthetic tissues. Values of fI are only known for a few plant species and have not been calculated for C4 species with different biochemical subtypes and other species such as ferns, liverworts and other gymnosperms. In line with previously published work, our study showed that fI for spinach is 0.5. For the C3 grass Panicum bisulcatum, fI was 0.4 and 0.5 in high and low light grown plants, respectively. Both high-light and shade-grown C4 species (Maize, Panicum antidotale, Panicum milaceum and Panicum maximum) had fI of 0.6. Values of fI from other species were also determined. Gingko biloba (gymnosperm), Marchantia polymorpha (liverwort) and Polypodium sp. (fern) had fI of 0.5, while, Wollemi nobilis (gymnosperm) had fI of 0.3. Using the new fI values increased ETR1 by 12% - 20% in C4 species and decreased ETR1 by 16% in the C3 grass. This also caused the calculated value for ΔFlux at 1,000 µmol m-2 s-1 to increase by 20% - 30% in C4 species and decrease by 30% in the C3 grass species. The method developed in this study to calculate fI appears to be reliable for screening several plant species. The values obtained can be used to correctly quantify CEF and further used for photosynthesis modelling.

CLIMATE CHANGE LEADS TO ALTERED FLOWERING PATHWAY

Samarth S.1, Jameson P.1, Kelly D.1, Lee R.2, Macknight R.2 and Turnbull M.1 1School of Biological Sciences, University of Canterbury, Private Bag 4800, Ilam Christchurch, New Zealand. 2Department of Biochemistry, University of Otago, Dunedin, New Zealand.

Mast flowering is synchronised highly variable flowering by a population of perennial plants such as Chionochloa (Poaceae), over a wide geographical area. The ΔT model hypothesizes that the size of the temperature difference between successive summers determines each year’s flowering intensity. Currently, in model plant species we know that the flowering process is regulated by various transcription factors and micro-RNAs in response to a temperature change. GIGANTEA-HD1-Hd3a is the main flowering pathway conserved among the monocot species. The current study deals with the identification of a possible pathway controlling temperature-mediated masting in plants. By translocating plants we demonstrated that giving a large positive ΔT produces heavy flowering in Chionochloa which is sometimes premature (winter instead of summer). Gene expression studies suggest that the premature flowering could be an attribute of GIGANTEA-dependent but HD1-independent flowering pathway. RNA-seq was employed to identify the key flowering gene(s) involved. The RNA-seq data were validated using RT-qPCR in samples from flowering and non-flowering plants over an altitudinal range across seasons. The study suggests that a large positive ΔT can provoke heavy flowering through two different pathways, one of which results in premature flowering. The change in the flowering response can decrease the reproductive competency and reproductive output of a plant species. Such changes can have also contrasting effects on the interactions between plant species and suggest that global change may skew flowering patterns in mast seeding plants.


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GENETIC ANALYSIS OF ROOT TRAITS ASSOCIATED WITH SALT-TOLERANCE IN A BARLEY MAPPING POPULATION

Brien C.1, Gilbert S.2, Mather D.3, Tyerman S.D.4 and Shelden M.C.4 1Australian Plant Phenomics Facility, The Plant Accelerator, University of Adelaide, Glen Osmond, SA, Australia. 2Adelaide Microscopy, The University of Adelaide, Adelaide, SA, Australia. 3School of Agriculture, Food and Wine, The University of Adelaide, Glen Osmond, SA, Australia. 4ARC Centre of Excellence in Plant Energy Biology, School of Agriculture, Food and Wine, University of Adelaide, Glen Osmond, SA, Australia.

Abiotic stresses are major causes of crop yield losses in agriculture significantly impacting on sustainability. Barley (Hordeum vulgare L.) is the most salt-tolerant cereal crop with excellent genetic resources and therefore is a good model to study salt tolerance mechanisms in cereals. Salinity results in a reduction in root growth, however, some species can maintain root elongation at salt concentrations that inhibit root growth; an adaptive mechanism to ensure seedling establishment and maintain water and nutrient uptake. We aim to identify the key genes and pathways in barley roots that are involved in both perceiving osmotic changes in the soil and influencing root elongation, ultimately to increase salinity tolerance in crops. Barley cv. Clipper (malting barley) and Sahara (North African landrace 3771), have previously been shown to have a contrasting root growth phenotype and metabolic profile in response to the early phase of salinity stress. To further characterise these two genotypes, we have developed a method using laser ablation inductively coupled proton mass spectrometry to map the distribution of Na+ and K+, in the root tip. The results show that in response to salt stress the distribution of Na+ differs developmentally along the root tip and between genotypes. To elucidate the genetic basis for these mechanisms, a Clipper x Sahara DH mapping population has been screened for shoot and root phenotypic traits in response to salt stress. We are currently conducting a genetic analysis of the mapping population using Quantitative Trait Loci analysis and RNAseq to elucidate the genes involved in the maintenance of root elongation in response to salt stress. This study highlights the importance of utilizing spatial profiling and will provide us with a better understanding of abiotic stress response in plants at the tissue and cellular level.

EFFECTS OF PHOSPHATE AND PH ON ARSENATE UPTAKE KINETICS AND ARSENIC TOXICITY IN TWO WHEAT (TRITICUM AESTIVUM L.) CULTIVARS OF DIFFERENT ARSENATE TOLERANCE

Shi G.L.1, 2, 3, Jiang Y.J.1, Ma H.X.1, Chen Y.L.3, Song G.C.1, Lou L.Q.2 and Rengel Z.3 1Institute of Food Crops, Jiangsu Academy of Agricultural Sciences, Nanjing, 210014, P.R. China. 2College of Life Sciences, Nanjing Agricultural University, Nanjing, 210095, P.R. China. 3The UWA Institute of Agriculture, and UWA School of Agriculture and Environment, The University of Western Australia, Perth, WA 6009, Australia.

Two wheat (Triticum aestivum L.) cultivars differing in As-tolerance were used to investigate the effects of phosphorus (P) concentration and nutrient solution pH on As(V) toxicity and As(V) uptake kinetics, and to illustrate the mechanism of As(V) tolerance in wheat seedlings. Low pH and low phosphate concentration enhanced wheat uptake of As, resulting in high As toxicity. The As(V)-tolerant cultivar MM45 exhibited higher relative root elongation than non-tolerant cultivar HM29 in all treatments, except that no genotypic difference was recorded for the solution P at 100 µmol L–1 or greater. Wheat seedling As(V) tolerance was positively correlated with P concentration in roots and shoots. In short-term (30 min) As(V)-uptake kinetics experiments, the maximum influx rate (Vmax) of As(V) increased with decreasing solution pH (from 7.0 to 6.0). Compared with HM29, although MM45 had lower Vmax, its Michaelis–Menten constant (Km) did not exceed that of HM29 in any of the treatments. The Vmax values of both cultivars were not significantly affected by phosphate treatments, except for HM29 which had significantly higher Vmax value in the presence of phosphate at pH 7.0. The Km values of the two cultivars increased by 9- to 20-fold when phosphate was present as opposed to absent from the uptake solution. This study showed that the Vmax values are mainly increased by high pH and As(V) uptake Km is mainly increased by phosphate presence. Decreased As(V) influx rates during early stages and increased P concentration in plant tissues are associated with increased As tolerance in wheat seedlings.

IS PEPTIDE TRANSPORT IMPORTANT FOR THE LEGUME: RHIZOBIA SYMBIOSIS?

Gavrin A.2, Brear E.M.2, Mohd Noor S.N.2, Loughlin P.C.2, Chen C.2, Wen Z.2, Kaiser B.N.2, Day D.A.3, Rentsch D.4, 2 and Smith P.M.C.1, 2 1La Trobe University, Bundoora, Victoria. 2University of Sydney, Sydney, NSW. 3Flinders University of South Australia, Adelaide, SA. 4University of Bern, Bern, Switzerland.

The symbiosome membrane is a plant-derived membrane that surrounds rhizobia in nitrogen-fixing nodules, separating them from the plant cytoplasm. The membrane controls the transport of compounds into and out of the symbiosome and is likely to be a key point of regulation for the interaction between legumes and rhizobia in the symbiosis. We recently completed a proteomic analysis of the soybean symbiosome membrane (Clark et al. 2015 MCP 14:1301-22.). Among the proteins we identified were many putative transport proteins including members of families encoding transporters for sulphate, calcium, and hydrogen ions, as well as aquaporins. A number of proteins were part of the nitrate transporter 1/ peptide transporter family (NPF), which includes a range of transporters for nitrogen containing compounds including nitrate, peptides, glucosinolates, and plant hormones. We have used yeast complementation to show dipeptide transport by one of the NPF members that is expressed specifically in infected cells of mature nodules. Another transporter identified on the symbiosome membrane has been characterised as an oligopeptide transporter from the Yellow-stripe-like family. When we silenced the genes encoding these peptide transporters nitrogen fixation was inhibited suggesting an important role for peptide transport in determinant nodules.

FUNCTIONAL CHARACTERIZATION OF A NEW AMMONIUM TRANSPORTER IN SOYBEAN

Uddin M.K., Wen Z. and Kaiser B.N. Centre for Carbon, Water and Food, School of Life and Environmental Sciences, The University of Sydney, Brownlow Hill, NSW, 2570.

Biological nitrogen fixation (BNF) is an alternative nitrogen source to commercial synthetic nitrogen fertilizers. Soybean (Glycine max) is a major legume crop used globally in rotation with non-nitrogen fixing plants and has a calculated annual N2 fixation capacity of 16.4 Teragram (Tg) N, representing 77% of the total nitrogen fixed by all grain legumes. Soybean like other legumes form a symbiotic partnership with nitrogen fixing soil rhizobia bacteria. The symbiosis results in the formation of the root nodule where invaded bacteria (bacteroids) express the enzyme nitrogenase, which reduces atmospheric N2 to NH3. Fixed NH3 is rapidly protonated to NH4

+ and assumed transported across cellular membranes and then assimilated into amino acids for export to the shoot. Ammonium transport proteins (AMTs and AMFs) are considered the primary mechanisms responsible for the transport of NH4

+ in plants; however their role in legume nodule remains poorly understood. To address this, we have begun the process of characterising select members of the AMT and AMF families shown to be expressed in soybean nodules. We have cloned the uncharacterised GmAMT2;1 transport protein from nodule cDNA. GmAMT2;1 was found to functionally complement a yeast strain deficient in ammonium transport while chemical flux analysis indicated GmAMT2;1 behaves as a high-affinity ammonium transport protein. Using real time qPCR, GmAMT2;1 was found broadly expressed across the plant but showed elevated expression in the nodule as plants matured and began to fix atmospheric nitrogen. The functional role of GmAMT2;1 will be tested against other AMT and AMF genes found expressed in nitrogen fixing nodules.


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NOVEL APPROACHES FOR VISUALISING AND CHARACTERISING MICRO ROUGHNESS OF SURFACTANT TREATED LEAF SURFACES

Vittal L.V.M.1, Jones P.W.3, Killick A.R.3, Boyd B.J.2 and Cahill D.M.1 1Schools of Life and Environmental Sciences, Deakin University, Geelong Waurn Ponds Campus, Victoria, Australia,. 2Drug Delivery Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University Parkville Campus, Victoria, Australia. 3Victorian Chemical Company, Coolaroo, Victoria, Australia.

Surfactants are agrochemical additives that enable penetration of active ingredients into foliar surfaces. Surfactants interact with epicuticular waxes (EW) on foliar surfaces and cause structural change which can compromise surface integrity. Surface structural changes are usually visualized using electron microscopy (EM) and are recorded by visually inspecting and allotting a ‘phytotoxicity’ score, subject to confirmation bias. The purpose of this study was to characterize changes in EW post treatment with four commercial surfactants on the leaf surfaces of canola (Brassica sp.), wild radish (Raphanus sp.), wheat (Triticum sp.) and wild oats (Avena sp.). Two different methods were used to analyse the leaf surfaces: 1) Treated/ untreated surfaces were imaged using EM and their lacunarity (fractal dimension) was calculated using FracLac, a 2D approach and, 2) surfaces were profiled using an optical profilometer, a 3D approach. Changes in the wax morphology was clearly observed using both techniques. Leaves treated with a high concentration of surfactant showed higher lacunarity. Change in profile roughness was measured for a section, Ra, as well as for the whole area of the profile. Sa. The optical profiler also doubled as a microscope allowing visualization of waxes in their native state without any sample preparation steps. We propose that these two techniques when used in tandem are reliable methods to physically evaluate surfactant phytotoxicity on leaf surfaces. The methods will also find use more broadly for plant surface analysis.

UNRAVELLING THE RESPONSE OF THE ALTERNATIVE PATHWAY OF MITOCHONDRIAL ELECTRON TRANSPORT TO ABIOTIC STRESS IN PLANTS

Waterman C.D., Sweetman C., Rainbird B.M., Day D.A., Jenkins C.L.D. and Soole K.L. College of Science and Engineering, Flinders University of South Australia, GPO Box 5100, Adelaide, SA, Australia.

Plant mitochondria are comprised of a complex network of respiratory proteins regulating metabolism and driving production of ATP. Among these are the well characterised cytochrome c (CytC) pathway enzymes. Lesser known is the alternative pathway (AP). Unlike the conventional CytC pathway, the AP role remains unclear. Where the CytC pathway contributes to a proton gradient and ATP production, the AP does not. Utilising NAD(P)H, the first enzymes in this pathway, (the type II dehydrogenases) do not pump protons. Analogous to cytochrome c oxidase, the alternative oxidase (AOX) oxidises ubiquinol but again contributes no protons across the membrane. This begs the question, why is a pathway with the potential to be wasteful not only conserved across the plant kingdom, but also conserved across many isoforms. Transgenic knockdown and overexpressing models have demonstrated across multiple organisms that some of these enzymes are crucial to an adequate stress response. In Arabidopsis, an isoform of AOX, AOX1a, has been shown to not only be important to the stress response but improve the plants ability to deal with various abiotic stressors when overexpressed. Recent work in our laboratory has shown that plants over-expressing both AOX and external NADH dehydrogenase NDB2 exhibit enhanced recovery from combined drought and high light stress. Here we have explored transcriptional and metabolic networks in these transgenic lines to investigate the basis to this capacity for enhanced stress tolerance.

DROUGHT RESISTANCE IN RYEGRASS INVOLVES MORPHOLOGICAL PLASTICITY- OSMOTIC ADJUSTMENT INTERPLAY

Weerarathne L.V.Y.1, Dong W.2, Nie M.2, Wang Y.2, Lopez I.1 and Matthew C.1 1School of Agriculture and Environment, Massey University, PB 11-222, Palmerston North, New Zealand 4442. 2Ningxia University, 489 Helanshan West Rd, Yinchuan, Ningxia Hui Autonomous Region, China 750021.

Recent Massey University research into improving water-use efficiency (WUE)-assisted drought resistance in commercial perennial ryegrass varieties is outlined. Two clonal replicates of 220 genotypes of three market-leading cultivars were evaluated to identify elite genotypes possessing high WUE (g DM/g H2O used). Plants aged two months, after root system development in soil at near field capacity, were grown on for a month under strong moisture deficit, and then water relations traits were measured, including: dry matter yield (DM, g), pot water use per gram of DM (WU, g/g), leaf water potential (LWP, bars), leaf osmotic potential (LOP, bars), leaf relative water content (RWC, %), relative growth rate (RGR, g/g/day), root mass (for 4–20 cm and 20–50 cm soil depths), soil moisture content at 30–40 cm depth and, post-cutting regrowth score. Data were subjected to a principal component analysis (PCA) to evaluate trait associations of drought-response. For selected contrasting groups of genotypes chosen by PCA scores, WU ranged from 278±12 g/g (averaged for the best 25 genotypes) to 578±49 g/g (averaged for the least efficient 15 genotypes). The elite germplasm was characterized by more negative LOP (–24 bars) and higher RWC (67%) than for least efficient genotypes, under low LWP (–11 bars); indicative of osmotic adjustment (OA). In this experiment, plants demonstrating high RGR in soil near field capacity, also formed comparatively high below ground root mass, which, coupled with turgor maintenance by OA, enhanced water acquisition as the drought resistance strategy in the elite perennial ryegrass genotypes. Key words: perennial ryegrass, WUE, OP, RGR.

INFECTION BY PHYTOPHTHORA PARASITICA INDUCES DISTINCT CLASSES OF SMALL RNA IN ARABIDOPSIS ROOTS

Zhong C.C.1, 2, Wang M.B.2 and Shan W.X.1 11. Northwest Agricultural and Forestry University, Yangling, Shaanxi, China. 2CSIRO Agriculture and Food, Clunies Ross Street, Canberra, ACT 2601, Australia.

Oomycetes, including Phytophthora, are eukaryotic microorganisms that are evolutionarily similar to algae, and can cause damages to both agricultural production and natural ecosystems. Small RNAs (sRNAs) are important regulators in eukaryotes, and have been implicated in plant defence responses to pathogens. However, how sRNAs contribute to plant-Phytophthora interactions remains unclear. In this study we used Arabidopsis thaliana-Phytophthora parasitica as a pathosystem to investigate the role of sRNAs in plant response to Phytophthora. We analysed sRNA profiles in the roots of Arabidopsis plants infected with P. parasitica at successive stages of infection. sRNA deep sequencing resulted in approximately 10 million reads for each infection stage, of which around 70% matched perfectly to the Arabidopsis genome. Comparative analysis of sRNA population between infected and uninfected roots revealed a general induction of 21-nt and 24-nt canonical sRNAs at the cysts germination (3 hpi) and haustoria appearing (6 hpi) stages, and a strong induction of a novel class of 28-nt sRNAs at abundant haustoria (12 hpi) and hyphae colonization (24 hpi) stages. Of the canonical sRNAs, we identified several differentially expressed miRNAs that have target genes involved in plant immunity and differentially expressed in infected roots. In addition, we identified 24-nt small interfering RNAs (siRNAs) that target promoters or introns of Arabidopsis genes that showed differential expression upon Phytophthora infection, suggesting the involvement of siRNA-directed DNA methylation in Phytophthora response. The 28-nt size class are derived mainly from intergenic regions, and their biogenesis appears to be linked with the RNA-directed DNA methylation pathway. Details of the findings will be presented.


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IMPROVING PLANT CARBON FIXATION THROUGH ENGINEERING RUBISCO IN E. COLI

Zhou Y., Conlan B. and Whitney S. Research School of Biology, Australian National University.Enhancing the light and carbon capture reactions of photosynthesis are seen as prime opportunities for improving crop productivity and yield. A significant research focus has been on identifying strategies to enhance the CO2-fixing potential of the photosynthetic enzyme Ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase (Rubisco). Identifying mutations that enhance the carboxylation properties of plant Rubisco has proven particularly difficult. A significant obstacle is the extensive cocktail of ancillary proteins (chaperonin/chaperones) needed to assemble the 8 large and 8 small subunits of the 520 kDa plant L8S8 holoenzyme. Fortunately, the cocktail of ancillary proteins required for producing bacterial L8S8 Rubisco isoforms is less than plant Rubisco. This enables some bacterial Rubiscos to be expressed in both E. coli and plant chloroplasts. My project seeks to take advantage of the “E. coli & chloroplast expression capability” of a Rubisco sourced from a photosynthetic bacterium. In the first research stage, the kinetics of the bacterial Rubisco are being engineered by directed evolution using a novel Rubisco Dependent E. coli (RDE) screen re-designed from that published (1). The new RDE3 screen incorporates features tailored for selecting Rubisco mutants with improved carboxylation properties. The second research stage of the project involves transplanting the evolved bacterial Rubiscos by plastome transformation into leaf chloroplasts to translationally test their capacity to improve photosynthesis and plant growth. Towards this objective tobacco lines producing compatible components for maximising the assembly and metabolic regulation of the bacterial Rubisco are being generated. An update on progress with both research stages will be presented. (1) Wilson R.H., et al. (2018). JBC, 293, 18-27.

ANCIENT ORIGIN OF AMNIOTE MUSCLE DEVELOPMENT

Boisvert C.A.1, 2, Tulenko F.2, Rios A.3, Sieiro D.4, Davis M.5, Uy B.6, Marcelle C.2 and Currie P.D.2 1MLS, Curtin University, Kent street, Bentley, 6102 WA, Australia. 2ARMI, Monash University, 15 Innovation walk, Clayton, Vic 3800, Australia. 3PMCPO, Heidelberglaan 25, 3584 CS Utrecht, Netherlands. 4Harvard Medical school, Harvard University, 25 Shattuck St, Boston, MA 02115, USA. 5MCB, Kennesaw state university, 1000 Chastain Road Kennesaw, GA 30144, USA. 6Caltech, Caltech,1200 EAST CALIFORNIA BOULEVARD, PASADENA, CA 91125, USA.

Zebrafish (actinopterygians), mice and chicks (amniotes) are useful developmental models to understand muscle development and human diseases. However, their early development mechanisms are different in timing, morphology and mechanisms. In zebrafish, the first muscle fibers form prior to somite formation (adaxial cells), this is followed by somite rotation and the establishment of the external cell layer (single cell layer) which contributes to myoblasts for further muscle formation. In contrast, amniotes develop their first myoblasts from the dermomyome, a multicellular epithelial derivative of the somite. In order to understand the evolutionary origin of muscle development in vertebrates and reconcile the divergent developmental modes of extant vertebrates, we immunostained growth series of the elephant shark (basal jawed vertebrate), the lamprey (jawless vertebrate) and paddlefish (basal actinopterygian). Building on our results that elephant sharks display an amniote like dermomyotome with four epithelial lips expressing Pax7, we are presenting novel findings on lamprey muscle development. Our results suggest that muscle development in lamprey is preceded by a single celled simplified dermomyotome whereas muscle formation in paddlefish is very similar to that of zebrafish. This suggests that the amniote dermomyotome is an ancient feature of vertebrates which evolved from a single celled dermomyome present in lampreys. Early formation of myocytes adaxial to the notochord is a feature of actinopterygians and could have evolved at the base of the lineage to speed up development.

EFFECT OF MATERNAL DIETARY GLYCEMIC INDEX ON OFFSPRING METABOLIC PROGRAMMING IN MICE

Campbell G.J., Lucic Fisher S.G. and Bell-Anderson K.S. Charles Perkins Centre, School of Life and Environmental Sciences, University of Sydney, Australia.

The glycemic index (GI) is a measure of the quality of carbohydrate with regards to its effect on blood glucose levels, with low GI diets being associated with improved metabolic health, both in humans and rodents. However, present research is unable to elucidate the mechanism(s) responsible, largely due to confounding factors associated with low GI diets. Isocaloric rodent diets differing only in the base sugar carbohydrate (high GI: glucose, sucrose; low GI: isomaltulose, fructose) were constructed such that fibre content was well controlled, but GI varied, as confirmed through in vivo GI testing in mice. To examine the effect of maternal dietary GI on offspring metabolic programming, embryos and pups born of C57BL/6 mothers fed these diets or chow were compared. Preliminary results show embryos of chow-fed mothers were significantly heavier than embryos from glucose and isomaltulose-fed mums, and had significantly heavier livers and spleens than embryos of all other groups. Male pups were heavier, leaner, and more glucose intolerant than females across all diets. While we observed no differences in bodyweight and other variables amongst female pups, male pups exhibited greater variation in these parameters and were affected by maternal and postnatal diet. Chow, glucose and sucrose male pups were heavier than isomaltulose and fructose pups from weaning to 12 weeks. Fructose and glucose male pups had higher insulin peaks at 12 weeks of age, while there was no difference in basal blood glucose levels. Maternal dietary sugars exhibit a limited effect on offspring metabolic health, but only in male pups. Compared with chow, the sugar-diet pups were lighter, fatter and less glucose tolerant.

TGIF1, A NEW PLAYER IN FEMALE GONADAL DEVELOPMENT

Estermann M.A., Major A.T., Hirst C.E. and Smith C.A. Department of Anatomy and Developmental Biology, Monash Biomedicine Discovery Institute, Monash University, Clayton, Victoria 3800, Australia.

