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ComBio2012 s Adelaide, South Australia s 23-27 September 2012 Page 99

POSTERS MONDAY

POS-MON-001 POS-MON-002


SEQUENCING AND DE NOVO ANALYSIS OF A SNAIL MUCUS GLAND AND DART SAC TRANSCRIPTOME: REVEALING REPRODUCTION RELATED FACTORS

Adamson K.J., Stewart M., Kuballa A. and Cummins S.F. University of the Sunshine Coast.

There are approximately 25,000 known species of terrestrial snails worldwide, many of which produce calcareous love darts. These darts are formed in the dart sac, and when successfully injected into partners during courtship, transfer mucus from the mucus gland. There have been many functions proposed for these love darts in reproduction of snails, including calcium transfer, sexual arousal, and an effect on the quantity of sperm produced. It has now been shown that these darts transfer mucus which increases paternity of the donor snail, by causing contractions in parts of the reproductive tract of the recipient. Theba pisana is native to the Mediterranean region, and is one of several species of snails established as a major pest in grain crops in the southern parts of Australia. We have investigated the transcriptome of the dart sac and mucus gland, and sequencing has revealed various neuropeptides and other secreted gene products. A number of these neuropeptides have been localized using in situ hybridization and PCR on selected tissue. A better understanding of all facets of the snail reproductive system could lead to novel new methods of control for this and other snail pests.

APPLICATION OF FLUORESCENCE IN SITU HYBRIDIZATION (FISH) TO THE ANALYSIS DYNAMICS OF SULFATE REDUCING BACTERIAL COMMUNITY IN AN OILY BENCH SCALE REACTOR

Alamri S.1 and Hesham A.2 1Biological Department, Faculty of Science, King Khalid University, Abha, Saudi Arabia. 2Genetics Department, Faculty of Agriculture, Assiut University, Assiut, Egypt.

Advances in the field of genomics and meta-genomics have led to rapid and accurate strategies for the monitoring of microbial biodiversity and have revealed its potential for biotechnological applications. In this study, fluorescent in situ hybridization (FISH) as a culture-independent molecular approach using specific fluorescein-labelled oligonucleotide probes, was used to study the dynamics of the sulfate reduction bacterial community (SRB) of the activated sludge from an oily wastewater treatment system. The relative abundance of members of the dominant bacteria in the oily water reactor was determined by FISH for 16S rRNA using EUB-338 probes, for detecting general eubacteria, and SRB-385 for targeting SRBs and major species of δ-Proteobacteria sulfate reducers. The percentage of cells hybridizing with probe EUB338 for the dominant bacteria decreased from 25.85% to 6.25%, while with the SRB385 probe for SRB bacteria, it increased from 7.21% to 10.20% of total cells during the reactor process. These data show that SRB bacteria dominated the active microbial community in the system. It is interesting that SRB bacteria occupied a high percentage and took place in an oily biological system under aerobic conditions.

BIOINFORMATIC ANALYSIS OF GDAP1 EXPRESSION

Algothmi K., Hoonhout C. and Shield A. University of Canberra.

Charcot-Marie-Tooth disease (CMT) is the most common hereditary disease and affects 1 in 2500 people. CMT causes damage in the peripheral nerves with degeneration in the lower legs and arms. A severe recessive form of CMT has been linked to mutations in the coding region of Ganglioside-induced Differentiation Associated Protein (GDAP1), a member of the glutathione transferase family. The specific function of this GDAP1 protein is currently unclear. The purpose of this study was to investigate the expression levels of GDAP1 using a bioinformatics approach. Data was collected from NCBI GEO profiles which are a public repository of microarray data. Initially GDAP1 expression was determined in normal healthy tissues; high levels of expression were found in brain, spinal cord and mammary tissue. Using a systematic approach specific terms were then used to search for conditions causing changes in the expression of GDAP1. Selection criteria were used for inclusion (or exclusion) of profiles for example, profiles containing less than four samples or only 2 control samples were excluded. Statistical analysis was undertaken for the data within the selected profiles to determine whether there was a change in GDAP1 expression. For example the search term (human GDAP1 and brain) found 9 profiles suitable for inclusion. In one of these studies GDAP1 expression was found to be significantly higher in grade IV compared to grade III gliomas (p= 0.00013). It is hoped this information will help us to develop hypothesis regarding the function of GDAP1 and how it contributes to CMT.

CELLULAR REPROGRAMMING TOWARDS THE MEGAKARYOCYTE LINEAGE

Artuz C.M.1, Funnell A.P.W.1, Pearson R.C.M.1, Gonda T.J.2 and Crossley M.1 1School of Biotechnology and Biomolecular Sciences, The University of New South Wales, NSW 2052. 2University of Queensland Diamantina Institute for Cancer, Immunology and Metabolic Medicine, Princess Alexandra Hospital, Queensland 4072.

Blood cells termed megakaryocytes produce platelets that are crucial to normal blood clotting. If platelet numbers decline, a life-threatening condition termed thrombocytopenia ensues. Current treatments include costly blood transfusions, and drugs that are not always effective and are associated with numerous side effects. To investigate alternative approaches, we are focusing on the megakaryocyte gene regulatory network and associated transcription factors. Inspired by the success of recent research showing that transcription factors can drive lineage reprogramming, our aim is to convert heterologous somatic cells, such as skin cells, into platelet producing megakaryocyte-like cells.


POSTERS MONDAY



IDENTIFICATION OF MODULONS IN BACTERIAL MICROARRAY EXPRESSION DATA: FIGHTING THE OVERPREDICTION

Permina E.A.1, Hedge S.H.2, Medvedeva Y.U.A.3, 1, Baeck P.M.4, Mande S.H.2 and Makeev V.J.1 1Vavilov Institute for General Genetics, Moscow, Russia. 2National Centre for Cell Science, Pune University, India. 3King Abdullah University of Science and Technology, Saudi Arabia. 4Upper Austria University of Applied Sciences, Hagenberg, Austria.

Understanding regulatory circuits controlling gene expression in bacteria is important for intelligent genetic engineering to harness the power of bacteria for biotechnology and bioremediation, as well as to find new drug targets in bacterial pathogens. Several large collections of gene expression values measured with microarrays in large sets of different conditions are available now. We developed an easy-to-handle software for calculating the cliques of potentially coexpressed genes and successfully predicted additional modulon participants in bacterial transcriptomes. We tried to take into consideration the several prerequisites including regulon/modulon heterogenity, allowing one gene to fall into several modulons and allowing some known regulon members to be excluded from the forming modulons. The consistency of the procedure has been tested on subsampling stability and in comparison the results to those obtained from several independent datasets. The results obtained by implementation of our software are consistent with the available experimental data. It is widely known that mass data on transcriptomics suffers from increased false positive rates which could originate either from computational or from lab method feature. We have tested the hypothesis that the disruption of DNA structure leads to increased numbers of positive and negative correlations (against no correlation), comparing the quality of modulon prediction for two sets of conditions. This work demonstrate an effective strategy for dealing with mass transcriptomics data including the methodics of prediction consistensy evaluation.

COMPARISON OF CISPLATIN ANALOGUE COMPOUNDS FOR DNA INTERSTRAND COVALENT CROSSLINKS BY FLUORESCENT QUANTIFICATION AND APPLICATION OF A NEWLY DEVELOPED DENATURING SLAB GEL ELECTROPHORESISBurgess M.W., Aldrich-Wright J. and Temple M.D. School of Science and Health, University of Western Sydney, Campbelltown Campus, Australia. The discovery of the anticancer properties of cis-diamineplatinum(II) (cisplatin) by Rosenberg resulted in a surge in metallo-drugs research with the aim of discovering compounds that could deliver maximum therapeutic outcomes whilst minimising drug resistance and toxic side effects for cancer patients. It is widely believed that the primary mechanism for cisplatin-induced cancer cell death arises from its ability to form covalent adducts with DNA purine bases. The formation of cisplatin-DNA adducts leads to a disruption of biological functions such as protein/DNA binding, DNA replication and transcription. The detection of inter-strand adducts by metallo-drugs was conventionally done using sodium hydroxide based denaturing gel electrophoresis and post-electrophoresis gel staining. These experiments can be problematic as sodium hydroxide can hydrolyse polysaccharides so that the marker dye and the gel matrix can float out of the cast plate during electrophoresis. Furthermore, inaccuracies in downstream densitometric analysis could occur as the presence of sodium hydroxide and platinum DNA adducts can inhibit ethidium bromide from binding with DNA. Work presented here, compares the effectiveness for forming covalent inter-strand bonds by cisplatin with other covalent adduct forming analogues such as cis-diammine(1,1-cyclobutanedicarboxylato)-platinum(II) (oxaliplatin), dichloro(2,2-bipyridine)-platinum(II) (DCBP), cis-dichlorobis(pyridine)-platinum(II) (DCRP) and dichloro(1,2-diaminocyclohexane)-platinum(II) (DACH), and metallointercalators analogues such as [(5,6-dimethyl-1,10-phenanthroline)(1,2-diaminoethane)platinum(II)]2+ (56MESS), [(5,6-dimethyl-1,10-phenanthroline)(1,2-diaminoethane)copper(II)]2+ (Cu56MESS) and [(3,4,7,8-tetramethyl-1,10-phenanthroline)(1,2-diaminoethane)platinum(II)]2+ (3478MEEN) and the DNA strand breaker phleomycin. We also present a novel application of a newly developed urea-based denaturing agarose gel electrophoresis. Additionally, a fluorescently labelled DNA template was used to quantitatively compare the effectiveness of these cisplatin analogues to form covalent inter-strand DNA crosslinks. Hydrogen-bond melting inhibition studies on denaturing slab gel revealed that cisplatin and DACH were the most effective at forming inter-strand crosslinks. As expected, DNA treated with the strand breaker phleomycin induced a degraded smear of cleaved DNA. Surprisingly, the DNA intercalator 3478MEEN also inhibited DNA strand separation at high concentrations.

RAPIDLY PROFILING THOUSANDS OF LARGE NON-CODING RNAS FROM NANOGRAM AMOUNTS OF TOTAL RNA USING A SINGLE MICROARRAY DESIGN

Lucas A.B., Swaminathan K., Kulkarni V., Mullinax B., Cher C., Srinivasan M., Leproust E. and Fulmer-Smentek S. Agilent Technologies, 5301 Stevens Creek Blvd, Santa Clara, CA.

Large intergenic non-coding RNAs (lincRNAs) are emerging as key regulators of diverse cellular processes. As researchers face the challenge of investigating the function of lincRNAs, there is a need for tools that can accurately and rapidly measure the expression of both lincRNAs and mRNAs simultaneously. A catalog of more than 8,000 human lincRNAs was recently annotated from more than 4 billion RNA-Seq reads across 24 tissues and cell types by scientists at the Broad Institute of MIT and Harvard. Using this new catalog of lincRNAs we have updated the content of the Human SurePrint G3 microarrays to enable systematic profiling and simultaneous detection of coding and non-coding gene expression from a single sample. We used low nanogram amounts of total RNA from matched tumor and adjacent normal tissues to detect both large and subtle differences in gene expression profiles that were consistent with the current literature. GeneSpring GX software rapidly identified differentially expressed lincRNAs and protein-coding RNAs resulting in expression measurements from total RNA in less than two days. Microarray data demonstrated good reproducibility, wide dynamic ranges and high sensitivity, and the tumor versus normal ratios generated from the microarrays showed high correlation with ratios generated from whole transcriptome sequencing of the same matched RNA samples. In this study we demonstrate that microarrays can provide accurate differential expression measurements of both protein coding and non coding RNA from very low amounts of total RNA rapidly providing expression data that is equivalent to whole transcriptome sequencing measurements.

HIGH MOBILITY GROUP PROTEINS IN NEUROGENESIS AND REGENERATION

Colquhoun D.E., Tang K.Y.K. and Kaslin J. Australian Regenerative Medicine Institute, Level 1, Building 75,Monash University Clayton Campus, Wellington road, Clayton, Victoria 3800, Australia.

High Mobility Group (HMG) proteins are highly expressed during development in undifferentiated cells but are lowly expressed in adult tissues. An aberrant level of High Mobility Group (HMG) proteins can cause developmental defects, cancer and an altered immune response. We are interested in the role of HMGA and HMGB proteins in neurogenesis and regeneration. We use zebrafish to study the role of HMGA and HMGB proteins in these processes because zebrafish is an excellent neurodevelopmental model. Furthermore, zebrafish is able to regenerate many tissues including parts of their central nervous system. We have cloned and studied the expression of HMGA and B proteins in the developing and adult brain of zebrafish. HMGA1 and HMGB2 are highly and widely expressed during development but their expression is restricted to stem and progenitor cells in the adult brain. HMGB1B has an expression pattern that is restricted to the notochord during development and the ventricular regions of the developing brain. Interestingly, HMGA1A and HMGB1B expression is specifically upregulated at lesion site after injury. We are currently examining the specific role of HMG proteins during regeneration and development by overexpression and knockdown approaches.


POSTERS MONDAY



ASSEMBLIGRATION: A SIMPLE, FAST AND SCALABLE CHROMOSOMAL INTEGRATION STRATEGY IN BACTERIACui L.1, St Pierre F.2, Priest D.1, Dodd I.1, Endy D.2 and Shearwin K.1 1Department of Biochemistry, School of Molecular and Biomedical Sciences, The University of Adelaide, SA, 5005, Australia. 2Department of Bioengineering, Stanford University, CA, 94305, USA. Chromosomal level DNA manipulation in bacteria can be achieved either by complete DNA synthesis of the chromosome [1] or more commonly, by smaller scale chromosomal DNA insertion and deletion. Here, we report the OSIP (One Step Integration Plasmid) system, a set of plasmids which enable a single step chromosomal integration protocol, making site specific integration of DNA sequences of interest into bacterial chromosome simple, rapid and scalable. Based on the two-plasmid CRIM system of Wanner et al [2], a series of bacteriophage integrases are used to direct integration into specific attP site in bacterial chromosome. Following integration, an optimized FLP mediated excision system can be used to remove all but the minimal terminator-protected sequences of interest from the chromosome. The pOSIP plasmids are modular in design, consisting of three easily exchangeable modules, the insertion module, the selection module and propagation module. Both step-by-step integration into multiple sites and simultaneous multi-site integration can be achieved by using the pOSIP system. By coupling the OSIP system with in vitro DNA assembly method [3], the technique of assembligration was developed. Assembligration is the chromosomal integration of an assembled pOSIP plasmid by direct transformation of assembly reaction product into competent bacterial cells. This approach bypasses the need for propagation of the plasmid, thus circumventing problems often encountered with multi-copy plasmids, such as, poor growth or accumulation of mutations. This new strategy makes chromosomal integration as simple as a single transformation, and may find widespread application in the area of synthetic biology. 1. Gibson, D.G., et al., Creation of a Bacterial Cell Controlled by a Chemically Synthesized Genome. Science, 2010. 2. Haldimann, A. and B.L. Wanner, Conditional-replication, integration, excision, and retrieval plasmid-host systems for gene structure-function studies of bacteria. J Bacteriol, 2001. 183(21): p. 6384-93. 3. Gibson, D.G., et al., Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods, 2009. 6(5): p. 343-5.

OPTX:LUC BASED CYCLIC NUCLEOTIDE SENSITIVE PROMOTER REPORTER SYSTEMS

Wheeler J.I., Freihat L.A. and Irving H.R. MONASH INSTITUTE OF PHARMACEUTICAL SCIENCES.

Cyclic nucleotides are second messengers with many physiological functions in both prokaryotes and eukaryotes. Currently cellular cyclic nucleotide levels can be assessed using antibody kits or mass spectrometry analyses, both of which have drawbacks. Our aim is to develop a rapid screening, non-invasive reporter system to measure cellular levels of cGMP (guanosine 3,5 - cyclic monophosphate). We designed a promoter system containing the OPTX promoter fused to a luciferase gene. OPTX is responsive to cGMP in plant cells (Maathuis Plant J 45:700-11, 2006), and surprisingly the OPTX promoter fragment contains the mammalian cGMP responsive element (RE) (Hum et al. Hypertension 43:1270-8, 2004). To enhance the promoter response to cGMP, we inserted three additional cGMP RE into the OPTX promoter. A third construct was designed containing five gibberellic acid response elements (GARE) (Bastian et al. Plant Signal & Behav 5: 224-232; 2010) that we postulated may be upregulated by cGMP since GA treatment raises cGMP levels in plant cells. Different concentrations of cGMP significantly altered the luciferase activity of the OPTX and GARE augmented promoter constructs in the plant protoplast system and endogenous ligands such as GA mimicked this result. However, cAMP also induced similar responses to cGMP. Significantly altered luciferase activity was observed with all promoters in bacterial cells which also responded to both cGMP and cAMP. Tests in mammalian cells (HEK 293T/17) revealed that the GARE augmented promoter effectively discriminated between cGMP and cAMP but the results for the other promoters were inconclusive. The OPTX promoter system presents an effective screening tool for determining nucleotide cyclase activity in different cell types by measuring the differences in luciferase activity.

TRANSLATIONAL CONTROL OF GENE EXPRESSION MEDIATED BY MRNA ELEMENTS

Garama D.1, Stevens S.1, Chen X.1, Biswas A.1, Sarman A.1, Chen A.1 and Brown C.1, 2 1Department of Biochemistry, University of Otago, Dunedin, New Zealand. 2Genetics Otago, University of Otago, Dunedin, New Zealand.

Recently large scale transcriptomic and proteomic datasets for human cells have become available. A striking finding from analysis these studies is that the level of an mRNA typically predicts no more than 40% of the abundance of protein. This is largely due to regulation of translation. Interestingly, our analysis and modelling of this data defines a large and distinct group of mRNAs where there is a good correlation between mRNA and protein levels across cells. For these genes expression is canonical - mRNA levels are good predictors of protein levels. On the other hand, there are a large number of proteins for which the translation efficiency varies greatly between cells. We analysed this data to examine hypotheses of the causes of significant deviations in different cell lines. We show indications of translational control for many genes that have not yet been tested or reported. We aim to identify all the RNA elements in mRNAs that contribute to this regulation. To do this we have developed curated sets of cis-regulatory RNA elements (CisRegRNA) and novel bioinformatic methodologies to detect similar elements using support vector machines (CisRNA-SVM) and tools to analyse mRNA data (scan for Motifs). Our methodology outperforms previous methods and has discovered new structural classes of candidate regulatory elements. We are also using high throughput proteomics (pSILAC) in parallel with mRNA profiling (RNA-Seq) to identify cis regulatory elements that mediate translational control in cancer cell lines. These experimental results are being combined with the bioinformatic analysis to define the functions of novel regulatory elements.

UTILIZATION OF THE BOMBYX MORI HSP70 PROMOTER FOR EFFICIENT TRANSGENE EXPRESSION

Goo T.W., Kim S.W., Kim S.R., Park S.W., Choi K.H., Kang S.W. and Yun E.Y. National Academy of Agricultural Science.

To identify more powerful promoter than previously reported BmA3 promoter, we isolated 9 clones that show stronger signal compared to BmA3 by dot blot hybridization. Among these 9 clones, we focused on one clone which has high amino acid homology (94%) with hsp70 gene of Trichoplusia ni. This clone, named bHsp70 was ubiquitously expressed in all tissues and developmental stage of B. mori, and up-regulated by thermal and ER stress. As result of promoter assay using dual luciferase assay system, we found the highest transcription activity region (-1003/+147) in the 5’-flanking region of bHsp70 gene, which has about 264 fold more intensive promoter activity than BmA3 promoter. Transcriptional activity of bHsp70 promoter under heat shock condition (42℃, 4 hr) was increased over 2 fold than normal condition. The bHsp70 promoter was normally regulated in Bm5, Sf9, and S2 cells. Moreover, the bHsp70-EGFP marker was detected ubiquitously in embryonic, larval, pupal, and adult tissues of transgenic silkworm. Therefore, it is possible to screen transgenic individuals before the G1 larval stage using this promoter, because the bHsp70-EGFP marker was expressed in embryos. As above results, we suggest that bHsp70 promoter may be used more effectively for the large-scale production of biologically active recombinant proteins and screening of transgenic silkworm.


POSTERS MONDAY



INVESTIGATION INTO THE ROLE OF URSODEOXYCHOLIC ACID IN THE REGULATION OF THE UDP GLYCOSYLTRANSFERASE 3A1 AND 3A2 GENES

Haines A.Z., Meech R. and Mackenzie P.I. Department of Clinical Pharmacology, Flinders University, School of Medicine, Flinders Medical Centre, Bedford Park, SA 5042.

The UDP glycosyltransferases (UGTs) are a super-family of enzymes involved in the metabolism of endogenous molecules and xenobiotics. Variations in UGT expression have been associated with certain disease states and changes in individuals’ responses to drug therapy, yet limited data is currently available on the regulation of the UGT3A family. There are a number of cases in which bile acids have been shown to be regulators of gene expression. The secondary bile acid ursodeoxycholic acid (UDCA) is a known substrate of UGT3A1, and it was hypothesised that UDCA may play a role in the regulation of the UGT3A family. As such, three cell lines (HepG2, HEK293T and T47D) were treated with UDCA, in the presence or absence of the histone deacetylase inhibitor trichostatin A (TSA). Changes in the endogenous levels of both UGT3A genes, UGT3A1 and UGT3A2, were tested. Treatment with UDCA did not lead to any significant changes in the endogenous levels of UGT3A1 transcripts in any of the three cell lines tested. However, in HepG2 cells, a dose-dependent increase in UGT3A2 transcripts was detected when cells were treated with UDCA in combination with TSA (P ≤ 0.001 – 0.010). UDCA had no significant effect on UGT3A2 expression levels in HEK293T or T47D cells. This study has identified UDCA as a potential regulator of UGT3A2 gene expression. Further experiments are currently being conducted to identify the nuclear receptor/s involved, and to elucidate the mechanism by which this up-regulation occurs.

REVERSE BACTERIAL TWO HYBRID: A NEW TOOL FOR STUDYING DIMERIC PROTEIN INTERACTIONSHao N.1, 2, Whitelaw M.L.1, 2, Shearwin K.E.1, Dodd I.B.1 and Chapman-Smith A.1, 2 1School of Molecular and Biomedical Science (Biochemistry), The University of Adelaide. 2Australian Research Council Special Research Centre for Molecular Genetics of Development.

The basic helix-loop-helix/Per-Arnt-Sim (bHLH/PAS) family of transcription factors function in early development, stress response and homeostasis. They include the Aryl hydrocarbon Receptor (AhR, xenobiotic metabolism), Hypoxia Inducible Factor-α (HIF-α, adaption to low oxygen levels), Single minded proteins (Sims, neurogenesis, appetite control), and CLOCK proteins (circadian rhythms), are key regulatory proteins involved in oxygen homeostasis, neurogenesis and circadian rhythm. Target gene induction by bHLH/PAS proteins depends in part on their partner protein choices, where dimerization with common partner factor Aryl hydrocarbon receptor nuclear translocator (Arnt) or BMAL is an essential step towards forming active DNA binding complexes. To gain a better understanding on the molecular basis of bHLH/PAS protein interactions, we developed a novel Reverse Bacterial two Hybrid (RevB2H) screen for positive selection of protein loss-of-interaction mutants. Using this system, we have identified 22 amino acids in the PAS.A region of Arnt that when mutated, significantly disrupted AhR/Arnt heterodimerization. It appeared that Arnt uses the same dimerization interface for both homo- and hetero-dimerization. However, several identified amino acids within this interface were selective only for Arnt/AhR interaction, since mutations at these positions had little or no effect with other Arnt partner proteins such as HIF-1α and Sim1. Mutagenic screen of AhR suggested that equivalent region of AhR is used when dimerizing with Arnt. Interestingly, dimerization between AhR and Arnt appeared to extend beyond just the PAS domains, as mutations at flanking N-terminal helices also markedly reduced AhR/Arnt heterodimerization. Consistent with this result, a recent crystallography study suggested that high affinity interaction between bHLH/PAS proteins CLOCK and BMAL is mediated by both PAS β-sheets and the flanking N-terminal α-helical caps.

TRANSCRIPTOME PROFILING OF BOVINE OVARIAN FOLLICULAR GRANULOSA CELLS IN VITRO AND IN VIVO

Hatzirodos N.1, Knight P.G.2, Glister C.2 and Rodgers R.J.1 1Robinson Institute, University of Adelaide, SA Australia. 2School of Biological Sciences, University of Reading, Hopkins Building, Whiteknights, Reading RG6 6UB, UK.