The embryonic gonad is the only organ inside our body with the ability to transform into two completely different organs: Testis or Ovaries. We use the chicken as a model organism to understand these processes. In developing chicken embryos, the gonads form on the medioventral surface of the mesonephric kidneys at embryonic day (E) 3, equivalent to Hamburger and Hamilton stage (HH) 19. At this stage, they are undifferentiated or bipotential. At E6 (HH29), gonads begin morphological differentiation into testes in ZZ embryos or unilateral ovary in ZW embryos. A previous gonadal sex differentiation RNA-seq experiment performed in our lab revealed that Transforming growth-induced factor 1 (TGIF1) is differentially expressed between males and females after the onset of sex differentiation, with higher levels in female gonads at embryonic day 6 (stage 29 HH). TGIF1 belongs to the superfamily of TALE homeodomain proteins that control many biological processes, including embryonic development, cell proliferation, and differentiation. To characterize the expression pattern of TGIF1 in the chicken gonads we performed whole mount in situ hybridization and evaluated transcripts localization in male and female urogenital systems at E4.5, E6.5 and E8.5. We found stronger gonadal staining in females than males after sex differentiation (E6.5 and E8.5). To evaluate the possible role in female gonadal differentiation processes we performed overexpression experiments. Preliminary results show that overexpression in male gonads turned them into a more female phenotype. This is the first report of TGIF1 playing a role in gonadal differentiation. We hypothesize that TGIF1 is one of the master genes controlling female gonad development.


POSTERS


POS-WED-054A POS-TUE-055

MECHANICAL STRAIN REGULATES THE HIPPO PATHWAY IN DROSOPHILA

Fletcher G.C., Diaz-De-La-Loza M.C., Borreguero-Munoz N., Holder M., Aguilar-Aragon M. and Thompson B.J. The Francis Crick Institute 1 Midland Rd London NW1 1AT United Kingdom.

Animal cells are thought to sense mechanical forces via the transcriptional co-activators YAP/TAZ, whose sole Drosophila homolog is named Yorkie (Yki). In mammalian cells in culture, artificial mechanical forces induce nuclear translocation of YAP/TAZ. Here we show that physiological mechanical strain can also drive nuclear localisation of Yki and activation of Yki target genes in the Drosophila follicular epithelium. Mechanical strain activates Yki by stretching the apical domain, reducing the concentration of apical Crumbs, Expanded, Kibra and Merlin and reducing apical Hippo kinase dimerisation. Overexpressing Hippo kinase to induce ectopic activation in the cytoplasm is sufficient to prevent Yki nuclear localisation even in flattened follicle cells. Conversely, blocking Hippo signalling in warts mutant clones causes Yki nuclear localisation even in columnar follicle cells. We find no evidence for involvement of other pathways such as the Src42A kinase in regulation of Yki. Finally, our results in follicle cells appear more generally applicable to other tissues, as nuclear translocation of Yki is also readily detectable in other flattened epithelial cells such as the peripodial epithelium of the wing imaginal disc, where it promotes cell flattening.

DEFINING THE ROLE OF BETA-CATENIN IN MUSCLE REGENERATION

Cui S.1, Li L.1, Makarenkova H.P.2, Downes M.3 and Meech R.1 1Department of Clinical Pharmacology, Flinders University, South Australia 5042, Australia. 2Department of Cell Biology, The Scripps Research Institute, La Jolla, CA 92037, USA. 3Gene Expression Laboratory, The Salk Institute, La Jolla, CA 92037, USA.

Satellite cells are the resident stem cells of skeletal muscle; their activation after injury generates transit-amplifying myoblasts that differentiate to replace damaged myofibers. Canonical Wnt signalling controls myoblast differentiation in vitro and in vivo. However, the role of the Wnt effector β-catenin in adult myogenesis has been contentious, and its mechanism(s) of action unclear. We examined the cell autonomous functions of β-catenin during differentiation using CRISPR-generated β-catenin null adult mouse myoblasts. β-catenin was essential for morphological differentiation and timely deployment of the myogenic gene program. β-catenin null myoblasts showed a rounded phenotype and increased rate of proliferation; after treatment with Wnt, they showed delayed acquisition of the myogenic gene expression program. Fusion, elongation and alignment were impaired, and myogenic gene expression was not coordinated with cytoskeletal and membrane remodelling events. We performed rescue studies with mutant forms of β-catenin that cannot interact with TCF/LEF factors or with α-catenin at membrane junction complexes. Interaction of β-catenin with TCF/LEF factors was not required for differentiation; whereas loss of interaction of β-catenin with α-catenin impaired differentiation. Genome-wide epigenomics showed that β-catenin is required for Wnt-mediated enhancement of MyoD binding to DNA and for activation of myogenic target genes. Overall, we propose that β-catenin acts in the nucleus through its binding partner MyoD to activate the myogenic differentiation program; the membrane-associated β-catenin pool may also control cell-cell interactions. While β-catenin-TCF/LEF complexes are not required for differentiation, they are likely to be involved in feedback to control levels of β-catenin to prevent precocious/excessive myoblast fusion. We also show how specific inhibition of different β-catenin-cofactor-coactivator complexes could be used to curtail pro-fibrotic signalling, whilst maintaining essential pro-differentiation functions.

PHENYTOIN INDUCED MALFORMATIONS, FETAL HYPERGLYCAEMIA: A ROLE FOR ARACHIDONIC ACID?

Howe A., Farrell E., Oakes D.J. and Ritchie H. School of Medical Sciences, Univeristy of Sydney.

Background: Phenytoin (Phe) controls epilepsy, so women must remain on the drug during pregnancy. It is a human teratogen causing cleft lip and maxillary hypoplasia. Using an animal model developed by the authors, a teratogenic dose of phenytoin was associated with acute maternal hyperglycaemia and an increased risk of malformations. A previous study demonstrated increased serum levels of arachidonic acid (AA) were associated with decreased malformations in offspring of diabetic rats (Reece et al., 1996. Am J of Obst and Gyn, 175(4): 818-823). Aim: To determine whether dosing with AA ameliorates the teratogenic effects of phenytoin. Method: Pregnant rats were given a teratogenic dose of Phe during the critical period of craniofacial development followed by an injection of arachidonic acid (PheAA) or insulin (PheIns). Control rats received either Phe or AA. Blood glucose levels were measured at 2, 4 and 24 hrs later. At the end of pregnancy, fetuses were examined for the presence of malformations. Malformations ranged from cleft lip to moderate or severe maxillary hypoplasia. Result: Phe and PheAA rats showed increased glucose levels (2 and 8 hrs) in the dams and fetus, unlike PheIns or controls (saline, or AA only) rats. Treatment produced cleft lip and maxillary hypoplasia in all the Phe exposed embryos. Co-administration of insulin (PheIns) reduced both clefting and severity of maxillary hypoplasia, whereas, co-treatment with AA (PheAA) reduced the rate but not severity of malformations. Conclusion:Results indicate reduced hyperglycaemia (by insulin) was associated with reduced rate and severity of Phe- induced malformations. AA did not reduce, but in fact, increased the severity of Phe- induced malformations. This suggests acute hyperglycaemia (and associated malformations) induced in our model is not related to the AA deficiency that occurs with the chronic hyperglycaemia reported in both in vitro and in vivo models of diabetic embryopathy.

WNT5A CONTROLS URETEROVESICAL JUNCTION FORMATION BY MODULATING SHH

Yun K., Hall M.D. and Perantoni A.O. National Cancer Institute, Frederick, MD 21702 USA.

Ureterovesical junction formation depends upon a series of morphogenetic events which begins with the insertion of the nephric duct into the cloacal epithelium and apoptotic degeneration of the common nephric duct at the urogenital sinus and ends with the repositioning of the ureter and its subsequent connection with the bladder. Mutations in Wnt5a or its predominant receptor Ror2result in hydronephrosis/hydroureter due to a failed ureterovesical connection, but how Wnt5a regulates the ureter maturation process has not been established. Here we determine the underlying mechanism responsible for this abnormality in the mouse. The initial insertion of the nephric duct into the cloacal epithelium occurred normally in Wnt5a mutants. However, deletion of Wnt5aprior to E10.5 in the cloacal region led to a persistent common nephric duct during ureter maturation by reducing cell death in the duct. Concomitant with Wnt5a loss, Shh expression in the cloacal epithelium was increased in mutants, and Shh haploinsufficiency rescued cell death in the common nephric duct and suppressed the hydronephrosis/hydroureter phenotype. Furthermore, constitutive activation of Shh activity in the cloacal region induced the hydronephrosis/hydroureter phenotype and caused a reduction of cell death in the common nephric duct, suggesting that Wnt5a functions, at least in part, through its inhibition of Shh. This study identifies a new mechanism for ureterovesical junction formation that is dependent on the regulation of cell death in the nephric duct and implicates Wnt5a in the repression of Shh in the cloacal epithelium to facilitate degeneration of the common nephric duct and ureter insertion.


POSTERS

POS-TUE-57

POS-WED-058 POS-TUE-59

POS-WED-056THE A/HEJ MOUSE: A DYSFUNCTION IN SEX DEVELOPMENT

Robinson J.1, 2, Graham A.1 and Kalitsis P.1, 2 1Murdoch Children’s Research Institute. 2University of Melbourne.

Expression of the mammalian Y chromosome gene, Sry, is the trigger for development of a testis in males. In mice, the action of this gene is tightly regulated in both space and time, following a distinctive wave pattern of expression across the gonad over a very brief window of embryonic development. Failure of Sry expression to adhere to this precise profile, by expression reduction or delay, can result in defects in the development of the gonad. This can present as sex reversal, producing an XY female, or in the development of a gonad which consists of both ovarian and testicular tissue (an ovotestis). The A/HeJ mouse strain has been previously shown to have a slight defect in testis determination, resulting in 4% of males developing as overtly intersex, and 17% having small testes which do not produce epididymal sperm. The cause of this developmental defect is unknown; we hypothesise that it is due to an interstitial deletion in a gene array lying between Sry and the Y chromosome centromere, exerting a positional effect on Sry. This study aims to determine the nature of this structural change on the A/HeJ Y chromosome, as well as characterise the epigenetic and gene expression changes to Sry that would exert the observed phenotype in this mouse strain.

EXPRESSION OF POLO-LIKE KINASE 1 OF MURINE SOMATIC CELL NUCLEAR TRANSFER EMBRYOS IN PRE-IMPLANTATION STAGE

Roh S. and Moon J. Seoul National University School of Dentistry.

Somatic cell nuclear transfer (SCNT) has various abilities of application in research, as well as the medical field and industry. However, the efficiency of SCNT is very low because the accurate mechanism of SCNT embryo development is still unknown. In previous studies, Polo-like kinase 1 (Plk1) has been found to be a crucial element in cell division including centrosome maturation, cytokinesis and spindle formation. Therefore, we investigated the role of PLk1 in SCNT embryos in this experiment. First, BI2536, a Plk1 inhibitor, was used to investigate the influence of Plk1 in 1-cell embryos during first mitotic division. Because the Plk1 was decreased by BI2536, the embryos failed to develop to the 2-cell stage. Next, we checked the Plk1’s localization and intensity by immunofluorescence. The SCNT embryos, which were not developed, have two types of different Plk1 expressions in comparison with normal developed embryos. One is a low expression pattern of Plk1 and the other is ectopic expression of Plk1. Therefore, this result shows that Plk1 also plays critical role in SCNT embryos.

GPR56: A NOVEL GENE ESSENTIAL FOR MULLERIAN DUCT FORMATION

Roly Z.Y., Major A.T. and Smith C.A. Monash Biomedicine Institute, Department of Anatomy and Developmental Biology, Monash University, Clayton, Victoria

The embryonic Mullerian ducts give rise to the female reproductive tract. While a number of genes have been implicated in duct formation and differentiation, many of the molecular details remain obscure. In a recent RNA-seq analyses of the embryonic chicken urogenital system, we identified the adhesion-associated receptor, G-Protein coupled Receptor 56 (GPR56). We found the gene up-regulated in the Mullerian duct and developing ovary. GPR56 has not previously been liked to duct formation. Whole Mount in situ hybridisation confirmed GPR56 mRNA expression in the Mullerian Duct Epithelium (MDE) in both sexes during invagination and the elongation phases of duct formation. Collagen type III (COL3A1) is a putative ligand of GPR56. We found that COL3A1 is expressed in Mullerian Surface Epithelium (MSE) and Mullerian Duct Mesenchyme (MDM), but not in Mullerian Duct Epithelium (MDE). Thus, we hypothesise that GPR56 may interact with COL3A1 at the basal membrane junction between MDM and MDE of Mullerian Duct. Over expression of GPR56 had no overt effect upon duct development whereas targeted knockdown using RNAi perturbed the formation of Mullerian duct. Ducts were truncated and the expression of marker genes such as PAX2, was retarded or absent. We conclude that GPR56 is essential for the Mullerian duct formation and elongation. We hypothesise that GPR56 plays a role specifically in the duct epithelium during elongation through interaction with mesenchymal COL3A1. Keywords: GPR56, Adhesion G-protein-coupled receptors, Mullerian Duct, Chicken.

SEX-DIFFERENTIAL EXPRESSION OF GENES AND MICRORNAS IN THE DEVELOPING BRAIN

Szakats S.K. and Wilson M.J. University of Otago, PO Box 56, Dunedin 9054, New Zealand.

Biological sex contributes to many pathologies. Investigating sex differences is therefore a powerful strategy for identifying mechanisms underlying idiopathic disorders with a sex bias, including neurodevelopmental disorders such as ADHD, schizophrenia and autism. Regulation of gene expression by sex may alter development of the brain, thus we investigated sex-differential gene expression in the embryonic (E15.5) mouse brain. Given the importance of microRNAs (miRNAs) in regulating developmental processes, we also postulated that their expression may differ between the sexes during neurodevelopment. RNA sequencing identified 98 genes with significantly different expression between males and females (n=3/sex, p adj.<0.05), while small RNA sequencing identified 67 sex-differentially expressed miRNAs (n=3/sex, FDR<0.05). RT-qPCR has confirmed differential expression several genes and miRNAs, and in situ hybridisation has been used to investigate spatial expression patterns of Aff2, Dicer1, Eif2s3y and miR-10a/b-5p from this data set. Finally, gene ontology analysis to compare over-represented pathways targeted by upregulated genes and miRNA target genes in males compared to females reveals sex differences in pathways with various developmental and neurological functions. Future experiments aim to establish how sex differences in the regulation of gene expression are induced, and identify how those genes and miRNAs identified to be sex dimorphic contribute to sex differences in neurodevelopmental disorders.


POSTERS



INVESTIGATING THE PATHOMECHANISMS OF FOXG1 SYNDROME

Tan C.S.D.1, 2, Ittner L.M.1, 2 and Delerue F.1, 2 1Dementia Research Unit, Department of Anatomy, School of Medical Sciences, Faculty of Medicine, University of New South Wales, Sydney, NSW, Australia. 2Transgenic Animal Unit, Mark Wainwright Analytical Centre, UNSW, Sydney, New South Wales, Australia.

FOXG1 syndrome is a neuro-developmental disorder that affects the early development of the telencephalon leading to severe cortical impairments. Patients typically present with post-natal microcephaly, severe mental retardation, apraxia and seizures. The disease is associated with mutations in the FOXG1 gene, which encodes a transcription factor belonging to the forkhead family. Here we report a novel mutation of the FOXG1 syndrome, a single nucleotide deletion c.946del (p.Leu316Cysfs*10) resulting in the premature truncation of the FOXG1 protein. We hypothesized that truncation of the FOXG1 protein leads to loss of potential interacting partners that may contribute to the pathology. To uncover these interacting partners, we performed immunoprecipitation of the FOXG1 protein and its mutant form in Neuro-2a cells. Subsequently, we performed mass spectrometry on purified extracts for identification. Our findings provide an insight into potential regulatory mechanisms of the physiological FOXG1 pathway and its pathological counterparts. Targeting the newly identified interaction partners could lead to new therapeutic strategies to abate the symptoms of the disease.

SYCON CAPRICORN AS A MODEL TO INVESTIGATE EARLY EVOLUTION OF ANIMAL BODY PLAN DEVELOPMENT

Vaughn E.E. and Adamska M.A. ARC Centre of Excellence for Coral Reef Studies and Research School of Biology, Australian National University, Canberra, ACT 0200, Australia.

Extensive radiation of gene regulatory networks has characterised the evolution of animal body plans, leading to the stunning morphological diversity within the animal kingdom. This diversity has long inspired researchers to investigate the evolution of animal development; yet, we know relatively little about the evolutionary origins of key animal body features such as multicellularity, axial polarity, body symmetry, and germ layer specification. We stand to gain great insight into the evolution of animal body plan development through the study of early branching animal lineages. Sponges, often argued to be the oldest animal clade, bridge the gap between our most recent unicellular ancestors and the more familiar “true” multicellular animals, cnidarians and bilaterians. Intriguingly, despite their relatively simple body plans, a majority of the key conserved developmental gene regulatory network components are found in sponge genomes. In my talk, I will describe a new sponge model system for the study of developmental gene regulatory networks. Sycon capricorn, a calcaronean sponge endemic to the temperate New South Wales coast, is well-suited as a model species due its remarkable regenerative capacity, a growing suite of molecular data resources, and ease of laboratory culturing. To elevate S. capricorn as powerful model, I have used a hybrid genome assembly approach incorporating massive short-read sequencing with cutting edge MinION long read sequencing. I will present the results of genome annotation using RNAseq data, which will be used as a reference for ChIP-Seq of DNA-binding targets of the Wnt signalling pathway transcription factor, Tcf.

DEVELOPMENT OF MOLECULAR MARKERS FOR STRAIN IDENTIFICATION IN THE FUNGAL PATHOGEN OF HONEY BEE

Brar K., Waters M., Grassl J. and Baer B. University of Western Australia.

Honey bees (Apis mellifera) are of central importance to agriculture as they provide pollination services for many food crops. Dramatic declines have occurred worldwide due to several biotic and abiotic factors but parasites and pathogens have been identified as key contributors. Nosema apis is a globally widespread fungal pathogen that can negatively impact honey bee health. More recent studies have investigated the complex relationship between N. apis and its honey bee host and have provided evidence that there is genetically based variation in the host susceptibility. We quantified the genetic variation with the two marker genes (RPBI and EF1α) in different bee populations throughout Western Australia. The findings we got will help in better understanding of honey bee immune responses to N. apis and in breeding more Nosema tolerant bees.

TRACKING MANNOSE-6-PHOSPHORYLATED GLYCOPROTEINS IN CANCER AND INFLAMMATION

Chatterjee S.1, Lee L.Y.2, Loke I.1, Ashwood C.1, Sakuma R.K.1, Hinneburg H.1, Palmisano G.3, Molloy M.P.1, Packer N.H.1 and Thaysen-Andersen M.1 1Department of Molecular Sciences, Macquarie University, Sydney, Australia. 2UISGlobal, Barcelona Centre for International Health Research (CRESIB), Hospital Clínic -Universitat de Barcelona, Barcelona, Spain. 3Institute of Biomedical Sciences, University of Sau Paulo, Brazil.

Mannose-6-phosphate (M6P) is a modification of N-glycoproteins facilitating the trafficking of lysosomal hydrolases via the ER-Golgi network. Recently, extracellular roles of M6P-glycoproteins were suggested including their involvement in cell-to-cell communication and cell growth as indicated by the surface expression of M6P-receptors in inflammation and cancer. However, solid structural data is unavailable to assess the global M6P-glycoprotein expression. Herein, we have utilized quantitative LC-MS/MS-based glycomics to map, in an unbiased way, the presence of M6P-modified N-glycans from extra- and intra-cellular human glycoproteins derived from various cancer (e.g. prostate and breast) and immune (neutrophil) cells, tissues and bodily fluids (e.g. cystic fibrosis sputum). This interrogation revealed a hitherto unknown heterogeneity of N-glycans receiving M6P modifications and demonstrated both mono- and di-M6P additions to extracellular and subcellular-specific glycoproteins. Collectively, these findings add support for the involvement of M6P-glycoproteins in processes relating to inflammation and cancer.


POSTERS



MUTATIONS IN THE DEATH DOMAIN AND GC CENTRE OF IRAK3 AND THE EFFECT ON DOWNSTREAM SIGNALLING

Galea C.A.1, Freihat L.A.1, 2, Nguyen H.T.2 and Irving H.R.1, 2 1Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC, Australia. 2La Trobe Institute for Molecular Science, Latrobe University, Bendigo, VIC, Australia.

Interleukin-1 receptor associated kinase 3 (IRAK3) plays an important role in maintaining homeostasis in the innate immune response and in preventing the development of autoimmune diseases. IRAK3 acts as a negative regulator of inflammation and is involved in inflammation-associated disorders such as lung injury, metabolic syndrome and tumour growth. Prior studies within our group identified IRAK3 as a potential novel guanylate cyclase (GC) catalyzing cyclic guanosine monophosphate (cGMP) synthesis. IRAK3 is predicted to be a mammalian representative of a new class of GCs originally identified in plants, containing a complex domain architecture where the GC centre is encapsulated within the pseudokinase domain. Here we report the effect of mutating amino acid residues of the GC catalytic site and death domain upon IRAK3’s GC activity and anti-inflammatory downstream signalling. HEKBLUE hTLR4 cells containing a SEAP reporter system were transfected with IRAK3 or IRAK3 mutant constructs and NFκB activity was monitored in the presence of lipopolysaccharide (LPS). Overexpression of IRAK3 significantly reduced LPS-induced NFκB activation while IRAK3 mutants possessing reduced GC activity failed to inhibit LPS induced NFκB function. Addition of cell-permeable cGMP restored IRAK3 function and significantly reduced NFκB activity in IRAK3 mutants with reduced cGMP-generating capacity. These findings are providing insight into hidden functions of IRAK3 and may assist in explaining its selectivity and functionality in the inflammatory signalling cascade. Understanding how this novel GC function impacts the anti-inflammatory activity of IRAK3 will aid in the development of novel therapeutics for the treatment of various inflammation-associated diseases.

THE MOLECULAR MECHANISM OF MANGANESE IMPORT IN STREPTOCOCCUS PNEUMONIAE

Carey Hulyer A.R.1, Begg S.L.1, Ween M.P.1, Counago R.M.2, O’Mara M.L.3, Maher M.J.4, Kobe B.2, McEwan A.G.2, Paton J.C.1and McDevitt C.A.1 1University of Adelaide, South Australia 5005, Australia. 2University of Queensland, Brisbane 4072, Australia. 3Australian National University, Canberra 2601, Australia. 4LaTrobe University, Melbourne 3080, Australia.

Streptococcus pneumoniae is a bacterial pathogen of global significance, responsible for more than 1 million deaths per year. Transition metal ion homeostasis is essential for S. pneumoniae pathogenicity. Consequently, the mechanisms associated with metal ion homeostasis are potential antimicrobial targets. The ATP binding cassette (ABC) transporter PsaBCA is the sole manganese acquisition pathway in vivo and is essential for pneumococcal virulence. The ABC permease complex comprises a homodimer of heterodimers (PsaB2C2), to which Mn2+ is delivered by the extra-cytoplasmic protein PsaA. Although PsaBCA only imports manganese ions, prior studies have shown that PsaA has highly promiscuous interactions with various divalent cations. Nonetheless, specificity of the transporter is achieved by slow release kinetics associated with non-cognate transition metal ions, such as Zn2+, from PsaA. However, these inferences are based on structural and in vitro experiments conducted in the absence of the ABC transporter. Building on this framework we sought to investigate how different PsaA-metal ion complexes influenced PsaBC function. Here, recombinant PsaBC was heterologously expressed in E. coli Lemo21(DE3). Membranes were isolated, the membrane protein complex solubilised by DDM and purified by affinity chromatography. Reconstitution of PsaBC was achieved using Triton X-100 mediated insertion into pre-formed liposomes, prior to detergent removal. Freeze-thaw cycling was employed to incorporate metal ions and PsaA into the proteoliposomes. PsaBCA complex was then assessed using an ATPase activity assay. This revealed that, in contrast to manganese, metal-PsaA complexes formed by non-cognate ions with slow release kinetics (i.e. zinc or copper), could not stimulate the activity of PsaBC. Hence, this work demonstrates how specificity of the S. pneumoniaemanganese transporter is achieved. This finding provides a mechanistic basis for how poorly abundant metal ions, such as manganese, can be selectively acquired from chemically complex extracellular environments.

A GENERAL SMALL-ANGLE X-RAY SCATTERING-BASED SCREENING PROTOCOL VALIDATED FOR PROTEIN-RNA INTERACTIONS

Chen P.1, Masiewicz P.1, Rybin V.1, Svergun D.2 and Hennig J.1 1EMBL Heidelberg, Meyerhofstrasse 1, 69117 Heidelberg, Germany. 2EMBL Hamburg, DESY, Building 25A, Notkestrasse 85, 22607 Hamburg, Germany.

We present a screening protocol utilizing small-angle X-ray scattering (SAXS) to obtain structural informatio on biomolecular interactions independent of prior knowledge, so as to complement affinity-based screening and provide leads for further exploration. This protocol categorizes ligand titrations by computing pairwise agreement between curves, and separately estimates affinities by quantifying complex formation as a departure from the linear sum properties of solution SAXS. The protocol is validated by sparse sequence search around the native poly uridine RNA motifs of the two-RRM domain Sex-lethal protein (Sxl). The screening of 35 RNA motifs between 4 to 10 nucleotides reveals a strong variation of resulting complexes, revealed to be preference-switching between 1:1 and 2:2 binding stoichiometries upon addition of structural modeling. Validation of select sequences in isothermal calorimetry and NMR titration retrieves domain-specific roles and function of a guanine anchor. These findings reinforce the suitability of SAXS as a complement in lead identification. In addition to the published data, we intend to present current progress in determining performance limits for test systems at ESRF Grenoble and DESY Hamburg.

THE STRUCTURAL BIOLOGY OF FABG PROTEINS FROM MULTIDRUG-RESISTANT ACINETOBACTER BAUMANNII

Cross E.M., Smith K.M. and Forwood J.K. Charles Sturt University Wagga Wagga NSW 2678.

Acinetobacter baumannii is a gram negative, nosocomial pathogen that is resistant to multiple drug classes, including carbapenems. In 2017, the World Health Organisation listed A. baumannii as priority one for the research and development of new antibiotics. Fatty acids are essential cell components, required for formation of phospholipids, lipopolysaccharides, and lipoproteins that help build the bacterial cell envelope. Bacterial type two fatty acid synthesis (FASII), is a potential target for the development of inhibitors and is favourable due to the distinct differences from eukaryotic type one fatty acid synthesis (FASI). In particular the first reductase enzyme, 3-oxoacyl-ACP reductase (FabG), is an attractive target for inhibition in the FASII pathway. FabG is a member of the short-chain dehydrogenase/reductase family, a class of proteins known to display a diverse range of functions whilst maintaining conserved sequence motifs and typical Rossmann folding patterns. BLAST results show multiple sequences annotated as FabG proteins for A. baumannii, which has been noted in other bacteria such as Mycobacterium tuberculosis and suggested to play a role in bacterial virulence. This project aims to find the structure of FabG homologs through recombinant protein expression, purification and crystallization. Diffraction data was collected at the Australian Synchrotron and used to solve the enzyme structure. The structural analysis was complemented with functional data, enabling the assessment of enzyme activity against different substrates. This was achieved spectrophotometrically by analysing the oxidation of the cofactor (either NADH or NADPH) and observing a decrease in absorbance at 340nm. Inhibitors designed to target FabG proteins and prevent fatty acid synthesis are currently under investigation, and may provide a platform for the potential use against multidrug-resistant Acinetobacter baumannii.


POSTERS



CALCIUM IS AN ESSENTIAL COFACTOR FOR METAL EFFLUX BY THE FERROPORTIN TRANSPORTER FAMILY

Deshpande C.1, 2, Ruwe T.3, 4, Shawki A.3, 4, Xin V.2, Vieth K.3, Valore E.5, Ganz T.5, Nemeth E.5, Mackenzie B.3, 4 and Jormakka M.2, 6 1Protein Production, Drug Discovery, Sydney Analytical, Core Research Facilities, The University of Sydney. 2Structural Biology Program, Centenary Institute, Sydney. 3Department of Pharmacology & Systems Physiology, University of Cincinnati College of Medicine, Cincinnati, Ohio, USA. 4Systems Biology & Physiology Program, University of Cincinnati College of Medicine, Cincinnati, Ohio, USA. 5Department of Medicine and 6Department of Pathology, David Geffen School of Medicine at University of California, Los Angeles, California, USA. 6Sydney Medical School, University of Sydney, Sydney, NSW 2006, Australia.

Iron is essential for almost all living organisms owing to its involvement in a number of metabolic and catalytic processes. Iron metabolism is critical in mammalian system and both iron deficiency and overload can account for some of the most common human diseases, such as iron-restricted anemia and hemochromatoses. To date, Ferroportin (FPN) is the only known mammalian iron exporter and is responsible for the entry of iron from intracellular storage into plasma for circulation. Despite its central role in iron metabolism, our molecular understanding of FPN-mediated iron efflux remains incomplete. We have shown by combining transport and biophysical studies, that FPN is a Ca2+ dependent iron transporter (1). In addition, we have determined the crystal structure of a Ca2+-bound BbFPN protein (a prokaryotic homolog of FPN), revealing a marked Ca2+-induced conformational change. Our results have demonstrated that Ca2+ is a required cofactor in FPN-mediated metal efflux. We also provide novel insights into the substrate binding pocket of FPN. Our findings provide important conceptual advancements in the understanding of FPN-mediated iron efflux and have fundamental implications for developing strategies to manipulate FPN therapeutically. {Reference 1) Calcium is an essential cofactor for metal efflux by the ferroportin transporter family. Chandrika N. Deshpande, T. Alex Ruwe, Ali Shawki, Vicky Xin, Kyle R. Vieth, Erika V. Valore, Bo Qiao, Tomas Ganz, Elizabeta Nemeth, Bryan Mackenzie, Mika Jormakka (Accepted in principle with final editorial revisions, Nature Communications. 2018)}.

FINDING THE ACHILLES HEEL IN PSEUDOMONAS AERUGINOSA

Impey R.E.1, Bock L.J.2, Sutton J.M.2, Perugini M.A.1 and Soares Da Costa T.P.1 1Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Bundoora, Victoria, Australia. 2Technology Development Group, National Infection Service, Public Health England, Salisbury, UK.

Pseudomonas aeruginosa is an emerging threat due to the increasing rate of multi-drug resistance. This highlights the need to both discover novel antimicrobial agents and identify promising new targets. One such target is the diaminopimelate (DAP) pathway that yields essential metabolites for bacterial survival, namely meso-diaminopimelate and L-lysine. The first committed step of the DAP pathway is the condensation of pyruvate and aspartate semi-aldehyde catalysed by the essential enzyme dihydrodipicolinate synthase (DHDPS). In bacteria, DHDPS is typically encoded for by a single dapA gene, but bioinformatic analysis of the P. aeruginosa genome reveals four putative dapA genes encoding 4 products; DapA1, DapA2, DapA3 & DapA4. In silico analyses show that only DapA1 and DapA2 contain the 7 signature residues known to be critical for DHDPS catalytic function. Not surprisingly, enzyme kinetic studies show that DapA1&2 are catalytically active, whereas DapA3&4 are void of DHDPS function. Despite similar secondary structures, analytical ultracentrifugation shows that DapA1 exists in a dimer-tetramer equilibrium, whilst DapA2 forms a stable dimer. Enzyme kinetic analyses reveal that DapA1 and DapA2 share similar substrate affinities; however, the two enzymes differ in their allosteric inhibition by L-lysine with DapA1 insensitive and DapA2 sensitive to L-lysine. Together, these studies show that P. aeruginosa contains two incorrectly annotated DHDPS enzymes (DapA3 & DapA4) and two bona fide DHDPS enzymes (DapA1 & DapA2) that interestingly differ in their L-Lysine inhibition. These results offer insight into rational approaches for the development of new antibiotic agents targeting DHDPS in P. aeruginosa.

SMALPS: BREAKING THE BARRIER TO STUDY ABCB1 IN ITS NATIVE LIPID ENVIRONMENT

Iqbal S.1, Skrzypek R.1, Kerr I.2 and Callaghan R.1 1Division of Biomedical Science and Biochemistry, Research School of Biology, The Australian National University, Canberra, Australia. 2School of Life Sciences, University of Nottingham, Queens Medical Centre, University of Nottingham, Nottingham, UK.

The multidrug resistance P-glycoprotein (ABCB1) has ability to bind and extrude a vast array of chemotherapeutic drugs out of cancer cells. ABCB1 has four pharmacologically distinct drug binding sites and recent work by our laboratory has provided preliminary locations for them [1]. The ongoing aim of our research is to provide detailed locations of the binding sites and their physical properties. Three of the drug binding sites are located at the protein-lipid interface and it is clear that the lipid environment has a significant influence on drug binding. Consequently, we intend to use the extraction procedure using styrene malic acid (SMA) co-polymer. SMA makes nanodiscs, termed as Styrene Malic Acid Lipid Particles (SMALPs). A key feature of SMALPs is their ability to encapsulate membrane proteins along with native lipids, particularly those at the annular region. SMALP based extraction has been compared with conventional method using the detergent dodecyl-maltoside based (DDM). The two methods were compared in terms of ABCB1 solubilisation efficiency, yield, purity and homogeneity at different stages during the purification procedure. DDM was more effective at solubilisation of ABCB1 and displayed higher affinity binding to the Ni-NTA chromatographic resin. Overall, the final purity between the two preparation procedures did not differ. A considerable advantage of SMALP based extraction is the ease of centrifugal concentration, with minimal losses compared to the DDM-solubilised protein. Finally, the final yield of purified protein did not differ between the two procedures. The SMALP system has been optimised for preparation of ABCB1 at high concentration with the crucial native lipid environment maintained. [1] R. Mittra, M. Pavy, N. Subramanian, A.M. George, M.L. O’Mara, I.D. Kerr, R. Callaghan, Location of contact residues in pharmacologically distinct drug binding sites on P-glycoprotein, Biochem Pharmacol 123 (2017) 19-28.

PROTEOMIC ANALYSIS OF THE UBIQUITIN LANDSCAPE IN THE TYPE II DIABETIC RAT MODEL

Lingam S.1, 2, Smith L.1, 2, Cordwell S.1, 2, 3 and White M.1, 2, 3 1Charles Perkins Centre, University of Sydney, Australia. 2Discipline of Pathology, School of Medical Sciences, University of Sydney, Australia. 3School of Life and Environmental Sciences, University of Sydney, Australia.

Type II diabetes mellitus (DM) remains one of the major causes of morbidity and mortality in the industrialized world. Several animal and human studies have identified increased generation of reactive oxygen species (ROS), a consequence of metabolic abnormalities in DM, as one of the key mediators of signal transduction during DM and DM-mediated diseases. In addition to redox-specific post-translational modifications (PTM), accumulating evidence suggests the involvement of ROS in accelerating non-specific ubiquitination PTMs of cardiac proteins as a label for protein degradation. Our group has defined changes in several protein PTMs in DM and begun to understand how these various PTMs cross-talk with each other. Here, we examined the ubiquitin-modified proteome in key metabolic tissues; adipose, brain, heart, liver, pancreas, kidney and skeletal muscle in DM. Rats were fed a standard chow citrate (CC) (12% fat) or high fat (HF) (42% fat) diet for 8 weeks, with DM induced in 50% of the animals after 4 weeks utilising a low dose of streptozotocin (STZ; 55mg/kg); a pancreatic β-cell toxin. At the cessation of the feeding protocol, animals were euthanised and organs were excised. Tissue lysates were subjected to western blotting using anti-ubiquitin antibodies. Western blots revealed gross changes in ubiquitination associated with diet and pathology. To identify sites targeted by these PTMs we next used immunoprecipitation to enrich for modified peptides, which were subsequently analysed by LC-MS/MS. These analyses further the scope of PTMs associated with DM and may further understanding of molecular mechanisms underlying metabolic disturbances of this disease.


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MULTIPLE PERSONALITIES: COMPETITIVE BINDING OF NURD SUBUNITS CAN LEAD TO DISTINCT FUNCTIONS

Low J.K.K.1, Silva A.P.G.1, Sharifi-Tabar M.1, Torrado M.1, Sana M.1, Parker B.L.1, Shepherd N.E.1, Landsberg M.J.2 and Mackay J.P.1 1School of Life and Environmental Sciences, University of Sydney, NSW, Australia. 2School of Chemistry and Molecular Biosciences, The University of Queensland, QLD, Australia.

The Nucleosome Remodeling and Deacetylase (NuRD) complex is a ~1-MDa 10-subunit transcriptional co-regulator that plays essential roles in normal development, stem cell renewal and DNA repair. A detailed understanding, however, of NuRD structure and function has proven to be challenging. Using a barrage of biochemical and biophysical techniques, we demonstrate that the NuRD complex is made up of two modules of roughly equal size: (i) a symmetric ~450 kDa MTA-HDAC-RBBP (MHR) module carrying the deacetylase activity driven by its HDAC component; and (ii) an asymmetric ~320 kDa MBD-GATAD2-CHD (mGC) module carrying the remodelling activity driven by its CHD component. This bifurcation of the two enzymatic activities into apparently stable modules is intriguing; especially in the light of new observations that another transcriptional co-regulator can compete directly with the mGC module for binding to MHR. This transcriptional co-regulator selectively recruits the MHR module to gene promoters to influence histone acetylation levels. Isoform-specific functions of NuRD have been previously described; MBD2- and MBD3-NuRD target different promoters, and CHD3-, CHD4-, CHD5-NuRD have distinct roles in cortical development. However, the MHR and mGC modules as separate entities, upon which other complexes are built upon (for MHR at least), are yet to be appreciated. It is possible the NuRD complex (and its isoform-specific versions) may in fact be just one example out of the many possible combinations of MHR-based complexes, and that each of these complexes have distinct functions and targets in the cell.

CHARACTERISATIONS OF A HOSPITAL-ACQUIRED CARBAPENEMASE FROM ENTEROBACTERIACEAEPrombhul S.1, 2, Pedroso M.M.1, 2, Lo A.W.1, 2, Nouwens A.1, Josh P.1, Neo I.Y.Y.1, Mcgeary R.P.1, 2, Schembri M.A.1, 2 and Schenk G.1, 2 1School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, Queensland 4072, Australia. 2Australian Infectious Diseases Research Centre, The University of Queensland, Brisbane, Queensland 4072, Australia.New Delhi metallo-β-lactamase (NDM) is a major health concern due to its efficient inactivation of antibiotics and its rapid spread across the globe [1]. This enzyme displays catalytic efficiency towards a broad spectrum of clinically used β-lactam drugs [2]. Here we constructed two variants of NDM-1, M27 and G36, as previously reported [1, 3-7], and probed the Zn2+ stoichiometry of the enzymes using mass spectrometry. The mass spectral data indicate that the M27 variant is predominantly present in bimetallic form, while for the G36 variant both bi- and monometallic as well as metal-free species are observed. This observed variation between the two variants suggests that the N-terminal domain may play a direct role in metal ion binding and possibly also in the protein solubility and stability. Moreover, we examined the effects of aztreonam (a monobactam derivative) on the catalytic efficiency of NDM-1; while it has an inhibitory effect for full-length NDM-1, the two variants with truncated N-terminal ends are not affected. Our results thus agree with previous findings that ascribed a role for the N-terminus of NDM-1 in substrate/inhibitor binding and specificity [8, 9]. Also, we recently presented evidence that copper may act as a sensitisation agent for clinical antibiotics [10]. Our results were consistent with a loss of NDM-1 activity in the presence of copper, indicating a noncompetitive mode of inhibition with an apparent inhibition constant (Ki) of ~10 μM. However, we observed that the enzymes were precipitated in the presence of CuCl2, this might be due to the occurrence of interprotein crosslinking between metal-ion bound and the accessible histidines, and leads to the formation of large and insoluble complexes. This is another step to explore the application of metal coordination complex as an attempt to replace predominant zinc metals of the enzyme and the importance of N-terminal sequence of protein. Furthermore, we currently explored the potential mode of binding of a compound 5 (N-(1-Benzyl-4,5-diphenyl-1H-imidazol-2-yl)benzamide) in the active sites of the enzyme to examine specificity of this inhibitor with respect to the two variants. Interestingly, the compound represented partial inhibitory activity towards the two variants of NDM-1 when penicillin G was used as substrate. The result showed a competitive mode of inhibition with an apparent inhibition constant (Kic) of 9.85 and 2.6 μM for M27 and G36, respectively. Further studies will focus more on investigating the inhibitory mechanism and visualising the binding mode(s) of the potential compounds to develop universal MBL inhibitors.

QUANTITATIVE PROTEOMICS OF CYSTEINE REDOX POST-TRANSLATIONAL MODIFICATIONS IN MYOCARDIAL ISCHEMIA / REPERFUSION (I/R) USING PARALLEL REACTION MONITORING MASS SPECTROMETRY

Rookyard A.W.1, 2, Li D.K.2, White M.Y.1, 2, 3 and Cordwell S.J.1, 2, 3 1School of Life and Environmental Sciences. 2Charles Perkins Centre. 3Discipline of Pathology.

Ischemic heart disease involves the occlusion of blood vessels resulting in a cessation of oxygenated blood flow to the heart. This hypoxia, and the necessary reperfusion to salvage surviving myocytes, induces cellular damage. Notably mitochondrial dysfunction occurs, increasing the production of reactive oxygen and reactive nitrogen species (ROS/RNS). This increase in ROS/RNS overwhelms cellular antioxidant defence mechanisms and can alter protein structure / function via various protein post translational modifications (PTMs). The most redox active amino acid is Cysteine (Cys) and Cys redox PTMs can be broken down into two classes, those that are biologically reversible (e.g. S-glutathionylation) or irreversible (sulfinic and sulfonic acid; Cys-SO2H/SO3H). Irreversible Cys redox PTMs occur with sufficient exposure to high levels of ROS/RNS and are associated with protein dysfunction and/or degradation. A mass spectrometry technique based on parallel reaction monitoring was employed to detect changes in irreversible Cys modification in a Langendorff model of myocardial ischemia reperfusion injury (I/R). Due to the low abundance of Cys, and Cys PTMs, an enrichment strategy was used to better profile the changes in irreversible Cys PTM. I/R significantly increased Cys-SO2H/SO3H-modified peptides from proteins involved in mitochondrial fatty acid biosynthesis and the tricarboxylic acid cycle. The addition of an aminothiol antioxidant MPG (N-2-mercaptopropionylglycine) in reperfusion attenuated irreversible modification of Cys and improved functional recovery of hearts.

WHO’S CONTROLLING THE CONTROLLERS? CONSTRUCTING THE REGULATORY NETWORK OF HISTONE METHYLATION IN YEAST

Separovich R.J. and Wilkins M.R. Systems Biology Initiative, School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Sydney, New South Wales 2052, Australia.

Methylation of histone proteins tightly regulates patterns of gene expression in the eukaryotic cell. The addition of methyl (CH3) groups to histone tails is catalysed by methyltransferase enzymes, while demethylase enzymes can reverse this modification. Together, the activity of these ‘writer’ and ‘eraser’ enzymes determines which histone methyl marks accumulate, and thus which genes are accessible for transcription. In the lower eukaryote, Saccharomyces cerevisiae, there are only four known histone lysine methyl writers (Set1, Set2, Set5, Dot1) and four erasers (Jhd1, Jhd2, Gis1, Rph1). While the catalytic activity and specificity of these enzymes have been established, knowledge of how they are post-translationally modified is surprisingly limited. Consequently, the regulatory network of histone methylation in yeast remains unknown. To this end, we aim to comprehensively characterise the eight writers and erasers in S. cerevisiae, with a focus on phosphorylation. This will be done through purification of these proteins, and their analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Through a series of gene knockout experiments, we will then seek to identify the upstream kinases responsible for the phosphorylation, and potential regulation, of the methyl writers and erasers. This will facilitate the integration of these enzymes into intracellular signalling pathways, and thereby enable the assembly of the first complete regulatory network of histone methylation in yeast. Given the evolutionary conservation of this histone modification, the foundational insights gained through this work will be relevant to other eukaryotes and may prompt similar in-depth analyses in these organisms.


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COMPREHENSIVE IDENTIFICATION OF CROSSLINKED PEPTIDES USING A MULTI-CROSSLINKER, FRAGMENTATION AND DATA ANALYSIS APPROACH

Smith D.L.1, Gotze M.2, Hart-Smith G.1 and Wilkins M.R.1 1Systems Biology Initiative, School of Biotechnology and Biomolecular Sciences, UNSW Sydney, NSW 2052, Australi. 2Institute of Biochemistry, Martin Luther University Halle-Wittenberg, Kurt- Mothes-Str. 3, D-06120 Halle (Saale), Germany.

Crosslinking mass spectrometry (XLMS) is a method to study protein-protein interactions. It combines chemical crosslinkers, protease digestion and tandem mass spectrometry, whereby fragmentation of the crosslinked peptides is used to discover intra- and inter-protein crosslinks. Early crosslinking studies used non-mass spectrometry cleavable crosslinkers such as BS3, BS2G and DTTSP. However, new mass spectrometry-cleavable linkers, such as DSSO, DSBU have helped address many of the prior limitations of crosslinking experiments, such as co-fragmentation of crosslinked peptides. They allow predictable generation of high-intensity reporter ions which new generation crosslink identification programs such as MeroX and XlinkX can use to increase speed, and confidence of identifications. Here, we have used a multi-crosslinker, fragmentation and program approach to address two major aims. Firstly, we investigated the enzyme/substrate interaction between Npl3p and its methyltransferase Hmt1p. Secondly, we used this combined analysis approach to understand how different crosslinkers (DSBU/DSSO), fragmentations (CID+ETD/SteppedHCD), programs (MeroX/XlinkX 2.0), and algorithms (Precursor and Reporter-Ion) impacted the results. From this study we have defined that the interaction between Npl3p and Hmt1p involves the intrinsically disordered “SRGG” region of Npl3p and the N-, C- termini and S-adenosyl methionine binding site of Hmt1p. For the first time, we have also shown direct evidence for Npl3p dimerization occurring at the “SRGG” region. We have also confirmed higher order multimerisation of Hmt1p in accordance to the known structure. Importantly, we demonstrate from our multi-crosslinker, fragmentation and program comparisons that one-type of analyses may not capture all identifiable crosslinks. This is attributed to a combination of biases inherent to either a MeroX/DSBU approach or an XlinkX/DSSO approach.

CRYO-EM STUDIES OF E. COLI ATP SYNTHASE

Sobti M.1, Smits C.1, Wong A.S.W.2, Ishmukhametov R.3, Stock D.1, Sandin S.2 and Stewart A.G.1 1Victor Chang Cardiac Research Institute, Australia. 2Nanyang Technological University, Singapore. 3University of Oxford, United Kingdom.

ATP synthase is an essential biological motor that synthesizes the bulk of cellular ATP, the energy currency of the cell. Here we present our cryo-EM maps of the intact ATP synthase complex from Escherichia coli (1). Three different states that relate to rotation of the enzyme were observed, with the central stalk’s ε subunit in an extended auto inhibitory conformation in all three states. For the first time in this rotary ATPase subtype, the peripheral stalk is resolved over its entire length of the complex revealing the F1 attachment points and a coiled-coil that bifurcates toward the membrane. The molecular models also provide a framework onto which the vast array of information available on the widely studied E. coli enzyme can be mapped. References: 1. Sobti M, Smits C, Wong AS, Ishmukhametov R, Stock D, Sandin S, et al. eLIFE. 2016;5: e21598.

UNDERSTANDING THE MOLECULAR MECHANISM OF KAP1-DEPENDENT TRANSCRIPTIONAL SILENCING

Stoll G.A.1, McLaughlin S.H.2 and Modis Y.1 1Department of Medicine, University of Cambridge, Medical Research Council Laboratory of Molecular Biology, Cambridge CB2 0QH, UK. 2Medical Research Council Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK.