Ovarian granulosa cells mature in ovarian follicles as follicles enlarge. Growth factors and hormones regulate this process. To examine this further we compared cells treated in vitro (small follicles 4- 6 mm; with and without Follicle-Stimulating Hormone and Tumour Necrosis Factor α; n = 4) with cell maturing or dying (atresia) in vivo (< 5 mm healthy and atretic and > 10 mm healthy; n = 4-5). RNA was transcribed and hybridised to Bovine Genome Affy arrays for differential gene expression analysis by ANOVA using Partek Genomics Suite (v6.5) software. TNFα, unlike FSH, significantly affected global gene expression. Differentially expressed genes (DEGs) were imported into Ingenuity Pathway Analysis (IPA) and GOEAST (Gene Ontology Enrichment Analysis Software Toolkit) for function and pathway determination. DEGs from the FSH treated group compared with the control showed significant up regulation of cell division and lipid metabolism. In contrast, DEGs from both TNFα treatment groups compared with the control mapped positively to fibrotic and inflammatory signalling pathways, although the TNFα treatment alone was biased toward protein degradation pathways. Comparison of the FSH treatment results with in vivo showed additional associations with guided cell migration mediated by integrin, ephrin receptors and extracellular matrix for large versus small in vivo. The TNFα treatment effect on gene expression was similar to atresia in small follicles, however TGFβ signalling was more strongly represented in the latter analysis. These results allow us to better dissect the molecular mechanisms underlying development and atresia of the ovarian follicle.

FLUORESCENCE LABELLING OF A TRANSCRIPTION FACTOR FOR SINGLE-MOLECULE IMAGING

Al-Zyoud W.1, Sobey T.1, Liu D.2 and Böcking T.1 1Centre for Vascular Research, University of New South Wales, Sydney, Australia. 2Department of Chemistry and Biochemistry, Loyola University, Chicago, USA.

The chimeric transcription factors of the GntR family in bacteria contain an N-terminal Winged Helix-Turn-Helix (WHTH) DNA-binding domain and a C-terminal Effector-Binding/Oligomerization (EB/O) domain. The GntR family regulator GabR controls the expression of genes essential for glutamic acid metabolism. It represents an unusual transcription factor in that its EB/O domain is homologous to type I aminotransferases and is thought to form a head-to-tail dimer. Simultaneous binding of effector molecules such gamma-aminobutyric acid and the co-enzyme pyridoxal-5’-phosphate lead to activation of gene expression. This regulation is expected to involve conformational changes and/or changes in the number of transcription factors bound to DNA. We aim to use a combination of biochemical and single-molecule imaging approaches to resolve the architecture and conformational changes of GabR at the operator. The aminotransferase domain of GabR contains three native cysteines such that labeling using thiol chemistry was not sufficiently site-specific despite poor reactivity of all three native cysteines. Here we investigate two recent approaches for site-specific fluorescence labelling of GabR. The first approach involves the generation of an N-terminal cysteine residue via tobacco etch virus (TEV) cleavage of a modified recognition site followed by a condensation reaction with a chemical fluorophore. The second approach utilises a genetically encoded aldehyde tag with a cysteine residue that is enzymatically converted to a formylglycine residue. This aldehyde group serves as a unique chemical handle for site-specific labeling using a hydrazide containing fluorophore.


POSTERS MONDAY



THE METAMORPHIC CLIC1 PROTEIN REQUIRES CHOLESTEROL FOR OPTIMAL CONDUCTION IN MEMBRANES

Alkhamici H.1, Carne S.2, Brown L.3, Cornell B.2 and Valenzuela S.1 1School of Medical and Molecular Biosciences, University of Technology Sydney, Australia. 2Surgical Diagnostics Pty Ltd, St Leonards, Australia. 3Department of Chemistry and Biomolecular Sciences,Macquarie University, Australia.

The Intracellular Ion Channels (CLIC) is a family of metamorphic human proteins consisting of six members that were found to exist as soluble in solution and as membrane bound proteins. CLIC1 protein has been reported to spontaneously insert into cell membranes to form chloride ion channels. However, factors that control the structural transition of CLIC1 from being soluble in solution into membrane bound protein in order to form functional ion channels have yet to be adequately described. Thus, the objectives of this work are to identify and characterize factors that are involved in protein insertion and assembly into bilayer lipid membranes. Monomeric CLIC1 (WT) protein was expressed in E-Coli and purified in the presence of the reducing agent TCEP (0.5mM), first by His-tag Ni2+-NTA high affinity chromatography column followed by size exclusion chromatography. Control proteins (boiled CLIC1), Listeriolysin O (Sapphire Bioscience), α-Hemolysin (Sigma Aldrich) along with reduced CLIC1 (WT) (incubated with 0.5mM TCEP) and oxidised (incubated with 2mM H2O2) were added to tBLM (tethered bilayer lipid membrane) and the resultant conduction from each protein was measured by impedance spectroscopy. tBLMs were formed using Hepes buffer (pH 6.5) and neutral lipids mixed with 50mol%, 25mol%, 12.5mol%, 6.25mol% and 0mol% cholesterol. Our findings suggest that in order for CLIC1 (WT) protein to insert into membranes and form conducting ion channels, cholesterol is required in the range of 10mole% to 50mole% relative to membrane phospholipids. A model is proposed in which CLIC1 (WT) protein inserts into bilayer membranes in a manner similar to the Cholesterol Dependent Cytolysin (CDC) protein family.

ANALYSIS OF SUBUNIT INTERACTIONS WITHIN THE NUCLEOSOME REMODELLING AND DEACETYLASE (NURD) COMPLEX

Alqarni S., Thong S. and Mackay J. School of Molecular Bioscience, University of Sydney, NSW, Australia.

The Nucleosome Remodeling and Deacetylase (NuRD) complex is a multi-protein transcriptional co-regulator which plays a major role in controlling the chromatin conformation through multiple mechanisms, including histone deacetylation, nucleosome remodeling and recruitment of other regulatory proteins (Denslow et al, 2007 and Ramirez et al, 2009). It is conserved across a wide range of plants and animals and expressed broadly in most or all tissues. This ~1.5 MDa complex comprises at least ten polypeptides, including Mi-2/CHD-family proteins, HDAC1/2, RbAp46, RbAp48, MTA-family proteins (MTA1, MTA2 or MTA3), MBD2 or MBD3, p66α and p66β. Despite its importance, a little is known about the structure, assembly or biochemical mechanism of action of this complex. RbAp46/RbAp48 and MTA1/MTA2 are core members of the complex and have been shown previously to interact with each other. To identify the residues of MTA proteins that are critical for mediating RbAp46/48 interactions. We generated a series of constructs encoding truncations and mutants of MTA1 and MTA2. Using pulldown experiments, we tested the interactions between RbAp46/RbAp48 and different truncations of MTA1 and MTA2. Our data indicate that RbAp46/RbAp48 proteins interact with both MTA1 and MTA2. We have identified two short motifs in the C-terminal region of MTA1 that appears to be necessary for the interaction with RbAp48. We have used a range of RbAp48 mutants to delineate the MTA1-binding surface of RbAp48. Together, these data could provide some detailed information on the inter-subunit interactions that facilitate the assembly of the NuRD complex, and also provide insight into the manner by which NuRD interacts with promoter-bound proteins to regulate gene expression.

THE ROLES OF ARG427 AND ARG472 IN THE ALLOSTERIC REGULATION OF RHIZOBIUM ETLI PYRUVATE CARBOXYLASE

Adina-Zada A.1, Sereeruk C.2, Jitrapakdee S.2, Zeczycki T.N.3, St Maurice M.4, Cleland W.W.3, Wallace J.C.5 and Attwood P.V.1 1The University of Western Australia, School of Chemistry and Biochemistry, 35 Stirling Highway, Crawley, WA 6009, Australia. 2Mahidol University, Department of Biochemistry, Bangkok 10400, Thailand. 3University of Wisconsin-Madison, Department of Biochemistry, Madison, Wisconsin 53726, USA. 4Marquette University, Department of Biological Sciences, P.O. Box 1881, Milwaukee, WI 53201-1881, USA. 5University of Adelaide, School of Molecular Biosciences, Adelaide, S. A., 5005, Australia.

Mutation of Arg427 and Arg472 in Rhizobium etli pyruvate carboxylase to serine or lysine greatly increased the activation constant (Ka) of acetyl CoA, with the increase being greater for the Arg472 mutants, indicating these residues are involved in the binding of acetyl CoA, with Arg472 being important than Arg427. The mutations had substantially smaller effects on the kcat for pyruvate carboxylation, part of which was to increase the Km for MgATP and the Ka for activation by free Mg2+ determined at saturating acetyl CoA concentrations. The inhibitory effects of the mutations on the rates of the enzyme-catalysed bicarbonate-dependent MgATP cleavage, carboxylation of biotin and phosphorylation of MgADP by carbamoyl phosphate indicate that the major locus of the effects of the mutations was in the biotin carboxylase (BC) domain active site. Even though both Arg427 and Arg472 are distant from the BC domain active site, it is proposed that their contacts with other residues in the allosteric domain, either directly or through acetyl CoA, affect the positioning and orientation of the biotin-carboxyl carrier protein domain and thus the binding of biotin at the BC domain active site. Based on the kinetic analysis proposed here it is proposed that mutations of Arg427 and Arg472 perturb these contacts and consequently the binding of biotin at the BC domain active site. Inhibition of pyruvate carboxylation by the allosteric inhibitor L-aspartate was largely unaffected by the mutation of either Arg427 or Arg472.

RMND5 PROTEINS ARE NOVEL E3 UBIQUITIN LIGASES THAT TARGET THE PROSTATIC TUMOUR SUPPRESSOR, NKX3.1

Louw A.1, 2, Thomas M.A.1, 2, Harvey J.M.2 and Bentel J.M.1, 2 1Anatomical Pathology, Royal Perth Hospital. 2School of Pathology & Laboratory Medicine, University of Western Australia.

Required for Meiotic Nuclear Division 5A (RMND5A) and RMND5B are uncharacterised proteins that contain putative Really Interesting New Gene (RING) domains, typically found in E3 ubiquitin ligases. Although their function has not been described previously, the RMND5A chromosomal locus is disrupted in a variety of cancer types and the RMND5B gene is located in an uncharacterised prostate cancer heritability locus. In in vitro ubiquitin assays, the RING domain of RMND5A was able to associate with UbcH2, UbcH5b and UbcH5c, while the RING domain of RMND5B interacted with UbcH5b and UbcH5c to mediate ubiquitin transfer. Supporting this evidence of E3 activity, RMND5A and RMND5B were both associated with ubiquitinated proteins in vivo in the LNCaP prostate cancer cell line. RMND5A and RMND5B interact with the prostatic tumour suppressor NKX3.1, and overexpression of either RMND5A or RMND5B induced dose-dependent declines in NKX3.1 protein levels that were partially restored following proteasome inhibition. NKX3.1 is ubiquitinated in LNCaP cells and overexpression of either RMND5A or RMND5B markedly increased NKX3.1 ubiquitination, indicating that NKX3.1 is a ubiquitination target of RMND5 proteins. In LNCaP cells, RMND5 proteins were diffusely distributed in the cytoplasm and nucleus, with RMND5B occasionally exhibiting a punctate appearance in the cytoplasm. When NKX3.1 and RMND5A or RMND5B were overexpressed in LNCaP cells, the RMND5 proteins were predominantly nuclear, while the nuclear levels of NKX3.1, a homeodomain transcription factor, were markedly reduced. These studies indicate that RMND5 proteins exhibit E3 ubiquitin ligase activity and target NKX3.1 for ubiquitination and proteasomal degradation, potentially regulating its levels, intracellular localisation and function as a transcription factor.


POSTERS MONDAY



STRUCTURE-FUNCTION RELATIONSHIPS OF UDP- GLUCURONOSYLTRANSFERASE UGT2B7: APPLICATION OF PROTEIN HOMOLOGY MODELLING, DOCKING AND MUTAGENESIS TO IDENTIFY CRITICAL RESIDUES INVOLVED IN COFACTOR BINDING

Chau N., Lewis B.C., Mackenzie P.I. and Miners J.O. Department of Clinical Pharmacology, Flinders University, School of Medicine, Flinders Medical Centre, Bedford Park, SA 5042.

UDP-glucuronosyltransferases (UGTs) are a superfamily of enzymes that primarily utilize UDP-glucuronic acid (UDP-GlcUA) as cofactor in the elimination of lipophilic drugs and endogenous compounds from the body. Recent data indicates that UGT2B7 catalyzes both morphine glucuronidation and glucosidation, the latter reaction utilizing UDP-glucose (UDP-Glc) as cofactor. Automated in silico docking with a 3D homology model of UGT2B7 and site directed mutagenesis was used to identify the critical residues that confer the cofactor binding selectivity of UGT2B7. UDP-GlcUA and UDP-Glc were docked into the active site of a UGT2B7 homology model using the SYBYL-X 1.1.2 Surflex-Dock software (Tripos). Docking studies showed that the UDP-moiety of both cofactors bind within the same domain of the C-terminus but residues involved in the binding of the sugar differed; aspartic acid 398 and glutamine 399 interact with glucose while asparagine 402 and tyrosine 33 contribute to the hydrogen bonding of glucuronic acid. Homology modelling also suggests that asparagine 378 is involved in the selective binding of the carboxylate moiety of UDP-GlcUA in UGT2B7. Based on the docking results Y33F, Y33L, D398A, D398L, Q399A, Q399L, N378A, N378L, N378H, N378Q, N402A, N402H, N402L and N402Q mutants were generated and recombinant enzymes expressed in HEK293T cells. Activity assays were conducted to characterise the effects of the mutations. Mutagenesis of C-terminal domain amino acids hypothesized to be involved in cofactor binding to UGT2B7 resulted in loss of enzyme activity. Mutation of the only N-terminal domain residue (Y33) retained activity and showed opposing trends between the glucosidation and glucuronidation of morphine and 4-methylumbelliferone.

PREDICTION OF VLP ANTIGEN STRUCTURE USING MOLECULAR DYNAMICS SIMULATION

Connors N.K.1, Anggraeni M.R.1, Chuan Y.P.1, Lua L.H.L.2, Zhang L.3, Sun Y.3 and Middelberg A.P.J.M.1 1Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Brisbane, QLD, Australia. 2Protein Expression Facility, The University of Queensland, Brisbane, QLD, Australia. 3Department of Biochemical Engineering and Key Laboratory of Systems Bioengineering of the Ministry of Education, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072,China.

Vaccination against influenza remains an enormous global challenge, with continuous genetic shift and drift resulting in influenza strains impervious to existing vaccines. Microbial modular virus-like particle (VLP) based vaccines hold promise to deliver rapid and cost-effective vaccine. Previous work demonstrated the ability to present antigenic proteins from other diseases onto the surface of a VLP, such as the Group A Streptococcus J8 protein, and the M2e Influenza protein. However, translation of the antigenic peptide into an efficacious vaccine largely depends on the structural conformation of the antigen once inserted into the modular VLP. Without native structural presentation, appropriate immunogenic response to the target pathogen is unlikely. In silico evaluation of peptide conformation by molecular dynamics (MD) simulation may assist in structure-based design of such immunogenic peptides. Here we present preliminary molecular dynamics simulation results on various influenza antigenic peptides. Two different designs of an H1N1 influenza receptor binding site peptide were inserted on the surface of murine polyomavirus VLPs, and significant differences in the biological efficacy between the two constructs was observed. GROMACS MD peptide simulations were performed to explain these differences and preliminary results show the ability of modular VLPs to raise appropriate immune response is a consequence of the structure of the antigen engineered on the VLP. Simulation of the modular VLP capsomeric protein provides further structural insights into antigen conformation.

POLYPLEX GRID ARRAY, ELISA AND IMMUNO-PCR FOR THE DIAGNOSIS OF MELIOIDOSIS

Cooper A.1, Williams N.2, Morris J.2, Ketheesan N.2, Norton R.3 and Schaeffer P.1 1School of Pharmacy and Molecular Sciences, James Cook University, Douglas QLD 4811, Australia. 2School of Veterinary and Biomedical Sciences, James Cook University, Douglas QLD 4811, Australia. 3Queensland Health Pathology Service, The Townsville Hospital, Douglas QLD 4811, Australia.

Melioidosis is an infectious disease caused by the Gram negative bacterium Burkholderia pseudomallei. The mortality rate in northern Australia is currently 16%, with immune-suppressed persons being at greatest risk. The current gold standard for diagnosis is culture, which often requires enrichment followed by several days incubation. As the seroprevalence of antibodies to B. pseudomallei in North Queensland is relatively low at approximately 2.5%, the use of serology in diagnosis could improve rapidity of diagnosis. However, the existing serological technique is an indirect haemagglutination assay (IHA) which has several disadvantages. Approximately half of patients later confirmed culture positive are not detected by IHA in early stages of the disease and a subset of patients are persistently seronegative with IHA. We have developed a serological assay capable of detecting antibodies in patient sera from a greater proportion of IHA-negative patient subsets. The assay is based upon antigenic fractions from various B. pseudomallei isolates, extracted using a novel technique. The assay can distinguish between different serotypes in polyplex using a grid assay format. Currently the assay has a sensitivity and specificity of 87.5% and 100% respectively, with 85.7% of culture positive, IHA-negative samples detected. An ELISA and immune-PCR have also been developed. More rapid and reliable serological testing for melioidosis is essential and will improve diagnosis and patient outcome, particularly in travelers and defence personnel returning from endemic areas, situations with unusual presentations or where melioidosis is suspected but the infection site is inaccessible or the organism fails to grow in culture.

INSIGHT INTO THE VENOM OF THE BENEFICIAL PARASITOID WASP, DIADEGMA SEMICLAUSUM

Cooper T.1, Asgari S.2 and Glatz R.1 1South Australian Research and Development Institute. Waite Campus, Urrbrae, SA, Australia. 2University of Queensland, School of Biological Sciences, St Lucia, QLD, Australia.

The venom of parasitoid wasps alters the immune responses and development of their hosts to benefit their own progeny’s development. We investigated the venom of Diadegma semiclausum which is an important beneficial insect in the control of a major pest of brassica crops, Plutella xylostella (Diamondback moth). We show that the venom consists of some higher molecular weight proteins but appears particularly abundant in low molecular weight peptides. When applied to insect hemocytes, venom was able to inhibit cell-spreading immune responses. Furthermore, a group of putative venom transcripts, cloned from the venom gland of D. semiclausum are described that show no significant sequence similarities with other database proteins. We propose that the proteins resulting from these transcripts that are highly expressed only in female wasps could be pre-proproteins that are further processed into bioactive peptides. We investigated the effects of synthesised peptides designed from these transcripts to find that a peptide from the C-terminus of DsVn3 appeared to accumulate within hemocytes and reduced their viability in vitro. This peptide also reduced the viability of Sf9 cells with an EC50 of 3.6 μM.


POSTERS MONDAY



STRUCTURAL AND FUNCTIONAL ANALYSIS OF THE GM-CSF:GM-CSF RECEPTOR ALPHA CHAIN BINARY COMPLEX

Dottore M.1, McClure B.1, Hercus T.R.1, Broughton S.E.2, 3, Nero T.L.2, 3, Taing H.H.1, Parker M.W.2, 3 and Lopez A.F.1 1Division of Human Immunology, Centre for Cancer Biology SA Pathology, Adelaide, South Australia. 2Biota Structural Biology Laboratory, St. Vincent’s Institute of Medical research, Fitzroy, Victoria. 3Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Victoria.

Granulocyte-macrophage colony stimulating factor (GM-CSF) is a pleiotropic cytokine that controls the production and function of blood cells and is implicated in multiple pathologies such as rheumatoid arthritis and leukaemia. GM-CSF signals through a heterodimeric receptor consisting of a ligand-specific α subunit (GMRα) and a β subunit (βc) which is shared with the interleukin-3 and interleukin-5 receptors. We recently determined the crystal structure of the human GM-CSF:GMRα:βc ternary complex that revealed a novel mode of receptor activation involving a higher-order dodecamer complex. Based on X-ray diffraction data collected to 2.8 Å resolution we have now generated a complete model of the GM-CSF:GMRα binary complex wherein the GMRα structure is strikingly reminiscent of the IL-13Rα1 and IL-13Rα2 subunits. Docking of the complete GMRα model within the GM-CSF:GMRα:βc dodecamer complex suggests a functional interaction between GMRα N-terminal domains from adjacent hexamers. We have used mutagenesis, ligand binding and functional studies to examine the contribution of residues in GMRα and GM-CSF that form the binding interface. This has allowed us to identify a number of residues that define GM-CSF binding to GMRα and are therefore involved in receptor complex assembly. This includes residues from all three domains of GMRα and may represent a different mode of binding to that seen for other cytokine receptors such as IL3Rα and IL5Rα.

CHARACTERISING THE ROLE OF FACTOR INHIBITING HIF (FIH) IN POXVIRUS INFECTION

Fabrizio J.A.1, Wilkins S.E.1, Chen D.2, Mercer A.2 and Peet D.J.1 1School of Molecular and Biomedical Science, University of Adelaide, Adelaide SA 5005, Australia. 2Department of Microbiology and Immunology, University of Otago, PO Box 56, Dunedin 9016, New Zealand.

Factor Inhibiting Hypoxia Inducible Factor-α (FIH) is an oxygen-dependent asparaginyl hydroxylase, important in the cellular response to hypoxia. It was first found to catalyse the hydroxylation of Hypoxia Inducible transcription Factors (HIFs). More recently a number of proteins containing ankyrin repeat domains (ANKs) have been identified as substrates for FIH, and can indirectly influence HIF activity. Of particular interest are specific ANK proteins in poxviruses. Poxviruses are a large family of enveloped double stranded DNA viruses that infect both vertebrates and invertebrates, and include the Orf Virus (ORFV) that infects small ruminants. ORFV encodes 5 ANK proteins identified as possible FIH substrates. This study aims to elucidate the functional interaction of FIH with poxvirus ANK proteins in viral pathogenesis. We demonstrate that ORFV ANK proteins appear to be substrates of human FIH using In vitro hydroxylation assays. Fluorescent microscopy of HeLa cells transiently transfected with tagged ORFV ANK proteins show that endogenous FIH colocalises with ORFV ANK proteins and is sequestered in specific subcellular locations. We have now cloned and expressed ovine FIH, demonstrated that it is a functioning hydroxylase and are currently comparing its ability to hydroxylate ORFV ANK proteins relative to human FIH. These studies are being extended to other poxviruses, specifically ectromelia and vaccinia, to determine whether this is a conserved mechanism, and the importance of FIH in poxvirus infection. Importantly, regulation of HIFs by other viruses, such as Hepatitis viruses, plays an important role in viral infection.

STRUCTURAL AND FUNCTIONAL CHARACTERISATION OF ACYL-COA THIOESTERASES

Forwood J.K. Charles Sturt University.

Acyl-coenzyme A (acyl-CoA) thioesterases play a crucial role in the metabolism of activated fatty acids, coenzyme A, and other metabolic precursor molecules including arachidonic acid and palmitic acid. These enzymes hydrolyse coenzyme A from acyl-CoA esters to mediate a range of cellular functions including β-oxidation, lipid biosynthesis, and signal transduction. We have crystallised and determined the structure of hexameric hot-dog domain-containing acyl-CoA thioesterases from bacteria and humans to provide a structural and comparative analyses to the apo- and coenzyme A-bound forms and identify key conformational changes induced upon ligand binding. We observed dramatic ligand-induced changes at both the hot-dog dimer and trimer-of-dimer interfaces; the dimer interfaces in the apo-structure differ by over 20% from one another, and decrease to about half the size in the ligand-bound state. We have also assessed the specificity of the enzymes against a range of fatty acyl-CoA substrates, and have identified a preference for short-chain fatty acyl-CoAs. The conformational changes are likely to be conserved from bacteria through to humans, and provide a greater understanding, particularly at a structural level, of thioesterase function and regulation.

EXTENDED EXPOSURE TO CAFFEINE AND SUCROSE HAS DIFFERENTIAL EFFECTS ON LOCOMOTOR RESPONSE AND THE PROTEOME OF ADULT AND ADOLESCENT SPRAGUE DAWLEY RATS

Franklin, J.F., Sauer, M., Wearne, T., Clemens, K.J., Homewood, J., Haynes, P.A. and Cornish, J.L. Departments of 1 Psychology and 2 Chemistry and Biomolecular Sciences, Macquarie University, North Ryde, NSW, Australia.

Caffeine is a psychostimulant that is commonly consumed by adults and children. During adolescence, enhanced consumption of caffeinated beverages is also associated with high levels of sugar intake via ‘energy drinks’. The neurobiological consequences of this combination are as yet unexplored. The aim of the current study was to investigate the effect of chronic exposure to oral caffeine (CAFF), sucrose (SUCR), or their combination on locomotor behaviour and proteomic profile of the prefrontal cortex (PFC) in adolescent and adult rats. Method: Male adolescent Sprague Dawley (SD) rats, (n=12 per group) were treated from postnatal day (PD) 35 until PD60 with either water, CAFF 0.6g/L, SUCR 10%, CAFF 0.6g/L+SUCR 10% or CAFF 0.3g/L+SUCR 5% in their drinking water. Adult SD rats (PD60+ on the first day of treatment, n=12 per group) were treated for the same time period. Locomotor behaviour was measured on the first and last day of treatment, then one week after treatment. Following final behavioural testing brains were rapidly removed; snap frozen in liquid nitrogen then stored at -80 until proteomic analysis of the PFC was conducted. Results: When tested drug free PD35 SUCR10%, PD60+ SUCR10% and CAFF0.6g/L animals were significantly more active than animals exposed to other treatments. Label free quantitative shotgun proteomic analysis of selected brain regions is currently ongoing, and data from these analyses will be presented. Our initial results suggest that exposure to caffeine and sucrose has the potential to change neural functioning deferentially depending on whether exposure occurs during adulthood or adolescence.


POSTERS MONDAY



DISCOVERY AND CHARACTERISATION OF NOVEL SUBSTRATES OF THE HIF HYDROXYLASE, FIH

Hampton-Smith R.J.1, Devries I.2, Mulvihill M.2, Komives E.2 and Peet D.1 1School of Molecular and Biomedical Science, University of Adelaide, Adelaide SA 5005, Australia. 2Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Drive La Jolla, CA 92093-0378, United States.

Cells have evolved complex machinery to sense and respond to hypoxia, the state in which oxygen demand exceeds supply. A candidate oxygen sensor is the asparaginyl hydroxylase, Factor Inhibiting HIF (FIH). This enzyme directly utilises molecular oxygen, and has a physiologically relevant Km for oxygen. The recent generation of FIH knock-out mice has revealed an important role for FIH in regulation of metabolism: the mice hyperventilate, have an elevated heart rate and insulin sensitivity, and are somewhat resistant to weight gain. At present, though, little is understood of how FIH deficiency causes these effects. FIH is known to have an important role in regulating Hypoxia-inducible Factor (HIF) transcription factors in an oxygen-dependent manner. HIF hydroxylation, however, explains little of the KO mouse phenotype, suggesting the existence of other FIH substrates. We have utilised a yeast two-hybrid assay with human FIH as bait to identify novel FIH targets. The results of this screen demonstrate a new role for FIH in the hydroxylation of a variety of ankyrin repeat domain (ARD)-containing proteins. Determining a role for hydroxylation of ARDs, however, has proven challenging. In collaboration with Betsy Komives (UCSD), we used a biophysical approach for analysing the effect of hydroxylation on FIH substrate, IκBα. We concluded from numerous in vitro analyses that hydroxylation has little effect on IκBα structure and function. Our current work is aimed at determining the relative contribution of ARD vs HIF hydroxylation to the metabolic and gene regulation effects seen in FIH KO mEFs.

CYOKINE REGULATION BY ALCOHOL EXTRACT FROM ALLOMYRINA DICHOTOMA AND ITS COMPOSITION OF GLYCOSAMINOGLYCAN

Ahn M.Y., Kim S.J., Han J.W., Hwang J.S. and Yun E.Y. Dept. Agricultural Biology, National Academy of Agricultural Science, RDA, South Korea.

Holotrichia diomphalia larvae is one of the most widely used Korean folk medical preparations and has been utilized as anti-hepatofibrosis, antidiabetic and anti-neoplastic agents. In this study, the effects of preventing or and treating fatty liver and hyperlipidemia, and the cytokine level (IL-6, NOS, No, VEGF, sPLA2, PGE2, MMP-2, and MMP-9), with the ethanol extract from the adult and larva of Protaetia brevitarsis (Cetoniidae), Allomyrina dichotoma (Dynastidae) and Serrognathus platymelus castanicolar (Lucanidae) were examined. Three days of administration of theses ethanol extract reduced free fatty acid levels (49.3% reduction) and increased the hyaluronic acid levels (213.3% increase) in the alcoholic fatty liver of rats. These alcohol extracts inhibited expression of VCAM-1 (56.3% inhibition) and ICAM-1 (82.3% inhibition) in HUVEC cells stimulated with TNF-alpha. The histological appearance of the liver sections was improved and a reduction in the number of fat cells with fat droplets was observed in db/db mice. These insect glycosaminoglycans were isolated and their monosaccharide compositions were analyzed by trimethylsilylated GC-MS.

EVALUATION THE BIOLOGICAL ACTIVITY OF A COMPUTATIONALLY DE NOVO DEIGNED PEPTIDE ON SKIN CANCER CELLS IN VITRO

Almansour N.1, 1, Pirogova E.2, 1, Colo P.1, 1, Cosic I.2, 1 and Istivan T.1, 2 1School of Applied Sciences, Health Innovations Research Institute, RMIT University, Melbourne, Australia. 2School of Electrical and Computer Engineering, Health Innovations Research Institute, RMIT University, Melbourne, Australia.

Anti-cancer therapeutic peptides have been applied in developing cancer therapeutics focusing on small molecular weight peptides with strong tumoricidal activity and low toxicity. RRM-MV is a bioactive peptide analogue for myxoma virus protein (M-T5) designed by the Resonant Recognition Model (RRM) as a linear short peptide. M-T5 is an ankyrin-repeat protein which is known to be critical for virus replication in the majority of human tumor cells. The non-bioactive peptide RRM-C was also designed by the RRM with a different inactive frequency, and was used as a negative control peptide. The objective of this study was to evaluate the biological influence of the bioactive peptide RRM-MV on cell viability of human skin cancer and normal cells in vitro. The toxic activity of RRM-MV was determined on melanoma cells (MM96L), carcinoma cells (COLO-16) and normal skin cells (HDF) at various doses and incubation times using the Prestoblue cell viability assay. The degree of cell recovery in the treated cells was also evaluated. Our results indicated that RRM-MV produced a time and dose dependent cytotoxic effect on the cancer cells, while it had a negligible effect on normal cells. However, the cancer cells were recovered after 16 h of the treatments, indicating the need for a second dose. Treatment of the cancer cells with a second dose of RRM-MV had a significant effect affected on cell growth. Interestingly, no effects on growth of either cancer or normal cells were detected following treatment with RRM-C. Therefore, it can be suggested that the RRM is a valid tool to design bioactive peptides with targeted therapeutic functions.

REGULATION OF BREAST CANCER PROGRESSION BY THE MICRORNAS, MIR-193B AND MIR-342-3P

Arnet V.K.1, 2, Roslan S.1, Johnstone C.T.3, Li X.1, Khew-Goodall Y.1, 4, Anderson R.L.3, Goodall G.J.1, 2, 4 and Gregory P.A.1, 2 1Division of Human Immunology, Centre for Cancer Biology, SA Pathology, Adelaide, SA, 5000, Australia. 2Discipline of Medicine, The University of Adelaide, Adelaide, SA 5005, Australia. 3Peter MacCallum Cancer Centre, St. Andrews Place, East Melbourne, Victoria, 3002, Australia. 4School of Molecular and Biomedical Science, The University of Adelaide, Adelaide, SA 5005, Australia.

Breast cancer is the leading cause of cancer related death in women and 90% of cancer deaths are associated with metastasis. Recent reports have indicated microRNAs, small noncoding regulatory RNAs, control primary breast tumours and pre-invasive breast cancer progression. Given the importance of microRNAs in gene expression regulation within signalling pathways that underlie cancer, it is important to examine the functional roles of microRNAs in metastatic breast cancer. MicroRNA profiling of mouse and human mammary carcinoma cell lines of varying metastatic capacities revealed differential expression of miRNA-193b and miRNA-342-3p. This study investigated whether these microRNAs influence cancer cell migration and invasion in vitro and aimed to determine their potential gene targets relevant to these processes. The results of the Transwell migration and scratch-wound healing assays revealed miR-193b and miR-342-3p were able to alter breast cancer cell migration. In addition, we were able to show the influence of miR-193b and miR-342-3p was independent of effects on in vitro proliferation or Matrigel invasion. Our findings warrant further investigation into the function of miR-193b and miR-342-3p in mediating breast cancer cell invasion and to identify their targets involved in metastasis. The results of this study will help to advance our understanding of how molecular signalling pathways regulate metastasis and assist in the development of improved cancer prognosis and treatments.


POSTERS MONDAY



INVESTIGATION INTO THE STRUCTURE AND FUNCTION RELATIONSHIP FOR BIOTIN TRANSPORTER FROM STAPHYLOCOCCUS AUREUS

Azhar A., Polyak S.W., Wiridja A., Baker P. and Booker G.W. Discipline of Biochemistry, School of Molecular and Biomedical Science, the University of Adelaide, South Australia 5005.

Uptake of the important micronutrient biotin into bacteria is mediated by a specific transport protein, BioY. The biotin importer belongs to a new class of ATP-dependent vitamin transporters that are distinct from the well-characterised, classical ATP-binding cassette (ABC) transporters1. Classical ABC importers use a soluble solute-binding protein to capture a substrate and a second membrane-bound protein to transport it across the membrane. In contrast the biotin transporter uses a single membrane-bound protein, BioY, for solute capture and import it into the bacteria1,2. The molecular details of solute binding and transport have not been well studied. Here we focus on BioY from the clinically important human pathogen Staphylococcus aureus. In order to delineate protein structure-function relationships, we recombinantly expressed S. aureus BioY in E. coli and developed a novel competitive binding assay using fluorescently labeled biotin. Here non-labeled biotin was shown to compete with binding of the tracer on the surface of the bacterial cells, thereby demonstrating the specificity of the assay. A range of biotin analogues were also tested, and revealed that the thiophane and ureido rings on biotin were important determinants in ligand binding. We also generated a molecular model of BioY using X-ray crystal structures of the thiamin and riboflavin transports. The molecular model assisted with proposing specific amino acids required for binding, and for designing mutagenesis studies to investigate this. We are now refining these approaches to determine the regions on BioY protein important for biotin binding. 1. Rodionov, D. A., P. Hebbeln, et al. (2009). J. Bacteriol. 191(1): 42-51. 2. Eitinger, T., Rodionov, D.A., et al., FEMS Microbiol. Rev. 35, 3–76 (2011).

PASSING THE BATON: NPAS4 RESPONDS AND DELIVERS UNDER STRESS

Bhakti V.L.D., Hao N. and Whitelaw M.L. School of Molecular and Biomedical Science, University of Adelaide.

The basic helix-loop-helix/Per-ARNT-Sim (bHLH/PAS) family of transcription factors are commonly involved in the regulation of gene expression in response to external stimuli. This is best demonstrated by the Hypoxia Inducible Factor α (Hifα), which responds to low cellular oxygen levels, and the Aryl hydrocarbon Receptor (AhR), which is activated by xenobiotic compounds. They heterodimerise with another bHLH/PAS protein, Aryl hydrocarbon Receptor Nuclear Translocator (Arnt), to form an active transcription factor complex. While these factors are expressed ubiquitously, other bHLH/PAS proteins display tissue-specific expression pattern, such as the neuronal PAS domain 4 (Npas4) and Arnt2, which are primarily expressed in the brain. Npas4 mRNA and protein levels are induced by neuronal activity, generalised seizure and ischemic insults. We are studying the signalling processes by which Npas4 expression is regulated in response to these stimuli, and how Npas4 confers neuroprotection through the activation of target genes such as Brain Derived Neurotrophic Factor (Bdnf). We have found that during neuronal differentiation, expression of Arnt is reduced concomitant with a sharp increase in Arnt2 levels, consistent with Arnt2 being the functional partner of Npas4. We are also investigating how tissue-specific regulation of Arnt2 expression is achieved and whether Arnt2 is absolutely required for Npas4 transcriptional activity.

DETERMINING THE ROLE OF TROPOMYOSINS IN TRIGGERING ANOIKIS

Desouza M., Gunning P. and Stehn J. Department of Anatomy, School of Medical Sciences, University of New South Wales, Sydney, Australia.

The actin cytoskeleton has been implicated as a sensor and mediator of apoptosis with changes in actin filament dynamics triggering an apoptotic cascade in numerous cell types. Tropomyosins are a family of proteins lying along the major groove of actin filaments providing structural stability and diverse actin filament function. The high molecular weight tropomyosin isoform Tm1 has been shown to increase sensitivity to apoptosis induced by cell detachment (anoikis) via an unknown mechanism. We aim to delineate the molecular mechanism by which Tm1 regulates sensitivity to anoikis. In this study, rat neuroblastoma cell lines over-expressing Tm1 were generated by stable transfection and their actin cytoskeleton morphology was characterised by immuno-fluorescent staining. Elevated Tm1 levels were demonstrated to increase the incorporation of Tm1 into filaments with a concomitant increase in actin filament bundles. To assess cell survival in an adhesion-independent environment Tm1 over-expressing cells were seeded onto plates coated with the adhesion-free compound polyHEMA and cell viability was measured using alamar blue. Tm1 over-expressing cells showed a reduced capacity to survive in an adhesion-independent environment when compared to Tm1 cells seeded onto uncoated plates (p<0.05). In conclusion, Tm1 over-expression alters actin filament dynamics leading to a more organized cytoskeleton which may increase a cells’ sensitivity to anoikis. Analysis of focal adhesions may reveal the potential mechanism by which Tm1 senses anoikis. Furthermore Tm1 may be triggering anoikis by activating the pro-apoptotic protein Bmf, which is sequestered within the actin cytoskeleton. To test this hypothesis, Tm1 over-expressing B35 cells will be treated with multiple apoptotic stimuli and the sub-cellular localisation of Bmf will be assessed by western blotting and immuno-fluorescent staining.

INTERMEDIATE SECRETED SUBSTRATES OF THE NOVEL SECRETION SYSTEM IN PORPHYROMONAS GINGIVALIS FORM MULTIMERIC COMPLEXES

Glew M.D., Veith P.D., Gorasia D.G., Chen D., Chen Y.Y. and Reynolds E.C. Oral Health CRC, Melbourne Dental School, and Bio21 Institute, The University of Melbourne, 3010, Australia.

Porphyromonas gingivalis is the major etiological agent causing chronic periodontitis in humans leading to supporting tissue/bone destruction and tooth loss. P. gingivalis is a Gram-negative bacterium that secretes abundant Arg-specific (RgpA and RgpB) and Lys-specific (Kgp) cysteine proteases called gingipains which are considered to be the major virulence factors of this pathogen. The gingipains, together with more than 30 other proteins are secreted to the cell surface by a novel secretion system that is restricted to members of the Bacteroidetes/Chlorobi phyla. All secreted substrates share a conserved C-terminal domain (CTD). N- and C-terminal signal sequences play pivotal roles in directing secretion through inner/outer membranes and subsequent attachment to anionic lipopolysaccharide (A-LPS) at the cell surface for covalent anchorage. Recently, we showed that one of the CTD proteins, PG0026, functions as the C-terminal signal peptidase which cleaves the CTD prior to A-LPS attachment. Additionally, pro and adhesin domains, if present, must also be proteolytically processed to produce the mature gingipains. In this study, 2-dimensional blue native polyacrylamide gel electrophoresis was employed to understand the nature of the intermediate CTD protein forms prior to CTD cleavage and A-LPS attachment. The secretion of RgpB was followed as a model substrate in two specific secretion mutants, blocked at two known stages of secretion, and compared to the wild type. The results reveal that intermediate cleaved RgpBs form multimeric complexes which were changing in size in all three strains. This suggests that both conformational and association state changes are taking place during secretion.


POSTERS MONDAY



TARGETING CHROMOSOMAL INSTABILITY

Shaukat Z.1, Wong H.W.-S.2, Nicolson S.1, Saint R.B.2 and Gregory S.L.1 1Molecular and Medical Biosciences, University of Adelaide. 2Dept. of Genetics, University of Melbourne.

We have set up a screen in Drosophila to target one typical feature of tumour cells – the defective regulation of chromosome segregation that gives rise to chromosomal instability (CIN). Our objective is to identify inhibitors that are useful against chromosomally unstable cells that can escape traditional mitotic disruption therapy. We are depleting genes by RNAi to find those that can generate the best level of apoptosis and minimum level of aneuploidy in cells that have genetically induced CIN. As a first step we have screened the kinase and phosphatase genes from the Drosophila genome and found that our best candidates affect the centrosome, including nek2, SAK and a subset of the JNK signalling pathway. Consistent with this, recent reports both in Drosophila and mammals have highlighted the role of centrosome regulation in controlling mitotic fidelity and preventing tumour progression. We are characterizing of two of our target genes, JNK and PASK, focussing on the relationship between apoptosis and unregulated progression through mitosis.

EVIDENCE FOR A ROLE FOR PROTEIN KINASE C IN IMPAIRED STORE OPERATED CALCIUM ENTRY IN STEATOTIC LIVER CELLS

Ali E.S.1, Wilson C.H.1, Scrimgeour N.R.2, Martin A.1, Rychkov G.Y.2 and Barritt G.J.1 1Department of Medical Biochemistry, School of Medicine, Flinders University, Australia. 2Department of Physiology, School of Medical Sciences, The University of Adelaide, Australia.

Store operated Ca2+ entry (SOCE) and changes in [Ca2+]cyt play essential roles in the mechanisms by which insulin and glucagon regulate liver carbohydrate and lipid metabolism. Store operated Ca2+ channels (SOCs) are composed of Orai1 and STIM1 proteins. Hepatocyte steatosis linked with non-alcoholic fatty liver disease can lead to hepatocyte insulin resistance and type 2 diabetes. Hepatosteatosis is associated with the activation of protein kinase C (PKC), generation of ROS, the ER (endoplasmic reticulum) stress response and impaired activity of the ER (Ca2++Mg2+ )-ATPase. The aim was to investigate the effect of steatosis on SOCE. H4IIE liver cells were loaded with lipid (confirmed by Nile red fluorescence) by pre-treatment with amiodarone. In fura-2 Ca2+ imaging experiments, steatotic H4IIE cells showed around 70% reduced Ca2+ release from ER stores (induced by DBHQ) and 80% decreased Ca2+ entry through SOCs, compared to non-steatotic control cells. Similar results were obtained with freshly-isolated rat hepatocytes and with H4IIE cells pre-loaded with lipid using palmitate and oleate. The amount of Ca2+ released by ionomycin from steatotic cells in the absence of extracellular Ca2+ was reduced by 50% compared with controls. IP3-initiated ISOC (SOCE current measured by patch clamp) was reduced by 70% in steatotic cells. GF109203X, a PKC inhibitor, reversed the impaired Ca2+ entry by 40% in steatotic liver cells. Immunoblotting showed that no significant change in expression of the Orai1 or STIM1 in steatotic cells, indicating that decreased expression of Orai1 and/or STIM1 is unlikely to make a significant contribution to the observed decrease in SOCE. It is concluded that steatosis leads to a substantial decrease in SOCE and this is mediated in part by PKC. Current experiments are directed towards evaluation of the role of PKC and Orai1 phosphorylation.

MEASURING PROTEIN THIOL OXIDATION IN VIVO

Arthur P.G., Armstrong A.E. and Fournier P.A. University of Western Australia, 35 Stirling Hwy, Crawley, W.A. 6009, Australia.

Oxidative stress, evident in many diseases and physiological conditions, can affect cell function by a number of different molecular mechanisms. One mechanism involves changes in protein function caused by the reversible oxidation of thiol groups of cysteine residues and has been well described in single protein studies and in vitro systems such as cultured cells and isolated tissue. However, direct evidence of the biological significance of protein thiol oxidation has not been established in vivo for disease pathology or normal physiology in animal models or humans. As a step towards establishing the in vivo impact of protein thiol oxidation, we developed a technique to directly measure the extent of protein thiol oxidation in tissue. Using this technique, we tested whether the level of protein thiol oxidation was affected in the red gastrocnemius muscles of exercising rats. Swimming rats (n=6) were subjected to a fatigue inducing exercise protocol for three minutes. Exercise did not affect the level of total protein thiols (PStot) calculated as the sum of reduced proteins thiols (PSred) and oxidised protein thiols (PSox). However, exercise did cause a 20% increase in oxidised protein thiols [PSox/(PSox + PStot)*100]. The level of thiol oxidation of myosin, actin, troponin and glycogen phosphorylase were readily quantifiable using 1D SDS PAGE. The level of thiol oxidation of myosin and glycogen phosphorylase increased by 15% and 8.7% respectively after exercise, but there were no significant changes for the other proteins. These data provide evidence that proteins are sensitive to reversible thiol oxidation under normal physiological conditions such as exercise.

THE THIOREDOXIN SYSTEM UNDER INTERMITTENT HYPOXIA IN CANCER CELLS

Bhatia M.1, 2, Karlenius T.C.1, 2, Di Trapani G.1 and Tonissen K.F.1, 2 1School of Biomolecular and Physical Sciences, Griffith University, Nathan, Qld, Australia. 2Eskitis Institute for Cell and Molecular Therapies, Griffith University.

The oxygen environment is an important determinant of carcinogenesis. Cancer cells are mostly in flux between low and high oxygen availability, experiencing cycling between hypoxia and reoxygenation, a phenomenon termed as intermittent hypoxia. The thioredoxin system, comprising thioredoxin and thioredoxin reductase, is an important antioxidant system that maintains the cellular oxygen homeostasis by eliminating harmful oxidants. It also regulates several important transcription factors. High levels of thioredoxin have been observed in cancer cells and are linked to their growth and progression. Due to its immense importance in the cancer cell biology, the thioredoxin system is also considered a potential anticancer target. Surprisingly, very few studies involving the thioredoxin system have been performed under intermittent hypoxia. We used MDA-MB-231, a model breast cancer cell line to characterize the expression of the thioredoxin system under intermittent hypoxia. Cells were exposed to prolonged hypoxia followed by different lengths of reoxygenation alongside normoxic conditions. Additionally, hypoxic cycling preconditioning prior to exposure to prolonged hypoxia and reoxygenation were also used. Cellular viability, ROS levels, thioredoxin protein levels and promoter activity were measured under all the conditions. We also observed the thioredoxin reductase specific activity under normoxia, hypoxia and hypoxia followed by 4 hours of reoxygenation. Our results indicate that reoxygenation provides cells with growth stability. A 4-hour reoxygenation significantly increases the ROS levels, thioredoxin protein levels and promoter activity, and thioredoxin reductase specific activity. Better understanding of mechanisms involved in the regulation of the thioredoxin system under different oxygen conditions will enable designing of improved therapeutics that are more effective in the in vivo tumor microenvironment.


POSTERS MONDAY



THE LOSS OF THE CDKN2A GENE IS A CRITICAL EVENT IN THE TRANSFORMATION OF LIVER PROGENITOR CELLS

Strauss R.1, Passman A.1, VanVuuren J.1, London R.1, Yeoh G.C.1, 2 and Callus B.A.1, 2 1School of Chemistry and Biochemistry, University of Western Australia. 2Centre for Medical Research, Western Australian Institute of Medical Research.

Worldwide, liver cancer is the fifth most prominent cancer and the third highest cause of cancer-related deaths. Evidence is mounting to suggest that liver progenitor cells (LPCs) are a source of liver cancers, however, the underlying mechanisms that transform LPCs to cause liver cancer remain largely unknown. We searched for significant molecular changes that associated with the spontaneous transformation of non-tumorigenic LPCs during routine passage. The p19Arf and p16Ink4A tumour suppressors were consistently undetectable in transformed cells. Investigating possible mechanisms to account for this we determined that the CDKN2A gene, which encodes both Arf and Ink4A, is consistently deleted in our panel of transformed LPC lines. To examine the effect of Arf loss on LPC transformation we generated LPC lines from Arf-/- mice and tested each line at every second passage for their ability to grow colonies in soft-agar as an indication of transformation. Arf-/- LPCs did not grow in agar at passage 9; however, by passage 21 the cells could clearly produce colonies compared with our non-transformed controls. These results indicate that Arf-/- LPCs have an increased propensity to transform. Preliminary studies examining Arf in LPCs in the livers of mice that were fed a choline-deficient, ethionine supplemented (CDE) diet have revealed that Arf is only expressed in a subset of LPCs induced as part of the regenerative response in chronically damaged liver. These results lead us to propose that Arf/Ink4A may have important roles in the LPC inductive phase of the regenerative response in chronic liver damage and that it is the subset of LPCs that lose Arf/Ink4A expression which transform resulting in liver cancer. Future studies are aimed at providing definitive evidence to support the hypothesis that mutations to the CDKN2A pathway in LPCs prompts these cells to assume the role of a liver cancer stem cell.

SMALL MOLECULES THAT RENDER 14-3-3 PHOSPHORYLATABLE AND INDUCE APOPTOSIS

Coolen C.1, Pitson S.1, Bittman R.2, Lopez A.1 and Woodcock J.1 1Centre for Cancer Biology, SA Pathology, Frome Rd, Adelaide, South Australia. 2Queens College, City University of New York, USA.

14-3-3 proteins, of which there are 7 mammalian isoforms, are up-regulated in many human cancers and are associated with poor prognosis and chemo-resistance, making 14-3-3 proteins potential targets for cancer therapy. 14-3-3 proteins are a highly conserved family of dimeric proteins, each comprised of two 30kDa monomer units. These monomers dimerise via their N-termini to form dual amphipathic grooves that bind to phospho-serine motifs in client proteins. Through this interaction 14-3-3 proteins regulate multiple signalling pathways including survival and proliferation by regulation of apoptosis-signal regulating kinase-1 (ASK-1), the BH3 protein BAD, and activation of Raf. Critically, it is the dimeric status of 14-3-3 which is important for these regulatory functions of 14-3-3 and disruption of the dimer leads to apoptotic signalling. We have discovered that the dimeric state of 14-3-3 is regulated; the naturally occurring lipid sphingosine renders 14-3-3 proteins phosphorylatable at a Ser at the dimer interface, disrupting the dimer and inducing apoptosis in a time and dose dependent manner. Likewise, the sphingosine analogues FTY-720 and cetrimonium bromide regulate cell fate through 14-3-3 dimer destabilisation and degradation. We can harness this regulatory mechanism as a novel anti-cancer approach and using our understanding of lipid: 14-3-3-interaction, we have identifed a family of small molecules that mimic sphingosine’s ability to regulate 14-3-3. We show here the ability of this unique family of compounds to render 14-3-3 phosphorylatable, leading to 14-3-3’s degradation. Significantly these compounds induce apoptosis in primary leukaemic blasts and in chemo-refractory leukaemic stem cells via mitochondrially-mediated cell death suggesting that they have anti-leukaemic properties.