Approximately half of the human genome consists of transposable elements (TEs), some of which are still replication-competent. TEs represent major drivers of evolution and there is mounting evidence that they fulfil important functions in early embryonic development. At the same time, they have the potential to severely damage the host genome if allowed to replicate unchecked. The activity of retrotransposons consequently must be tightly controlled. A major factor contributing to the repression of potentially harmful retroelements in mammals is the transcriptional regulator KRAB-associated protein 1 (KAP1). Following its recruitment to retrotransposons by KRAB domain-containing zinc finger proteins (KRAB-ZFPs), KAP1 induces epigenetic silencing of these elements by coordinating the assembly of a large repressor complex comprising the histone methyltransferase SETDB1, heterochromatin-associated protein 1 (HP1) and the Nucleosome Remodelling and Deacetylase (NuRD) complex. Despite the essential role of KAP1 in preserving genome integrity, structural and biophysical data on this protein is scarce and consequently, many aspects of this process remain poorly understood. To elucidate the molecular basis of KAP1-dependent transcriptional regulation, we characterized assembly and stoichiometry of KAP1 complexes. Multi-angle light scattering (MALS) and analytical ultracentrifugation (AUC) revealed that KAP1 is dimeric in solution but assembles into higher-order oligomers with increasing protein concentration. We subsequently determined the affinity of KAP1 self-association and mapped the protein domains mediating oligomerization. Possible functional consequences of this behavior are discussed. Furthermore, we present our progress towards high-resolution structures of KAP1-containing repressor complexes using a combination of X-ray crystallography and electron cryomicroscopy (cryo-EM).

A STRUCTURAL INSIGHT OF NRZ MEDIATED APOPTOSIS REGULATION IN ZEBRAFISH

Suraweera C.D.1, Caria S.1, Jarva M.1, Hinds M.G.2 and Kvansakul M.1 11Department of Biochemistry & Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria 3086, Australia. 2Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Parkville, Australia.

Bcl-2 family proteins play a crucial role in regulating apoptosis, a process critical for development, eliminating damaged or infected cells, host-pathogen interactions and in disease. Dysregulation of Bcl-2 proteins elicits an expansive cell survival mechanism promoting cell migration, invasion and metastasis. Through a network of intra-family protein-protein interactions Bcl-2 family members regulate the release of cell death factors from mitochondria. NRZ is a novel zebrafish pro-survival Bcl-2 orthologue resident on mitochondria and the endoplasmic reticulum (ER). However, the mechanism of NRZ apoptosis inhibition has not yet been clarified. Here, we examined the interactions of NRZ with pro-apoptotic members of the Bcl-2 family. We show that NRZ binds almost all zebrafish pro-apoptotic proteins and displays a broad range of affinities. Furthermore, we define the structural basis for apoptosis inhibition of NRZ by solving the crystal structure of both apo-NRZ and a holo form bound to a peptide spanning the binding motif of the proapoptotic zBad, a BH3-only protein orthologous to mammalian Bad. The crystal structure of NRZ revealed that it adopts the conserved Bcl-2 like fold observed for other cellular pro-survival Bcl-2 proteins and employs the canonical ligand binding groove to bind Bad BH3 peptide. Our findings provide a detailed mechanistic understanding for NRZ mediated antiapoptotic activity in zebrafish and suggest that NRZ likely occupies a unique mechanistic role in zebrafish apoptosis regulation.


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PROTEIN PRODUCTION FACILITY

Torrado M.1, 3, Deshpande C.N.1, 3, Kassiou M.2, Lay P.A.1, 2 and Mackay J.P.3 1Sydney Analytical. The University of Sydney, NSW 2006. 2School of Chemistry, The University of Sydney, NSW 2006. 3School of Life and Environmental Sciences. The University of Sydney, NSW 2006.

The Protein Production Facility (PPF) belongs to the recently established “Sydney Analytical” core facility at The University of Sydney, and is one of the platforms created under the University’s Drug Discovery Initiative (DDI). The PPF’s aim is to assist researchers, wherever they come from, with the expression, purification and characterisation of their proteins of interest. We work with three different expression hosts (bacterial, insect, and mammalian cells), and use highly specialised protein purification equipment. Since our opening a few months ago, we have been involved in several projects helping our users to extend the scope of their research with protein production solutions. Examples of these include the expression and purification of: - Cas9 protein, to give our user access to a more affordable and efficient method for CRISPR/Cas9 gene editing in living worms. - dCas9 protein, to allow our user to perform modern sequence-targeted genomics and proteomics methods. - Histone-modifying proteins, of central interest for one of our users. This user can now work with purified versions of these enzymes, and therefore gain a more detailed insight into their function. - Large amounts of an enzyme involved in fungal metabolism, with the aim of designing drugs targeting it in our associated Fragment Based Drug Design (FBDD) platform. We are also producing sufficient quantities of this protein for crystallisation studies. In this poster I will show the results of case examples like these and, in this way, how the PPF can benefit a broad range of researchers.

STRUCTURAL BASIS FOR IMPORTIN ALPHA 3 SPECIFICITY OF W PROTEINS IN HENDRA AND NIPAH VIRUSES

Smith K.M.1, Tsimbalyuk S.1, Edwards M.R.1, Cross E.M.1, Batra J.2, Soares da Costa T.P.3, Aragão D.4, Basler C.F.2 and Forwood J.K.1 1School of Biomedical Sciences, Charles Sturt University. 2Institute for Biomedical Sciences, Georgia State University. 3Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University. 4Australian Synchrotron, Australian Nuclear Science and Technology Organisation.

Seven human isoforms of importin α mediate nuclear import of cargo in a tissue- and isoform-specific manner. Understanding how nuclear import adaptors differentially interact with cargo harbouring the same NLS remains poorly understood since the NLS recognition region is highly conserved. Here, we provide a structural basis for the nuclear import specificity of W proteins in Hendra virus (HeV) and Nipah virus (NiV). We determine the structural interfaces of these cargo and importin α1 and α3, identifying a 2.4-fold more extensive interface and >50-fold binding affinity for importin α3. Through the design of importin α1 and α3 chimeric and mutant proteins, together with structures of cargo-free importin α1 and α3 isoforms, we establish that the molecular basis of specificity resides in the differential positioning of the armadillo-repeats 7 and 8. Overall, our study provides mechanistic insights into a range of important nucleocytoplasmic transport processes reliant on isoform adaptor specificity.

CHARACTERISING METAMORPHIC MOONLIGHTING CLIC PROTEINS WITHIN AGEING EPIDERMAL TISSUE

Turkewitz D.R.1, Gorrie C.A.1, Bishop D.P.2 and Valenzuela S.M.1 1School of Life Sciences, University of Technology Sydney, NSW, Australia. 2Elemental Bio-imaging Facility, University of Technology Sydney, NSW, Australia.

The chloride intracellular ion channel (CLIC) family consists of six evolutionarily conserved protein members in vertebrates with evidence emerging that CLICs exhibit enzymatic properties in addition to their traditional ion channel roles. This could serve as an important component for the protection of the intracellular environment against oxidation. We aim to confirm the presence of CLIC proteins in human and mice skin tissue and examine their role in aging and cell cycle processes. CLIC family expression and localisation in 5µm thick commercially available human (n=1; Ages=17 male, 55, female, and 96 female years) and donated mice (n=3; Ages=1 male, 4 female, 8 female months), skin tissue was determined using a combination of classical histology and immunohistochemistry. Spatiotemporal analysis of human skin tissue reveals a thinner epidermis with age, due to a flattened interface between the dermis and epidermis. CLIC1 and CLIC4 stained throughout the epidermis. CLIC1 had slight stratum corneum staining while CLIC4 had much stronger staining with co-localisation in the stratum corneum and granulosum. As the donor tissue ages, there is a significant decrease in CLIC4 protein levels and a change in localisation. CLIC4 with ageing relocates from a strong presence across the entire epidermal network to a more restricted localisation, focused in the stratum corneum and granulosum, where differentiation of keratinocytes to corneocytes occur. A spatiotemporal study shows that CLIC1 and CLIC4 proteins are present within the epidermal network of human and mice skin in high concentrations with CLIC4’s expression level and localisation changing with age regardless of the donor’s species and sex.

A FRAGMENTED APPROACH TO DRUG DISCOVERY

Wilkinson-White L.1, Headey S.2, Scanlon M.2, Szyszka T.3, Wai D.3, Kwan A.3 and Mackay J.3 1Drug Discovery, Sydney Analytical Core Facility, University of Sydney. 2Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University. 3School of Life and Environmental Sciences, University of Sydney.

In fragment based drug discovery (FBDD), a protein for which an inhibitor is sought is screened against a library of low molecular weight compounds known as fragments. The best fragment hits are then subsequently elaborated into higher affinity, larger ligands. Since the number of theoretical compounds increases exponentially with molecular weight, the screening of these smaller fragments allows for a much greater exploration of chemical space – meaning that fewer compounds need to be tested in order to identify initial hits. The Sydney FBDD platform is part of the Sydney Analytical Core Facility at the University of Sydney, and works in close collaboration with the Monash Fragment Platform (MFP) founded by A/Prof Martin Scanlon and colleagues at Monash Institute of Pharmaceutical Sciences (MIPS). We currently have several on-going projects targeting interactions for mammalian, bacterial and fungal proteins. One such project involves the ET domain of Bromodomain 3 (Brd3-ET). Brd3-ET plays a role in interacting with chromatin remodeling complexes, and the disruption of this interaction could lead to the development of novel cancer therapeutics. We have screened a fragment library curated by the MFP against Brd3-ET using an NMR Saturation-Transfer-Difference (STD) approach. Validation of hits has then been carried out using a combination of Triple-Ligand-Detect NMR, HSQC and Surface Plasmon Resonance. We have designed and tested analogues for our best validated hits, and are now in the process of combining structural information and medicinal chemistry to undertake further fragment elaboration, with the aim of developing therapeutics with high affinity and specificity.


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INVESTIGATION OF THE UNDERLYING ACTIVITIES AND MECHANISMS OF CHROMODOMAIN HELICASE DNA BINDING PROTEIN 4 (CHD4)

Zhong Y.1, Paudel B.2, Mackay J.1 and Van Oijen A.2 1School of Life and Environmental Science, University of Sydney, Australia. 2School of Chemistry, University of Wollongong, Australia.

Nucleosome is the fundamental unit of eukaryotic genome, each of which generally consists of a histone octamer core with 147 bp DNA wrapping around, allowing the whole genome can be efficiently packed into a small nucleus. On the other hand, the presence of nucleosome remodelers ensures the genetic information still remains accessible to regulatory factors. Although it was known for a long time that these remodelers can weaken the interaction between the DNA and histones in an ATP-dependent manner to expose part of the DNA sequence, the underlying mechanisms have not been fully explored, especially for the CHD remodeler family. In the project, we will assemble recombinant nucleosomes and will use them as a substrate to test the remodeling ability of CHD4 and also NuRD (Nucleosome Remodeling Deacetylase) complex, using single molecule FRET (fluorescence resonance energy transfer) experiments. The FRET experiment involves fluorescent labelling of one of the histones and also of the DNA, which allows the monitoring of the translocation of the nucleosome in real time. Other experimental approaches, including bulk experimental measurement of CHD4 remodeling rate in real-time and gel-based remodeling assays, will also be carried out. Together with CryoEM structural studies, these experiments will be used to delineate the biochemical mechanism of remodelling by CHD-family chromatin remodelling proteins.

LONG NON-CODING RNA-PROTEIN INTERACTIONS AND BUTYRATE SENSITIZATION OF COLORECTAL CANCER CELLS

Ali S.R., Orang A.V., McKinnon R.A. and Michael M.Z. Flinders Centre for Innovation in Cancer, Flinders University, Flinders Medical Centre, Adelaide, South Australia.

Colorectal cancer (CRC) is the second most common cause of Australian cancer related deaths. The development of CRC is associated with epigenetic alterations including altered histone acetylation patterns and dysregulated long non-coding RNA (lncRNA) expression. Butyrate, a short-chain fatty acid, produced from the fermentation of dietary fibre in our gut, has been shown to alter CRC cell behaviour through epigenetic mechanisms. Butyrate can alter CRC gene expression, including lncRNA expression, via histone deacetylase inhibition activity, resulting in decreased proliferation and increased apoptosis. lncRNAs regulate gene expression through various mechanisms including epigenetic modifications, lncRNA-miRNA, lncRNA-mRNA, lncRNA-protein interactions and their ability to produce regulatory ncRNAs, such as miRNAs. lncRNAs have been shown to regulate cell growth and apoptotic pathways in CRC. The effect of exposing CRC cells to the anti-tumorigenic molecule, butyrate, in combination with lncRNA knockdown has yet to be investigated. High throughput functional screens were used to systematically identify oncogenic lncRNAs, which when knocked down resulted in the sensitization of CRC cells to butyrate (enhanced anti-proliferative and pro-apoptotic effects). Knockdown of some lncRNAs resulted in enhanced apoptosis in the presence of butyrate. Pathway and network analyses assisted in identification of predicted key lncRNA-protein interactions involved in apoptosis. Further investigation of lncRNA knockdown and their protein interactors in the context of butyrate is required. Identification of oncogenic lncRNAs, and protein interactors, with the ability to sensitise CRC cells to butyrate when suppressed, may reveal the potential chemo-preventive or therapeutic value of these biological molecules.

THE PROTEIN-PROTEIN INTERACTION NETWORK OF THE EUKARYOTIC NUCLEUS

Bartolec T.K. and Wilkins M.R. Systems Biology Initiative, School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Sydney, NSW 2052, Australia.

In proteomics, mass spectrometry has traditionally been used to profile the identity, abundance and modification state of proteins within complex biological samples, providing a systems-level means to examine cellular function. Until very recently, interrogation of protein-protein interactions on a global scale has not been technically possible, with the tools of choice being low-throughput and high-noise techniques such as affinity-based pulldowns (AP-MS) and proximity labelling (such as BIO-ID). Cross-linking mass spectrometry (XL-MS) couples the use of novel cross linkers such as DSSO (membrane permeable, amino acid reactive, covalent bond forming, mass-spec cleavable) with high mass accuracy/resolution tandem mass-spectrometry. XL-MS can generate high-throughput, unbiased and rich protein interaction networks from complex samples. It provides information beyond just identifying protein interactors or complex components, but also structural information at the protein and complex level. However, the dynamic range of protein expression in the eukaryotic cells is very large - functionally important yet low abundance proteins, such as those found in the nucleus, are usually underrepresented in proteomic and hence XL-MS studies. This project aims to generate the first high coverage and high confidence interaction network of a eukaryotic nucleus, to provide fundamental insights into the architecture and dynamics of nuclear processes. Nuclei from actively dividing yeast were isolated by enzymatic cell wall digestion, mechanical cell disruption and sucrose density gradient centrifugation. SILAC-based quantification was used to calculate enrichment and purity of nuclear proteins relative to a heavy labelled whole cell lysate. Nuclei were highly enriched and showed significant depletion of common cytosolic and other organellar proteins. XL-MS data will be presented to show a draft of the eukaryotic nuclear protein-protein interaction network.

A SEARCH FOR PREDICTIVE BIOMARKERS OF OVINE PRE-PARTUM VAGINAL PROLAPSE

Brown S.C. and Norris G.E. Institute of Fundamental Science, Massey University, Palmerston North, New Zealand.

Ovine pre-partum vaginal prolapse (known as bearings in sheep) occurs within a few weeks prior to lambing. Rates of prolapse vary from 0.1 to 5% per annum, varying between season and farms. Much research has been undertaken over many years to determine the cause of this condition but no clear etiology has emerged. In this study plasma samples were collected prior to prolapse occurring and were subjected to 2D DIGE (differential in gel electrophoresis) to determine if there were differences between the protein profile of ewes that were about to prolapse and control ewes. An improved method for running 2D gels was developed resulting in improved resolution and sensitivity. Results will be presented and discussed.


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GENOME REGULATION IN CONTEXT: EXPANDING UNDERSTANDING OF GENOME REGULATORY PROGRAMS BY INTEGRATIVE ANALYSIS OF SIGNALLING PATHWAYS AND CELLULAR RESPONSES CONTROLLED BY THE PLANT HORMONE JASMONATE

Lewsey M.G.1, Zander M.2 and Ecker J.R.2, 3 1AgriBio, La Trobe University, Melbourne, VIC, Australia. 2Plant Biology Laboratory, Salk Institute, La Jolla, CA, USA. 3Howard Hughes Medical Institute, Salk Institute, La Jolla, CA, USA.

We are investigating the genome regulatory network that controls responses to the hormone jasmonate in seedlings. There is much interest currently in understanding the systems-level actions of transcription factors. However, such studies can provide much greater biological insight if set in the context of the signal transduction pathway that initiates gene expression changes and the downstream organismal phenotypes they control. We are generating an integrated framework of the response to jasmonate that spans from signal perception and transduction, through activity of master and secondary-regulatory transcription factors, to gene expression outputs and chromatin remodelling. We make use of genome-wide target maps for hundreds of transcription factors, dynamic histone modification analysis, massively multiplexed protein-protein interactome data and time series transcriptome analysis. This enables us to predict previously unknown components of the jasmonate regulatory mechanism and validate them through targeted mutant studies. The result is a comprehensive understanding of how a plant hormone remodels cellular function and plant behaviour.

THE E3 UBIQUITIN LIGASE RNF20 WRITES A CENTRAL HISTONE MODIFICATION, INFLUENCING THE CHROMATIN LANDSCAPE AND DEMONSTRATING POTENTIAL FOR TARGETING AS A CANCER THERAPEUTIC

Dickson K.-A.1, Cole A.J.1, 2, Clifton-Bligh R.J.1 and Marsh D.J.1 1Hormones and Cancer Group, Kolling Institute, University of Sydney, Sydney, NSW, Australia. 2Department of Internal Medicine, University of Michigan Comprehensive Cancer Center, Ann Arbor, MI, USA.

E3 ubiquitin ligases are responsible for the final stage of the enzymatic ubiquitination cascade, where they transfer ubiquitin from an E2 conjugating enzyme to a lysine on the recognised substrate. The most common family of E3 ligases are RING (Really Interesting New Gene) domain proteins. Using immunohistochemistry, we have shown that the ring finger protein RNF20 is strongly expressed in 87% (379 of 424) of high-grade serous ovarian cancer (HGSOC) (1). RNF20 functioning in a heterodimer with RNF40 is the main E3 ligase complex responsible for monoubiquitination of histone H2B at lysine 120 (H2Bub1). This active histone mark leads to an open chromatin configuration favouring DNA access by transcriptional complexes and DNA repair proteins. We have down-regulated RNF20 in ovarian cancer cell lines to investigate the potential of targeting this E3 ligase to overcome resistance to platinum therapies that is a major problem for the clinical management of women with ovarian cancer. Cells were studied as monolayers and spheroid cultures. We have shown that down-regulation of RNF20 decreases levels of H2Bub1, consistent with a closed chromatin configuration. Clonogenic cell survival assays following the treatment of ovarian cancer cell lines with cisplatin showed an impaired ability of cells in which RNF20 had been down-regulated to form colonies. This work provides preliminary evidence that developing strategies to therapeutically down-regulate RNF20 may increase the efficacy of standard-of-care chemotherapy for women with ovarian cancer. (1) Dickson KA et al., Hum Mol Genet (2016).

ANALYSIS OF POLYMORPHISMS IN 59 POTENTIAL CANDIDATE GENES FOR ASSOCIATION WITH HUMAN LONGEVITYMorris B.J.1, 2, 3, Donlon T.A.1, 4, 5, Chen R.1, Masaki K.H.1, 2, Allsopp R.C.6, 7, Willcox D.C.1, 2, 8, Elliott A.1 and Willcox B.J.1, 2

1Department of Research, Honolulu Heart Program/Honolulu-Asia Aging Study, Kuakini Medical Center, Honolulu, Hawaii, USA. 2Department of Geriatric Medicine, John A Burns School of Medicine, University of Hawaii, Honolulu, Hawaii. 3School of Medical Sciences and Bosch Institute, University of Sydney, NSW. 4Department of Cell and Molecular Biology, John A. Burns School of Medicine, University of Hawaii, Honolulu, Hawaii, USA;. 5Department of Pathology, John A. Burns School of Medicine, University of Hawaii, Honolulu, Hawaii, USA;. 6Institute of Biogenesis Research, John A. Burns School of Medicine, University of Hawaii, Honolulu, Hawaii. 7Cancer Center, John A. Burns School of Medicine, University of Hawaii, Honolulu, Hawaii. 8Department of Human Welfare, Okinawa International University, Okinawa, Japan.

This study tested single nucleotide polymorphisms (SNPs) in genes differentially expressed during caloric restriction in mice for association with human longevity. Subjects were American men of Japanese ancestry recruited in the mid-1960s for the Honolulu Heart Program and followed until the present or death as the Honolulu-Asia Aging Study. The longevity group comprised 440 men who survived to age ≥ 95 years and 374 men who had an average lifespan. Using leukocyte DNA from blood collected in 1991 to 1993, we tested 459 SNPs in the human homologues of 46 genes found to be differentially expressed in calorically-restricted mice (Estep et al. PLoS One 2009;4:e5242), 4 that were differentially expressed, but not significantly so, and 8 other genes of interest, for association with human longevity. All SNPs chosen had a minor allele frequency ≥ 5%. SNPs were genotyped at the University of Hawaii Cancer Center on the Illumina GoldenGate platform, which performs high-throughput SNP genotyping on universal bead arrays. Based on a dominant model of inheritance, an association with longevity at the P ≤ 0.05 level was seen for SNPs in 13 of the genes. Testing by all possible models increased the number of genes to 16. After correction for multiple testing, SNPs in 4 genes retained significance, namely, MAP3K5 (P=0.00004), SIRT7 (P=0.00004), SIRT5 (P=0.0007), and PIK3R1 (P=0.01). In a dominant model, association with longevity was seen for multiple adjacent SNPs within two of these genes (MAP3K5 and PIK3R1), as well as in FLT1, consistent with linkage disequilibrium with a causative variant in the vicinity of each respective SNP set. Haplotypes of MAP3K5 and FLT1 were associated with longevity. In conclusion, the present study implicates variation in MAP3K5, FLT1, PIK3R1, SIRT7 and SIRT5 in human longevity. These findings may merit further study in other populations and age-related conditions.

CONNECTING THE DOTS OF RNA MODIFICATIONS AND SMALL RNA PROFILES IN ARABIDOPSIS THALIANA

Ng P.1, Zhao J.1, Nguyen V.1, Morische M.2 and Searle I.1 1The University of Adelaide, South Australia, Australia. 2School of Life Science Hamburg gGmbH, Hamburg, Germany.

Small RNAs (sRNAs), such as microRNAs, have important regulatory roles in plants and animals despite their short sequences. While the biogenesis pathways of some small RNAs is clear, for other regulatory sRNAs, like tRNA-derived sRNAs, it is less clear in plants. Now it is becoming clear that many diverse cellular RNAs in plants have post-transcriptional RNA modifications and their function is only starting to be elucidated. Here we address the question whether two RNA modifications, N6-methyladensoine (m6A) and 5-methylcytosine (m5C), play a role in sRNA biogenesis in Arabidopsis thaliana. To answer this question, we Illumina-sequenced sRNAs from wild type and the m6A and m5C methyltransferase mutants mta and trm4b, respectively. Our bioinformatics analysis identified 163 sRNA loci that were increased in abundance and 11 sRNA loci that were decreased in abundance in the mutants when compared to the wild type control. Interestingly, we identified 154 sRNA loci produced only in trm4b and this suggests m5C may protect long precursor RNAs from subsequent RNA processing. We are currently characterizing a candidate ribonuclease that may cleave m5C depleted RNAs. In summary, our results suggest that RNA modifications do contribute to sRNA biogenesis in Arabidopsis thaliana.


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HARNESSING MIRNAS TO ENHANCE THE ANTI-CANCER PROPERTIES OF METFORMIN IN COLORECTAL CANCER

Orang A.V., McKinnon R.A., Petersen J. and Michael M.Z. Flinders Centre for Innovation in Cancer, Flinders University, Flinders Medical Centre, Adelaide, South Australia 5042.