WNT AND NK GENES IN SPONGE BODY PLAN PATTERNING

Adamska M.A., Adamski M., Bergum B., Fortunato S., Guder C. and Leininger S. Sars International Centre for Marine Molecular Biology, Thormohlensgt. 55, N-5008 Bergen, Norway.

Wnt and antennapedia class homeobox genes are two major codes patterning body plans of eumetazoans from cnidarians to vertebrates. In the siliceous sponge Amphimedon queenslandica, expression of one of three Wnt genes is limited to the posterior end of the larva, consistent with its function in axis specification. In contrast, developmental expression of Amphimedon NK genes suggests involvement in cell type specification rather than body plan patterning. Since sponges are a diverse clade, we set up to investigate expression of Wnt and NK genes in calcareous sponges, developmentally distinct and distantly related to siliceous sponges. As our model system we selected Sycon ciliatum, which retains a single axis of symmetry in the adult form. Genome sequencing revealed presence of 21 Wnt genes and 9 antennapedia class genes. Three Wnt genes are expressed in the posterior end of the larvae, in staggered domains reminiscent of Wnt expression in cnidarians. Significantly, several Wnts are also expressed in staggered domains at the apical end of the adult, indicating involvement in patterning of both larval and adult body plans. Three NK genes are expressed in domains forming a set of stripes along the larval body axis. Two others are expressed during development of larval cruciform cells, which have been suggested to act as photoreceptors. In adults, several NK genes are expressed in cell-type specific patterns. Thus, NK genes appear to be involved in both body plan patterning and in cell type specification. Our results indicate that conserved usage of the two systems extends beyond eumetazoans, and likely originated in the last common ancestor of all multicellular animals.

LONGITUDINAL STUDY OF NUCLEOTIDE METABOLITES IN INFANTS’ SALIVA COMPARED TO ADULTS AND MAMMALSAl-Shehri S.1, Henman M.2, Cowley D.C.2, Shaw P.N.1, Liley H.2, Tomarchio A.2, Charles B.G.1, Wright J.R.3 and Duley J.A.1, 2 1School of Pharmacy, The University of Queensland, Brisbane 4072,Australia. 2Mater Medical Research Institute & Health Services, Brisbane 4101, Australia. 3School of Veterinary Science, The University of Queensland, Gatton Q 4343, Australia.

Background: Human saliva has been extensively described for its composition of major proteins, electrolytes, cortisol, melatonin and some metabolites such as amino acids. Little is known, however, about nucleotide precursors in human saliva. Nucleotides are essential for synthesis of DNA/RNA, supplying energy, regulating G-protein signalling, and biosyntheses. Methods: Saliva swabs were collected from full-term neonates, aged 1-3 days, 6-weeks, 6-months and 12- months. Unstimulated fasting (morning) saliva samples were collected directly from adults. Samples were extracted and ultrafiltered, then nucleotide metabolites were analysed by RP HPLC with UV-photodiode array and ESI-QTrap mass spectrometry. Results: Median concentrations (μM) of salivary nucleobases and nucleosides in infants for neonates/6-weeks/6-months/12-months respectively were: uracil 5.3/0.80/1.4/0.70, hypoxanthine 27/7.0/1.1/0.80, xanthine 19/7.0/2.0/2.0, adenosine 12/7.0/0.90/0.80, inosine 11/5.0/0.30/0.40, guanosine 7.0/6.0/0.50/0.40, uridine 12/0.80/0.30/0.90. Deoxynucleosides, dihydropyrimidines and dihydroxyadenine concentrations were essentially negligible. The concentrations in human adults/cows/horses/dogs respectively were: uracil 0.80/3.8/81/12, hypoxanthine 2.0/0.20/7.0/0.20, xanthine 2.0/0.40/34/1.2, adenosine 0.10/0.20/0.10/19, inosine 0.20/0.50/9.4/3.4, guanosine 0.10/0.30/10/30, uridine 0.40/0.40/27/4.0. Salivary deoxyuridine (13μM), deoxyinosine (10μM), deoxyguanosine (7μM), and thymidine (18μM) were unusually high in horses. Saliva from cats, goats, sheep and camel were also assayed. Discussion: Salivary concentrations of purine nucleotide precursors such as hypoxanthine, xanthine, adenosine, inosine and guanosine were surprisingly high in neonates and higher than in plasma. Transition from human neonate to adult levels occurred during the first year. These precursors in saliva may be useful biomarkers for diagnosis of inborn errors of nucleotide metabolism and the investigation of some pharmacogenetic disorders.


POSTERS MONDAY



DYNAMIC REMODELLING OF THE LYMPHATIC VASCULATURE DURING POSTNATAL MOUSE MAMMARY GLAND MORPHOGENESIS IS MEDIATED VIA EPITHELIAL-DERIVED LYMPHANGIOGENIC STIMULIBetterman K.L.1, 2, Paquet-Fifield S.3, Asselin-Labat M.-L.4, Visvader J.E.4, 5, Butler L.M.2, 6, Stacker S.A.3, Achen M.G.3 and Harvey N.L.1, 2 1Division of Haematology, Centre for Cancer Biology, SA Pathology, Adelaide, Australia. 2Discipline of Medicine, University of Adelaide, Adelaide, Australia. 3Peter MacCallum Cancer Centre, East Melbourne, Victoria, Australia. 4The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia. 5The University of Melbourne, Parkville, Victoria, Australia. 6Dame Roma Mitchell Cancer Research Laboratories, Hanson Institute, SA Pathology, Adelaide, Australia. Lymphatic vessels are of fundamental importance to breast cancer patients. Lymphatic vessels not only provide a key route of transport for the metastasis of breast cancer cells, but their damage during axillary lymph node resection results in secondary lymphoedema. Mammary tissue undergoes major, dynamic remodelling in response to hormonal signals during puberty, pregnancy, lactation and the oestrous cycle. The morphogenesis of mammary gland epithelial and stromal components is accompanied by dynamic growth and regression of the blood vasculature. While blood vascular remodelling during mammary gland morphogenesis has been well documented, little is known about the growth and development of lymphatic vessels in this tissue. We have developed a three dimensional imaging approach to investigate lymphatic vessel growth and patterning during mouse mammary gland morphogenesis and reveal that lymphatic vessels in the postnatal mouse mammary gland share an intimate spatial association with epithelial ducts and large blood vessels. Moreover, we demonstrate that the lymphatic vasculature is dynamically remodelled during mammary gland morphogenesis; growth of the lymphatic vessel network accompanies expansion of the mammary epithelial tree during pregnancy and is followed by regression during involution. We demonstrate that epithelial cells, in particular myoepithelial cells, are a rich source of pro-lymphangiogenic stimuli including VEGF-C, VEGF-D and FGF2, and that expression of these growth factors is temporally regulated during mammary gland morphogenesis. Our work sheds new light on the mechanisms by which the lymphatic vasculature is patterned during development, reveals that the mammary epithelial tree provides a key source of growth factors driving lymphangiogenesis during mammary gland morphogenesis and provides a new explanation for the propensity of metastatic breast tumour cells to gain access to the lymphatic vasculature.

CELL SYSTEMS FOR THE STUDY OF ENDOGENOUS LEUCINE-RICH REPEAT PROTEINS DURING DEVELOPMENT

Gaskin S.M. and Haines B.P. Discipline of Biochemistry, School of Molecular and Biomedical Science, The University of Adelaide, North Terrace, Adelaide, S. A. 5005, Australia.

Many families of single-pass leucine-rich repeat (LRR) transmembrane proteins exist in the mammalian genome. These proteins contain varying numbers of extracellular leucine-rich repeats, a transmembrane domain, and a short cytoplasmic tail, and also may contain extracellular fibronectin and/or immunoglobulin-like domains adjacent to the membrane. Many of these genes show highly specific developmentally regulated expression, including the Flrt and Nlrr families, which are expressed during neurogenesis and somitogenesis. The Flrt proteins can perform multiple functions including regulation of fibroblast growth factor (Fgf) signalling, cell migration and synaptogenesis. Most functional experiments on Flrts have been carried out using over-expression systems or global knock-out, resulting in complex phenotypes. Therefore, there is a need for cell systems to study endogenous Flrt and Nlrr proteins. P19 embryonic carcinoma (P19 EC) cells induce robust endogenous Flrt3 expression when differentiated into neural precursors using retinoic acid. Flrt3 also shows strong induction of expression with RA differentiated embryonic stem cells. The C2C12 muscle satellite cell line results in the up-regulation of Nlrr1 upon differentiation in a similar manner to its in vivo expression. We will utilize these cell model systems to study the regulation of Flrt3 and Nlrr1 expression and function.

A TASMANIAN DEVIL GROWTH FACTOR SUPPORTS PLURIPOTENT STEM CELLS IN VITRO

Maclennan I.O.A., Reiger J., Chan C.-K. and Hart A.H. Department of Genetics, La Trobe Institute of Molecular Sciences, La Trobe University, Bundoora, Victoria 3086, Australia.

The Tasmanian Devil (Sarcophilus harrisii) is a highly endangered Australian dasyurid marsupial. There has been an approximate 90% decline in the wild population over the last 15 years, mainly due to the highly contagious devil facial tumor disease. Hence preservation of this iconic marsupial has become a high priority. Our ultimate aim is to produce pluripotent stem cells from the Tasmanian devil, both for species preservation and with the goal of engineering resistance to the devil facial tumor disease. With this in mind, we have utilized the recently completed Tasmanian Devil genome sequence to generate and test the bioactivity of recombinant Tasmanian devil growth factors capable of sustaining pluripotent stem cells in cell culture. Here we describe the identification, cloning, recombinant protein production, and bioactivity of a Tasmanian Devil growth factor with a potent anti-differentiation activity on murine embryonic stem cells.

REGULATION OF DROSOPHILA SPERMATOGENESIS VIA ENDOCRINE SIGNALLING

Dominado N., Siddall N.A., Chan F.L. and Hime G.R. Department of Anatomy and Neuroscience, University of Melbourne, Parkville VIC 3010, Australia.

Gametes are produced throughout the adult life of Drosophila and as a consequence the adult Drosophila testis contains germ cells at different stages of development. Different signals are shared between germ cells and somatic support cells at different stages of spermatogenesis in order to facilitate the production of functional spermatozoa from germ line stem cells. The ordered differentiation of cells observed in the Drosophila testis makes it an excellent system for identifying signals that are utilized during germ cell differentiation. A number of signalling pathways, including EGFR, BMP, JAK-STAT, have been shown to regulate differentiation of germline stem cells and spermatogonia. For many years it has been known that tissue remodelling during development is dependent upon signalling via ecdysone, a steroid hormone, that interacts with a heterodimeric nuclear receptor complex comprised of the Ecdysone Receptor (EcR) and an ortholog of the Retinoid X Receptor called Ultraspiracle (USP). We have shown that different isoforms of EcR show specific expression patterns in maturing somatic and germ cells of the testis. Knockdown of EcR function using an inducible dominant-negative transgenic model has demonstrated that EcR signalling regulates exit from spermatogonial mitoses and meiotic cytokinesis.


POSTERS MONDAY



USP9X IS A NOVEL INTELLECTUAL DISABILITY GENE THAT REGULATES NEURONAL MIGRATION

Homan C.1, Wood S.2, Gecz J.1, 3 and Jolly L.1, 3 1University of Adelaide, SA, Australia, 5005. 2Griffith University, Qld, Australia, 4111. 3SA Pathology, SA, Australia, 5006.

USP9X encodes a substrate-specific deubiquitylating enzyme, known to regulate neural progenitor cells of the developing brain. We identified 3 unique changes in USP9X in three families with intellectual disability (ID) that we predicted to be deleterious. To test their functional significance and to identify potential pathological mechanism(s) we utilised a Usp9x knock-out mouse model. Loss of Usp9x resulted in a significant size reduction of the hippocampal formation and neurite defects in the cortex. Because these structures function in learning and memory, we investigated their respective neuronal cell types further in vitro. In the absence of Usp9x, isolated hippocampal neurons displayed reduction in primary axonal growth as well as neurite arborisation. To identify critically deregulated Usp9x substrates during neuronal growth we preformed 2D-DIGE followed by mass spectrometry on isolated cortical neurons. 23 unique proteins were identified. Consistent with being potential Usp9x substrates, all displayed features targeting them for ubiquitylation, and 22/23 were down-regulated in the absence of Usp9x. Functional annotation of these genes revealed pathways relevant to neuronal development and neuronal migration in particular. Subsequently a 43% reduction of in-vitro neuronal migration was identified in Usp9x null neurons. The three unique USP9X variants all alter the C-terminus of the protein, which is known to interact with the regulator of neuronal migration, and epilepsy-associated protein DCX. We tested whether these variants also altered neuronal migration in vitro. When overexpressed in Usp9x null neurons, 2/3 USP9X variants were unable to rescue the migration defect. Together our data reveal a new role of USP9X in neuronal development as well as identifies USP9X as a novel intellectual disability gene.

INVESTIGATING SEMAPHORIN 3A MEDIATED NEURAL CREST CELL MIGRATION USING EX VIVO AND CELL CULTURE MODELS

Kabbara S., Scherer M.A. and Schwarz Q.P. Center for Cancer Biology, SA Pathology Adelaide.

Neural crest cells are a transient population of stem cells that form at the dorsal ridge of the neural tube. Following delamination from the neuroepithelium neural crest cells migrate under the tight regulation of several guidance cues to ensure correct positioning of the developing sympathetic and sensory nervous systems. We have recently shown that a member of the class 3 semaphorin family, semaphorin 3A (Sema3A), is expressed in distinct zones within the sclerotome and that it controls migration of neuropilin-1 (Nrp-1) expressing neural crest cells. Thus, in Nrp1 or Sema3A mutant embryos the sympathetic and sensory nervous systems are disrupted as a result of neural crest migration defects. To mediate downstream signalling pathways Nrp1 binds obligate co-receptors such as A-type plexins and Vascular Endothelial Growth Factor receptors. However, the co-receptors recruited by Nrp1 in neural crest cells are currently unknown. We are employing several ex vivo models to determine the co-receptors and downstream signalling pathways controlling Sema3A neural crest cell guidance. For these studies we have purified large quantities of a biologically active form of Sema3A (fused to secreted alkaline phosphatase) to conduct functional assays in primary neural crest cells and cell lines. We have found that many traditional migration assays are unable to provide robust and repeatable outcomes with primary cells and have now modified the scratch assay and spot assays to dissect the molecular mechanism of cell signalling and functional outcomes for the cell.

CELL WALL INVERTASE POSITIVELY REGULATES POLLEN GERMINATION AND ELONGATION AND CONFERS HEAT TOLERANCE

Astija S., Wang L., Liu Y., Offler C.E., Patrick J.P. and Ruan Y.-L. School of Environmental and Life Sciences, University of Newcastle, NSW 2308, Australia.

Successful pollination is essential for fruit- and seed-set, hence realization of yield potential in most crop species (1). Pollination is highly vulnerable to abiotic stresses such as heat that often lead to fruit- and seed-abortion and hence yield loss (1). Sucrose metabolism is central to pollen maturation but less is known about its possible role in pollen germination and elongation. Here we show that suppressing cell wall invertase (CWIN) in transgenic tomato (2) reduced pollen germination and elongation in comparison with wild-type plants in medium containing sucrose as the sole carbon source. Importantly, enhancing CWIN activity by silencing its inhibitor (3) increased pollen germination and elongation under both optimal and heat stress conditions on sucrose medium. Interestingly, the pollen phenotype observed in the two sets of transgenic plants was absent in medium containing glucose or fructose or both. The data demonstrate an elevated CWIN activity confers heat tolerance to the processes of pollen germination and subsequent pollen tube elongation. The observations also suggest that CWIN regulates pollen germination and pollen tube elongation mainly through sugar signaling rather than metabolism. References (1) Ruan et al (2012) Trends in Plant Sciences (in press). (2) Zanor et al (2009) Plant Physiol. 150, 1204-1218. (3) Jin et al (2009) Plant Cell 21, 2072–2089.

DOES THE GROWTH RESPONSE OF WOODY PLANTS TO ELEVATED CO2 INCREASE WITH GROWTH TEMPERATURE? A META-ANALYSIS

Baig S. and Medlyn B.E. Department of Biological Sciences, Macquarie University, North Ryde, 2109 NSW, Australia.

Trees respond to elevated carbon dioxide (eCO2) because CO2 and O2 compete for Rubisco, the primary enzyme for photosynthesis. eCO2 levels in atmosphere favour the carboxylation reaction, whereas high temperatures alone favour oxygenation reaction which leads to photorespiration. Vegetation models therefore predict that eCO2 responses of photosynthesis and Net Primary Productivity (NPP) should be higher at higher temperature. In particular, a stronger response is predicted for warm tropical forests as compared to boreal forests. To test this hypothesis we carried out two Meta-analyses. Firstly we tested for an interactive effect in factorial eCO2 X temperature experiments. This analysis revealed correlation of 0.09 (equivalent to 9%), confirming a small but significant interaction between eCO2 and temperature. The second meta-analysis examined 48 eCO2 experiments on woody plants across the globe to test for a gradient response to Mean Annual Temperature (MAT). The second meta-analysis showed a positive correlation between the log response ratio and MAT. A comparison between experimental NPP data relative to model predictions shows strong consistency, implying that a higher response should be expected at tropics as compared to boreal systems. It is therefore important to incorporate a CO2 X temperature interaction effect in models that predict forest productivity under climate change.


POSTERS MONDAY



LEAF INTERNAL CONDUCTANCE TO CO2 DIFFUSION IN C4 PLANTSBarbour M.M.1, Evans J.R.2, Simonin K.A.1 and Von Caemmerer S.2 1Environmental Science Department, Faculty of Agriculture and Environment, The University of Sydney, Private Bag 4011 Narellan, NSW 2567. 22. Research School of Biology, The Australian National University, Acton, ACT 0200.

Photosynthetic rate is limited by the rate of CO2 diffusion inside leaves of both C3 and C4 species. A number of techniques have been developed to quantify internal conductance to CO2 (gm) in C3 plants, including combined measurement of leaf gas exchange and carbon isotope discrimination (Δ13C), and combined measurement of gas exchange and chlorophyll fluorescence. Unfortunately these techniques are not possible for plants with C4 photosynthetic pathway, so gm has rarely been measured in C4 plants. We have developed a novel, on-line, real-time measurement system for gm in C4 plants by coupling traditional gas exchange with laser absorption spectrometers capable of measuring the stable carbon and oxygen isotope composition of CO2, and the stable oxygen isotope composition of transpired water vapour. Photosynthetic oxygen isotope discrimination (Δ18O) relates to equilibration between CO2 and leaf water, catalysed by carbonic anhydrase (CA). In C3 plants isoforms of CA are located in the chloroplast, the cytosol, the mitochondria and the plasma membrane. The high concentrations and location of CA in the plasma membrane of C3 plants suggests that Δ18O-derived estimates of gm in C3 plants should be higher than Δ13C-derived estimates. Our measurements on tobacco demonstrate this suggestion; Δ13C-gm was 0.62 mol m-2 s-1 bar-1 while Δ18O-derived gm was 1.97 mol m-2 s-1 bar-1 at high light and 400 ppm CO2. In C4 plants CA is located in the cytosol of the mesophyll cells, where it catalyses the first step of C4 photosynthesis. Our Δ18O-derived estimates of gm in the two C4 plants Setaria and Flaveria were 0.69 mol m-2 s-1 bar-1 and 1.12 mol m-2 s-1 bar-1. This suggests that gm and/or CA activity limit photosynthesis in Setaria (the CO2 concentration dropped from 142 ppm to 77 ppm between the intercellular spaces and the mesophyll cells) but gm and CA are less limiting in Flaveria (the CO2 concentration dropped from 192 ppm to 157 ppm between the intercellular spaces and the mesophyll cells).

A SEED BASED ASSAY FOR MEASURING MEIOTIC RECOMBINATION IN ARABIDOPSIS

Barrell P.J.1, Gatehouse M.L.1 and Conner A.J.2 1New Zealand Institute for Plant & Food Research Ltd, Private Bag 4704, Christchurch, New Zealand. 2AgResearch Ltd, Grasslands Research Centre, Private Bag 11008, Palmerston North, New Zealand.

Recombination during meiosis produces new combinations of DNA sequences in resulting gametes. A key requirement for analysis of genetic recombination occurring during meiosis is a convenient test system to measure changes in the frequency of genetic recombination. We use a seed-based assay reporter system for recombination in Arabidopsis thaliana that utilises green fluorescent protein (GFP) and red fluorescent protein (RFP) genes expressed under the seed-specific napin promoter from Brassica napus. This recombination ‘test-bed’ exists as homozygous lines with GFP and RFP markers linked on different chromosome arms at known genome locations in the A. thaliana ‘Columbia’ genetic background. Most importantly the same phenotypic markers can be used to assay recombination at different genome regions by simply using different tester lines with the markers on different chromosomes. The scoring of segregating progeny produced on heterozygous plants allows the frequency of meiotic recombination to be conveniently estimated soon after embryo formation. The ratio of RFP, GFP, or non-fluorescent seeds is distorted by linkage and the frequency of recombination between the loci. The frequency of recombination (r) can be determined by simply counting the proportion of only RFP and GFP seeds. We use an LED-based microscope to independently examine the RFP and GFP expression in seeds. This allows the automated examination of several hundred seed in a short time. Different ecotypes of Arabidopsis have been crossed into the test system and are being analysed for meiotic recombination frequencies.

MIXED-LINKAGE GLUCANS IN SORGHUM

Betts N.S., Byrt C.S., Nguyen H., Ermawar R., Collins H.M., Fincher G.B. and Burton R.A. ARC Centre of Excellence in Plant Cell Walls, Waite Campus, University of Adelaide, Urrbrae, SA 5064.

Within the Sorghum genus, there is great diversity in biomass production and carbon allocation. High biomass and high sugar-yielding sorghum germplasm is a valuable source of carbon for use in biofuel production (Byrt et al., 2011 J. Integrative Plant Biology 53:120). Cell wall composition is an important factor influencing the yield of bioethanol that may be generated from a given quantity of biomass. One polysaccharide of interest as a source of fermentable hexose sugar is (1,3;1,4)-β-D-glucan or mixed linkage β-glucan (MLG) (Burton et al., 2011 Plant Biotechnology J. 9:117). We have investigated the amount, properties, distribution and biosynthesis of MLG in diverse sorghum lines and observe that these traits vary significantly depending on the genotype of the material.

GLUCAN, WATER DIKINASE DOWN REGULATION IN WHEAT ENDOSPERM ALTERS GERMINATION CHARACTERISTICS

Bowerman A.F.1, 2, Howitt C.A.1, Gubler F.3, Pogson B.4, Morell M.K.1 and Ral J.-P.1 1CSIRO Food Futures Flagship, Canberra, Australia. 2Research School of Biology, The Australian National University, Canberra, Australia. 3CSIRO Plant Industry, Canberra, Australia. 4ARC Centre of Excellence in Plant Energy Biology, Research School of Biology, The Australian National University, Canberra, Australia.

Glucan, Water Dikinase (GWD) is an essential kinase responsible for phosphorylating the C6 carbon of starch α-D-Glucan residues. This phosphorylation is important in determining the functionality of starch, and its susceptibility to degradation. Reduction of GWD specifically in the wheat grain has previously been achieved using an RNAi construct driven by a seed specific promoter. This reduction leads to pleiotropic modification of plant growth and grain development in the subsequent generation, with a notable increase of α-amylase expression and activity in mature grain. Here we described the effect of this down-regulation during germination. We find that in contrast to the situation during grain development, a decrease in α-amylase expression and activity is observed approximately 48 hours post imbibition. Germination is slightly delayed in these lines, and exhibits sensitivity to exogenous sugars at imbibition. Alteration of the expression of other genes is demonstrated during germination, suggesting a modification of sugar sensing mechanisms. Further work is necessary to further determine the nature and mechanism of this effect of GWD down-regulation in developing seed on germination.


POSTERS MONDAY



IS PLASTOQUINONE INVOLVED IN THE RED-LIGHT RESPONSE OF STOMATAL REGULATION?

Busch F.A. and Von Caemmerer S. Research School of Biology, Australian National University, Canberra, Australia.