Colorectal cancer (CRC) is the third most prevalent cancer in the world. The diabetes medication, metformin, is linked to cancer prevention and may selectively repress cancer proliferation. MicroRNAs are small non-coding RNAs involved in most cellular processes. Although metabolic consequences of metformin treatment have been investigated, detailed analysis of the resultant changes in gene expression is still required. Also, the effect of metformin treatment in combination with anti-cancer miRNAs has yet to be explored. Full transcriptome and small RNA next generation sequencing were performed for CRC cells treated with metformin. Following differential expression, functional enrichment and network analyses, CRC cells were transfected with miRNA mimics to explore the anti-cancer effect of differentially expressed (DE) miRNAs. In addition, unbiased high throughput functional screens of a synthetic miRNA library, in combination with metformin treatment, were used to identify additional miRNAs that impact the metformin response. Potential protein-protein interactions, within specific biological pathways that are affected by metformin treatment, were extrapolated from DE mRNAs and miRNAs and used to build system networks. Also, metformin treatment resulted in downregulation of some pro-proliferative and upregulation of some anti-proliferative miRNAs. Furthermore, miRNAs that sensitize CRC cells to the anti-cancer effect of metformin were experimentally validated. Identification of miRNAs that sensitise CRC cells to metformin, and their potential transcript targets, are early steps in the design of innovative therapeutic strategies.

NUCLEOSOME POSITIONING AS A POTENTIAL DRIVER OF TRANSCRIPTOMIC ALTERATIONS IN PROSTATE CANCER

Raina A.S., Brettingham-Moore K., Holloway A.F. and Taberlay P.C. School of Medicine, College of Health and Medicine, University of Tasmania, Hobart, TAS, Australia. 7000.

Epigenetic marks remain dynamic in most somatic cells to enable flexible gene activity. Simultaneously, this creates a cellular environment prone to “epigenetic mistakes” and this is most evident in diseases, including cancers. It is well established that cancer initiation and progression is caused by a complex series of genetic and epigenetic changes encompassing altered patterns of DNA methylation, histone modifications and the physical chromatin structure (e.g. nucleosome positions and higher-order chromatin). The ultimate consequence of these collective changes is an abnormal gene expression signature that acts as a catalyst for disease. We have utilized a model of normal prostate epithelial cells (PrEC), localized tumour (22RV1) and two metastatic prostate cancer cell lines (LNCaP and PC3) and have examined common and rare transcriptomic changes including alternative splicing, exon skipping, and differential gene expression. Integration of these data with nucleosome occupancy and methylation patterning will enable us to determine whether nucleosome positioning is likely to play a role in all or some of these transcriptomic changes.

AN OPTIMISED CHROMATIN IMMUNOPRECIPITATION (CHIP) METHOD FOR MATURE LEAVES OF NICOTIANA BENTHAMIANA TO STUDY EPIGENETIC LANDSCAPE OF AN ALLOTETRAPLOID PLANT

Ranawaka B.1, Tanurdzic M.2, Waterhouse P.1 and Naim F.1 1Centre for Tropical Crops and Biocommodities, Queensland University of Technology, Brisbane, Australia. 2School of Biological Sciences, The University of Queensland, Brisbane, Australia.

Histone modifications are involved with mitotic and meiotic inheritance of active and silent gene variants in plants. These epigenetic modifications play an important role in physiological and phenotypic features of a plant. Nicotiana benthamiana is an important biotech tool and a model plant for economically important crop family Solanaceae. To study the epigenetic landscape of this allotetraploid plant, we optimised a ChIP protocol using mature leaves of N. benthamiana. We first tested a number of published ChIP protocols, however high concentration of starch was unfavourable in obtaining good quality of nuclei. In this study, we optimised nuclei isolation, storage and chromatin shearing steps to develop a consistent ChIP method for N. benthamiana. We found that Covaris M220 was more efficient compared to conventional DNA shearing using BioRuptor. ChIP was performed using antibodies for H3K4me2/3 and H3K9me2 histone modifications and success of protocol was determined by PCR and next generation sequencing. This optimised method is applicable to ChIP of starchy tissues followed by high-throughput DNA sequencing to map epigenetic landscapes.

NOT AVAILABLE AT TIME OF PRINTING


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IS THIS THE WAY?

Tu Y.H., Ahn M., Patchett M., Rakonjac J. and Norris G. Institute of Fundamental Sciences, Massey University, Palmerston North, New Zealand.

Sheepskin, a by-product of the meat industry, is then often processed to leather, primarily for the clothing industry. Where the tanneries are distant from the abattoirs and freezing works, the raw skins have to be transported long distances to be processed. In warm weather, there is the potential for putrefaction of the skins which then have to be disposed of at a cost. The first step of leather processing is to remove the wool from the skin (depilate) without damaging it. Conventional depilation involves the use of strong alkali and sulfide, chemicals that are harmful to the environment and also to the tannery workers due to the potential production of hydrogen sulfide, a toxic, flammable gas. To solve this problem, scientists have been looking to depilate using enzymes as these are environmentally friendly. Various enzymes, such as collagenase, keratinase, and proteases have been shown to be able to remove hair from skin, but unfortunately usually damage it. Furthermore, at present they are not cost-effective at the industrial level. We have found a simple solution, a derivative of a dairy by-product, that prevents putrefaction, preserving the skin for days at room temperature. In addition, it allows easy removal of the wool from the skin. Scanning electron microscopy showed there was no obvious damage to the depilated skin and that the wool is cleanly removed from the hair follicle. To assess any less visible damage that may have occurred as a result of soaking the skin, biochemical analyses were carried out to measure changes to the amino acid composition, crosslink and proteoglycan concentrations of the depilated skins. Culture dependent methods used to isolate the microorganisms present in the solution after depilation, showed that only four main species were consistently found in the depilation fluid and on the skins. Metagenomic analysis, confirmed these findings and is being used to follow the changes in the microbiome during the course of depilation. This presentation will describe the progress that has been made to understand the science behind these observations and compare the properties of skins depilated using this method with those depilated using the traditional beamhouse process.

A CONSERVATION STRATEGY FOR NEW ZEALAND HOP (HUMULUS LUPULUS)

Wiedow C.1, Templeton K.2, Pathirana R.1 and Beatson R.A.2 1The New Zealand Institute for Plant and Food Research Limited, Private Bag 11 600, Palmerston North 4442, New Zealand. 2The New Zealand Institute for Plant and Food Research Limited, 55 Old Mill Road.

Hop (Humulus lupulus L.) genetic resources, the backbone of the NZ$27.4 million industry, is maintained in field collections in Nelson Province. None of the diseases or the insect pests, which have a significant impact on hops production in many parts of the world, has been reported in New Zealand. These diseases and pests could have a devastating impact on the New Zealand hop industry if there was an incursion in to the country, as was experienced in the kiwifruit industry during the Pseudomonas syringae pv. actinidiae outbreak in 2010. Considering the vulnerability of field collections, we have initiated long-term gene-banking strategies, including cryopreservation, to secure horticultural germplasm resources already in New Zealand. In this context, we have taken the first steps to ‘back-up’ and conserve hop genetic resources as well. These comprise 95 accessions of European and North American origin which includes New Zealand-bred cultivars. To identify the accessions for ex situ conservation by cryopreservation, it is necessary to collate and analyse genotypic, phenotypic and geographic data using specialised software, which leads to defining the core collection. First results of genetic relationships (distance/similarity) in the hop germplasm using SSR showed that cultivars bred and developed in New Zealand are a mix of both North American & European H. lupulus gene pools. This insight into the diversity of the collection can help with breeding decisions, and more importantly will provide us with a core set covering the genetic diversity, for long-term conservation through cryopreservation.

VARIABLE DEPTH FOREST: A MORE RANDOM RANDOM-FOREST FOR HETEROGENEOUS DISEASE GENETICS

Bayat A., Wilson L., O’Brien A.R., Szul P., Dunne R. and Bauer D.C. Transformational Bioinformatic Team CSIRO.

Genome-wide-association studies (GWAS) nowadays often apply Random-Forest for its capability to consider the interactions between genes. Random-Forest is an ensemble technique that adds randomness to Decision Trees, which are individual predictive machine learning models that capture interaction between genomic loci (features). This randomness allows the evaluation of a larger solution space for associated loci in GWAS-style analyses. One of the important parameters in the Random-Forest is the number of features to be evaluated at each node of each tree (mtry). This parameter directly controls the randomness in the model and substantially affects the performance by potentially limiting the exhaustive exploration of the solution space. This is especially crucial for multi-gene diseases, where sets of features may only incrementally obtain strong disease-association (deep trees) and thereby initially compete with individual features of moderate association (shallow trees). There have been efforts in the literature to find the optimal value of mtry. However, the optimal value highly depends on the dataset and its characteristics. In our work, we propose a method in which the value of mtry varies during the training process. Thus, not all trees are built using the same mtry allowing the creation of trees with diverse depths. The ensemble hence captures the strongest individual but importantly also sets of features associated with the disease. Furthermore, we evaluate changing the value of mtry at the node level, which allows an even more comprehensive search of the solution space. We assess our approach on Bone Mineral Density (BMD) case/control datasets.

A CAROTENOID-DERIVED SIGNAL CONTROLS ROOT LENGTH AND ANCHOR ROOT FORMATION IN ARABIDOPSIS

Anwar S.1, Nayak P.1, Alagoz Y.1, Watkins J.2, Hou X.2, Pogson B.2 and Cazzonelli C.1 1Hawkesbury Institute for the Environment, University of Western Sydney, Hawkesbury Campus, Bourke Street, Richmond, NSW AUSTRALIA 2753. 2Australian Research Council Centre of Excellence in Plant Energy Biology, College of Medicine, Biology and Environment, Research School of Biology, The Australian National University, Canberra, ACT 2601.

Carotenoids are organic pigments that are essential for animal and human health. They provide a precursor for Vitamin A and required for the prevention of eye diseases and certain types of cancer. In plants, they facilitate photosynthesis and photoprotection in chloroplasts. Carotenoids serve as substrates for the production of phytohormones (e.g. strigolactone and abscisic acid) and apocarotenoids (e.g. β-cyclocitral and β-ionone) that facilitate cellular acclimation to environmental stress, promote root-mycorrhiza interactions, maintain shoot dormancy and control nuclear gene expression. A function for a carotenoid-derived signal in controlling root architecture remains to be elucidated. We report the characterisation of a carotenoid mutant displaying a shorter primary root phenotype with enhanced anchor root formation. Carotenoid mutant root tissues accumulate upstream cis-carotenes, yet lack the accumulation of downstream xanthophyll carotenoids. Transcriptomic analysis of carotenoid mutant root tissues revealed a significant enrichment of genes involved in; 1) glucosinolate and glycoside metabolic process, 2) tetrapyrrole and heme binding related functions, and 3) controlling root meristem, nodule and cell division in response to nitrogen stress. A forward genetics approach identified revertants of the carotenoid mutant displaying a wild type-like root architecture. Roots from these revertants displayed an altered sensitivity to chemical inhibitors that impair carotenoid (e.g. norflurazon) and apocarotenoid (e.g. D15) biosynthesis, as well as phytohormone treatments (e.g. strigolactone, abscisic acid, auxin, brassinosteroid and ethylene). A simultaneous mapping and mutation identification by next generation sequencing approach is underway to identify genetic loci that perturb apocarotenoid signal processes that control root architecture. We discuss how a novel apocarotenoid signal pathway controls root architecture, and in particular the formation of anchor roots that provide support and enhance nutrient uptake.


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REGIONAL AND CELLULAR DISTRIBUTION OF THE TRANSLOCATOR PROTEIN 18 KDA (TSPO) IN THE NORMAL BRAIN

Betlazar C.1, 2, Harrison Brown M.1, 2, Middleton R.J.1, Banati R.1, 2 and Liu G.J.1, 2 1Australian Nuclear Science and Technology Organisation, New Illawarra Road, Lucas Heights, NSW 2234, Australia. 2Faculty of Medicine and Health, Brain and Mind Centre, University of Sydney, 94 Mallett Street, Camperdown, NSW 2050, Australia.

The inducible expression of the mitochondrial translocator protein 18 kDa (TSPO) by activated microglia is a prominent, regular feature of acute and chronic-progressive brain pathology. This expression is also the rationale for the continual development of new TSPO binding molecules for the diagnosis of neuroinflammation by molecular imaging. However, there is in the normal brain an ill-defined, low-level constitutive expression of TSPO. Taking advantage of healthy TSPO knockout mouse brain tissue to validate the specificity of TSPO antibodies, this study uses immunohistochemistry to systematically investigate the distribution and abundance of TSPO in the normal mouse brain. High magnification fluorescence microscopy reveals punctate TSPO immunostaining in vascular endothelial cells throughout the brain. Constitutive TSPO expression is also observed in neurogenic niches, the olfactory nerve layer and glomeruli of the olfactory bulb, and in cerebellar Purkinje cells. In contrast, there is no clearly discernible TSPO immunostaining in resting microglia and astrocytes of the normal brain. We conclude from these systematic observations the presence of a parenchymal-vascular expression of TSPO in the normal brain that is expected to give rise to a low baseline signal in molecular imaging studies using TSPO ligands. Mapping TSPO expression across the normal mouse brain is expected to expand future exploration into TSPO function in specific brain cell subtypes, and inform the interpretation of the growing number of studies utilising TSPO as an imaging biomarker of brain pathologies.

REVERSIBLE CELL FILAMENTATION OF UROPATHOGENIC ESCHERICHIA COLI (UPEC) AND THE ROLE OF THE CEDA GENE IN URINARY TRACT INFECTIONS

Blair T.A., Iosifidis G., Mediati D.G. and Duggin I.G. Faculty of Science, University of Technology Sydney, PO Box 123, Broadway NSW 2007, Australia.

Urinary Tract Infections (UTIs) are one of the most common and significant bacterial infections worldwide. Unfortunately, with the rise of antibiotic resistance by most bacteria, the treatment for UTIs is becoming less effective and research into new methods of treatment as well as prevention are necessary. During UPEC’s invasive lifecycle it undergoes remarkable morphological changes, including extensive cell filamentation—caused by a block to cell division with ongoing bacterial cell growth and elongation. It has been noted that the filamentation observed during UTI accompanies the rupture of the infected bladder cells. Re-initiation of division at multiple sites along the length of the filaments generates the short rod-shaped bacteria. The regulatory mechanisms that control filamentation and its reversal to allow for reinfection are unresolved. This project aims to test our hypothesis that the E. coli cedA gene (for cell division activator) is involved in regulating filamentation in UPEC. Previous work showed that overexpression of cedA prevents filamentation in a mutant strain of E. coli, called dnaA(cos), which conditionally over-replicates the chromosome and becomes filamentous. We found that dnaA(cos) filamentation is dependent on the RecA and SlmA DNA-binding proteins, consistent with a model in which the SlmA-DNA inhibits division under these conditions. Similarly, cedA was recently implicated in counteracting the effects of an antibiotic drug that blocks cell division. In UPEC, cedA was previously shown to be under positive selection, and was also found to be upregulated 8.4-fold during the filamentation stage of the UPEC infection cycle in cultured human bladder cells. We are currently determining the types of cell division blockage that can be prevented by cedA expression, and we are characterizing the phenotypic and transcriptional impact of cedA in vitro and in experimental models of UTI.

EXTRACTS FROM CEREAL GRAINS INDUCE APOPTOSIS IN COLORECTAL CANCER CELLS

Chinkwo K.1, 2., L, Rao S.1, 2o, L, Santakumar A.1, 20, L and Blanchard C.1, 2, L 1School of Biomedical Sciences, Charles Sturt University, Wagga Wagga, 2650, New South Wales, Australia. 2Australian Research Council (ARC) Industrial Transformation Training Centre (ITTC) for Functional Grains, Graham Centre for Agricultural Innovation, Charles Sturt University, Wagga Wagga, 2650, NSW.

Several plant extracts have been studied for anticancer properties, however the exact mechanisms how these compounds affect tumour growth is largely unknown. One possible mechanism is apoptosis, which is the body’s natural mechanism to eliminate abnormal cells such as cancer cells. Apoptosis is characterised by morphological changes, which include cell shrinkage, protein fragmentation and DNA degradation followed by rapid engulfment of cell debris by macrophages. This study examines the effect of phenolic extracts from four cereal grains including rice, barley, oats and sorghum to induce apoptosis in cancer cells. Whole grain aqueous extracts were used to induce apoptosis on SW480 colorectal cancer cell lines. Morphological observation and monitoring with other biological assays were conducted to study apoptosis. Extracts from pigmented cereals which include yunlu 29, purple rice, shawaya short black 1, IS11316 sorghum, showed a significant reduction in cancer cell proliferation at p<0.005. The pigmented rice extracts reduced cancer cell viability at a concentration of 500μg/mL, while normal cells were not affected. Further analysis to study the mechanism of action with APOPercentage assay, Muse and Flow cytometry showed positive for apoptosis. The results from Muse flow confirmed the activation of multiple caspases 3 and 7 as possible pathways for the induction of apoptosis. This preliminary findings suggest that, whole grain cereals pigmented rice have a potential to prevent cancer growth. Keywords: Apoptosis; Cytotoxicity; Colorectal cancer; Polyphenols; Antioxidant activity; Cancer therapy.

A NEUROPEPTIDE-COPPER COMPLEX CONTRIBUTES TO NUCLEAR COPPER UPTAKE VIA A RECEPTOR-MEDIATED ENDOCYTOSIS PATHWAY: TOWARDS UNDERSTANDING COPPER DYSREGULATION IN NEUROLOGICAL DISEASE

Christofides K., Jones M.R. and Jones C.E. Western Sydney University, School of Science and Health, Locked bag 1797, Sydney, New South Wales, Australia, 2153.

Our central nervous system employs strict regulation of the trace element copper, and dysregulation is implicated in the neuropathology of many neurodegenerative and neurocognitive diseases. Recent interest into the contribution of copper dysregulation to neurological disease has exposed our limited understanding of neuronal copper homeostasis. For instance, known uptake mechanisms do not completely account for cellular copper concentrations, suggesting alternate mechanisms remain to be found. Past work has established that some neuropeptides can bind copper, with the tachykinin neuropeptide, Neurokinin B (NKB), able to form an unusual complex with copper. Other recent approaches focusing on NKB and its corresponding receptor NK3R, identified the endogenous ligand-induced translocation of NK3R to the nucleus. Further, the newly identified copper-NKB complex has been shown to not inhibit receptor uptake, however intracellular localisation and interactions are unknown. To identify copper-NKB complex induced changes intracellularly, we used Confocal microscopy, Spectral phasor analysis, and Inductively Coupled Plasma-Mass Spectrometry to determine receptor localisation and copper concentrations in astrocyte and neuronal cell lines. Our results indicate that receptor–mediated endocytosis (RME), particularly of the [Cu(NKB)2]/NK3R complex contributes to cellular copper levels. Additionally, nuclear trafficking of the receptor and nuclear copper concentrations increase when exposed to the [Cu(NKB)2] complex. Furthermore, results illustrate that this process is clathrin-mediated. This is indicative of a novel CNS-specific copper trafficking pathway, due to the CNS-specific expression of NKB. This study establishes a foundation for future neurobiological research into the underlying role RME plays in copper regulation within the CNS.


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THE POTENTIAL OF CLIC PROTEINS AS BIOMARKERS FOR THE DETECTION AND SCREENING OF OVARIAN CANCER

D’Amario C. and Valenzuela S.M. UTS - University of Technology Sydney.

Exosomes (30-100 nm) are extracellular vesicles released by a diverse number of cells, including cancerous body cells. They carry molecular constituents from their cells of origin, principally proteins and RNA (including mRNA and miRNA). It is expected that part of this exosomal cargo would be membrane proteins, including ion channels such as CLICs. When exosomes fuse with the membranes of their target cells, changes in the membrane conductance are expected as their cargo of spontaneously inserting CLIC proteins enter the target cell membrane. This project aims to investigate the profile of such conductance changes using impedance spectroscopy and tethered membranes, and use this knowledge to develop a diagnostic screening platform for cancers that overexpress CLICs. For this study, two ovarian serous adenocarcinoma cell lines were used. As a first step, exosomes secreted by these cells were isolated using a series of ultracentrifugation. The nature of the exosomes particles needs to be verified by identifying the presence of the exosome surface marker CD9, as well as performing NTA (Nanoparticle Tracking Analysis) to measure the effective size of the isolated particles. Western Blot is used to investigate the presence of CLIC proteins in the microvesicles secreted by these ovarian cancer cell lines. For the moment, results from these preliminary steps will be presented.

A METHOD TO DETECT MICRORNAS AND VIRAL RNAS IN HUMAN SALIVA

Deutsch F.1, Khoury S.1 and Tran N.1, 2 1School of Biomedical Engineering, Faculty of Engineering and IT, University of Technology Sydney. 2Sydney Head and Neck Cancer Institute, Sydney Cancer Centre, Royal Prince Alfred Hospital.

The field of salivary diagnostics is growing because of its non-invasive collection and its potential as a biological fluid for biomarker discovery studies. Our lab is interested in using non-coding RNAs found in saliva as biomarkers for the early detection of oral cancer. This study will provide a robust methodology for the isolation of total RNA from the saliva of patients with OC. We then demonstrated that the RNA can be used to detect a range of long and short ncRNAs using RT-qPCR. Our approach utilises 1mL of whole saliva collected from 12 control volunteers and 10 patients with oral cancer. Total RNA was extracted from supernatant saliva utilising a liquid-based guanidine isothiocyanate reagent. This modified approach yielded high quantities (200ng/μl) of RNA material. We found that cell-free salivary supernatant proved a more consistent medium for total RNA yields than whole saliva or cell pellets. With this total RNA, we were able to detect specific species of microRNAs such as miR-21 and other mRNAs. Furthermore, we were able to detect viral RNA in the saliva. With this approach, we hope to develop an assay for the detection of aberrant miRNAs or HPV using the saliva of oral cancer patients. In summary, we have optimised a methodology which can provide sufficient quantities of total RNA from human spit. This material can be utilised for diagnostic purposes such as the detection of miRNA or viral biomarkers. It is a cost-effective method which most laboratories can adopt and only uses minimal amounts (1mL) of saliva.

RESCUE OF THE EPIGENETICALLY SILENCED MICRORNA-493-5P INHIBITS CANCER PROGRESSION

Gailhouste L.1, Liew L.C.1, 2, Hatada I.3 and Ochiya T.1 1National Cancer Center Research Institute Tokyo, JAPAN. 2The University of Tokyo, JAPAN. 3Gunma University, Maebashi, JAPAN.

Alteration of microRNAs (miRNAs) has been widely reported in cancer. However, little is known about the mechanisms that lead to the abnormal expression of these small noncoding RNAs during tumorigenesis. Our group previously showed that DNA methylation is a major biological phenomenon that controls the expression of specific miRNAs, which are associated with the maintenance of a hepatospecific phenotype (Gailhouste et al., 2013 and 2018). In this new study, we aimed to identify potential tumor-suppressor miRNAs silenced by aberrant CpG methylation using a human hepatocellular carcinoma (HCC) cell model. Here, we highlighted a major tumor-suppressor miRNA, miR-493-5p, which was found to be silenced by DNA hypermethylation in HCC cells. Interestingly, knockdown of the DNA methyltransferase (DNMT) 1 enzyme was associated with the demethylation of miR-493-5p promoter region and restauration of its expression. Furthermore, we demonstrated that miR-493-5p rescue suppressed HCC cell proliferation, motility, and invasion in vitro. Next, we identified insulin-like growth factor 2 (IGF2) and miR-483-3p oncomir as direct targets of miR-493-5p. In addition, the silencing of these targets was able to mimic the effect of miR-493-5p overexpression in HCC cells. Remarkably, miR-493-5p reexpression promoted a significant anticancer response in vivo by repressing HCC tumor growth. In conclusion, our results demonstrate that miR-493-5p is epigenetically silenced by CpG hypermethylation in liver cancer cells. After rescue, this miRNA acts as a tumor-suppressor by inhibiting IGF2 and miR-483-3p expression. Combined, these findings suggest the therapeutic potential of miR-493-5p for the treatment of solid tumors.

THE NEW ONCOGENIC FACTOR C11ORF67/AAMDC LINKS RAB/PIK3/MTOR SIGNALING WITH METABOLIC REPROGRAMING IN A SUBTYPE OF AGGRESSIVE HORMONE RECEPTOR POSITIVE BREAST CANCER

Golden E.1, 2, Sgro A.1, 2, Woodward E.1, 2, Rashwan R.1, 2, Cuyas E.1, 3, 4, Duffy C.1, 2, Curtis C.5, Jain M.6, Redfern A.1, 2 and Blancafort P.1, 2 1The Harry Perkins Institute of Medical Research, Nedlands, Western Australia, Australia. 2The University of Western Australia, Perth, Western Australia, Australia. 3Girona Biomedical Research Institute, Girona, Catalonia, Spain. 4Catalan Institute of Oncology, Girona, Catalonia, Spain. 5Stanford Cancer Institute, Stanford, CA, USA. 6University of California San Diego, CA USA.

Recent genomic analysis of breast cancer tumors has identified subtypes of cancers with distinct genetic and molecular profiles which drive variable responses to treatments. Of these subtypes, the intercluster 2 showed one of the poorest prognosis for patient survival, along with amplification of a region of chromosome 11 containing several oncogenic genes. Within this region, C11orf67 was also significantly amplified, although little is known about its function or its role in oncogenesis. We found that expression of C11orf67 is correlated with increased expression of folate cycle enzymes (particularly MTHFD1L), activation of the PI3K signaling pathway, and expression of the transcription factors ATF-4 and c-MYC. C11orf67 also increases susceptibility of cells to the mTOR inhibitors dactolisib and everolimus suggesting that C11orf67 promotes a reliance of the cell on 1C-metabolism for survival. C11orf67 interacts with the Rab GTPase, RABGAP1L. Rabs tightly control vesicle trafficking in the cell and several have been implicated in the regulation of mTORC1activity. These results suggest that C11orf67 supports proliferation and survival of tumour cells through upregulation of the PI3K signaling pathway by targeted vesicular trafficking, resulting in increased ATF-4 and c-MYC-mediated expression of 1C- metabolic enzymes leading to increased amino acid and nucleotide synthesis to support cell growth and proliferation. This study provides a rationale for targeted treatment of these intclus2 cancers through inhibition of PI3K signaling enzymes, folate cycle enzymes and C11orf67 activity through disruption of the interaction with RABGAP1L.


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POS-WED-110 POS-WED-111

FUNCTIONAL ANALYSIS OF PROTEIN DISULFIDE ISOMERASE P5 IN GLIOBLASTOMA CELLS BY BIOLUMINESCENCE IMAGING METHOD AT SINGLE CELL LEVEL

Horibe T. and Kawakami K. Department of Pharmacoepidemiology, Graduate School of Medicine and Public Health, Kyoto University, Japan.

It is known that P5 is a member of protein disulfide isomerase (PDI) family proteins and has isomerase and chaperone activity. However, the detailed physiological roles and relationships of P5 to the other PDI family proteins in the cells still remain obscure. In order to understand the significant roles of P5 in cancer cells, we examined the expression levels of P5 on normal or cancer cell lines by flow cytometry analysis using affinity purified anti-P5 antibody. It was found that the expression levels of P5 was increased on the surface of many types of cancer cells such as glioblastoma, breast, colon, ovary and uterine cervix cancer, and leukemia compared with that of normal cells. When we performed the knock down experiment of P5 by siRNA in glioblastoma cells, it was found that the knock down affected the Bip promoter activation during the cancer cell growing using real-time monitoring method by bioluminescence imaging at single cell level and inhibited significantly the cancer cell growth and migration. We also performed the immunoprecipitation by anti-P5 antibody in cancer and normal cells, and it was found that vimentin was one of the binding proteins to P5 and bound to this enzyme predominantly in glioblastoma cells. The knock down of P5 in glioblastoma cells did not affected the expression level of vimentin protein but affected several epithelial mesenchymal transition (EMT) markers such as snail and slug proteins in the cells. These results suggest that P5 have significant roles for cancer cell growth and might be attractive and potent target for therapy of glioblastoma.

HOMER1C AND CASR INTERACTION DIRECTING TO THE PHOSPHORYLATION OF AKTS473

Islam K.S.1, Brennan S.C.1, Rybchyn M.S.2, Mason R.S.2 and Conigrave A.D.1

1University of Sydney, School of Life and Environmental Sciences. 2University of Sydney, Discipline of Physiology, School of Medical Sciences.

Homer proteins form multivalent complexes that bind proline-rich motifs in group 1 metabotropic glutamate receptors, thereby coupling these receptors in signalling complexes (1). We hypothesized that the Homer1c scaffolding protein could interact with the calcium-sensing receptor (CaSR) leading to phosphorylation of the downstream protein kinase Akt at the site S473 (AktS473). First we examined whether increases in extracellular Ca2+ concentration (Ca2+o) might induce phosphorylation of AktS473 in HEK-293 cells that stably express the CaSR (HEK-CaSR cells), a cell line that does not express the Homer1c protein endogenously. In the absence of Homer1c, activation of the CaSR by Ca2+o (0.1 mM to 8.0 mM) for 2-30 min had no effect on pAktS473 level as determined by Western Blotting. We next investigated the behaviour of HEK-CaSR cells that had been transiently transfected with Homer1c for 24-72 h and observed that in cells that had been transiently transfected with Homer1c for 48 h elevated Ca2+o induced phosphorylation of AktS473 in the range 0.1 to 5.0 mM, with a maximal increase of 3-fold at 3.0 mM compared to control ( Ca2+o 0.1 mM; p 0.05; n = 4). This effect was not observed in HEK-CaSR cells transfected with vector control (pcDNA3.1) or in control HEK-293 cells that did not express the CaSR. These experiments demonstrate that the interaction between the CaSR and Homer1c is necessary for phosphorylation of AktS473. We next examined the mechanism by which Homer1c interacts with CaSR, leading to the downstream phosphorylation of AktS47. We first showed that we can replicate the effects seen in HEK-CaSR cells, using HEK-293 cells transiently transfected with both Homer1c and the wild-type CaSR. In these cells, pAktS473 levels were increased by two-fold in cells exposed to 3.0 mM Ca2+o compared to 0.1 mM (p 0.05, n = 6). We next co-transfected Homer1c and a mutant CaSR truncated at residue 866, which lacks the entire C-terminus. The effect was preserved intact demonstrating that the CaSR C-terminus is not required (n = 4). Further experiments indicate that receptor intra-loops 2 and 3 are required for Ca2+o-dependent AktS473 phosphorylation. The present study demonstrates that Ca2+o-induced CaSR-mediated phosphorylation of AktS473 requires a novel interaction between a class C GPCR and Homer1c with potential implications for the role of the CaSR in bone formation.

NEW INSIGHTS INTO THE REGULATION OF AKT BY ITS HYDROPHOBIC MOTIF

Kearney A.L., Cooke K.C., Burchfield J.G., Krycer J.R., Fazakerley D.J. and James D.E. The University of Sydney, Charles Perkins Centre, Faculty of Science, School of Life and Environmental Sciences, Sydney, New South Wales, Australia.

A common hallmark amongst complex diseases such as diabetes and cancer is an aberration in cell signalling. Often implicated is the protein kinase Akt, which coordinates numerous physiological processes from cell growth to metabolism. As such, Akt is one of the most highly studied kinases, with decades of in vitro experiments dedicated to understanding its regulation, primarily by phosphorylation. For instance, Akt possesses a hydrophobic motif in its C-terminal domain that requires phosphorylation at Ser474 for it to possess kinase activity in vitro. However, we and others have new evidence that this is not the case within the cell. To demonstrate this, we generated cells ectopically expressing Akt mutants which are resistant to the Akt inhibitor MK2206. Following acute inhibition of endogenous Akt with MK2206 we analysed the ability of these mutants to facilitate insulin signalling. An Akt Ser474Ala phosphomutant facilitated the phosphorylation of several Akt substrates and enabled Akt-dependent processes such as protein synthesis and GLUT4 translocation. To dissect the mechanism by which Akt retains its activity without Ser474 phosphorylation in the cell, we performed a mutational analysis of the Akt C-terminal domain. Contrary to previous in vitroevidence, the presence of the hydrophobic motif drastically increased Akt activity even in the absence of Ser474 phosphorylation. These findings pinpoint the hydrophobic motif as a key regulator of Akt kinase activity. Furthermore, they highlight the discrepancy between the activity of kinases in vitro and in vivo, having implications for translating drug screens against kinases in diabetes and cancer research.

DIFFERENTIAL GALANIN EXPRESSION BY CHEMORECEPTOR NEURONS IN MOUSE FOLLOWING LONG TERM HYPERCAPNIA CHALLENGE

Kumar N.N. and Dereli A.S. Dept Pharmacology, UNSW Sydney.

The primary role of breathing in mammals is to maintain blood gases within a narrow range. Glutamatergic chemoreceptor neurons in the retrotrapezoid nucleus (RTN) are critical in mediating the central respiratory chemoreflex, the primary homeostatic mechanism used by mammals to control blood carbon dioxide (CO2) levels. RTN neurons extensively project to the ventral respiratory column (VRC), which is located in the ventrolateral medulla (VLM) and generates the rhythmic breathing pattern. Interestingly, RTN neurons also express the inducible neuropeptide transmitter galanin and previous studies have demonstrated that injection of galanin into the VRC induces apnoea by inhibiting breathing and ventilatory chemoreflex responses. We hypothesise that long-term exposure to elevated environmental CO2 elicits chronic chemoreceptor stimulation, resulting in altered regulation of galanin neurotransmission in the RTN, which adjusts the central respiratory chemoreflex. Here we demonstrate the distribution of preprogalanin (ppGal) and galanin receptor 1 (GalR1) mRNA in the adult C57Bl6 mouse RTN and VLM respectively, using qualitative PCR and in situ hybridisation. We also measured ppGal mRNA expression in the RTN, VLM, NTS, and cerebellum, during exposure to room air (0% CO2) or hypercapnia (5% CO2, balance room air) for 3, 6 or 8 hours (short term challenge) or continuously for 10 days (long term challenge). Our main qPCR findings were that ppGal mRNA levels increased by 62% in the RTN after long term hypercapnia (10 days) compared to room air (p<0.001). No changes in gene expression were observed in the RTN following short term hypercapnia. Conversely, ppGal expression in the VLM decreased by 32% following short term hypercapnia (p<0.05) however no changes were observed after long term challenge. In summary we have identified a role for peptidergic circuits in plasticity arising in the RTN, during long term adaptation of breathing to respiratory stressors.


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INVESTIGATING NEUROKININ B-NK3R RECEPTOR MEDIATED ENDOCYTOSIS AS A NOVEL COPPER TRAFFICKING MECHANISM

Menon R. and Jones C.E. School of Science and Health, Western Sydney University, Locked bag 1797, Sydney, New South Wales, Australia, 2153.

Neurokinins are a family of neuropeptides which have a broad role in cognition, emotion and reproduction. Several neurokinins are also known to bind copper and one, neurokinin B (NKB), was shown to facilitate copper uptake into astrocytes suggesting a role in copper homeostasis. Copper homeostasis is critical for normal brain function and dyshomeostasis is a feature of several neurodegenerative disorders, including Alzheimer’s disease. Little is known about the mechanisms and pathways by which neurokinin B can traffic copper into astrocytes and participate in cellular responses. To begin to address this, we used fluorescein-labelled NKB (FNKB) to track the peptide in the cell, and immunofluorescence to follow the NKB receptor (NK3R) after activation by NKB and copper-bound NKB. We show that FNKB is taken into the cell and localises to perinuclear vesicles. Intracellular concentrations are increased in the presence of the recycling inhibitor monensin, suggesting that NKB is actively recycled in these cells. FNKB and the NKB receptor do co-localise, but only in vesicles close to the plasma membrane. The addition of copper as [CuII(NKB2)] causes an increase in cellular copper that rises in the presence of monensin. We predict that receptor mediated endocytosis accounts for the increased copper concentration. This mechanism has not previously been observed for any metal other than iron.

HYPOXIA REGULATES DPP4 EXPRESSION AND PROTEOLYTIC SHEDDING OF INACTIVE ENZYME FROM THE SURFACE OF OVARIAN CANCER CELLS

Moffitt L.R.1, 2, Bilandzic M.1, 2, Wilson A.1, 2, Chen Y.1, 2, Plebanski M.3, Gorrell M.D.4 and Stephens A.N.1, 2, 5 1Monash University, VIC, Australia. 2Hudson Institute of Medical Research, VIC, Australia. 3RMIT University, VIC, Australia. 4University of Sydney, NSW, Australia. 5Epworth Research Institute, VIC, Australia.

Ovarian cancer is the leading cause of death from a gynaecological malignancy. There is an urgent need to develop targeted therapies to improve outcomes for patients. Dipeptidyl peptidase 4 (DPP4) is a serine protease with a diverse role in tumour progression; involving mediating cell-cell interactions, immune modulation and degradation of the extracellular matrix. DPP4 is overexpressed within the hypoxic microenvironment of solid ovarian tumours and tumour-derived spheroids, which drive metastasis of the disease. Whilst DPP4 and its activity is regulated by hypoxia in other cell types, the mechanism is cell-type dependent and has never been investigated in ovarian cancer. In this study we examined the effect of hypoxic growth on DPP4 expression and activity in human ovarian cancer cell lines OVCAR4, SKOV3 and CaOV3. Cells grown under chronic hypoxic stress exhibited upregulation of DPP4, which was correlated with an increase in enzyme release from the cell surface. DPP4 in culture media was inactive, however, and its presence correlated with simultaneous increases in mRNA and protein expression of the matrix metalloproteinases; MMP-1, MMP-10 and MMP-13. We provide the first evidence of the hypoxic regulation of DPP4 in ovarian cancer cell lines and identify MMP-1, MMP-10 and MMP-13 as possible mediators for the shedding of inactive DPP4 from the cell surface. Our study offers a novel insight into the potential role of DPP4 in epithelial ovarian cancer, and highlights some possible therapeutic approaches targeting DPP4 expression or activity.

PHOSPHOPROTEOMIC SCREENING OF EXERCISE-LIKE TREATMENTS REVEALS DRUG INTERACTIONS REGULATING MYOKINE SECRETION

Needham E.J.1, Humphrey S.J.1, Cooke K.C.1, Duan X.1, Parker B.L.1 and James D.E.1, 2 1Charles Perkins Centre, The University of Sydney, Sydney, NSW, Australia. 2School of Medicine, The University of Sydney, Sydney, NSW, Australia.

Exercise improves health through adaptive metabolic and mechanical remodelling, mediated by a network of kinases in response to homeostatic stress. Secretion of factors from exercising muscle causes systemic benefits. The exercise kinase network provides an approach to understand the regulation of myokine secretion as we predict that activation of a subset of the kinases will trigger secretion pathways. The phosphoproteome of acutely exercised human skeletal muscle provides a blueprint of kinase regulation, however, an in vitro model of exercise is needed to dissect molecular mechanisms. We performed a phosphoproteomic screen of 10 candidate exercise-like treatments in rat L6 myotubes quantifying 20,249 unique Class I phosphopeptides. Of the regulated phosphosites in exercised human skeletal muscle that mapped to the in vitro treatments, 74% were regulated by at least one treatment. Computing in silico combinations predicted that isoproterenol combined with thapsigargin would most closely reproduce the exercise phosphoproteome. The phosphoproteome of the combined treatments had a greater overlap with exercise than the in silico prediction by 16%, highlighting the importance of interactions between stimuli. As exercise involves multiple stimuli, their interactions may be required for myokine secretion. As the combined treatments recapitulated a sizable proportion of exercise signalling, we measured the secretion of exercise factors to test if exercise-related phenotypes were also reproduced. Global mass spectrometry-based secretome analysis of treated L6 myotubes revealed known secreted factors in exercise and novel candidates. The phosphoproteome provides a resource to determine the upstream regulation of myokine secretion.

COMPARING THE EFFECT OF DIFFERENT GROWTH FACTORS ON MELANOMA CELL SIGNALLING PATHWAYS

Chan X.Y., Osman N. and Piva T.J. School of Health & Biomedical Sciences, RMIT University, Bundoora, Vic 3083.

Vemurafenib (PLX4032) is often used to treat melanomas that possess the BRAFV600E mutation. While the tumours are shown initially to regress, they become resistant to the drug and the patient relapsed and eventually dies. It has been shown that the growth factors secreted by adjacent cells activated signalling pathways in these melanoma cells. Using specific signalling pathway inhibitors, we investigated the growth factor-activation of intracellular signalling pathways in four melanoma cell lines. MM418-C1 (1° tumour) and C32 (2° tumour) cells harbour the BRAFV600E mutation, while MM329 (1° tumour) and D24 (2° tumour) cells do not. Growth factors (HGF or TGFα) were added to cells that had been serum starved for 24 h in low serum (0.5% FBS) containing media, and the expression of p-BRAF, p-Akt, p-ERK1/2, p-MEK, p-p38, p-JNK1/2 were quantified using Western blots. From preliminary data, we observed that HGF signalled through BRAFV600E in MM418-C1 cells, but TGFα signalled through BRAFWT in D24 cells. However, expression of other downstream signalling intermediates (p-ERK1/2, p-p38 and p-JNK1/2) of BRAF was increased when either HGF or TGFα was added to the cell lines. We also observed the activation of p-Akt (Ser473 and Thr308) by both growth factors which suggests that the PI3K-AKT-mTOR pathway is active in these cell lines. The effect of these growth factors and/or specific signalling pathway inhibitors on these signaling intermediates and cell migration were examined and the significance of the results will be discussed.


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MIMICKING THE BIOMECHANICAL FEATURES OF THE BRAIN FOR IMPROVED IDENTIFICATION OF EFFECTIVE TREATMENTS FOR BRAIN CANCERPrior V.G.1, 2, 5, Vessey J.Y.1, Griffin K.R.1, Sarker F.A.1, 2, Grundy T.J.1, 5, Turner K.1, Bradbury P.1, Mitchell C.B.1, Day B.W.3, 4and O’Neill G.M.1, 4, 5 1Children’s Cancer Research Unit, Kids Research at the Children’s Hospital at Westmead NSW, Australia. 2Department of Anatomy & Histology, School of Medical Science, University of Sydney, Sydney, Australia. 3Immunology in Cancer & Infection Laboratory, QIMR Berghofer Medical Research Institute, Brisbane, Queensland, Australia. 4Brain Cancer Discovery Collaborative, Australia. 5Discipline of Paediatrics & Child Health, University of Sydney, Sydney, Australia.Mechanopharmacology is a recently coined term that reflects the role that the physical forces contained within tissues and organs in the body play in proliferation and invasion and, ultimately, cellular response to drugs. Seminal studies in the tissue engineering field revealed that culturing stem cells on matrices which match the softness of brain induced the cells to differentiate into neuronal lineages, independently of any other external factors. While the role for biomechanical forces has begun to be addressed in other solid cancer types, to date there has been limited consideration of this parameter in brain cancer. We hypothesized that mechanosensing (cellular sensing of mechanical forces contained within the surrounding tissue) may alter the biology and response of gliomas to anti-cancer treatments. This was tested using combinations of kinome screening and materials engineered to recapitulate the brain’s soft features, together with primary patient-derived glioblastoma (GBM) and diffuse intrinsic pontine glioma (DIPG) cells cultured in serum-free media to maintain in vivo characteristics. As a key feature of high grade gliomas is their propensity to migrate and invade widely throughout the brain we used time-lapse imaging and cell tracking to quantify cell migration on a range of soft brain-like matrices. This revealed that the majority of the patient lines exhibited mechanosensitive migration. Kinome screening next identified the putative therapeutic target Aurora Kinase B (AURKB) as a mechanosensitive kinase. Importantly, we have demonstrated that mechanosensitive patient lines are significantly resistant to AURKB inhibitors when cultured on soft surfaces versus plastic. Our studies suggest that the brain’s biomechanical milieu is indeed an important determinant of glioma biology and response to anti-cancer treatments. We propose that assessment of the mechanopharmacology of putative treatments for gliomas should be considered as part of the preclinical assessments in the progression of novel treatments to clinical trial.

EGF EXACERBATES TGF-BETA-INDUCED EPITHELIAL-MESENCHYMAL TRANSITION IN THE OCULAR LENS: A NOVEL MECHANISM IN CATARACT FORMATION

Shu D.Y.1, 2 and Lovicu F.J.1, 2 1Discipline of Anatomy and Histology, Bosch Institute, University of Sydney, Camperdown, NSW, Australia. 2Save Sight Institute, Sydney Medical School, Sydney, NSW, Australia.

The ocular lens is surrounded by a cocktail of growth factors that differentially influence cellular behaviour. While many of these promote normal physiological processes, such as EGF, TGFβ induces lens pathology, namely, epithelial-mesenchymal transition (EMT) leading to cataract and subsequent visual impairment. This study seeks to decipher how EGF impacts on TGFβ-induced EMT in the lens. Lens epithelial cells (LECs) in explants prepared from 21-day-old Wistar rats were treated with either 200 pg/ml TGFβ2, 5 ng/ml EGF, or a combination of these, with or without pre-treatment with PD153035 (EGFR inhibitor), U0126 (MEK inhibitor) or SIS3 (Smad3 inhibitor). Co-treatment with TGFβ2 and EGF not only resulted in a more pronounced morphological elongation and transdifferentiation of LECs into myofibroblastic cells, compared to TGFβ2 alone, but had higher protein expression levels of mesenchymal markers (α-SMA and tropomyosin). Adding EGF to a less potent dose of TGFβ2 (50 pg/ml) induced LECs to undergo EMT similar to treatment with a standard dose of TGFβ2 at 200 pg/ml over 5 days culture. EGF alone did not induce EMT in LECs. Co-treatment with EGF and TGFβ2 activated a complex and integrated network of Smad2/3-, ERK1/2- and EGFR-signalling pathways. Inhibition of EGFR-signalling using PD153035 blocked the EMT response induced by co-treatment with EGF and TGFβ2. Taken together, our data show that EGF can exacerbate the TGFβ2-induced EMT in LECs, highlighting the importance of EGFR-signalling in cataract formation. By directly blocking EGFR signalling, the activity of both EGF and TGFβ2 can be simultaneously suppressed, thus serving as a potential drug target for treating cataract.

ELUCIDATION OF THE TYPE OF CELL DEATH AT THE ONSET OF NON-ALCOHOLIC STEATOHEPATITIS (NASH)

Tsurusaki S.1, 2, Matsuda M.1, Shimizu S.3, Nakano H.4, Miyajima A.2 and Tanaka M.1, 2 1National Center for Global Health and Medicine, Tokyo, Japan. 2Institute for Quantitative Biology, The University of Tokyo, Tokyo, Japan. 3Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan. 4Toho University School of Medicine, Tokyo, Japan.

Non-alcoholic steatohepatitis (NASH) is a liver disease with which the number of patients has recently increased. Although the pathogenesis of NASH is still unclear, hepatic cell death triggered by abnormal accumulation of fat is considered as an etiology of NASH. In general, the types of cell death have been classified into apoptosis and necrosis. However, recent accumulating evidences about the new type of cell death “programed necrosis” have highlighted the role and significance of multiple types of cell death in human diseases. In this study, we aimed to identify the type of hepatic cell death involved in the initial onset of NASH. To address the issue, we used choline-deficient, ethionine-supplemented diet (CDE diet) model, which is a murine model of NASH. By monitoring the serum alanine aminotransferase (ALT) levels as a liver damage marker and the type of cell death in the progression of NASH, we found that massive hepatic cell death accompanied by apoptotic and necrotic hepatocytes was induced two days after feeding of CDE diet. To further investigate the type of cell death triggering the onset of NASH, we tested the inhibition of specific type of cell death by using chemical inhibitors or genetically modified mice. Consequently, ferroptosis inhibitors dramatically suppressed the onset of hepatic cell death in CDE-induced liver injury, suggesting that ferroptosis is a crucial cell death and a promising target for the pathogenesis and treatment of NASH.