Stomata regulate the uptake of CO2 and the loss of water in leaves of higher plants. Plants that maximize their CO2 uptake by opening the stomata are faced with an increased rate of transpiration, which is problematic especially under conditions of water limitation. Therefore, stomatal movements are tightly regulated to maximize the plant’s water use efficiency. The guard cells, controlling stomatal opening, respond to a number of different environmental stimuli, such as water status and CO2 concentration. The response of stomata to light follows two independent pathways: Blue light is sensed by the photoreceptor phototropin. Red light also stimulates stomatal opening, but the underlying sensing mechanisms are not know. Experimental evidence suggests contrasting conclusions, such as the existence of an independent red-light receptor, to it being an indirect effect of photosynthesis via a decrease of the intercellular CO2 concentration. Here we examined the hypothesis that red light is sensed by the redox state of plastoquinone, a molecule becoming more reduced at higher light intensities. We used a combination of chlorophyll fluorescence and gas exchange techniques in combination with photosynthetic electron transport inhibitors to test the involvement of plastoquinone in the stomatal red-light response of Arabidopsis and tobacco. Our results show that this response is not an indirect effect and indeed independent of photosynthesis. The redox state of plastoquinone strongly correlates with stomatal conductance independent of experimental condition, which makes plastoquinone a possible candidate as the sensing element for the stomatal red-light response.

AGAVE BIOMASS COMPOSITION

Byrt C.S.1, Karem G.1, Betts N.S.1, Fincher G.B.1, Beck-Oldach C.1, Chambers D.2 and Burton R.A.1 1ARC Centre of Excellence in Plant Cell Walls, Waite Campus, University of Adelaide, Urrbrae, SA 5064. 2AusAgave, PO Box 32, Aldgate, SA 5154.

We are interested in hardy crops that can generate high yields of sugar and biomass on marginal lands with minimal requirement for water and fertilizer. Agave is efficient at using water, since it employs the crassulacean acid metabolism (CAM) photosynthetic adaptation where carbon dioxide is fixed at night. Agave also has the potential to generate lots of sugar in semi-arid environments (Davis et al., 2011); however, little is known of the composition of the biomass. We have investigated cell wall composition in the leaves of the two agave varieties A. americana and A. tequilana. Traits of interest include the crystalline cellulose and lignin content. We also examined anatomical features of agave leaves and visualised the deposition of a number of cell wall polysaccharides with fluorescence and transmission electron microscopy using specific antibodies. Davis, SC, Dohleman FG and Long SP (2011) The global potential for Agave as a biofuel feedstock. GBC Bioenergy 3:68-78.

ROLE OF BETALAINS IN VEGETATIVE PLANT TISSUES

Calcott K.E.1, 2, Gould K.S.2, Lewis D.H.1, Davies K.M.1 and Schwinn K.E.1 1Plant and Food Research, Private Bag 11 600, Palmerston North 4442, New Zealand. 2Victoria University of Wellington, PO Box 600, Wellington, New Zealand.

Betalain and anthocyanin pigments appear very similar, yet no plant has been found to contain both pigment types. Due to this, it thought that betalain pigments functionally replace anthocyanins in many Caryophyllales. However, there has been minimal research conducted to support this replacement theory, resulting in the function of betalains in vegetative plant tissues being somewhat overlooked. Anthocyanins have been shown to mitigate stress exacerbated by abiotic factors such as high light, low temperature, and salinity. The induction of betalain pigment production has been associated with many of the same abiotic stressors. This project is investigating whether betalains ameliorate the effects of environmental stressors in the same way as anthocyanins. As a first step, we determined where betalains occur within ten different species through both sectioning and pigment analyses. Histological analyses revealed similarities between species in the tissue- and cell-specific localisation of pigment. HPLC and in situ absorbance spectra, however, found uneven distribution of betalanic compounds between and within organs on the same plant. This tissue specific betalain pigmentation may be indicative of essential functions within betalanic plants. We will also be directly comparing anthocyanin and betalain function in vegetative tissues, using transgenic Arabidopsis thaliana lines capable of producing anthocyanins, betalains or both. These plants will be exposed to the stressors of excess light, ultraviolet radiation, and chilling. The effect of these stressors on plant growth and survival will be monitored. Results from these experiments will hopefully shed some light on why some plants produce one pigment type and not the other.

TRANSGENIC HULLESS BARLEY OVER EXPRESSING THE CELLULOSE SYNTHASE-LIKE GENE (HVCSLF6) SHOWS AN INCREASE IN (1,3;1,4)-β-D-GLUCAN LEVELS AND MODIFIED GRAIN MORPHOLOGY

Collins H.M.1, 2, Kibble N.A.J.1, 2, Singh R.R.1, 2, Burton R.A.1, 2 and Fincher G.B.1, 2 1CSIRO Food Futures Flagship, High Fibre Grains Cluster. 2ARC Centre of Excellence in Plant Cell Walls, School of Agriculture, Food and Wine, University of Adelaide, Waite Campus, Glen Osmond, South Australia, 5064, Australia.

The (1,3;1,4)-β-D-glucans are the major cell wall polysaccharide in the starchy endosperm of barley. They have been associated with numerous health benefits, including the lowering of plasma cholesterol and a reduction in the risk of cardiovascular disease and colorectal cancer. It is now recognised that the cellulose synthase-like (Csl) gene family is involved in the biosynthesis of β-glucan in cereals, particularly the HvCslF and HvCslH genes. Recently a study showed that by over expressing the HvCslF6 gene in transgenic barley one could achieve an increase of up to 80% in (1,3;1,4)-β-D-glucan levels and an increase of up to 50% in dietary fibre content. In this study the covered barley variety Golden Promise was used, but this variety is poorly adapted to growing conditions in Australia and is not suitable to be used as food barley. In the future if barley with improved health benefits is to be released for public consumption it will need to be in a suitable background. For this reason transformation techniques have been developed for use with the hulless barley variety Torrens and a number of hulless barley lines that have been produced are over expressing the HvCslF6 gene. As expected these lines are showing an increase in grain (1,3;1,4)-β-D-glucan content. They are also showing a modified morphology with reduced grain size and a shrunken appearance.


POSTERS MONDAY



FIELD TO FUEL: CONVERSION OF LIGNOCELLULOSIC BIOMASS TO BIOETHANOL

Corbin K.R.1, Byrt C.S.1, Betts N.S.1, Fincher G.B.1, Grbin P.R.2, Jiranek V.2, Liccioli T.2 and Burton R.A.1 1ARC Centre of Excellence in Plant Cell Walls, Waite Campus, University of Adelaide, Urrbrae, SA 5064. 2School of Agriculture, Food and Wine, The University of Adelaide, PMB1, Glen Osmond, SA 5064, Australia

Lignocellulosic biomass is likely to be a major source of the carbon needed to synthesize future chemical fuels. Biomass consists predominantly of a heterogeneous matrix of polysaccharides, which are not readily available for bioconversion due to the recalcitrant nature of the cell wall (Burton et al, 2010). Existing methods for processing biomass to increase the availability of cell wall polysaccharides are summarized here. Pre-treatment techniques alter the biomass by using physical, chemical (such as dilute sulphuric acid) and/or thermal means. Next, an enzymatic cocktail is used to increase the liberated monosaccharides for fermentation. Sugar composition is dependent not only on the biomass used, but also on the methods in which it was conditioned. The final fermentation step converts the monosaccharides into ethanol. The feedstocks we are working with include monocot and dicot plants encompassing great diversity in cell wall composition. We are testing and optimizing pre-treatment and fermentation methods that are applicable to our biomass feedstocks of interest at an industrial scale. Fermenting microorganisms are being investigated that are able to ferment both hexose and pentose sugars liberated from the feedstock. Burton, R.A., Gidley, M.J. and Fincher, G.B. (2010) Heterogeneity in the chemistry, structure and function of plant cell walls. Nature Chemical Biology , 6, 724-732.

CEREAL (1,3;1,4)-β-D-GLUCANS: A STUDY INTO THE MECHANISM OF BIOSYNTHESIS AND CSLF6 FUNCTION

Dimitroff G.1, 1, Burton R.A.1, Little A.1 and Fincher G.B.1 1ARC Centre of Excellence in Plant Cell Walls, University of Adelaide, Waite Campus, Urrbrae SA 5064, Australia. 2Grains Research and Development Corporation, 40 Blackall Street, Barton ACT 2600, Australia.

The non-cellulosic polysaccharide constituents of plant cell walls provide numerous attributes that effect wall structure and development as well as human health and industry. (1,3;1,4)-β-D-glucan is a non-cellulosic polysaccharide that accumulates in the cell walls of cereals such as barley (Hordeum vulgare (Hv)), rice (Oryza sativa) and sugar cane (Saccharum officinarum). The CELLULOSE SYNTHASE LIKE F (CslF) gene family has been implicated in (1,3;1,4)-β-D-glucan biosynthesis, and in several cereals, the CSLF6 gene has been identified as the most influential family member. The exact biochemical process by which (1,3;1,4)-β-D-glucan is synthesised remains unknown, and in the absence of a crystal structure and proteomic analyses, it remains unclear as to how CSLF6 interacts with other proteins or how it may bind substrates and contribute to (1,3;1,4)-β-D-glucan biosynthesis. To address this, we are assessing the role of different CSLF6 structural domains and their role in (1,3;1,4)-β-D-glucan biosynthesis. SbCSLF6 (Sorghum bicolor) and HvCSLF6, two enzymes that produce differing amounts and structures of (1,3;1,4)-β-D-glucan in Nicotiana benthamiana, will be analysed by domain swapping and transient expression. By analysing the resultant (1,3;1,4)-β-D-glucan, this method should permit the identification of ‘domains’ that influence content and fine structure.

DOUBLE-STRANDED RNA BINDING PROTEINS DRB2, DRB3 AND DRB5 FUNCTION IN A TISSUE-SPECIFIC, NON-CANONICAL MICRORNA PATHWAY IN ARABIDOPSIS

Eamens A.L.1 and Waterhouse P.M.1, 2 1University of Sydney. 2CSIRO Plant Industry.

The Arabidopsis thaliana (Arabidopsis) double-stranded RNA (dsRNA) binding (DRB) protein family consists of five members, DRB1 to DRB5. MicroRNAs (miRNAs) are an endogenous class of small RNA that regulate the expression of developmentally important genes. In the plant cell nucleus, DRB1 is required by DICER-LIKE1 (DCL1) for efficient processing of miRNA dsRNA precursor transcripts transcribed from miRNA genes. However, we recently demonstrated that DRB2, also a nuclear protein, is required for the production of specific miRNA subsets in specific tissues, namely the shoot apical meristem region. Through the use of artificial miRNA (amiRNA) vectors expressed in different DRB loss-of-function plant lines (drb mutant plants), we report here that DRB2-associated miRNAs also require the activity of DRB3 and DRB5, two cytoplasmic proteins, for efficient RNA silencing of their target genes. Taken together, our results indicate that in Arabidopsis DRB2, DRB3 and DRB5 function in a tissue-specific, non-canonical miRNA pathway.

ROLE OF ENDOSOMAL SODIUM-PROTON ANTIPORTERS IN SEED PROTEIN PROCESSING

Ernest J.R. and Gendall A.R. LaTrobe University.

Transmembrane sodium-proton antiporters have been shown to be associated with diverse physiological processes such as cell expansion and differentiation, salt tolerance and vesicle trafficking. Two closely related Na+-H+ exchangers (NHX), NHX5 and NHX6, act redundantly to regulate cellular ion homeostasis and plant development in Arabidopsis. During maturation, the seeds of higher plants store long-term reserves of nitrogen in the form of storage proteins, which accumulate in large central vacuoles known as Protein Storage Vacuoles (PSVs). In order to stably and densely package nitrogen reserves, which may protect the viability of the seed over long periods of dormancy, the synthesis and post-translational modification of conserved seed storage proteins and formation of PSVs is highly specialised. The phenotype of the nhx5 nhx6 double mutant suggests that NHX5 and NHX6 are indirectly associated with regulation of seed viability, development and PSV formation. Here we describe a PSV and seed storage protein phenotype in nhx5 nhx6 double mutant embryos.


POSTERS MONDAY



DROUGHT INCREASES HEAT TOLERANCE OF LEAF RESPIRATION IN EUCALYPTUS GLOBULUS SAPLINGS GROWN UNDER AMBIENT AND ELEVATED ATMOSPHERIC [CO2] AND TEMPERATURE

Gauthier P.P.G.1, Crous K.Y.1, 2, Ayub G.1, Duan H.2, Ellsworth D.S.2, Tissue D.T.2, Tjoelker M.G.2, Evans J.R.1 and Atkin O.K.1 1Division of Plant Sciences, RSB, ANU. 2Hawkesbury Institute for the Environment, UWS.

We examined the effects of elevated atmospheric CO2, warming and drought treatments on the temperature response of leaf respiration. Seedlings of the fast growing Eucalyptus globulus were grown under 8 treatment combinations: ambient and +240ppm CO2, ambient temperature and warmed +3°C, with well watered or two cycles of water limitation. The response of leaf respiration to temperature from 15 to 65°C was measured at the end of the second drought period. Drought reduced stomatal conductance and the rate of CO2 assimilation, but respiration was not significantly affected. The ratio of respiration to CO2 assimilation increased under drought, irrespective of growth conditions. The temperature response of respiration was not affected by warming or CO2 enrichment. However, drought increased both the temperature at which respiration reached a maximum as well as the maximum rate.

A CELLULAR ULTRASTRUCTURAL VIEW OF MANGANESE STRESS RESPONSES IN TOLERANT AND SENSITIVE WHEAT VARIETIES

Fernando D.R.1, Moroni J.S.2, Lynch J.P.3 and Marshall A.T.4 1Department of Botany, La Trobe University, Bundoora, Vic 3085, Australia. 2School of Agricultural and Wine Sciences, Charles Sturt University, Wagga Wagga, NSW, 2678, Australia. 3Department of Horticulture, The Pennsylvania State University, University Park, PA 16802, USA. 4Analytical Microscopy Laboratory, La Trobe University, Bundoora, Vic 3085, Australia.

Plant manganese (Mn) toxicity in Australian agriculture has been documented in the scientific literature since the 1960s; however, local research into the problem has steadily declined over the last two decades. A majority of important food crops are grown in eastern Australia where Mn-rich acidic soils and/or certain climatic effects often render soil-Mn highly plant-available, and thus toxic to plants (phytotoxic). Past studies have identified this problem in a range of crop species, investigated sensitive and tolerant varieties, and also described environmental conditions that give rise to Mn phytotoxcity. The mediation of excess Mn in plants is generally highly variable interspecies compared to other mineral nutrients. This aspect of plant-Mn physiology remains poorly understood, as does the variability in Mn tolerance between certain crop cultivars. In the light of current knowledge about global climate change and recent understanding about Mn-toxicity induced oxidative stress, this study revisits past research linking ambient temperature to plant-Mn tolerance, and seeks to examine these induced responses at the cellular ultrastructural levels in both Mn-sensitive and –tolerant varieties of wheat ( Triticum aestivum ).

HOW DO ODD SOCS REGULATE FLOWERING TIME IN MEDICAGO TRUNCATULA?

Fudge J.B. and Macknight R.C. Department of Biochemsitry, University of Otago, PO Box 56, Dunedin, New Zealand.

In Arabidopsis, the floral transition takes place when the floral integrator genes, FLOWERING LOCUS T (FT, or ‘florigen’) and SUPPRESSOR OF OVEREXPRESSION OF CO 1 (SOC1) are turned on in leaves and the shoot apex, respectively. FT expression is induced in leaves in response to long days (LD) and vernalisation. FT protein is the mobile flowering signal, a florigen component, which moves to the shoot apex to activate SOC1 and thereby promote flowering. SOC1 is also activated by vernalisation and gibberellin in the absence of LD. Like Arabidopsis, the legume Medicago truncatula is a vernalisation-responsive, long-day plant. FTa1 has recently been shown to act as Medicago ‘florigen’, yet the downstream SOC1 targets remain unknown. In Medicago there are three SOC1 genes (SOC1a, b and c). 35S:MtSOC1a rescues the late-flowering soc1-2 Arabidopsis mutant, meanwhile 35S:MtSOC1b could partially complement yet 35S:MtSOC1c could not; however the extent appears dependent upon transgene expression levels. Vernalised WT Medicago plants grown in non-inductive short-day (SD) conditions show up-regulation of MtSOC1a and MtSOC1b when moved to just three LD then back to three SD, consistent with induction of FTa1 in leaves and nodes in LD, suggesting FTa1 may act as an activator of SOC1a and/or SOC1b expression to promote flowering. Current work includes transient expression assays with dual reporters to confirm trans-activation by FTa1 and the downstream targets of the Medicago SOC1 genes. Exogenous application of GA3 under SD conditions to WT and late-flowering fta1-17 mutants will determine if MtSOC1 induction is requisite for floral induction via the gibberellin pathway and whether this is independent of the FT module.

SIGNALLING IN PLANTS: LESS A CUE, BUT CLUELESS?

Gagliano M.1, 2 1Centre for Evolutionary Biology, University of Western Australia, Crawley, Australia. 2Centre for Microscopy, Characterisation and Analysis, University of Western Australia, Crawley Australia.

Current knowledge suggests that the mechanisms by which plants communicate information take numerous forms. Previous studies have focussed their attention on communication via chemicals, contact and light; other methods of interaction between plants have remained speculative. In this study we tested the ability of young chilli plants to sense their neighbours and identify their relatives using alternative mechanism(s) to recognised plant communication pathways. We found that the presence of a neighbouring plant had a significant influence on seed germination even when all known sources of communication signals were blocked. Furthermore, despite the signalling restriction, seedlings allocated energy to their stem and root systems differently depending on the identity of the neighbour. These results provide clear experimental evidence for the existence of communication channels between plants beyond those that have been recognized and studied thus far.


POSTERS MONDAY



CALIFORNIA POPPY COLOUR VARIANTS AS A MODEL SYSTEM TO SEARCH FOR CANDIDATE REGULATORS OF CAROTENOID ACCUMULATION

Gatehouse M.1, Conner A.2 and Barrell P.1 1New Zealand Institute for Plant & Food Research Limited, Private Bag 4704, Christchurch, New Zealand. 2Present address: AgResearch Ltd, Grasslands Research Centre, Private Bag 11008, Palmerston North 4442, New Zealand.

We are using California poppy (Eschscholzia californica Cham.) as a model system to search for candidate genes affecting carotenoid accumulation. Pigmentation by carotenoids is responsible for the intense yellow to orange colour of the petals and pollen of California poppy. An intense orange colour is typical; however flower colour variants ranging from white to yellow and orange also occur. The inheritance of petal and pollen colour in 24 variants with yellow and white-coloured flowers was investigated through complementation analysis, and these variants were resolved into five loci (b, s, f, i and x). Each of the mutations is recessive at the genetic level. Biochemical assays showed reduced carotenoid levels in petal tissue of four yellow s, f, i and x loci and a virtual absence of carotenoids in the petal tissue of the white (b locus) variant. The mutation causing the white phenotype is hypothesised to be a regulatory gene controlling the initiation of carotenoid biosynthesis. We employed a mapping strategy to search for candidate genes causing this phenotype. A mapping population was generated by crossing white variant b locus plants to wild-type orange-flowered plants. High throughout transcriptome sequencing was then carried out to search for candidate regulators of carotenoid accumulation. Candidate sequences will be assessed for their effect on carotenoid accumulation.

THE EFFECT OF COLD STRESS ON PROTEIN VARIATIONS AND TISSUE CULTURE OPTIMIZATION OF 3 TOMATO VARIETIES (SUPERQUEEN, NEMADOR AND UC-105)

Ghasempour H. and Hosseininia S. Department of Biotechnology and Chemical engineering, Science and Research branch, Islamic Azad University, Kermanshah, Iran.

Low temperature is one of the most important environmental factors that regulate plant growth and development and limit plant production. This stress causes significant economic losses to producers in scattered geographic regions. Many species that originated in tropical and subtropical regions are susceptible when temperatures fall below 15ºC. Tomato (Lycopersicon esculentum Mill.) is a classic example of a chilling-sensitive plant and after potato, the most frequently consumed vegetable in the world. In this study, the effect of cold stress on proteins variations and also optimization of the tissue culture of 3 tomato varieties (Superqueen, Nemador and UC-105) was investigated. In the first experiment, cold stress (4˚C), proteins variations in 7- and 21-days conditions was analyzed. The proteins variations were investigated by SDS-PAGE. In the second experiment, By using following growth regulators 0, 1 and 2 mg/l 2,4-D with 0 and 0.5mg/l Kin, callus induction was occurred from hypocotyl and root explants on MS media. Results of the first experiment indicated that cold stress decreased the amount of proteins and were destroyed some of the protein bindings in 3 varieties. Therefore, the results of first experiment indicates that stressed Superqueen variety shows less changes in amount of proteins of bands in SDS-PAGE compared to two other varieties. As a result, Superqueen is more tolerant than the other two varieties. Also, results of the second experiment indicated that, the highest callus induction was achieved at 1 mg/l 2, 4-D +0.5 mg/l Kin in hypocotyl and root explants. Keywords: Cold stress, Tomato, SDS-PAGE, Tissue culture.

HIGH-THROUGHPUT PHENOTYPING OF SORGHUM USING THE PLANT ACCELERATOR

Neilson E.1,5, Berger B.2, Blomstedt C.1, Edwards A.1, O’Donnell N.1, 3, Neale A.1, Hamill J.1, 4, Møller B.L.5 and Gleadow R.1 1Monash University. 2Australian Plant Phenomics Facility. 3Purdue University. 4The University of Melbourne. 5University of Copenhagen.

The high growth rate and water use efficiency of Sorghum bicolor make it an increasingly attractive option as a climate-change ready crop but it can be toxic to stock during droughts due to enhanced concentrations of the nitrogen-containing cyanogenic glucoside, dhurrin. Through EMS mutagenesis we have generated sorghum lines that are either totally cyanide deficient or become cyanide deficient as they transition to the adult phenotype. Our hypothesis was that changes in allocation of nitrogen to defence would improve nitrogen use efficiency, photosynthetic rate, growth and drought tolerance. To test this hypothesis, we used the automated imaging and watering system at The Plant Accelerator to compare the two acyanogenic lines with elite parental lines under well-watered and water-limited conditions. Trade-off between growth and defence appear to be vary with plant age and genotype. In a second experiment, we compared the growth and development of two commercial varieties grown at low (2 mM ) and high (10 mM) nitrogen. For both experiments, the non-destructive imaging during plant growth was complemented with destructive end-point analyses. Actual leaf area and plant size were highly correlated with values derived from the image analysis. ‘Greenness’ was found to be a good indicator of chlorophyll concentration. The usefulness of NIR imaging for estimating water status was investigated. We conclude that the Plant Accelerator is an excellent tool for rapid non-destructive phenotyping of plants and offers the potential to screen large mutant populations, accelerating the rate at which new plant varieties can be developed.

BIOMINERALISATION IN AUSTRALIAN DESERT ACACIA SPECIES: MUCH MORE TO EXPECT BEYOND CALCIUM OXALATEHe H.1, Kuo J.2, Bleby T.1, Veneklaas E.1 and Lambers H.1 1School of Plant Biology, The University of Western Australia. 2Centre for Microscopy, Characterisation & Analysis, The University of Western Australia.

Biomineralisation, the formation of minerals by living organisms, is a widespread phenomenon displayed by most forms of life on Earth, including plants. Calcium, is the predominant cation associated with biomineralisation for most organisms, and calcium-bearing minerals represent about 50% of known biominerals. In the plant kingdom, the most common type of crystals is calcium oxalate. Some proposed functions of biomineralisation in plants include bulk element (mainly calcium) regulation and detoxification of aluminium and heavy metals. By means of optical and scanning electron microscopy (SEM), together with energy-dispersive X-ray spectroscopy (EDS), crystals of various morphologies and elemental compositions were observed in phyllodes (modified petioles that function as leaves) of four Acacia species native to the Telfer region in the Great Sandy Desert in north-western Australia, i.e. Acacia stipuligera F. Muell., Acacia ancistrocarpa Maiden & Blakely, Acacia stellaticeps Kodela, Tindale & D. Keith and Acacia robeorum Maslin. In phyllodes of the four species grown in their natural habitat, in addition to calcium, other elements including magnesium, strontium, barium, and potassium were also precipitated. EDS revealed that these elements were precipitated together with carbon and sulfur, most likely in forms of oxalate and sulfate. In addition to the above-mentioned elements, other elements such as sodium, aluminium, iron, manganese, copper, titanium, and vanadium were sequestered in phyllodes of two species grown in proposed rehabilitation substrates collected from a mine site. The results proved that calcium oxalate was not the only form of biominerals formed in these species. It was found that elements precipitated differed between substrates, plant species, and tissues within an individual plant; the precipitation was also related to tissue age, indicating that, in most cases, biomineralisation is biologically induced for these species.


POSTERS MONDAY



IMPROVEMENT SALINITY TOLERANCE OF COMMERCIAL RICE CULTIVARS BY EXPRESSING SODIUM TRANSPORTER GENES IN SPECIFIC CELL TYPES

Hairmansis A.1, Berger B.2, Tester M.1, 2 and Roy S.1 1Australian Centre for Plant Functional Genomic and The School of Agriculture Food and Wine, University of Adelaide, PMB1 Glen Osmond, SA 5064. 2The Plant Accelerator, University of Adelaide, Glen Osmond, SA 5064.

Rice is the most susceptible cereal to saline environments. Improvement of salinity tolerance in rice is crucial to maintain sustainability of rice production in salt affected areas. The control of sodium transport by expressing sodium transporters in specific cell types has been shown to improve a plant’s ability to cope with salt stress by effectively reducing salt accumulation in the shoot. However, the work so far has been limited to either model plants or technologies incompatible with future field trials, such as enhancer trap systems. Therefore, we need to transfer and test this approach in commercially relevant cultivars of popular rice varieties, using native promoters to drive the desired expression pattern. Two elite Indonesian cultivars, Fatmawati and IR64, have been selected as genetic backgrounds to express sodium transporter genes in specific cell types. Cell specific promoters have been identified from a variety of resources and have been used to drive the expression of a range of sodium transporter genes. The alterations to sodium transport are targeted to minimize sodium influx into the root, maximise sodium retrieval from the xylem, and/or increase sodium compartmentalization in root tissues. Here, we report on the progress of the generation of salinity tolerant rice with cell type-specific expression of a variety of sodium transporter genes.

THE ROLE OF HD-ZIP I TRANSCRIPTION FACTORS IN THE DROUGHT RESPONSE OF WHEAT (T. AESTIVUM L)

Harris J.C.1, Eliby S.N.1, Sivasankar S.2, Tingey S.3, Lopato S.1 and Langridge P.1 1Australian Centre for Plant Functional Genomics, PMB 1, Glen Osmond, SA 5064, Australia. 2DuPont Ag Biotechnology, Johnston, IA 50131, USA. 3DuPont Ag Biotechnology, Wilmington, DE 19880, USA.

Adaptation of plant growth to abiotic stresses involves HD-Zip I TFs. Three closely related drought inducible HD-Zip I wheat genes have been isolated. Transgenic wheat has been produced for phenotyping and assessment under water deficit. Transient assays were used to investigate the mechanism of activation of promoters of two HD-Zip I genes and to assess activator/repressor properties of HD-Zip I TFs. Three drought inducible HD-Zip I genes possess different induction characteristics under drought, cold, salt and ABA. Their products interact in Y2H assays and two of them activate transcription of an artificial promoter through the HD-Zip I cis-element. Constitutive overexpression of the HD-Zip I genes negatively influences phenotypes of transgenic wheat and barley plants. When over-expressed under a drought inducible promoter they have no impact upon drought tolerance or yield under drought. Also, the three closely related HD-Zip I TFs appear to act in different stress response pathways.

BETALAIN PRODUCTION IN ANTHOCYANIN-PRODUCING PLANT SPECIES BY EXPRESSION OF DOPA-DIOXYGENASE AND L-DOPA FEEDING

Harris N.N.1, 2, Javellana J.2, Davies K.M.2, Lewis D.H.2, Jameson P.E.3, Deroles S.C.2, Calcott K.E.2, 4, Gould K.S.4 and Schwinn K.E.2 1Commonwealth Scientific and Industrial Research Organization, Ecosystem Sciences, Glen Osmond, South Australia 5064, Australia. 2New Zealand Institute for Plant & Food Research Limited, Private Bag 11600, Palmerston North, New Zealand. 3School of Biological Sciences, University of Canterbury, Private Bag 4800, Christchurch, New Zealand. 4Victoria University of Wellington, PO Box 600, Wellington 6140, New Zealand.

Betalains are a minor class of pigments that occur in certain families within the order Caryophyllales, from which the more common anthocyanin pigments are absent. This mutually exclusive nature of the betalain and anthocyanin biosynthetic pathways in the plant kingdom is a curious phenomenon and the evolutionary mechanism behind this is yet to be determined. The betalain biosynthetic pathway is relatively simple with putatively few reactions being enzyme catalaysed. While the anthocyanin biosynthetic pathway has been extensively studied and metabolic engineering carried out in various species, betalain biosynthesis is yet to be fully characterised. This study was aimed at identifying whether betalain production is possible in anthocyanin-producing species. The introduction of a single enzyme, dihydroxyphenylalanine (DOPA) dioxygenase (DOD), and feeding of DOD substrate (L-DOPA) was sufficient for the synthesis of significant levels of both types of betalains, the yellow/orange betaxanthins and the red/purple betaxyanins in various species. This work demonstrates that the introduction of just two enzymes, DOD and the as yet uncharacterised enzyme converting tyrosine to L-DOPA would be sufficient to engineer betalain production in anthocyanin-producing species. Furthermore, the stably transformed 35S:DOD Arabidopsis thaliana plants provide a valuable resource to study the physiological effects of having both betalain and anthocyanins in the same plant tissue.

GERMANIUM AS A CHEMICAL ANALOGUE FOR BORON IN BARLEY AND WHEAT

Hayes J.E.1, Pallotta M.1, Baumann U.1, Berger B.2, Langridge P.1 and Sutton T.1 1Australian Centre for Plant Functional Genomics, School of Agriculture, Food and Wine, University of Adelaide, Urrbrae, South Australia 5064, Australia. 2The Plant Accelerator, The University of Adelaide, Waite Campus, Urrbrae, South Australia 5064, Australia.

Germanium (Ge) is a metalloid element that is chemically similar to boron (B). In this study, we mapped leaf symptoms of Ge toxicity tolerance in a barley doubled haploid population (Clipper X Sahara 3771). Two QTL associated with Ge tolerance were identified, located on chromosomes 6H and 2H. These QTL co-located with two of four B toxicity tolerance loci previously mapped in the same population. The B toxicity tolerance gene underlying the 6H locus encodes HvNIP2;1, an aquaporin with permeability to Ge, silicon and arsenic, as well as to B. Targeted mutations in HvNIP2;1 coding sequence can reduce the permeability of this transporter to Ge. We propose that germanium may be useful for the study of particular aspects of B toxicity tolerance in plants, including targeted mutation analyses and screening wheat and barley populations for altered function of HvNIP2;1 and related aquaporins.


POSTERS MONDAY



COMPARISON OF THE BIOACTIVITIES OF PEEL AND FLESH EXTRACTS OF THREE MANGO VARIETIES

Hoang V.L.T.1, 2, Pierson J.-T.1, Curry M.C.1, Flanagan B.2, Dietzgen R.G.1, 2, Gidley M.J.2, Roberts-Thomson S.J.1, Shaw P.N.1 and Monteith G.R.1 1School of Pharmacy, The University of Queensland, QLD 4072. 2Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, Queensland 4072.

Mango (Mangifera indica L.) is a rich source of phytochemicals and has been the focus of recent studies aiming to identify potential benefits to human health. Improving the nutritional and health value of mango fruits is also an emerging area of mango breeding. This study aimed to compare the bioactivities of peel and flesh crude extracts of three different mango varieties - Kensington Pride, Irwin and Nam Doc Mai. Antioxidant activity was determined using the Folin-Ciocalteu method. The results showed significant differences in the total phenolic antioxidant content between peel and flesh extracts of each mango variety. Differences were also observed between mango varieties with the highest levels present in Nam Doc Mai. A specific Nam Doc Mai peel extract was associated with inhibition of MCF-7 breast cancer cell proliferation as assessed using an MTS assay, with an IC50 value of 56 μg/mL. Assessment of the effects of extracts on cell death indicated that some extracts affect the viability of MDA-MB-231 breast cancer cells. Nam Doc Mai peel extract significantly induced cell death at a concentration of 100 μg/mL. Collectively, our studies indicate that mango peel extracts appear to have more pronounced bioactivities than flesh extracts. Our studies also suggest that Nam Doc Mai may represent a mango variety with particularly high levels of bioactivity. Future studies will assess variation of secondary metabolite genes that may be responsible for the different bioactivities observed between mango varieties.

VARIETAL DIFFERENCES IN GRAPE CELL WALL MORPHOLOGY AND NUTRIENT ACCUMULATION

Hocking B.J.1, 2, 3, Burton R.1, 2, Tyerman S.D.1, 3 and Gilliham M.1, 3 1School of Agriculture, Food, and Wine, University of Adelaide, Glen Osmond, South Australia 5064, Australia. 2ARC COE Plant Cell Wall Biology. 3ARC COE Plant Energy Biology.

In grapes, cell wall properties are key determinants of grape and wine quality; cell wall failure prior to harvest is associated with increased pathogen susceptibility, berry splitting and berry shrivel (1, 2). Calcium nutrition impacts cell wall strength by cross-linking de-methylated pectins (1). This research aims to further understanding of cell wall strength and Ca2+ accumulation in grapes, and investigate the potential for calcium as a crop protection agent in wine grapes. Clear differences in skin cell morphology between red, white and table grape varieties were revealed by light microscopy. Varieties with high skin break energy (e.g. Grenache) generally have higher [Ca]skin than weak skin varieties (e.g. Thompson Seedless). Thompson Seedless exhibits the hardest grapes, highest [Ca]pulp and highest [Ca]apoplast, probably related to maintenance of post-veraison calcium in-flow through the xylem and a greater proportion of vital cells (2, 3). In varieties where cell wall degradation and loss of cell vitality was occurring, [Ca]apoplast proved difficult to estimate. These results demonstrate that differences between grape varieties in Ca2+ uptake, cell wall composition, pectin modification and cell vitality can be linked to physical and qualitative traits (2, 3, 4). Further studies to ascertain the effects of calcium application on bunch quality and cell wall properties including pectin modification are currently being undertaken. (1) Cabanne and Doneche. (2002) Microbiol. Res. 157: 183-189. (2) Fuentes et al. (2010) Aust. J. Grape Wine Res. 16: 327-336. (3) Tilbrook and Tyerman. (2009) Funct. Plant Biol. 36(6): 541-550. (4) Rogiers et al. (2006) Vitis. 45(3): 115-123.

A MOLECULAR TOOL FOR QUANTIFYING VIABLE RESTING SPORES OF SPONGOSPORA SUBTERRANEA - AN RT-qPCR ASSAY

Agarwal A., Wiechel T.J., Richardson F., Wardzynski M., Verstraten J., Auer D., De Boer R.F. and Edwards J. Department of Primary Industries, Biosciences Research Division, Knoxfield, Victoria 3180, Australia.

Powdery scab caused by the biotrophic pathogen Spongospora subterranea is a significant soilborne disease of potatoes. Disease symptoms of galls on roots and stolons and scab-like eruptions on the surface of tubers contain multicelled sporosori, conglomerations of 150-1500 dormant resting spores that can survive in the soil for up to 20 years. Under favourable conditions, each resting spore releases an infective uninucleate primary zoospore. Quantitative PCR (qPCR) of DNA used as a predictor of disease potential in field soils may overestimate inoculum, especially as resting spores in a sporosorus probably germinate progressively over many years. This study aims to develop a molecular tool using mRNA to quantify viable resting spores in S. subterranea sporosori. Primer and probe pairs were designed for four genes - actin, polyubiquitin, 18S rRNA and elongation factor-like (EFL) specific to S. subterranea. RNA extracted from 2009 and 1982 sporosori collections, from Plasmodiophora brassicae resting spores (a related pathogen) and from infected tomato roots (zoosporangial stage) was reverse transcribed into cDNA and a two-step real-time quantitative reverse transcriptase PCR (RT-qPCR) conducted. PCR analysis confirmed the expression of all four genes in the S. subterranea 2009 resting spores and in the infected roots. P. brassicae spores was not detected using any of the primer-probe combinations designed in this research confirming the specificity of the primer and probe pairs to S. subterranea. Detection and quantification of 18S rRNA and EFL genes will be used in further studies to quantify viable resting spores of S. subterranea.

A STRUCTURAL AND FUNCTIONAL INVESTIGATION INTO THE HOP EFFECTOR PROTEINS FROM PSEUDOMONAS SYRINGAE

Tan K.S., Gabriel K. and Clements C.S. Dept. of Biochemistry & Molecular Biology, Monash University, Clayton, 3800.

Pseudomonas syringae is one of the most destructive agricultural pathogens, infecting over 80 plant species worldwide, including many of economic importance. There are nearly 50 identified pathovars of P. syringae, each with a narrow host range. One of the most studied pathovars is P. syringae pv. tomato, the causative agent of bacterial speck in tomato. It is a common model system for three reasons: it is easily cultured and genetically manipulated; its host, tomato, is similarly amenable to genetic manipulation; and it is also pathogenic on the model plant Arabidopsis thaliana. P. syringae pv. tomato uses a type III secretion system to translocate approximately 30 different effectors into the host. These effectors are designated Hop (Hrp outer protein) or Avr (avirulence). Recently two effectors, HopAA1-1 and HopG1, have been shown to target the mitochondria of host plants. A further five effectors, HopAD1, HopAM1, HopE1, HopF2 and HopX1 may also target the mitochondria. We are undertaking a structural and functional study of these effectors using three approaches: 1. Determination of the subcellular localisation of the Hop effectors; 2. Identification of the compartment to which mitochondrial-localised effectors are targeted; 3. Characterisation of the structures of the Hop effectors using X-ray crystallography. Together this information will allow us to determine the mechanism of action of these Hop effectors. This will provide a greater understanding of the pathogenesis of P. syringae, and ultimately lead to strategies that may be used to control disease outbreaks.


POSTERS MONDAY



SECONDARY METABOLISM IN STAGONOSPORA NODORUM: A METABOLOMIC APPROACH

Gonzalez M.J. and Solomon P.S. Research School of Biology, Australian National University.

Stagonospora nodorum is the causal agent of Stagonospora nodorum blotch, a wheat disease associated with significant yield losses in Australia. Fundamental advances have been recently made on identifying the genetic basis of the disease, although the pathogen itself remains relatively poorly understood. One such aspect is that of secondary metabolism. A bioinformatics analysis of the Stagonospora nodorum genome has identified 19 polyketide synthase (PKS), 8 non ribosomal peptide synthase (NRPS) and one hybrid PKS-NRPS genes highlighting the significant potential for this fungus to produce novel compounds (Hane, Lowe et al. 2007). Nevertheless, only ten metabolites have been reported to date for Stagonospora nodorum: Devys et al. first identified 8 secondary metabolites produced by S. nodorum in 1994 and thirteen years subsequent to this study the mycotoxin alternariol was identified reviving the curiosity on the potential of S. nodorum to produce secondary metabolites (Tan, Trengove et al. 2009). In this poster we present promising results towards the elucidation of the Stagnonspora nodorum secondary metabolome using a metabolomics approach. An LC/MS analysis of mycelial and secreted secondary metabolites produced by Stagonospora nodorum under different nutritional and light conditions have revealed a host of compounds not previously identified in this fungus. References: Devys, M., M. Barbier, et al. (1994). Isolation of the (-)-(3R)-5-hydroxymellein from the fungus Septoria nodorum. Phytochemistry 35(3): 825-826. Hane, J. K., R. G. T. Lowe, et al. (2007). Dothideomycete-plant interactions illuminated by genome sequencing and EST analysis of the wheat pathogen Stagonospora nodorum. Plant Cell 19(11): 3347-3368. Tan, K. C., R. D. Trengove, et al. (2009). Metabolite profiling identifies the mycotoxin alternariol in the pathogen Stagonospora nodorum. Metabolomics 5(3): 330-335.

IDENTIFICATION OF FLAGELLAR MASTIGONEME PROTEINS FROM PHYTOPHTHORA

Hee W.Y., Blackman L.M. and Hardham A.R. The Australian National University.

Motile, flagellated zoospores of Phytophthora and Pythium species play a key role in pathogen dissemination and the initiation of infection of host plants. The diseases these pathogens cause are highly destructive and result in extensive losses in agriculture and natural ecosystems worldwide. Tripartite tubular hairs called mastigonemes on the anterior flagellum of Phytophthora and Pythium and other protists in the Stramenopile taxon are responsible for cell motility by reversing the thrust of flagellar beat. Immunoprecipitation experiments using antibodies directed towards one mastigoneme protein, PnMas2, in Phytophthora nicotianae have facilitated the identification of two similar PnMas proteins. The genes for these three proteins, PnMas1, PnMas2 and PnMas3, have been cloned and their expression analysed. The expression of all three genes is up-regulated during asexual sporulation, a period during which many zoospore components are synthesized. Unexpectedly, the expression of PnMas1 is also up-regulated in 3-h germinated cysts and that of PnMas3 in vegetative hyphae. Analysis has revealed that the PnMas proteins are similar in sequence to mastigoneme proteins in other Stramenopile protists including the algae Ochromonas danica, Aureococcus anophagefferens and Scytosiphon lomentaria and the diatoms Thalassiosira pseudonana and T. weissflogii. They have an N-terminal secretion signal and contain four cysteine-rich epidermal growth factor (EGF)-like domains. Previous studies found that PnMas2 encodes for a protein in the shaft of tripartite mastigoneme. Evidence from non-denaturing gels indicates that PnMas2 forms large oligomeric complexes, most likely through disulphide bridging. Liquid chromatography-mass spectrometry and peptide mass fingerprinting of polypeptides co-precipitated by the PnMas2 antibody have identified proteins that are potentially associated with PnMas2 including PnMas1, PnMas3, Hsp70, actin, annexin and a protein transport Sec23/24-like protein.

WHOLE GENOME AVERAGE INTERVAL MAPPING OF AGRONOMIC AND METABOLIC TRAITS TO IDENTIFY NOVEL DROUGHT TOLERANCE RELATED QUANTITATIVE TRAIT LOCI IN BREAD WHEATHill C.B.1, Taylor J.D.2, Edwards J.3, Mather D.2, 4, Bacic A.5, 6, 7, Langridge P.4 and Roessner U.1, 5 1Australian Centre for Plant Functional Genomics, School of Botany, The University of Melbourne, Victoria 3010, Australia. 2School of Agriculture, Food and Wine, Waite Research Institute, The University of Adelaide, PMB 1, Glen Osmond, South Australia 5064, Australia. 3Australian Grain Technologies, Roseworthy Campus, Roseworthy, South Australia 5371, Australia. 4Australian Centre for Plant Functional Genomics, The University of Adelaide, PMB1, Glen Osmond, South Australia 5064, Australia. 5Metabolomics Australia, School of Botany, The University of Melbourne, Victoria 3010, Australia. 6Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Victoria 3010, Australia. 7ARC Centre of Excellence in Plant Cell Walls, School of Botany, The University of Melbourne, Victoria 3010, Australia. The survival and reproduction of plants is dependent on their ability to adapt to an unpredictable and dynamic environment. Many of the traits that are important in crop breeding, such as abiotic stress tolerance, have complex genetic control and are highly sensitivity to environmental factors. Their low heritability limits the response to selection in plant breeding programs. High-throughput “omics” technologies, including metabolomics, are increasingly being applied to study the function of genes and/or cellular networks for both the understanding and manipulation of plant genes and traits. We are using metabolomics as part of a functional genomics approach to improve our understanding of the mechanism(s) used by plants to either tolerate or to adapt to different abiotic stresses. In this study we utilize metabolomics to define metabolic differences in a doubled haploid (DH) population from a cross between drought-intolerant (Kukri) and -tolerant (Excalibur) wheat cultivars grown in the field under a drought regime that mimics Australia’s climate of cyclic drought. Metabolite profiling of the complete DH population was carried out using GC-MS to determine and semi-quantify a total of 205 known and unidentified metabolites. Genetic linkage maps and marker scores enabled us to map 217 QTL’s affecting 95 metabolite traits, including novel metabolites, onto the wheat genome. We related these to QTLs identified for 29 individual plant growth and yield traits for the lines grown under drought stress. The co-location and genetic correlation of metabolic and yield and yield-related traits allowed us to identify specific combinations of metabolites as potential drought biomarkers. This approach will serve as a tool to identify candidate genes involved in drought-related metabolite changes in wheat and for identifying novel biomarkers for agronomically important traits under drought-stress conditions.

PHOMA KOOLUNGA, BLACKSPOT PATHOGEN OF FIELD PEA: IS THE FUNGUS SEED-BORNE?

Khani M.1,2, Davidson J.3, Sosnowski M.3 and Scott E.1 1School of Agriculture, Food and Wine, University of Adelaide, Australia. 2College of Agriculture and Natural Resources, Shiraz University, Darab, Iran. 3South Australian Research and Development Institute, Adelaide, Australia.

Phoma koolunga was described in 2009 as the fourth pathogen in the ascochyta blight (blackspot) complex on field pea (Pisum sativum). Little is known about the epidemiology of this pathogen on field pea. The aims of this research were to determine 1) if P. koolunga can be seed-borne, 2) whether the fungus can be transmitted from seed to seedling and 3) the most effective fungicides for seed treatment. Seed samples (cv. Kaspa) were collected in 2010 from the following sources; 18 National Variety Trials (NVT) in South Australia (SA), Victoria and Western Australia, nine agronomy experiments with three times of sowing in SA and four Pulse Breeding Australia (PBA) trial sites in SA. A modified Standard Agar Plate Test to isolate seed-borne fungi was used to determine percentage of seed in each sample infected by P. koolunga. Most seed samples yielded Mycosphaerella pinodes, although Phoma medicaginis var. pinodella was detected occasionally. P. koolunga was isolated from some NVT samples from Victoria and SA, and a few PBA samples, but not from seed from the agronomy experiments. This is the first report that P. koolunga can be seed-borne, albeit infrequently. Sowing of seeds artificially inoculated with conidial suspension of P. koolunga showed that the disease can be transmitted to seedlings. Therefore, sowing healthy pea seed and treatment with fungicides would reduce seed to seedling transmission. The efficacy of six fungicides for seed treatment against P. koolunga was tested by sowing inoculated seeds coated with fungicide. The results of this study are presented.


POSTERS MONDAY


POS-MON-087

THE EFFECT OF FORMATIVE AND SUMMATIVE E-ASSESSMENT TASKS ON STUDENT LEARNING IN THE SCIENCES

Riggs K.M., Loveys B.R., Scott E.S., Schultz C.J. and Able A.J. School of Agriculture Food and Wine, The University of Adelaide, Waite Campus, Glen Osmond, SA, Australia 5064.

E-assessment is being used by university teachers to increase student engagement and enhance student learning. On-line tasks allow flexible delivery of topic material because they can be completed anywhere at any time and encourage self learning outside the classroom environment. We investigated the effect of on-line formative and summative assessment tasks on student learning in second year microbiology and genetics courses. Students completed either formative or summative on-line quizzes. The formative quizzes were designed as a revision tool with built-in feedback, unlimited attempts and no grade weighting. Summative quizzes were designed with restrictions to attempts and accounted for 2% each of the topic grade. After the quizzes, students completed an anonymous survey about issues such as student motivation, task weighting, feedback and time spent completing the quizzes. Preliminary analysis of survey results indicated that the majority of students sought a grade incentive to engage in formative assessment tasks. Both forms of quizzes assisted students to revise for summative assessment tasks such as exams, improving overall learning. A comparison of grades for traditional and on-line assessment methods in the genetics course indicated that the introduction of e-assessment tasks increased the number of students able to obtain ≥75% for the summative practical component while reducing the number of students who failed. The exam performance for those students who did or did not complete the formative on-line assessment tasks will be compared. We will also discuss the impact of e-assessment on general learning outcomes for different cohorts within the school.

ASSESSING RELEVANCE AND SUCCESS OF INDEPENDENT RESEARCH IN LEVEL 2 PLANT SCIENCE

Loveys B.R., Riggs K., Kaiser B.N. and Able A.J. School of Agriculture Food and Wine, The University of Adelaide, Waite Campus Glen Osmond, Adelaide SA, 5064.

Assessment through project-based learning is increasing due to evidence of positive outcomes for enhanced student learning including the development of teamwork, communication, critical thinking and problem solving skills. Project-based learning enables students to use theory (learned through lectures) to answer questions practically. This learning approach may also allow the development of research skills in the plant sciences. Our aim was to determine if independent, student-centred learning was appropriate for level 2 plant science. Foundations in Plant Science, a level 2 course, began in 2011 within the Agricultural Sciences and Viticulture and Oenology degrees. Part of the course is an independent research project running over 7 weeks. Groups of students were given freedom to plan an experiment, collect and analyse data independently. The learning outcomes addressed include that students’ should be able to design and manage an experiment, to analyse and present the data clearly; and develop skills in designing experiments, collection, analysis, interpretation and presentation of data. We assessed the relevance and success of the project in providing those learning outcomes, through formal Student Evaluation of Learning and Teaching as well as asking students to evaluate their learning using a 5-point Likert survey. The group project was generally a positive experience for students with their perceived ability in skills pertinent to writing scientific reports increased during the project. However, students were challenged by preparation of experiments and identifying published papers to support their hypothesis. We will determine if project-based learning has improved learning outcomes for level 2 degree students and prepared them for applied studies in plant sciences.

BROADENING STUDENT LEARNING EXPERIENCES VIA INTEGRATION OF CROSS-DISCIPLINARY EDUCATION PROGRAMS

Speed C.J., Kleiner A. and Macaulay J.O. Monash University, Wellington Rd, Clayton, Victoria 3800.