FIBROBLAST ACTIVATION PROTEIN (FAP) PROMOTES LIVER FIBROSIS IN MICE

Lay A.J.1, Xiang M.S.W.1, Liu Y.1, Wetzel S.1, Lv Y.1, Hamsom E.J.1, Zhang H.E.1, Chowdhury S.1, McCaughan G.W.1, 2 and Gorrell M.D.1, 2, 3 1Centenary Institute, NSW. 2A.W. Morrow Gastroenterology and Liver Centre, Royal Prince Alfred Hospital, NSW. 3University of Sydney, Faculty of Medicine and Health, NSW.

Chronic liver diseases resulting in cirrhosis are characterised by progressive inflammation and fibrosis, with continual extracellular matrix (ECM) remodelling resulting in scar tissue formation and eventual loss of function. However, the exact mechanisms underlying these changes are not well understood. Fibroblast activation protein (FAP) is an extracellular serine protease within the dipeptidyl peptidase IV (DPP4) family. FAP expression is known to regulate multiple processes including cell proliferation, adhesion and migration, which may impact upon ECM remodelling. Intrahepatic FAP expression has previously been shown to correlate positively with liver fibrosis severity in humans, implicating it in chronic liver disease. In our present study, we aimed to investigate the role of FAP during liver fibrosis and the underlying mechanisms. Using our unique FAPgki (gene knock-in) mouse model, we show that after 16 weeks of thioacetamide-induced fibrosis, mice deficient in FAP enzyme activity (FAPgki) developed significantly less liver fibrosis compared to WT littermate controls, as measured by digital image analysis of Sirius Red stained sections. This difference in fibrosis was accompanied by a significant decrease in intrahepatic leukocyte clusters. Furthermore, we showed that FAP modulated the abundance of proteins involved in ECM remodelling, including collagen, ECM-1 and fibronectin. Our data suggests that FAP promotes liver fibrosis, possibly though modulating ECM, and may be an important therapeutic target for the treatment of chronic liver disease.


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CHARACTERIZATION OF RAGC PHOSPHORYLATION SITES REVEALS SELF-REGULATION OF MTOR COMPLEX 1

Yang G.1, Murashige D.2, Francis D.1, Wang Q.1, 3, Humphrey S.1, Neely G.1 and James D.1 1The University of Sydney, School of Life and Environmental Sciences, Charles Perkins Centre, Sydney. 2University of Pennsylvania, Perelman School of Medicine, Philadelphia, PA 19104, USA. 3Sun Yat-sen University, School of Pharmaceutical Sciences (Shenzhen), Guangzhou 510275, China.

The mechanistic target of rapamycin complex1 (mTORC1) controls cell growth, proliferation and metabolism in response to diverse stimuli. Two major parallel pathways are implicated in mTORC1 regulation including a growth factor-responsive pathway mediated via TSC2/Rheb, and an amino acid-responsive pathway mediated via the Rag GTPases. Here we identify and characterize three highly conserved growth factor-responsive phosphorylation sites on RagC, a component of the Rag heterodimer, implicating cross talk between amino acid and growth factor-mediated regulation of mTORC1. We find that RagC phosphorylation is associated with destabilization of mTORC1, and is essential for both growth factor and amino acid-induced mTORC1 activation. Functionally, phosphorylation of RagC suppresses starvation-induced autophagy, and genetic studies in Drosophila reveal that RagC phosphorylation plays an essential role in regulation of cell growth. Finally, we identify mTORC1 as an upstream kinase of RagC on Ser21. Our data highlight the convergence of amino acid and growth factor pathways at the level of the RagC GTPase, and identify a previously unappreciated auto-regulatory mechanism of mTORC1 activity.

IS ALIX A COFACTOR/ADAPTOR FOR ABCG1/ABCG4 AND NEDD4-1

Alrosan A.1, Sharpe L.2, Yang A.1, Brown A.J.2 and Gelissen I.C.1 1School of Pharmacy, Faculty of Medicine and Health, University of Sydney. 2School of Biotechnology and Biomolecular Sciences, University of NSW, Sydney.

Several ABC transporters, including ABCA1, ABCG1 and its close relative ABCG4, are essential regulators of cellular lipid homeostasis. ABCA1 and ABCG1 are expressed ubiquitously and are known in the context of macrophage lipid homeostasis and atherosclerosis, with substrates including cholesterol and phospholipids. ABCG4 is expressed almost exclusively in the brain, with potential substrates including cholesterol, oxysterols and cholesterol synthesis intermediates. ABCG4 has been linked to Alzheimer’s disease due to its additional role in amyloid-beta peptide export from brain cells. The protein half-lives of these transporters are relatively short (in the vicinity of 1-5 hours, dependent upon isoform variations), and it has been suggested that they are highly regulated at the post-translational level. We have previously shown that the HECT-domain E3-ubiquitin ligase, NEDD4-1, regulates the protein stability and activity of ABCG1 and ABCG4. E3-ligases are becoming increasingly of interest as therapeutic targets, hence elucidating these pathways is of interest. NEDD4-1 has a number of other identified targets, and can make use of a cofactor. Using peptide-mass spectrometry, we identified a potential cofactor/adaptor protein that might facilitate the interaction between ABCG1 and NEDD4-1, named Alix (also known as Programmed cell dead 6-interacting protein, AIP1 or ALG-2-interacting protein). Intriguingly, Alix has independently been identified as a possible plasma marker for Alzheimer’s disease. We hypothesised that Alix may facilitate the interaction between NEDD4-1 and the ABC transporters. Silencing of Alix in CHOK1 cells overexpressing ABCG1 increased transporter protein levels. Co-immunoprecipitation experiments were able to pull down all three proteins, i.e ABCG1, Alix and NEDD4-1. Further experiments are currently performed with ABCG4 as well as effects of Alix silencing on transporter activity. These studies will elucidate whether Alix plays a role in regulating the activity of ABCG1 as well as ABCG4.

A NOVEL REGULATORY STEP IN CHOLESTEROL BIOSYNTHESIS REVEALS A POTENTIALLY CRUCIAL E3 LIGASE IN CHOLESTEROL METABOLISM AND STABILIZATION OF MAMMALIAN STEROL REDUCTASES

Capell-Hattam I.M., Prahbu A.V. and Brown A.J. School of Biotechnology and Biomolecular Sciences, UNSW Sydney, NSW 2052, Australia.

Cholesterol homeostasis requires a careful balance of cholesterol uptake, efflux and synthesis. The biosynthesis of cholesterol consists of over 20 enzymes and is tightly regulated at the gene and protein level. Here we investigated the post-translational regulation of two enzymes involved in cholesterol biosynthesis that share a high level of sequence identity – 14-Dehydrocholesterol Reductase (DHCR14) and the Lamin-B Receptor (LBR). These enzymes are 3β-hydroxysterol Δ14-reductases, which catalyse the same reaction in the pathway. These enzymes also share a high level of homology with one of the terminal enzymes of cholesterol biosynthesis, DHCR7. Loss of function mutations in DHCR7 lead to the devastating developmental disorder Smith-Lemmli-Opitz Syndrome (SLOS) We found that despite the high similarities between these enzymes, LBR protein levels were stable over time, whereas DHCR14 protein was rapidly turned over. We establish that the cholesterol mediated degradation of DHCR14 occurs via the proteasome and is triggered by cholesterol, as well as sterol intermediates.

INVESTIGATION OF RADIATION-INDUCED BYSTANDER EFFECTS ON CANCER CELLS USING NMR METABOLOMICS

Chen B.Q.1, Rogers L.J.2, Esteves A.I.S.2, McKenzie D.R.3, Suchowerska N.2 and Kwan A.1 1School of Life and Environmental Sciences, University of Sydney, NSW, Australia. 2Department of Radiation Oncology, Chris O’Brien Lifehouse, Camperdown, NSW, Australia. 3School of Physics, University of Sydney, NSW, Australia.Cancer is a leading cause of death and is often treated with radiotherapy. Typically, a uniform radiation dose to the target volume is prescribed, but periodically modulated doses have been shown to enhance cancer cell death relative to normal cell survival [1]. This effect has been attributed to soluble signalling molecules known as radiation-induced bystander factors [2]. Currently, these factors are not well characterised and their effects are not considered in treatment planning. Identification of these factors will shed light on the mechanism that underlies the bystander effect and improve treatment plans and patient outcomes. Metabolomics is a technique that has been frequently used for the identification and quantification of small molecules (< 1500 Da) in biofluids, but has not been directly used to study the bystander effect. We use Nuclear Magnetic Resonance (NMR) spectroscopy to examine the changes in metabolite profiles of cancer cells irradiated under uniform and spatially modulated fields, compared to unirradiated cells.We have optimised the protocol for cell collection and extraction, including comparing different extraction methods, plus NMR acquisition parameters. Differential expression of observed metabolites informs the nature of the mechanisms driving bystander effects. 1. Peng, V., et al., Grid therapy using high definition multileaf collimators: realizing benefits of the bystander effect. Acta Oncologica, 2017. 56(8): p. 1048-1059. 2. Prise, K.M. and J.M. O’Sullivan, Radiation-induced bystander signalling in cancer therapy. Nat Rev Cancer, 2009. 9(5): p. 351-60.


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EXPLOITING THE ALTERED BIOENERGETIC METABOLISM TO OVERCOME CANCER MULTIDRUG RESISTANCE

Gao X., Aguanno D. and Callaghan R. Department of Biomedical Science and Biochemistry, Research School of Biology, The Australian National University, Canberra, ACT, 0200 Australia.

A major reason for the failure of cancer chemotherapy is the development of multidrug resistance, which may result from many cellular factors. One of the most prevalent factors is the over-expression of the transporter P-glycoprotein (P-gp). This protein mediates the ATP-dependent efflux of drugs from resistant cancer cells against considerable concentration gradients and this activity may impact the cellular ATP pool. Generation of ATP in cancer cells is primarily rely on the catabolism of glucose through the glycolytic pathway, preventing full utilisation of the energy from glucose. This strategy in cancer cells ensures a balance between energy production and sufficient biomass production to sustain proliferation. Does the ATP utilisation caused by active P-gp perturb this balance and initiate a distinct metabolic strategy for resistant cells? The investigation will focus on the metabolic strategy of P-gp over-expressing cells in the presence of drugs stimulating the transport activity of P-gp. We have demonstrated that resistant cells display higher basal levels of ATP, glucose uptake and lactate production, but significantly suppressed mitochondrial respiration. Drug treatment rapidly lowered the cellular ATP levels, causing further enhanced glucose uptake. However, this observation was not associated with an increase in lactate production or glycolytic flux. How do these resistant cells restore the ATP homeostasis? Additionally, we have demonstrated that cellular respiration was elevated to the maximal rate after drug addition, causing a significant increase in ROS levels. In summary, stimulation of P-gp transport activity affected ATP homeostasis, thereby forcing the resistant cells to produce more ATP through mitochondrial oxidative phosphorylation. This adaptive response perturbs ROS homeostasis in cells and may cause apoptosis.

THE E3 UBIQUITIN LIGASE MARCH6 IS STABILISED BY CHOLESTEROL

Howe V., Sharpe L.J. and Brown A.J. UNSW Sydney, UNSW, 2052.

The E3 ligase membrane-associated RING finger (C3HC4) 6 (MARCH6) helps control protein levels of the two rate-limiting enzymes in cholesterol synthesis, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) and squalene monooxygenase (SM). However, little is known about how MARCH6 itself is regulated. Considering MARCH6’s role in controlling cholesterol synthesis, we hypothesised that cholesterol may regulate MARCH6. Indeed, we found that cholesterol stabilises MARCH6 protein. Preliminary evidence suggests that Insigs also play a role in regulating MARCH6’s stability. Our ongoing investigations focus on the nature of the MARCH6-Insig interplay, and whether other intermediary proteins are involved. This work provides new insights into the complex feedback mechanisms underlying cholesterol homeostasis.

PYRUVATE CARBOXYLASE SUPPORTS BIOSYNTHETIC PATHWAYS REQUIRED FOR GROWTH AND SURVIVAL OF HIGHLY INVASIVE BREAST CANCER MDA-MB-231 CELLS

Phannasil P.1, Rattanapornsompong K.1, Akekawatchai C.2, El-Azzouny M.3, Ansari I.H.4, Longacre M.J.4, MacDonald M.J.4 and Jitrapakdee S.1 1Department of Biochemistry Faculty of Science, Mahidol University, Bangkok, Thailand. 2Department of Allied Health Sciences, Thammasat University, Bangkok, Thailand. 3Department of Internal Medicine, University of Michigan, Ann Arbor, MI, USA. 4School of Medicine and Public Health, University of Wisconsin, Madison, WI, USA.

Pyruvate carboxylase (PC), an anaplerotic enzyme, plays an essential role in various cellular metabolic pathways including gluconeogenesis, lipogenesis and amino acid synthesis. PC is up-regulated in human breast cancer tissue and its expression is correlated with tumor size and aggressive phenotype. Here we investigate the role of PC in supporting cell growth and survival using gene silencing approach combining with metabolomics analysis. We generated multiple stable PC knockdown cell lines from the MDA-MB-231 cell line and used mass spectrometry with 13C6-glucose and 13C5-glutamine to discern the pathways that use PC in support of cell growth. Cells with severe PC knockdown showed a marked reduction of proliferation rate associated with apoptosis, suggesting the perturbation of biosynthetic pathways that required for cell growth. Strong PC suppression lowered glucose incorporation into downstream metabolites of oxaloacetate, the product of the PC reaction, including malate, citrate and aspartate. Levels of pyruvate, lactate, the redox partner of pyruvate, and acetyl-CoA were also lower suggesting the impairment of mitochondrial pyruvate cycles. Serine, glycine and 5-carbon sugar levels and flux of glucose into fatty acids were decreased. ATP, ADP and NAD(H) levels were unchanged indicating that PC suppression did not significantly affect mitochondrial energy production. The data indicate that the major metabolic roles of PC in invasive breast cancer are primarily anaplerosis, pyruvate cycling and mitochondrial biosynthesis of precursors of cellular components required for breast cancer cell growth and replication.

WE ARE WHAT WE EAT: IDENTIFYING A REGULATORY CROSSTALK BETWEEN CENTRAL CARBON METABOLISM AND CELL DIVISION IN BACTERIA

Mann R.1, Bottomley A.L.1, Monahan L.G.1, Sonenshein A.L.2 and Harry E.J.1 1The ithree Institute, University of Technology Sydney, Ultimo, Australia. 2Tufts University, Boston, Massachusetts, United States of America.

Cell division, an essential process in bacteria, is driven by a cytoskeletal ring structure - the Z ring, whose formation at midcell site must be tightly regulated to ensure faithful cell division. Several mechanisms have previously been described that influence the positioning and timing of Z ring assembly, but one important yet poorly understood aspect of cell division regulation is the need to coordinate division with cell growth and nutrient availability. We have demonstrated for the first time the role of central carbon metabolism (CCM) in Z ring formation in the bacterium Bacillus subtilis. A mutant of the glycolytic enzyme pyruvate kinase (pyk), which produces pyruvate in the final step of glycolysis, was shown to form acentral Z rings. This Z ring placement defect, however, was rescued when pyruvate was added to the growth medium, signifying pyruvate to be a specific key metabolite in coordinating cell growth with division by regulating midcell Z ring formation. Along with validating a previous observation that the pyk mutant displays a DNA replication underinitiation phenotype, we have also found that the addition of pyruvate rescues this underinitiation defect of Δpyk cells. This observation correlates with the Z ring placement rescue observed after pyruvate addition and raises the possibility that pyruvate is involved in cell division regulation via its effect on the DNA replication process. Overall, we hypothesize that the metabolic regulation of the division process is indeed an outcome of the effect that metabolic perturbation has on the DNA replication process. Understanding this interconnection between various cell cycle events not only helps us answer some of the age-old basic biology questions, but also uncovers a new area of interest for possible exploitation for the development of antimicrobial agents.


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WILD AUSTRALIAN YEAST: TO BEER AND BEYOND

Kerr E.D.1, Howes M.2 and Schulz B.L.1 1School of Chemistry and Molecular Biosciences, The University of Queensland. 2Newstead Brewing Co.

Yeast is essential for making beer, as it produces ethanol and contributes heavily to its flavour and sensory properties. Commercial breweries typically use Saccharomyces cerevisiae for production of ales, and Saccharomyces pastorianus for lagers. The most notable exceptions to this are sour, lambic, and American coolship ales, where wort is exposed to a mix of environmental yeast and bacteria, rather than commercially cultivated Saccharomyces strains. Spontaneous environmental inoculation for beer fermentation is becoming more common and rather trendy. However, such wild ferments have unpredictable rates, efficiencies, and end products. We propose instead to isolate clonal strains of wild yeast, to allow efficient and predictable production of beers with interesting and diverse flavour profiles. Here, we describe a method for isolating and characterising wild yeast for brewing unique and interesting beers. Yeast is collected from the wild and grown on solid selective media to isolate clonal strains. These clones are identified by internal transcribed region (ITS) PCR and Sanger sequencing. Each clone is grown in liquid wort to measure growth on oligomaltose, and to identify fermentation end products. Gas chromatography mass spectrometry (Headspace-GCMS) is then used to quantify ethanol and other fermentation end products from fermentation. Strains that efficiently grow and produce ethanol are then fermented, and flavour profiles are measured using Headspace-GCMS. Intermediate upscaling of strains with efficient growth, ethanol production, and pleasant or interesting smelling ferments is then performed in a food grade brewery to allow sensory evaluation. This workflow provides the tools needed go from environmental yeast isolation to producing brewery-quality beer. This workflow also has uses beyond brewing, allowing quick and robust identification of wild yeasts and identification of compounds related to flavours in diverse beverages or industrial applications.

THE USE OF BIOID TO IDENTIFY NEW SUBSTRATES OF THE E3 UBIQUITIN LIGASE MARCH6

Scott N.A. and Brown A.J. School of Biotechnology and Biomolecular Sciences, UNSW Sydney, NSW 2052, Australia.

BioID is a new method used to detect interacting proteins within cells. This technique works by fusing a promiscuous biotin ligase, BirA, onto the protein of interest to tag nearby proteins with biotin. Because biotinylation is a rare modification we can isolate and identify the proteins via streptavidin-based approaches. Our protein of interest is MARCH6, an E3 ubiquitin ligase that targets two key cholesterol synthesis enzymes and several others outside of this for degradation. We have used BioID to identify new candidate substrates and interacting partners for MARCH6. Insight into these interacting partners will lead to a better understanding of the function of MARCH6 and its network of interactors, including novel substrates. All E3 ubiquitin ligases interact with complex networks of proteins, however, these networks are poorly understood. As key regulators of protein stability, E3 ubiquitin ligases themselves are of interest as pharmaceutical targets in the treatment of numerous diseases.

IDENTIFICATION OF A NOVEL PLAYER IN INSULIN-MEDIATED LIPOLYSIS INHIBITION

Zadoorian A., James D.E. and Stoeckli J. Charles Perkins Centre, University of Sydney.

Responsible for releasing free fatty acids (FFA) from triglycerides (TG), lipolysis is a catabolic process essential for maintaining whole-body energy homeostasis. It is activated by β-adrenergic stimulation during starvation or prolonged exercise, and is inhibited by insulin following nutrient intake. Dysregulated lipolysis, as is the case in insulin resistance, can lead to increased circulating FFA and ectopic TG accumulation, which exacerbates insulin resistance, leading to type 2 diabetes and non-alcoholic fatty liver disease. Despite its importance in such metabolic disorders, the mechanism governing insulin’s inhibition of lipolysis remains controversial. Currently, the prevailing model hinges on the enzymatic function of the Akt substrate, phosphodiesterase 3B (PDE3B) in explaining insulin’s antilipolytic action. However, several studies have challenged this and suggested that whilst PDE3B is essential, its function alone cannot explain this regulation. Instead, we implicate another Akt substrate, α/β-hydrolase domain-containing protein 15 (ABHD15), as a novel player in insulin’s inhibition of lipolysis. Through stable knockdown or knockout of ABHD15 in 3T3-L1 or brown adipocytes, we show an impairment in insulin-mediated inhibition of lipolysis that is rescued upon ABHD15 re-expression. Moreover, through the generation of ABHD15 and PDE3B mutants lacking novel insulin-regulated phosphorylation sites previously identified by our lab, we have explored a putative mechanism by which these proteins facilitate insulin’s inhibition of lipolysis. In summary, we have identified a novel regulator of lipolysis that will likely shed light on the mechanism of this important process.

LIVING LIVER BIOPSIES: 3D ORGANOID CELL CULTURE MODELS OF LIVER DISEASE FOR CLINICAL MEDICINE

Collins S.1, 2, 3, Bryant K.1, 2, 3 and Shackel N.1, 2, 3 1Ingham Institute for Applied Medical Research. 2UNSW Sydney. 3South Western Sydney Local Health District.

Liver disease and cancer represent a significant disease burden worldwide. In vitro liver models have gained significant insights into the basic science of liver disease. A 3D organoid cell culture is an in vitro model defined as a collection of cells with several cell types, that develop from stem cells or organ progenitors which self-organise through cell sorting and spatially restricted lineage commitment, similar to organogenesis in vivo. This self-renewing primary cell culture has tissue-specific architecture, intercellular heterogeneity, mechanical and biochemical cues, cell-cell signalling and can be passaged into large data sets or frozen down for long term storage. We hypothesise that liver disease including hepatocellular carcinoma can be modelled ex vivo with 3D liver organoid cell cultures. The aim of this study is to establish and characterise mouse liver organoids for future use as a model for investigating liver injury. Mouse LGR5+ stem cells were isolated from dissociated liver tissue using fluorescence-activated cell sorting and viability assessed. LGR5+ cells were cultured in vitro under growth factor-defined conditions that included noggin and R-spondin-1 and were found to expand and self-organised into 3D structures which had the ability to differentiate into functionally mature hepatocytes. Mouse liver organoids were characterised for mature hepatocyte function using phase-contrast microscopy, hematoxylin and eosin staining, and immunofluorescence analysis. This research will establish methodology for culturing liver organoids, which can be used as an ex vivo assay of liver injury increase our understanding of the relationship between liver injury and the progression of fibrosis, cirrhosis and liver cancer.


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IRRIGATION WITH NOVEL MEMBRANES FOR IMPROVED WATER USE EFFICIENCY

Lima V.1, Keitel C.2, Sutton B.2, 1 and Leslie G.1 1School of Chemical Engineering, UNESCO Centre for Membrane Science and Technology, The University of New South Wales, Sydney 2052, Australia. 2School of Life and Environmental Sciences, Sydney Institute of Agriculture, University of Sydney, Australia.

We tested the potential of a novel subsurface membrane irrigation system to sustain the growth and productivity of crop plants. The membranes are commercially available and designed for use in reverse osmosis (RO) and forward osmosis (FO) applications, but when buried underground can react to changes in water-potential in the root zone, thereby supplying sufficient water for growth without the need of substantial positive water pressure on the supply side. Common bean plants (Phaseolus vulgaris) were grown in pots with RO and FO membranes and compared to conventional surface irrigation (control). We measured various physiological and agronomic parameters to assess productivity and stress, including assimilation rate (A), stomatal conductance (gs), the efficiency of photosystem II reaction centres (Fv/Fm), stable isotope composition of leaves and pods, as well as biomass and yield. A and gs was highest in the control, closely followed by the FO; in contrast, A and gs were significantly reduced in the RO due to the added resistance of this membrane to the water pathway. No difference was found between treatments for leaf internal carbon concentration (ci), Fv/Fm and carbon isotope composition, suggesting no significant effect of membrane irrigation on the production of photosynthates. Yield was significantly reduced for RO, but statistically equivalent between control and FO, with a 25% increase in plant water use efficiency for the latter. These results indicate that membrane irrigation may deliver a significantly improved water-use efficiency for crop growth without compromising yield.