This study explored the impact on student learning and engagement through the integration of cross-disciplinary education programs. The initial program was developed for art and anatomy students as a collaborative initiative between Monash University and Florence University, Italy. A cohort of medical, physiotherapy and fine arts students from Monash University and medical and physiotherapy students from Florence University attended the program based in Prato, Italy. Students engaged in a three week program which explored the historical relationship between art and anatomy. The program entitled AnaRtomy gave students the opportunity to study historical art and anatomy through visits to museums, art history and anatomy lectures and life drawing. By introducing a cross-disciplinary approach to learning, the medical, physiotherapy and fine arts disciplines all benefited from each other. The learning experiences of the students were enhanced through interactions with international students of varying disciplines. The cohort of medical students from Monash University undertook the AnaRtomy program as part of their Student Project Case (SPC), which is a core component of year two medicine. The conventional SPC program provides an opportunity for students to research and present a medical case or disease. Students research and submit a written document and the SPCs culminate with oral presentations. A modified education program was developed allowing the medical students to meet the SPC learning objectives and the aims of the AnaRtomy program. A research study was performed to evaluate the AnaRtomy program in terms of meeting the SPC learning objectives, impact on student engagement, broadening students learning experiences and whether it is a valuable substitute for the conventional SPC program.

POS-MON-088HALOPLEX PCR TECHNOLOGY: DATA AND APPLICATIONS

Belluoccio D.1, Isaksson M.2, Eriksson P.2, Forsmark L.2, Zetterman K.2, Skarpas B.2, Rojmyr M.2, Roos F.2, Johansson H.1 and Dahl F.2 1Agilent Technologies, 679 Springvale Rd, Mulgrave 3170, VIC, Australia. 2Agilent Technologies, Dag Hammarskjölds väg 54A, SE 751 83 Uppsala, Sweden.

Agilent Technologies introduces HaloPlex PCR for targeted resequencing, which enables amplification of thousands of targets in one single tube. HaloPlex libraries can be run on the most widely used desktop NGS systems (Ion Torrent PGM and Illumina MiSeq) as well as on the HiSeq instrument. To enable high throughput and short turn-around times, we have developed a HaloPlex protocol that can be manually carried out in less than a day. For ultrahigh throughput a fully validated automation option on the BRAVO liquid handling system can be used. The technology eliminates the time-consuming and labor-intensive sequencing library preparation and does not require any dedicated instrumentation, such as DNA shearing instruments or robotics. As part of the protocol, HaloPlex PCR directly incorporates all sequencing primers and sample barcodes that are required for subsequent sequencing for both the Illumina and Ion Torrent platforms. The workflow is thus simpler and faster than both hybrid capture and microfluidic PCR approaches. The presented data and application examples include: - 10 minutes from data input to finalized design - A 6hr protocol : Manual preparation of a sequencing-ready library of 96 DNA samples within a day (16 for PGM) - Target capture size up to 5.0Mb - Input DNA down to 50ng - 96 barcodes - Superior coverage and specificity.


POSTERS MONDAY

POS-MON-089 POS-MON-090DYNAMIC CHROMATIN STATES REGULATE MICRORNA-200 FAMILY GENE EXPRESSION IN EPITHELIAL-MESENCHYMAL-TRANSITIONAttema J.L.1, Lim Y.1, 2, 3, Bert A.G.1, Smith E.4, Drew P.5, Khew-Goodall Y.1, 6 and Goodall G.J.1, 2 11Division of Human Immunology, Centre for Cancer Biology, SA Pathology, Adelaide, SA, 5000, Australia. 2Discipline of Medicine, The University of Adelaide, Adelaide, SA 5005, Australia. 3Institute of Biological Sciences, Faculty of Science, University of Malaya, Kuala Lumpur, Malaysia. 4Discipline of Surgery, The University of Adelaide, Adelaide, SA 5005, Australia. 5School of Nursing and Midwifery, Flinders University, Bedford Park, SA 5042, Australia. 6School of Molecular and Biomedical Science, The University of Adelaide, Adelaide, SA 5005, Australia. The major reasons for breast cancer mortality are complications arising from metastatic disease. A significant fraction of breast cancer patients have micro-metastases at diagnosis, which can develop into clinically relevant lesions and become refractory to current therapies. At the cellular level, there is compelling evidence that Epithelial-Mesenchymal Transition (EMT) of transformed breast epithelial cells leads to cancer metastasis, a process activated by key signalling pathways including Transforming Growth Factor β (TGFβ, Notch, and Fibroblast Growth Factor (FGF). We have previously shown that EMT is induced by the loss of expression of the microRNA-200 gene family, which enable maintenance of the epithelial phenotype due to their regulation of the co-repressor transcription factors, Zeb1 and Zeb2. Recent studies have revealed that the microRNA-200 gene expression is under epigenetic control, and that these processes are also commonly disrupted in cancer. In our current work, we show that the microRNA-200 genes undergo dynamic epigenetic regulation during EMT of breast epithelial cells including DNA methylation and post translational histone modifications. Using chromatin immunoprecipitation coupled to quantitative PCR, we have mapped a panel of epigenetic histone modifications, chromatin modifying complexes and the transcriptional co-repressor, Zeb1 in TGFβ-mediated EMT based on the human mammary epithelial cell in vitro model system. We found a cell type specific repression by distinct and complementary epigenetic mechanisms. The microRNA-200b~a~429 gene on Chromosome 1 was silenced by Polycomb Group proteins and Zeb1 whereas DNA methylation and G9a-mediated activities were linked to silencing of the microRNA-200c~141 gene on Chromosome 12. These results highlight the importance of epigenetic regulation in the maintenance of miR-200 family expression in epithelial cells, and their aberrant silencing in EMT and breast cancer metastasis.

ABNORMAL RETENTION OF ENDOSOME MARKERS DURING THE MATURATION OF HELICOBACTER PYLORI PHAGOSOMES

Borlace G.N., Keep S.J., Butler R.N. and Brooks D.A. Mechanisms in Cell Biology and Diseases Research Group, School of Pharmacy and Medical Sciences, Sansom Institute for Health Research, University of South Australia.

Helicobacter pylori (H. pylori) is a human gastric pathogen that is estimated to have infected over half of the global population. Despite eliciting a strong innate and adaptive immune response in the host, H. pylori is able to persist in its gastric niche for decades resulting in chronic gastritis. Large numbers of neutrophils, macrophages and dendritic cells are recruited to the infected mucosa and successfully ingest the bacterium, however H. pylori is able to avoid phagocytic killing. In an in vitro model of infection using primary human macrophages, we have previously demonstrated that H. pylori is able to avoid phagocytic killing for up to 48 hours, and that there are disruptions to the normal phagosome maturation process. We have employed markers specific for early endosomes (Rabex5, Rab5, Rabaptin5, PI(3)P and EEA1) and late endosomes (Rab7, RILP, Rab9, TIP47 and CD63) to define the time course of H. pylori phagosome maturation. While the recruitment of early endosome markers Rabex5 and Rab5 to H. pylori phagosomes was consistent with that seen for control latex bead phagosomes, their downstream effectors Rabaptin5 and EEA1 appeared to be recruited earlier and retained for longer on H. pylori phagosomes. Similarly, the late endosome markers appeared to cycle on and off of the latex bead phagosomes consistent with other models of phagosome maturation, whereas they maintained their association with H. pylori phagosomes throughout the time course. This represents a significant departure from the normal phagosome maturation process, indicating that H. pylori phagosomes sequester and retain earlier endosome machinery despite recruiting later effectors.


POSTERS TUESDAY

POS-TUE-001 POS-TUE-002


MOLECULAR CHARACTERIZATION AND APOPTOTIC MECHANISMS IN HUMAN LEUKEMIA CELLS OF DEFENSIN-LIKE PEPTIDE, HARMONIASIN FROM HARMONIA AXYRIDIS

Hwang J.S., Kim I.W., Lee J.H., Kwon Y.N., Yun E.Y., Nam S.H. and Ahn M.Y. Department of Agricultural Biology, National Academy of Agricultural Science, RDA, Suwon 441-100, Republic of Korea.

We investigated antibacterial activity of Harmoniasin analog(HaA4, IGGYCSWLRL-NH2) that exhibited potent antibacterial activities against Gram negative and positive bacteria strains and it also evidenced no hemolytic activity. And we also investigated whether HaA4 had the apoptosis inducing properties in human leukemia cell lines U937, Jurkat, and AML-2. HaA4 caused the cell growth inhibition and the DNA fragmentation in these cancer cells. These results indicate that HaA4 selectively caused a marked decrease in cell viability in Jurkat, U937 cells. And fragmentation of DNA, a marker of apoptosis, was also confirmed in the leukemia cell lines. HaA4-induced apoptosis in leukemia cells was mediated by Caspase 7, 9 and PARP (poly (ADP-ribose) polymerase) indicating induction of a caspase-dependent signaling pathway. And we checked that increase of cell viability to depend on Z-VAD-FMK, a pan-caspase inhibitor concentration. But AIF (Apoptosis inducing factor) was not changed.

HEPATOCYTE NUCLEAR FACTOR-4ALPHA REGULATES PYRUVATE CARBOXYLASE GENE EXPRESSION THROUGH THE NON-CLASSICAL DR MOTIF LOCATED IN THE PROXIMAL PROMOTERChavalit T., Rojvirat P. and Jitrapakdee S. Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok 10400, Thailand.

Pyruvate carboxylase (PC), a biotin-dependent carboxylase, catalyzes the ATP-driven carboxylation of pyruvate to oxaloaceate and is one of the four key regulatory enzymes of hepatic gluconeogenesis in mammals. In non-gluconeogic tissues, PC is involved in lipogenesis in adipocytes and glucose-induced insulin secretion in pancreatic β-cells. The PC gene possesses two promoters namely, the proximal (P1) and the distal (P2) promoters. The P1-promoter is active in gluconeogenic and lipogenic tissues while the P2-promoter is active in pancreatic β-cells. In this study we report that the proximal promoter of the murine pyruvate carboxylase gene contains three binding sites for the hepatocyte nuclear factor-4α (HNF4α). These binding sites include the -386/-374 dual peroxisome proliferator activated-receptor responsive element (PPRE)/HNF4α binding site, the -116/-92 E-box and the non-classical DR (HNF4α-specific binding motif, H4-SBM) (-28/-15). Under basal conditions, neither mutation of the dual PPRE/HNF4α site nor HNF4-SBM affects reporter gene activity while mutation of the -116/-42 E-box decreases reporter gene activity by 60%. Over-expression of HNF4α in a mouse hepatocyte cell line, AML12, results in a several fold increase in expression of the proximal promoter-luciferase chimeric reporter gene. However, mutation of only the HNF4-SBM site markedly attenuates the HNF4α-mediated transcriptional activation of the chimeric reporter gene activity by 65-90%, depending on the length of the promoter. Electrophoretic mobility shift assays reveal that these three putative HNF4α binding sites are capable of binding to endogenous HNF4α or recombinant human HNF4α with different affinities. Chromatin immunoprecipitation assays conducted in AML12 cells and mouse hepatocytes reveal that HNF4α is indeed associated with these three sites in vivo. Suppression of HNF4α expression by 90% in AML12 cells results in a 50% reduction in PC expression indicating that PC is a target of HNF4α.

THE ROLE OF GATA2 IN THE DEVELOPMENT AND MAINTENANCE OF LYMPHATIC VASCULATURE

Kazenwadel J.S.1, Secker G.A.1, Chong C.E.2, 3, Lee Y.2, Sutton D.L.1, Hahn C.N.2, 3, Scott H.S.2, 3, 4 and Harvey N.L.1, 3 1Division of Haematology, Centre for Cancer Biology, SA Pathology, Adelaide, Australia. 2Department of Molecular Pathology, Centre for Cancer Biology, SA Pathology, Adelaide, Australia. 3School of Medicine, University of Adelaide, Adelaide, Australia. 4School of Molecular and Biomedical Science, University of Adelaide, Adelaide, Australia.

Recent work has established that heterozygous germline GATA2 mutations predispose carriers to familial myelodysplastic syndrome (MDS)/acute myeloid leukaemia (AML), “MonoMAC” syndrome and DCML deficiency. We and others have also reported that a subset of patients who have mutations in the GATA2 locus, also exhibit primary lymphedema. Primary lymphedema occurs as a result of aberrations in the development and/or function of lymphatic vessels, spurring us to investigate whether GATA2 plays a role in the lymphatic vasculature. We demonstrate here that GATA2 protein is present in the lymphatic endothelium and is expressed at high levels in lymphatic vessel valves. We also show that GATA2 controls the expression of genes important for programming lymphatic valve development, in particular the transcription factor Prox1, the master regulator of lymphatic cell identity. Our data expand the phenotypes associated with germline GATA2 mutations to include predisposition to primary lymphedema and reveals a crucial role for GATA2 in lymphatic vascular development.

PAPILIOCIN, A FAMILY OF ANTIFUNGAL PEPTIDE FROM THE SWALLOWTAIL BUTTERFLY, PAPILIO XUTHUS

Kim S.R., Park S.W., Choi K.H., Goo T.W., Kang S.W. and Hwang J.S. Department of Agricultural Biology, National Academy of Agricultural Science, Rural Development Administration, Suwon 441-100, South Korea.

Papiliocin is a 37-residue cecropin-like peptide which was isolated recently from the swallowtail butterfly Papilio xuthus. This peptide was shown to contain potent antimicrobial activities against several human pathogenic bacterial and fungal strains. We investigated the structure and mechanism of action towards human pathogenic fungi. Circular dichroism analyses revealed that papiliocin was disordered structure in water but folded to form a relatively high alpha-helical structure in membrane-mimetic HFIP, SDS, DPC and LPS Solutions. This finding is further verified by NMR spectroscopy. The tertiary structure of papiliocin revealed that show a typical alpha-helical amphipathic structure in a micellar environment, a structural prerequisite in permeabilization of the bacterial membrane. To understand the antifungal mechanisms of papilocin, flow cytometric analysis using propidium iodide (PI) staining and 1,6-diphenyl-1,3,5-hexatriene (DPH) fluorescence analysis were conducted against Candida albicans cells. The results demonstrated that papiliocin perturbed and disrupted the fungal plasma membrane. Furthermore, we confirmed that C. albicans cells treated with papiliocin showed several diagnostic markers of yeast apoptosis, such as accumulation of intracellular ROS, phosphatidylserine exposure, active metacaspase, dissipation of the mitochondrial membrane potential. In addition, nuclear fragmentation, which is important marker of late stage apoptosis, was seen by DAPI and TUNEL assay. These evidence strongly support antifungal property of papiliocin by promoting apoptosis in C. albicans.


POSTERS TUESDAY



IDENTIFICATION OF A NOVEL PU.1 ISOFORM AND ITS POTENTIAL ROLE IN REGULATING ERYTHROID DIFFERENTIATION

Mak K.S., Funnell A.P.W., Pearson R.C.M. and Crossley M. School of Biotechnology and Biomolecular Sciences, University of New South Wales, NSW 2052.

Haematopoiesis is governed by cross-regulation of various lineage specific transcription factors. Differentiation of myeloid cells is promoted by the master myeloid regulator Pu.1 while simultaneously inhibiting lineage commitment towards the erythroid pathway. Our lab has always been interested in understanding the biological functions of the Krüppel-like Factor family member: Klf3, particularly in the context of haematopoiesis. Through the search of potential Klf3 targets, we have identified a novel Pu.1 isoform under the regulation of an alternative internal promoter that is responsive to Klf3. The presence of this alternative promoter has not been previously documented. We have performed transfection work and reporter assays to test the functional role of this Pu.1 isoform and results indicate that it may be involved in certain aspects of erythroid differentiation. These findings suggest that the phenotypes observed in various Pu.1 mutant and knockout mouse models may in part be attributed by the effect of this newly identified isoform.

IMPLEMENTATION OF A HIGH-THROUGHPUT GENOMIC PIPELINE FOR DISCOVERING DISEASE CAUSING MUTATIONS IN MENDELIAN AND COMPLEX DISEASES

Marshall M., Leo P., Mukhopadhyay P., McInerney-Leo A., Duncan E. and Brown M. University of Queensland Centre for Clinical Genomics, Diamantina Institute, University of Queensland.

A high-throughput sequencing pipeline has been implemented at UQCCG for the analysis of mendelian and complex diseases. The raw output from our three Illumina HiSeq is aligned to a reference genome and the Genome Analysis ToolKit (GATK) developed by the Broad Institute is used to fix sequencing and alignment errors. The GATK UnifiedGenotyper is then used to detect SNPs (Single Nucleotide Polymorphism) & INDELS (Insertions/Deletions) in the samples. Further analysis is done to see whether they are a true genetic variant versus a sequencing or data processing artefact. Allele Frequency, read quality, mode of inheritance, functional affect of SNP and whether it is in a coding region are used to determine if this is a disease calling mutation. Skeletal Dysplasia are rare bone diseases (3-4 per 1000 births) where defects of bone formation is caused by abnormalities of cartilage and bone growth which results in abnormal shape and size of the skeleton, resulting in disproportion of the long bones, spine, and head, ossification. These diseases with a MAF<0.5 cannot be detected in GWAS studies so instead exome sequencing is used. DNA samples have been collected from affected and unaffected members of 140 families. The UQCCG pipeline has allowed us to detect de-novo, dominant & recessive mutations in these families. Variations in bone density can result in a susceptibility to fractures with age, sex & whether the individual has osteoporosis being contributing factors. 1000 samples were collected from females, 500 high-density and 500 low-density. SNP-chips and GWAS have previously been used to identify common variants so exome sequencing is now being used to detect the rare variants.

DEVELOPMENT OF AN EXPRESSION SYSTEM TO MANIPULATE ORGANIC ACID METABOLISM IN VITIS VINIFERA

Marshall T.C.1, Sweetman C.2, Ford C.M.2, Jenkins C.L.D.1 and Soole K.L.1 1School of Biological Sciences, Flinders University, Bedford Park, South Australia. 2School of Agriculture, Food and Wine, The University of Adelaide, Urrbrae, South Australia.

Organic acids are essential components of grape berries and are involved in numerous metabolic pathways. The most predominant acids of Vitis Vinifera , tartaric and malic acids, are differentially regulated during grape berry development and can be altered by changes in environmental conditions. In particular, levels of malic acid can be lower in fruit that are exposed to warmer temperatures, which affects the acid balance of the fruit and wine and can add costs to the winemaking process. Therefore there is some interest in the investigation of genes involved in temperature-sensitive malic acid metabolism of grape berries. However, one of the difficulties of working with grapevine systems is the long gestation time, which limits the use of genetic modification as a technique for gene discovery. This project aims to test a transient agroinfiltration technique with grapevine leaves, whilst simultaneously exploring potential heat-responsive promoters from the Vitis vinifera genome. The identification of heat-responsive elements (HRE) for future use in heat-inducible expression vectors may result in the development of a vector which can control the metabolism of grape berry components such as malic acid degradation when vines are exposed to high temperatures. The heat-responsiveness of a putative promoter sequence from a grape heat-shock gene will be investigated. Constructs contain a luciferase reporter gene under the control of different regions of the putative heat-responsive promoter. Expression of the reporter gene will be assessed in leaves exposed to different temperatures.

INVESTIGATING THE TRANSCRIPTIONAL REGULATION OF HIF-2 AND ITS RELEVANCE TO MULTIPLE MYELOMA

Martin N.M.1, Hewett D.2, Zannettino A.C.W.2 and Peet D.J.1 1School of Molecular and Biomedical Science, University of Adelaide, Adelaide SA 5005, Australia. 2Myeloma Research Laboratory, Bone and Cancer Laboratories, Department of Haematology, Centre for Cancer Biology, SA Pathology, Adelaide, Australia.

Cellular survival within the body is dependent on oxygen for generation of energy which in turn powers metabolism. As such, it is important for cells to respond to insufficient oxygen levels, termed hypoxia, through mechanisms such as transcriptional regulation. Key proteins activated in hypoxia are the Hypoxia Inducible transcription Factors (HIFs), which in turn upregulate target genes whose products aid in metabolic responses and delivery of oxygen to cells. The HIFs are important for normal physiology and play roles in major human diseases such as cancer. Both HIF-1 and HIF-2 are regulated predominantly by the canonical post-translational hydroxylation pathway, which consists of prolyl and asparaginyl hydroxylases that act as “oxygen sensors” and are active in normoxia, and inactive in hypoxia. However, protein expression profiles in specific cell types show a delayed induction of HIF-2α compared to the rapid induction of HIF-1α that is characteristic of regulation by the HIF prolyl hydroxylases, demonstrating an alternative mechanism for HIF2α regulation in these cells. Protein and mRNA analysis confirmed this delayed HIF-2α induction in hypoxia represents a novel mechanism of HIF-2α regulation at the mRNA level. These data are of particular interest in the context of Multiple Myeloma, where this delayed HIF-2 induction is also observed and associated with increased expression of disease marker CXCL12, raising interesting questions about the role of HIF-2α in multiple myeloma. We are further characterising this mechanism of HIF-2 mRNA regulation in multiple myeloma, accompanied by a directed study into the role of HIF-2 compared with HIF-1 induction in multiple myeloma disease progression using an established mouse model.


POSTERS TUESDAY



ASSESSMENT OF PHYSIOLOGICAL AND TRANSCRIPTIONAL CHANGES IN YOUNG SUGARCANE PLANTS EXPOSED TO WATER STRESS

Mingossi F.B.1, 2, Silva-Filho M.C.2 and Casu R.E.1 1CSIRO Plant Industry, Queensland Bioscience Precinct, 306 Carmody Road, St. Lucia, Qld, Australia. 4067. 2University of Sao Paulo, ESALQ-USP, Avenida Padua Dias, 11, Piracicaba, Sao Paulo, Brazil. 13418-900.

Sugarcane is a high biomass tropical C4 grass crop which accumulates large quantities of sucrose which is also used for the production of bioethanol, a low-carbon emission fuel. Crop productivity can be significantly impacted by abiotic constraints, especially water availability, because it can severely impair plant growth and performance. Understanding the molecular basis for this loss in productivity will aid in identifying strategies for mitigation. We report here a preliminary experiment investigating physiological parameters and transcriptional profiles of genes with acknowledged roles in abiotic stress. Sugarcane cultivar Q208 was grown under controlled temperature and humidity in the Controlled Environment Facility at CSIRO Plant Industry for approx. 40 days after germination and establishment in a temperature-controlled glasshouse. These plants were then subjected to water deprivation for one week. During this period, photosynthesis rate, stomatal conductance, intercellular CO2 concentration and leaf transpiration were evaluated on the 0, 3rd, 5th and 7th days and the leaves (+1) were collected to measure leaf Relative Water Content and gene expression. Results of this work indicated that termination of irrigation resulted in measurable physiological effects in young sugarcane plants and analysis of the expression of the chosen stress-response genes revealed significant differential expression between the control and treatment groups. This will inform further research on water use efficiency in sugarcane, leading to identification of sugarcane varieties with improved tolerance to adverse environmental conditions.

INFLUENCE OF GIBBERELLINS ON THE TRANSCRIPTIONAL ACTIVITY OF THE SOYBEAN NODULE TRANSCRIPTION FACTOR GMSAT1

Mohammadi-Dehcheshmeh M.1, Chiasson D.1, Mazurkiewicz D.1, Ebrahimie E.2, Tyerman S.1 and Brent K.1 1Plant Research Center, School of Agriculture, Food and Wine, Waite Campus, University of Adelaide, Australia. 2School of Molecular and Biomedical Science, University of Adelaide, Australia.

GmSAT1 is a membrane bound bHLH transcription factor(Kaiser et al., 1998), which plays a key role in legume nodulation where loss of activity causes a reduction in nodule development and nitrogen fixation. The regulation of GmSAT1 activity is poorly understood. The aim of the present study was to identify underlying regulatory mechanism(s) of GmSAT1 function. We found a relatively high frequency of gibberellin-responsive regulatory elements on the promoter region of GmSAT1, which led us to examine whether signaling exists between GmSAT1 and Gibberellins. qPCR analysis revealed a significant negative correlation between GmSAT1 expression and short or long-term application of GA3 to nodulated roots. Interestingly, transcriptome analysis of GmSAT1 RNAi silenced nodules significantly down-regulated Gibberellin-regulated genes. All together, this study suggests that GmSAT1 activity in soybean nodule development may involve regulation by GA3.

CHARACTERISING THE MUTATION IN THE OVINE HERITABLE CATARACT

Morton J.D.1, McEwan J.C.2, Lee H.Y.Y.1, Anderson R.M.2, Wilson G.R.1, Gately K.1 and Brauning R.2 1Department of Wine,Food and Molecular Biosciences, Lincoln University, Lincoln, New Zealand. 2Invermay Agricultural Centre, AgResearch, Mosgiel, New Zealand.