POSTHARVEST STORAGE OF LOOSELEAF LETTUCE (LACTUCA SATIVA L.); THE ROLE OF LIGHT, NUTRIENTS, BIOACTIVE LEVELS AND POTENTIAL HEALTH BENEFITS

Kerr C.B. and Burritt D.J. Department of Botany, University of Otago, 464 Great King St, North Dunedin, Dunedin 9016.

Up to one third of food grown for human consumption is lost or wasted. By improving post harvest storage techniques food waste may therefore be reduced. Once harvested, vegetables continue to sense and respond to environmental stimuli, the potential for photosynthesis is generally retained and secondary metabolites can still be produced. Leafy lettuce (Lactuca sativa) is a popular vegetable worldwide with many health promoting compounds, but it is also highly perishable. Lettuce is generally stored in the dark and this can potentially influence the levels of health promoting compounds present at the time of harvest. We are investigating whether storage of leafy lettuce cultivars in the light influences senescence, nutritional value and in particular the levels of health promoting compounds. Newly developed broad spectrum light emitting diodes (LED’s) that have a spectrum that more closely matches natural day light are being used. These have a low heat output, a long shelf life, low cost and are more environmentally friendly than traditional light sources. Two cultivars of leafy lettuce have been investigated so far. Data on bioactive metabolite levels, plant physiological responses, nutritional contents, and the ability if simulated human digests to protect human cells (Caco-2) from oxidative damage has been gathered. Storage duration and seasonal variations have also been investigated. It was found that lettuce digests stored under an appropriate lighting regime had a higher capacity to protect Caco-2 cells from hydrogen peroxide induced oxidative damage than digests from lettuce stored in the dark.

ESTABLISHMENT OF A CRYOPRESERVED GENEBANK OF POTATO (SOLANUM SPP.) IN NEW ZEALAND

Pathirana R.1, Mathew L.1, Monaghan K.2, Fletcher P.2, Morgan E.1 and Baldwin S.2 1The New Zealand Institute for Plant & Food Research Limited, Batchelar Road, Palmerston North 4474, New Zealand. 2The New Zealand Institute for Plant & Food Research Limited, 74 Gerald Street, Lincoln 7608, New Zealand.

Potato is the world’s fourth most important crop in terms of production. It has been grown in Aotearoa (New Zealand) by Maori since the 18th century. The New Zealand Institute for Plant and Food Research Ltd (PFR) has a strong potato breeding programme holding over 700 accessions. Historically, this germplasm has been maintained in the field, except for ~120 accessions maintained in tissue culture after virus elimination. Clonally propagated crops, especially annual crops like potato, require significant resources to maintain genetic material. Cryopreservation offers a robust, cost-effective solution to long-term conservation of such germplasm. In 2017 we initiated a programme to cryopreserve PFR’s collection, to reduce risk of loss due to biotic and abiotic threats. We initiate tissue cultures from tubers that have sprouted, photograph the phenotype for reference and confirm genotype identity using simple sequence repeat markers. We have adopted a modified droplet vitrification protocol for cryopreservation, which includes excision of 120 fully formed axillary buds (0.5 mm) after micropropagation, their treatment in liquid Murashige & Skoog media with 0.3 and 0.7 M sucrose over two days, followed by treatment with a plant vitrification solution (PVS2, 30 min). The explants are then mounted on aluminium foil, transferred to liquid nitrogen and 20 buds are tested for viability after one month. So far we have conserved 126 accessions, with an average viability of 79%. We will discuss our future plans for this cryopreserved collection.

IN VITRO CHARACTERIZATION OF NEXT GENERATION PHOTOSENSITISER PHOTOSOFTTM FOR PHOTODYNAMIC THERAPY IN OVARIAN CANCER

Chen Y.1, 2 and Stephens A.N.1, 2, 3 1Hudson Institute of Medical Research, Australia. 2Molecular and Translational Biosciences, Monash University, Australia. 3Invion Ltd, Australia.

Ovarian cancers have >70% 5-year mortality, and patients almost universally develop recurrent, chemo-resistant disease; new therapies are urgently needed. Photodynamic therapy (PDT), combining a photo-sensitising compound and light to generate destructive reactive oxygen species (ROS), is an alternative, FDA-approved clinical strategy for direct tumour ablation. We have commenced in vitro characterisation of PhotosoftTM, a photosensitiser with improved safety profile and enhanced cytotoxic capacity, for development as an ovarian cancer therapy. The spectral characteristics and generation of reactive oxygen species (ROS) by Photosoft were examined in vitro. Cellular uptake and clearance were monitored using fluorescence (ex405, em650) in multiple ovarian cancer cell lines (2D and 3D culture), with sub-cellular localization determined using specific organelle staining (ER, Golgi, lysosomes, mitochondria). Cell death and IC90 were determined following exposure to red light (652nm). The mechanism of induced cell death was determined using antibody arrays. Photosoft excitation/emission spectra were characteristic of chlorin-based photosensitisers, and light-induced activation at 652nm efficiently produced ROS. Uptake into ovarian cancer cells was time and concentration dependent, with discrete localization to lysosomes and the ER. In the absence of activation, Photosoft remained non-toxic even at high concentrations (up to 100mg/ml); following activation, rapid cell death occurred dependent on time, concentration and light energy. Both caspase-dependent and –independent cell death were observed, dependent on total light energy delivered. Moreover, Photosoft penetrated effectively into 3D spheroids to induce cell death, suggesting its suitability for ablation of established, solid tumours. Our data demonstrate that Photosoft efficiently produces ROS to destroy tumour cells in vitro, and that modulation of light energy can be used to control the mechanism of induced cell death. Further work will focus on preclinical trials of Photosoft as an indication for chemoresistant, solid ovarian tumours.


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POS-WED-136 POS-MON-137

POS-MON-138 POS-WED-139

MEASURING THE EFFECTS OF AMBER INITIATOR TRNA EXPRESSION ON A GENOMICALLY RECODED ORGANISM

Vincent R.M., Wright B.M. and Jaschke P.R. Department of Molecular Sciences, Macquarie University, Sydney, NSW.

Bacterial translation initiation occurs at conserved AUG start codons recognized by the initiator tRNA anticodon. Since this codon-anticodon complementarity is sufficient for translation initiation, a mutant initiator tRNA (amber initiator) with the anticodon that recognizes an amber (UAG) stop codon has been shown previously to initiate translation at UAG start codons. Despite excellent prior work on characterizing the amber initiator, questions remain around the dynamic and population level behavior of cells carrying the amber initiator. To understand how the amber initiator impacts a cell with a reduced genetic code, I created a new modular set of two plasmids to be expressed in a genomically recoded organism E. coli strain C321.ΔA.exp. The plasmid set consists of an inducible system to express amber initiator and series of fluorescent reporter plasmids. This plasmid set has enabled me to show for the first time dynamic population-level characteristics of amber tRNA translation initiation. Fluorescence measurements confirmed that amber initiator starts translation from UAG start codon with 200-fold increase on inducing amber initiator compared to the repressed condition with 30-fold increase in reporter expression from an AUG start codon in similar conditions. Time-course measurements indicate different initiation effects as amber initiator matures. Surprisingly, proteomic analysis of cells expressing amber initiator showed no evidence of translation initiation from genomic UAG codons. These data suggest that the reduced genetic code of E. coli strain C321.ΔA.exp and conserved elements of amber initiator effectively prevent arbitrary initiation events from a UAG codon. However, proteomic analysis did reveal dramatic up-regulation of RNA-binding proteins. Lastly, I show that expressing amber initiator led to a reduced growth rate and reduced maximal cell density, revealing a link between phenotypic and proteomic effects.

HARMONISING THE TEACHING OF SCIENTIFIC COMMUNICATION SKILLS THROUGH THE DEVELOPMENT OF AN E-LEARNING TOOL

Willems-Jones A.J.1 and Russell J.2 1The Department of Biochemistry & Molecular Biology, School of Biomedical Sciences, The University of Melbourne. 2Department of Medical Education, Melbourne Medical School, The University of Melbourne.

Effective communication is a fundamental graduate attribute. University graduates are expected to be critical thinkers who can apply their knowledge and research skills to solve complex problems in a range of contexts. Developing and applying these skills requires a consistent approach to teaching and assessment of scientific communication at the undergraduate level. Despite being taught by leading academics with an abundance of scientific communication experience, anecdotal student feedback suggests that instruction across disciplines varies; creating student confusion and a lack of confidence in scientific literacy more broadly. Given the importance of preparing research-ready graduates, our project adopted a multidisciplinary approach to harmonise teaching scientific communication skills across subjects in the School of Biomedical Sciences. This was achieved through the development of an e-learning module designed to teach the requisite skills that can be applied across all forms of scientific communication. The module is innovative in enhancing the scientific literacy skills of students. Through the completion of the activities embedded within the two-level module, students can develop, practice and demonstrate their critical thinking and communication skills, as well as improve their understanding of scientific writing. An added benefit of the module is a strengthening in student understanding of scientific communication (and associated skills) more broadly, thereby improving student employability.

USING AN INQUIRY-BASED LEARNING MODULE TO IMPROVE CONTENT KNOWLEDGE IN A SECOND-YEAR BIOCHEMISTRY PRAC CLASS: AN UPDATE

Willems-Jones A.J., Szambelanczyk Orval I. and Hu W. Department of Biochemistry & Molecular Biology, School of Biomedical Sciences, The University of Melbourne.

Critical thinking and trouble-shooting skills, combined with theoretical knowledge in a content area linked to practical application, are key attributes of science graduates. In practical-based tertiary education, teaching of these attributes is often addressed through the process of scientific investigation. Unfortunately, there are distinct limitations to providing wet-lab inquiry-based learning (IBL) opportunities in large classes within the early stages of a degree due to the logistical and financial impost intrinsic to such activities. To address this limitation, a student-centred IBL e-module was developed to integrate knowledge of theory with practical application, and to examine student understanding of experiment design and analysis. The IBL e-module covers the common biochemistry and molecular biology techniques taught in BCMB20005: Techniques in Molecular Science at The University of Melbourne. Students must interrogate and examine one of three genes provided to overexpress and purify the encoded protein. Questions and unique feedback are provided along the way to aid students in their decision-making process. In this presentation, I will present an update of the completed product with data from pre- and post- student questionnaires from the first implementation of the 3-stage module.

GENETIC ANALYSIS AND MAPPING OF RESISTANCE IN BARLEY TO NON-ADAPTED PUCCINIA GRAMINIS ISOLATES

Haghdoust R., Singh D., Park R.F. and Dracatos P.M. The University of Sydney, Plant Breeding Institute, Cobbitty, Private Bag 4011, Narellan, NSW, 2567, Australia.

Stem rust, caused by Puccinia graminis, is an important disease that affects many economically significant cereals and can cause great concern for global cereal production. Different formae speciales of P. graminis have co-evolved, which primarily infect specific hosts. This host specificity is not always complete and some genotypes of other closely related cereals can also be infected. Barley is a host to the wheat and cereal rye adapted formae speciales of P. graminis (Puccinia graminis f. sp. triticiand Puccinia graminis f. sp. secalis), and a near nonhost to the formae speciales adapted to rye grass [P. graminis f. sp. lolii(Pgl)] and oat [P. graminis f. sp. avenae (Pga)]. This study aimed to determine the genetic basis of resistance in barley to Pgland Pga, to map resistance to both pathogens, and to compare the histology of resistance in barley to Pgl with the common host (Lolium perenne L). We tested over 400 diverse barley accessions with Pgl and Pga and determined that less than 10% were fully compatible at the seedling stage, suggesting barley is a near nonhost to these two formae speciales. The Oregon Wolf Barley (OWB) doubled haploid (DH) population was used to characterize and map resistance to Pgl and Pga. The resistance observed at seedling stages to three diverse Pga isolates and to one isolate of Pgl was polygenically inherited and due to QTL on chromosomes 1H, 2H, 4H, 5H, 6H and 7H with both overlapping and distinct specificities. Microscopic examination 14 days post inoculation suggested that the immunity observed within resistant lines was most likely prehaustorial.


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CITRATE EFFLUX FROM WHEAT ROOTS AFFECTS THE ROOT MICROBIOME AND ENHANCES NODAL ROOT DEVELOPMENT

Kawasaki A.1, Dennis P.G.2, Raghavendra A.K.H.2, Delhaize E.1, Richardson A.E.1, Mathesius U.3, Gilliham M.4, Watt M.5and Ryan P.R.1 1CSIRO Agriculture and Food, Canberra, ACT, Australia. 2School of Earth and Environmental Sciences, Faculty of Sciences, The University of Queensland, St Lucia, QLD, Australia. 3Research School of Biology, Australian National University, Canberra, ACT, Australia. 4ARC Centre of Excellence in Plant Energy Biology, School of Agriculture, Food and Wine, Waite Research Institute, University of Adelaide, Glen Osmond, SA, Australia. 5Forschungszentrum Juelich GmbH, Juelich, Germany.

Plants release a wide range of organic compounds from the roots. These exudates alter the physical, chemical and biological properties of the rhizosphere and sometimes these changes can benefit plant survival. A well-studied example is the release of malate and citrate anions from the root apices of some wheat genotypes in acidic soils. Malate release is controlled by TaALMT1 gene and citrate release is controlled by TaMATE1B gene. These organic anions protect the growing root tips from toxic Al3+ cations prevalent in acidic soils and serve as carbon sources which can influence the growth and composition of soil microbes. We tested the effect of citrate efflux on the root-colonizing microbes using near-isogenic wheat lines that vary in citrate efflux (Citrate_line and Null_line). These lines were grown in contrasting soils and the bacterial communities analysed at different positions of seminal and nodal roots. We found that the root colonizing bacterial community was influenced by soil type (acidic or non-acidic), root type (seminal or nodal) and position on the root (tip or base). Citrate efflux from the root tips also affected the microbiome but only in the non-acidic soil. These results demonstrate that the “root microbiome” is not fixed but instead depends on environment, root type and even position along the root. Furthermore we show that the microbiome can be manipulated by specific root exudates. In other experiments we observed a link between the TaMATE1B locus (controlling citrate efflux) and nodal root development in acidic soils. The Citrate_line produced 30-40% more nodal roots and longer nodal roots than the Null_line in both acidic soil and in hydroponics with toxic concentrations of Al3+. These differences were smaller or absent in non-acidic soil or in hydroponics without Al3+ toxicity. We are currently assessing the value of the TaMATE1B locus to wheat production in acidic soils.

LIVE IMAGING OF ACENTROSOMAL MICROTUBULE DYNAMICS CONTROLLING EARLY MAMMALIAN DEVELOPMENT

Zenker J.1, White M.1, Templin R.2, Parton R.2, Thorn-Seshold O.3, Alvarez Y.1, Gasnier M.1, Bissiere S.1, Biro M.4 and Plachta N.1 1IMCB, A*STAR Singapore. 2IMB, Centre for Microscopy and Microanalysis, University of Queensland, Australia. 3Department for Pharmacy, Center for Drug Research, Ludwig-Maximilians University, Munich, Germany. 4EMBL Australia, Single Molecule Science node, School of Medical Sciences, UNSW, Sydney, Australia.

The spatial and temporal configuration of the microtubule cytoskeleton is pivotal for a cell’s function. Whereas in most animal cells the centrosome is the primary microtubule organizing center (MTOC), the regulation of microtubule growth and dynamics in the preimplantation mouse embryo lacking centrosomes remained elusive. Using live imaging, we discovered a new site of non-centrosomal microtubule nucleation required for mammalian embryogenesis. Instead of undergoing stereotypical abscission, the cytokinetic bridge is maintained during interphase and transformed into a non-centrosomal MTOC. Accumulation of the microtubule minus-end protein CAMSAP3 enables the nucleation of non-centrosomal microtubules directing intracellular transport and the formation of the pluripotent inner cell mass. Moreover, acentrosomale and anastral spindles of the embryo establish a polar microtubule network at the end of cell division excluding F-actin from the apical cortex of outer cells. The resulting apical actin rings expand towards cell-cell junctions, zipper and seal the embryo to allow blastocyst cavitation.

ANTI-PRION COMPOUNDS FROM MARINE INVERTEBRATES

Jennings L.K.1, 2, Ahmed I.3, 4, Munn A.L.3, 4 and Carroll A.R.1, 2, 5 1School of Environment and Science,. 2Environmental Futures Research Institute,. 3School of Medical Science,. 4Menzies Health Institute Queensland, Griffith University, Parklands Drive, Southport, QLD, 4222, Australia. 5Griffith Institute for Drug Discovery, Griffith University (Brisbane Innovation Park), Don Young Road, Nathan, QLD, 4111, Australia.

Prions (infectious protein folds) are the etiological agent of a number of neurodegenerative disorders characterised by an accumulation of misfolded forms of normal proteins as insoluble aggregates in the brain. The neurodegenerative diseases caused by prions are invariably fatal and there is a lack of curative treatments. Therefore, there is an urgent need to identify small molecules that have the ability to enter cells and cure prions. Screens using animals or cell lines infected by prions are expensive and time-consuming. However, the discovery that baker’s yeast (Saccharomyces cerevisiae) also harbours prions enables the use of yeast as a tool to facilitate the screen for anti-prion compounds. We have used two yeast prions [PSI+] and [URE3] to optimise previous high-throughput yeast-based anti-prion screens. This has enabled the application of such screens to natural extracts. We screened extracts from >500 marine invertebrates collected from temperate waters in northern NSW and identified 24 that cure yeast cells of the yeast prion [PSI+]. Of these, 6 extracts also cure the unrelated yeast prion [URE3]. Bioassay-driven chemical investigation of one of these 6 bioactive sponge extracts showed that a group of bromotyrosine derivatives were active. The active compounds in the other bioactive extracts have also been identified. Our findings highlight the value of a first stage screen of natural products using yeast strains infected with prions for the identification of chemically-diverse anti-prion compounds.

MULTIDIMENSIONAL FREE ENERGY LANDSCAPES REVEAL MECHANISM OF METAL ION DISCRIMINATION IN PSAA

MacDermott-Opeskin H.I. and O’Mara M.L. Australian National University.

Manganese homeostasis is crucial for the viability of S. pneumoniae, protecting against oxidative stress and aiding cellular metabolism. Manganese uptake is mediated uniquely by the PsaBCA ATP Binding Cassette import system, whose substrate binding component, PsaA, controls selective uptake of manganese. PsaA lacks a metal chelating cofactor and faces significant competition from other d block metal species. Competitive and irreversible binding by other d block metals has been identified as a mechanism for bacterial susceptibility to zinc and cadmium. In this work we conduct a range of molecular dynamics experiments including free energy calculations to reveal mechanisms of cognate and competitive metal binding to PsaA. We demonstrate that manganese is scavenged more effectively than competing ligands from solution via a series of mobile carboxylates. We also demonstrate that ligand exchange of bound metals with water control reversibility of binding and that propensity to exchange ligands differentiates between metal species. We rationalise experimentally observed affinities and kinetics for multiple metal species on converged two dimensional free energy landscapes. We also compare and benchmark a range of molecular dynamics ion models as well as conducting quantum mechanical calculations to probe model ligand substitution reactions at the PsaA active site. Our work reveals that the coordination chemistry of PsaA is tightly balanced to provide selectivity and reversibility, utilising ligand exchange reactions and favourable kinetics to achieve transport of manganese despite significant competition. These atomic resolution insights into metal ion selectivity have implications for the de novo design of metalloproteins as well as for the development of novel inhibitors for metal uptake in S. pneumoniae.


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POS-TUE-146

RNASE III FUNCTION IN BACTERIA - INSIGHTS GAINED FROM SINORHIZOBIUM MELILOTI

Goodfellow S.1, Zhang D.1, 2, Wang M.B.2 and Zhang R.1 1Wollongong University, Northfields Ave, Wollongong NSW 2522, Australia. 2CSIRO Agriculture, Clunies Ross Street, Canberra, ACT 2601, Australia.

The RNAse III superfamily is a diverse group of enzymes involved in the maturation, processing and degradation of double-stranded or secondarily structured RNA species in both Eukaryotes and Prokaryotes. Previous studies in Escherichia coli have shown that while RNAse III plays a central role in ribosomal RNA maturation and post-transcription regulation, deletion is non-fatal - in spite of noticeable aberrations in rRNA subunit maturation. This importance necessitates further research in terms of extending existing knowledge of the detailed molecular function, and also the diversity of these RNAse III enzymes and their roles in other bacterial taxa. This study intends to address this by using a homologous recombination-based strategy to delete RNAse III in the model nitrogen fixation bacterium - Sinorhizobium meliloti. This will be followed by an investigation of the resultant effects on growth and RNA metabolism in comparison with E. coli. An RNAse III deletion strain has been isolated and the mutant - S. meliloti RmL7, is viable showing no noticeable defects in terms of growth. Work is in progress to uncover the global post-transcriptional regulatory effect of the deletion in both S. meliloti and E. coli in addition to further documentation of rRNA biogenesis.

AN ARABIDOPSIS PLANT NATRIURETIC PEPTIDE INTERACTS WITH CATALASE2 TO MODULATE CELLULAR H2O2 HOMEOSTASIS

Turek I.S.1, 2, Wheeler J.3, Gehring C.1, 4 and Irving H.R.2 1King Abdullah University of Science and Technology, Division of Biological and Environmental Sciences and Engineering, Thuwal, Saudi Arabia. 2La Trobe Institute for Molecular Science, La Trobe University, Bendigo, VIC, Australia. 3AgriBio, La Trobe University, Bundoora, VIC, Australia. 4Department of Chemistry, Biology and Biotechnology, University of Perugia, Perugia, Italy.

Plant natriuretic peptides (PNPs) comprise a novel class of systemically acting peptidic hormones that share some sequence similarity in the active site with their animal analogues regulating salt and water balance. One aspect of understanding the function of these compounds is their potential biotechnological application in conferring increased stress tolerance to plants. Although PNPs modulate many physiological responses, including plant responses to stress, their molecular mode of action still remains unclear. Since H2O2 is a key component of abiotic and biotic stress responses in plants, we set out to investigate if the Arabidopsis thaliana PNP (AtPNP-A) can directly or indirectly affect H2O2 homeostasis. To this end we screened AtPNP-A for interacting partners and identified catalase2 (CAT2), an enzyme capable of H2O2 decomposition, as a candidate direct interactor of AtPNP-A by yeast two-hybrid (Y2H) assay and cross-linking followed by mass spectrometric (MS) analysis. Surface plasmon resonance (SPR) revealed that the biologically active part of AtPNP-A binds specifically to CAT2 in vitro, while a biologically inactive peptide does not. Furthermore, zymograhic analyses revealed that AtPNP-A enhances CAT2 activity in vitro. We also noted that CAT2 activity if lower in homozygous atpnp-a knockdown plants compared with wild type plants. Finally, bimolecular fluorescence complementation (BiFC) revealed that CAT2 interacts with AtPNP-A in chloroplasts, consistent with the organellar localization of the CAT2, and modulates H2O2 levels having implications for plant stress responses.

GENERATION AND MAINTENANCE OF MESODERM BIASED HUMAN PLURIPOTENT STEM CELLS

Enver T. UCL Cancer Institute, London.

Human Pluripotent Stem Cells (hPSC) exist in heterogeneous populations when grown in standard culture. We utilised an hPSC reporter line for MIXL1 coupled with stem cell surface antigen, SSEA-3, to identify MIXL1(+)/SSEA-3(+) substate. While the substate is apparent when grown on a mouse embryonic feeder layer, it is virtually non-existent in defined culture systems. We developed “Primo” medium, balancing pro-self renewal and pro-differentiation factors to promote the presence of a MIXL1(+)/SSEA-3(+) substate. The cells grown in Primo exhibit a functional bias in terms of their differentiation potential, in particular, “neutral” Embryoid Bodies produced enhanced mesoderm populations. Bulk and single cell transcriptomics demonstrated that cells correlated with the populations seen in standard culture. Cells could be maintained in this lineage biased state for multiple passages, with a normal karyotype and normal pluripotency associated marker expression. Cells can also be transitioned back, with transcriptional changes reverting back to normal hPSC expression. Here we show the first long term maintenance of a lineage biased pluripotent state which could provide a tool to improve translational medicine for mesodermal derivatives.

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