Ovine Heritable Cataract (OHC) is an autosomal dominant mutation that typically leads to progressive development of cataracts over the first ten months. It has been used as an animal model for testing potential treatments for human cataract. The phenotype includes a rise in intracellular calcium and a breakdown of cytoskeletal proteins, but the initial mechanism is unknown. Linkage mapping had previously narrowed the locus for OHC to a region of less than one centimorgan on chromosome 6. In the current research 7 affected sheep and 7 related sheep with normal eyes were genotyped with the ovine 50K Single Nucleotide Polymorphism (SNP) chip to confirm this result and delineate the breakpoint region. This was followed by sequencing the entire genome of a carrier ram to the equivalent of 13 times coverage with Illumina technology. The reads have been aligned to OARv2.0. There is evidence of both indels and SNPs which would change the amino acid sequence in candidate genes within the linked region. Comparing the mutations identified with those present in 75 non-carrier sheep sequenced to similar depth as part of the International Sheep Genomics Consortia allows novel mutations to be identified. These will then be analysed for possible biological relevance.

STEMFORMATICS: A BIOLOGY-CENTRIC RESOURCE FOR INTUITIVE EXPLORATION OF GENE EXPRESSION IN STEM CELLS AND THEIR DIFFERENTIATED PROGENY

Mosbergen R.1, Choi J.2, Korn O.3, Seidenman N.2, Matigian N.3, Degraaf C.2, Anderson M.4, Sargeant T.2, Miotto A.3 and Wells C.1 1Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, St Lucia Brisbane 4072 Australia. 2The Walter & Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, Victoria 3050, Australia. 3Australian Centre for Adult Stem Cell Research, Eskitis Institute for Cell and Molecular Therapies, Griffith University, Nathan Brisbane 4111 Australia. 4The Institute for Molecular Biosciences, The University of Queensland, St Lucia Brisbane 4072 Australia.

Stemformatics.org was built for stem cell researchers with minimal experience in bioinformatics, providing a centralised portal with an intuitive, gene-centric search interface, transparent access to primary references, and high-quality graphics. It was developed from the recognition of the value of published gene expression signatures, and acknowledged the difficulty in identifying, searching, comparing or analysing this data in generic array repositories. All data has been curated, and includes quality metrics for each dataset and array platform. The workbench area provides a secure area to upload and analyse user-defined lists, and steps users through simple analysis workflows including differential expression, correlated gene lists and clustering. Statistical approaches use GenePattern as the analysis-engine, and a translation feature encourages facilitates more sophisticated analyses on external analysis sites. Gene list annotations include predictions on protein domains, GO annotations, subcellular localization and pathway membership. Highlights include biology-centric queries of public stem cell datasets, workbench analysis area for comparison or correlation of samples/genes, secure upload of proprietary gene lists with annotation features, file ‘translation’ for easy upload into external gene expression analysis tools.


POSTERS TUESDAY



GENETIC ASSOCIATIONS OF ACUTE ANTERIOR UVEITIS IDENTIFIED BY GENOMEWIDE ASSOCIATION STUDY

Mukhopadhyay P.1, Claushius D.1, Leo P.1, Hadler J.1, Bradbury L.A.1, Evans D.M.2 and Brown M.A.1 1University of Queensland Diamantina Institute. 2MRC Centre for Causal Analyses in Translational Epidemiology.

Anterior uveitis (AU) is the most common form of ocular inflammatory condition affecting the iris and/or ciliary body, with an incidence of 17 per 100,000 per year. AU is the most common extra-articular complication of Ankylosing Spondylitis (AS), affecting almost 30% of AS patients. Both AS-associated and independent form of AU is strongly associated with the presence of HLA-B27. Epidemiological studies have shown that genes other than HLA-B27 influence the risk of developing AU in AS patients. The pathogenesis of Uveitis involves shared genetic factors with Ankylosing Spondylitis. So far no genes have been convincingly shown to be associated with AU independently of association with AS. In order to identify genes associated with risk of developing AU both in cases with AS and in its absence we undertook a genome-wide association study on 1105 AU cases and 1404 AS cases without AU. Ankylosing Spondylitis patients (with or without uveitis) were obtained from the Welcome Trust Case Control Consortium 2 (WTCCC2) and Australo-Anglo-American Spondyloarthritis Consortium (TASC). Control genotypes from 5667 healthy Britons were obtained from the WTCCC2 AS genome-wide association study. The study demonstrates substantial overlap between AU and AS genes. Our results indicates that HLA-B27, ERAP1, ANTXR2 and gene deserts 21q22 are even stronger associated to patients suffering from AU and AS than AS alone.The results generated in this study also points that the genetic background of AU in AS as well as AU in general is different between HLA-B27 positive and negative patients. We anticipate that our study will provide a valuable resource to understand the genetics involved in the AU as a whole, as well as a specific type of AU, AU in AS.

THE ROLE OF EZH2 IN MESENCHYMAL STEM CELL GROWTH AND MULTI-DIFFERENTIATION

Hemming S.E.1, 2, 3, Cakouros D.1, 3, 4 and Gronthos S.1, 2, 3 1Mesenchymal Stem Cell Group, Department of Haematology, SA Pathology, Adelaide, 5000. 2The University of Adelaide, Adelaide,5000. 3Robinson Insitute, North Adelaide,5006. 4Centre for Stem Cell Research,Adelaide, SA, 5000.

The methyltransferase Enhancer of Zeste homolog 2 (Ezh2) is known to repress gene expression through histone 3 lysine 27 tri-methylation (H3K27me3) modifications on chromatin. However, the role of Ezh2 on differentiation and life-span of human bone marrow-derived mesenchymal stromal/stem cells (MSCs) is still unclear. Differentiation studies were used to determine the multi-differentiation capacity of Ezh2 enforced MSCs and DZNep treated MSCs in vitro. The proliferation and life-span potential was demonstrated by BrdU and Senescence assays. The gene expression profiles of differentiated cells were assessed by RT-PCR. This study demonstrated that MSCs express known MSC surface antigen markers and can differentiate into adipocytes, osteoblasts and chondrocytes under differentiation conditions in vitro. Enforced expression of Ezh2 in MSCs enhanced the adipogenic and inhibited the osteogenic differentiation potential of MSCs in vitro. Conversely, inhibition of Ezh2 H3K27me3 activity by DZNep resulted in a decrease in adipogenic differentiation and an increase in osteogenic differentiation in MSCs under in vitro. Further investigation into the role of Ezh2 is needed to conclude if Ezh2 is a negative or positive regulator of MSC chondrogenic differentiation and whether Ezh2 has a role in extending the life-span of MSCs. These findings implicate Ezh2 as a possible positive regulator of adipogenic differentiation and a negative regulator of osteogenic differentiation by MSCs in vitro.

INSECT OLFACTORY PROTEINS

Hill K.1, 2, Stevenson B.J.1, 3, Anderson A.1, 3, Glatz R.V.1, 2 and Chyb S.3 1CRC National Plant Biosecurity. 2SARDI, South Australia. 3CSIRO Food Futures Flagship and Division of Ecosystem Sciences, ACT.

While there is currently good agreement on the molecular events in vertebrate and nematode olfaction, debate still surrounds the olfactory signalling mechanism in insects. This project investigates proteins which are vital to insect olfaction and key in sustaining insect populations. We aim to apply this knowledge to practical solutions in areas such as detection and control. Understanding the mechanisms and proteins involved in the sensory communication between highly diverse invertebrate species could inspire the integration of their chemosensory proteins, or mimics thereof, into sensitive sensing devices which could aid in detection of an array of compounds that are important for diverse applications. We have focussed on proteins which may aid in creating such a detector by investigating the receptors used in pheromone communication in the red rust flour beetle (Tribolium castaneum), which is a stored grain pest worldwide. We hypothesised sex pheromone receptors would show sex-biased transcription and compared individual OR transcription in male and female beetles. Real-time quantitative PCR has uncovered three male-specific ORs and one female-specific OR and these receptors are targets for further functional investigations. In addition to receptors, another approach involves investigation into the odourant binding proteins (OBPs) found in the beetle as another class of potential detector molecules. We have expressed OBPs using E.coli and purified the recombinant OBPs for functional investigation. It is envisioned that research into insect proteins involved in olfaction could not only have applications in sensing devices, but also potentially aid in development or refinement of integrated control measures.

IMPROVING PROTEIN DETECTION USING IR-BASED QUANTITATION

Utzat C.1, Strug I.1, Amara R.1, Goncalves N.1, Lento J.1, Nadler T.1 and Kraschnefski M.2 1EMD Millipore Corporation, 290 Concord Rd, Billerica, MA 01821, USA. 2Merck Millipore, 207 Colchester Rd, Kilsyth, Vic, 3137.

The Direct Detect IR-based quantification system represents an innovation in biomolecule quantitation. By directly measuring amide bonds in protein chains, the Direct Detect system accurately determines an intrinsic component of every protein without relying on amino acid composition. Infrared (IR) spectroscopy is one of the oldest and well established experimental techniques for the analysis of polypeptides and proteins structure. It is convenient, requires minimal sample preparation and can be used under a wide variety of conditions. IR spectroscopy exploits the fact that molecules absorb specific frequencies that are characteristic of their structure. Protein primary structure is formed by a long chain of amide bonds that are represented by nine IR absorbance areas. In order to determine protein concentration Direct Detect IR-based technology utilizes amide I band, which is assigned to the C=O stretching vibration within the peptide bond. Within minutes and without any bio- or immuno-chemical staining, protein concentration in an extremely wide range from about 0.2 mg/mL to >20 mg/mL can be determined directly from the undiluted solution. In addition, amide bond quantitation is not subject to interference from many common buffer components such as detergents, reducing agents and chelators. This allows for direct quantitation of proteins without the need to run Bradford, BCA or other colorimetric assays.


POSTERS TUESDAY



DETERMINATION OF GLYCAN COMPOSITION AND SITE OCCUPANCY OF SOLUBLE RECOMBINANT HENDRA VIRUS (HEV) AND NIPAH VIRUS (NIV) STRUCTURAL PROTEINS

Shiell B.J.1, Colgrave M.2, Snelling H.1, Feng Y.3, Chan Y.3, Broder C.C.3 and Michalski W.P.1 1CSIRO Australian Animal Health Laboratory, Geelong, VIC, Australia. 2CSIRO Queensland Bioscience Precinct, St Lucia, QLD, Australia. 3Microbiology and Immunology, Uniformed Services University, Bethesda, USA.

Hendra virus (HeV). and Nipah virus (NiV) are closely related members of the family Paramyxoviridae. HeV has been isolated only in Queensland and New South Wales, Australia while NiV appears more widespread, and has been found in the Malay Peninsula, Cambodia, Bangladesh and India. Both viruses are zoonotic Biosafety Level 4 (BSL4) pathogens that continue to cause significant morbidity and mortality. HeV and NiV possess two structural membrane glycoproteins that mediate infection of host cells: the attachment glycoprotein (G) and the fusion protein (F), which are essential for receptor binding and virion-host cell fusion, respectively. N-linked glycosylation of these proteins are critical post-translation modifications that are important for structure and function. Recombinant HeV G glycoprotein is currently being trialled as vaccine in horses. Deciphering the glycan composition and structure on these glycoproteins may also assist in the development of glycan-targeted therapeutic intervention strategies. Site occupancy and glycan composition of recombinant HeV and NiV G glycoproteins from a variety of expression systems (HEK293, HEK293 vaccinia virus; HeLa and HeLa vaccinia virus) has been undertaken using a proteomics approach. This included two-dimensional electrophoresis, glycan identification with lectin based assays and mass spectrometry methods linear ion trap, triple quadrupole and a TOF-TOF. A targeted glycopeptide approach with precursor ion scanning (PIS) to identify marker ions of choice was the most successful and will be presented and discussed. Methods developed allowed us not only to assess overall glycan composition but also the compositional heterogeneity at specific glycan attachment sites in both HeV and NiV G proteins.

C-JUN N-TERMINAL KINASE 1 (JNK1) PHOSPHORYLATION UNDER CONTROL AND STRESS CONDITIONS: PHOSPHORYLATION SITE IDENTIFICATION BY MASS SPECTROMETRY

Misheva M.1, Williamson N.A.2, Yeap Y.Y.C.1 and Bogoyevitch M.A.1 1Department of Biochemistry and Molecular Biology. 2Mass Spectrometry and Proteomics Facility (MSPF). Bio21 Institute, University of Melbourne, Victoria, Australia 3010.

c-Jun N-terminal kinase 1 (JNK1) is a member of a conserved sub-family of mitogen-activated protein kinases (MAPKs). JNKs have been implicated in a number of physiological processes, including embryonic development, and cell proliferation, but are also known to be activated in response to a diverse range of stress conditions. The best-characterised phosphorylation events for JNK1 include phosphorylation of key threonine and tyrosine residues of its activation loop (T183-P-Y185) that result in the increased activity of JNK towards its substrate proteins. Although other phosphorylation sites on JNK have also been identified in mass spectrometric analyses of the cellular phosphoproteome, a comprehensive and focused investigation of the phosphorylation profile of JNK1 itself under control and stress conditions has not been undertaken. Here we report our use of mass spectrometry to identify JNK1 phosphorylation sites in vivo under control and stress conditions. We have transiently transfected c-myc tagged JNK1 in JNK1/2-/- mouse embryonic fibroblast (MEF) cells and subjected these cells to control and hyperosmotic stress (0.5 M sorbitol) conditions. Following immunoprecipitation, c-Myc-tagged JNK1 was subjected to in-gel trypsin digestion. Phosphorylation sites under control and stress conditions were identified using the selected reaction monitoring (SRM) approach where precursor and fragment m/z values for selected peptides are scanned by the mass spectrometer. Our analysis reveals the complexity of phosphorylation events directed towards JNK1 under both control and stress conditions, and paves the way for investigating the roles that these might play in the maturation, protein-protein interactions, trafficking and activation of JNK1.

GROWTH DIFFERENTIATION FACTOR-9/BONE MORPHOGENETIC PROTEIN-15 SYNERGISM: MECHANISM AND SIGNIFICANCE

Mottershead D.G.1, 2, Martin G.A.1, 2, Shi J.Y.1, 2 and Gilchrist R.B.1, 2 1Research Centre for Reproductive Health, Robinson Institute, University of Adelaide. 2Discipline of Obstetrics & Gynaecology, School of Paediatrics and Reproductive Health, University of Adelaide.

Growth differentiation factor-9 (GDF9) and bone morphogenetic protein-15 (BMP15) are two lesser known members of the transforming growth factor beta (TGF-β superfamily, both of which are selectively expressed in the oocyte and are essential for fertility. These two proteins are critically important for paracrine signaling between the oocyte and the surrounding granulosa cells, which is of great importance for successful oocyte maturation. GDF9 and BMP15 are structurally unique within the TGF-β superfamily in that they lack the conserved Cys residue within their respective mature regions which gives rise to the inter-monomer covalent linkage in the other members of this protein family. This leads to the suggestion that GDF9 and BMP15 signal as non-covalent dimers. Both proteins activate canonical SMAD signaling pathways within granulosa cells, GDF9 activates the traditional TGF-β/activin regulated SMAD2/3 pathway, whereas BMP15 activates the traditional BMP regulated SMAD1/5/8 pathway. Recently, we reported that the purified mature regions of GDF9 and BMP15 potently synergise on mouse granulosa cells (Mol. Hum. Reprod. (2012) 18:121-128). We have since determined that there are clear differences in signaling potential of mouse and human GDF9, and the relative importance for function of BMP15 synergism for these two proteins. Our current understanding of the mechanism and significance of GDF9/BMP15 synergism will be presented.

STRUCTURE-FUNCTION STUDIES OF THE POTENT PROKARYOTIC TRANSCRIPTIONAL ACTIVATOR CII

Murchland I. and Shearwin K.E. School of Molecular & Biomedical Science, University of Adelaide, South Australia.

The CII protein from the temperate bacteriophage 186 is a helix-turn-helix DNA-binding protein that activates the lysogeny-promoting pE promoter. We are interested in the way CII acts at the pE promoter primarily for two reasons. Firstly, the inverted-repeat half-sites to which CII binds are separated by 20 base pairs, or two full turns of the DNA helix, in contrast to the single turn usually associated with other helix-turn-helix activators. Secondly, activation by CII yields an increase in pE activity of approximately 400-fold over basal levels, placing CII among the most potent prokaryotic activators known. Using a dual-selection genetic screen, we isolated mutants of CII that retain wild-type DNA-binding yet are deficient in their activation of pE. Analysis of these mutants by suppression genetics using a combination of in vivo pE reporter assays and in vitro transcription reveals that CII acts via two distinct interactions with the sigma and alpha sub-units of RNA polymerase. A structural model of CII was obtained using the Rosetta suite of programs and docked onto pE DNA using HADDOCK. A complete structural model of CII and RNA polymerase at the promoter was developed which is consistent with a range of experimental data. Based on the model, we hypothesise that the potent activity of CII is related to the unusual arrangement of its binding sites. By reducing physical occupation of the DNA immediately adjacent to the polymerase, greater accessibility of the promoter is afforded to the mobile C-terminal domain of the polymerase alpha sub-unit (alpha-CTD), leading to greater occupation of this site by alpha-CTD and hence greater activation.


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FUNCTIONAL AND STRUCTURAL PROPERTIES OF A BARLEY BORATE TRANSPORTERNagarajan Y.1, Shadiac N.1, Mertens H.2, Tyerman S.3, Sutton T.1 and Hrmova M.1 1Australian Centre for Plant Functional Genomics, University of Adelaide, Glen Osmond, SA 5064, Australia. 2Australian Synchrotron, SAXS/WAXS beamline, 800 Blackburn Road, Victoria, Australia. 3University of Adelaide, School of Agriculture and Wine, Glen Osmond, SA 5064, Australia. Boron, present in the form of boric acid in the soil has a narrow-range between deficiency and toxicity in plants. In order to tackle the problem associated with boron toxicity, a boron tolerant gene HvBot1 was identified in highly boric acid tolerant barley landrace ‘Sahara’ (1). This tolerant variety contains four times as many HvBot1 gene copies as intolerant varieties and produces elevated levels of the Bot1 gene transcript. The efflux of excess of boric acid in the form of borate ions from plants in tolerant varieties occurs through the involvement of a membrane-localised borate transporter HvBot1. To date, no work has been reported that describes the underlying molecular mechanism of borate transport by HvBot1 or by other related transporters. We report the functional and structural properties of HvBot1 synthesized through a wheat germ-based cell free system. To study the functional properties of HvBot1, the protein was prepared by co-translational insertion into a variety of liposomes and purified by density floatation (2). A transport assay was developed by using the pH dependent fluorescent BCECF [2’-7’-bis(carboxyethyl)-5(6)-carboxyfluorescein] dye and a stopped-flow Rayleigh light scattering. We observed an increase in the fluorescence signal due to transport of borate ions, which altered the pH of the liposomal lumen as a function of time, when compared to the HvBot1-free liposomes. The structural properties of HvBot1 were studied by small angle X-ray scattering, using synchrotron radiation to determine the size and low resolution shape properties of HvBot1. The data obtained in this work will answer fundamental questions as to how plant borate transporters mediate their function at a molecular level. References: 1. Sutton T, Baumann U, Hayes J, Collins NC, Shi, B-J, Schnurbusch T, Hay A, Mayo G, Pallotta M, Tester M, Langridge P (2007) Science 318:1446-1449. 2. Periasamy A, Shadiac N, Amalraj A, Garajová S, Nagarajan Y, Sampath R, Waters S, Mertens HD, Hrmova M (2012) Biochimica et Biophysica Acta - Biomembranes. In the Press.

CHARACTERIZATION OF THE ALVEOLATE MITOCHONDRIAL ATP SYNTHASE COMPLEX

Naqvi A.Z.1, 2, White I.1, Herbert J.D.K.1, Nisbet R.E.R.3 and Menz R.I.1 1Flinders University, Sturt Road, Bedford Park, South Australia. 2Department of Microbiology, University of Karachi, Karachi, Pakistan. 3Sansom Institute for Health Research, University of South Australia, Adelaide, Australia.

The Alveolata are a diverse range of organisms which include several human pathogens such as Toxoplama gondii and Plasmodium falciparum. Currently, very little is known about oxidative phosphorylation in these organisms. Oxidative phosphorylation is a potential drug target for these pathogens. In eukaryotes, electron transfer along the mitochondrial electron transport chain (mETC) creates a proton gradient which drives the rotary motor of the ATP synthase and allows the phosphorylation of ADP to ATP. The classical mitochondrial ATP synthase is split into the F0 domain (consisting of the c subunit ring and the stator (a, b, d, F6 and Oligomycin sensitivity conferring protein)) and the F1 domain (consisting of α, β, γ, δ and ε subunits). The F0 domain contains a proton pore at the interface of ‘a’ and ‘c’ subunits. Proton translocation causes rotation of the c subunit ring, driving the conformational changes of F1 required to phosphorylate ADP. Although alveolates have been shown to have functional ATP synthase, they lack all of the classical F0 subunits except subunit ‘c’. This suggests that these organisms contain novel proteins involved in proton translocation. These proteins may be good drug targets. Here, we describe the purification of ATP synthase from the model alveolate Tetrahymena thermophila and the characterization of the component subunits by tryptic digestion and mass spectrometry.

IDENTIFICATION AND CHARACTERIZATION OF BI-THIAZOLE-2,2’-DIAMINES AS KINASE INHIBITORY SCAFFOLDS

Ngoei K.R.1, Ng D.C.H.1, Fairlie D.P.2, Stoermer M.J.2 and Bogoyevitch M.A.1 1Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, Victoria 3010, Australia. 2Division of Chemistry and Structural Biology, Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland 4072, Australia.

Based on bioinformatics interrogation of the genome, >500 mammalian protein kinases can be broadly clustered within seven different groups. Of these kinases, the mitogen-activated protein kinase (MAPK) family forms part of the CMGC group of serine/threonine kinases that includes the extracellular signal regulated kinases (ERKs), the cJun N-terminal Kinases (JNKs), and the p38 MAPKs. With the JNKs considered attractive targets in the treatment of pathologies including diabetes and stroke, efforts have been directed to the discovery of new JNK inhibitory molecules that can be further developed as new therapeutics. Capitalizing on our biochemical understanding of JNK, we performed in silico screens of commercially available chemical databases to identify JNK1-interacting compounds and tested their in vitro JNK inhibitory activity. We showed that the compound, 4’-methyl-N~2~-3-pyridinyl-4,5’-bi-1,3-thiazole-2,2’-diamine (JNK Docking (JD) compound 123) inhibited JNK1 activity towards a range of substrates whereas a related compound (4’-methyl-N~2~-(6-methyl-2-pyridinyl)-4,5’-bi-1,3-thiazole-2,2’-diamine; JD124) did not inhibit JNK in vitro. Molecular docking and enzyme kinetic analyses revealed both ATP- and substrate-competitive inhibition of JNK by JD123. In characterizing JD123 further, we noted its ATP-competitive inhibition of the related p38-γ MAPK, but not ERK1, ERK2, p38-α, p38-β or p38-δ. Further screening of a broad panel of kinases using 10 μM JD123 identified inhibition of kinases including Protein Kinase Bβ (PKBβ/Aktβ). Appropriately modified thiazole diamines, as typified by JD123, thus provide a new chemical scaffold for development of inhibitors for the JNK and p38-γ MAPKs as well as other kinases that are also potential therapeutic targets such as PKBβ/Aktβ.

THE SRC HOMOLOGY 3 DOMAIN OF DIHYDROPYRIDINE RECEPTOR β1A INTERACTS WITH THE α1S II-III LOOP

Norris N.C., Karunasekara Y., Dulhunty A.F., Board P.G. and Casarotto M.G. JCSMR, ANU, Australia.

Excitation-contraction (EC) coupling in skeletal muscle requires association between the dihydropyridine receptor (DHPR), which is the L-type voltage-gated calcium channel located in the surface membrane, and the ryanodine receptor (RyR1) calcium release channel located in the sarcoplasmic reticulum calcium store. The C-region in the II-III loop of the α1s-subunit is essential for EC coupling. This suggests that the C-region communicates with RyR1, leading to calcium release and skeletal muscle contraction. However, the C-region interaction site on RyR1 is unknown. One unexplored possibility is that the C-region and RyR1 do not interact directly but communicate through an intermediate “messenger” protein. We hypothesize that the β-subunit of skeletal DHPR, β1a, is the messenger. The β1a-subunit is essential for EC coupling. It contains a Src Homology 3 (SH3) domain of unknown function. SH3 domains typically interact with other proteins by binding to polyproline motifs. The structure of the β1a-subunit has not been experimentally determined. However, the SH3 polyproline-binding site is occluded in all available structures of other β-isoforms, leading to the assumption that all β-isoforms, including β1a, cannot bind polyproline motifs. Based on a comparison of the β-isoform SH3 sequences, we predicted that key amino acid differences in β1a may allow binding to the DHPR α1s C-region. Here we use intrinsic protein fluorescence binding assays to demonstrate that the β1a SH3 domain binds to polyproline motifs located in the α1s C-region. One of these polyproline motifs has been shown to be essential in EC coupling. Notably, we show that the cardiac β2a-isoform does not bind the α1s C-region. Therefore, we propose that β1a could be a messenger from the α1s-subunit to RyR1 during EC coupling.


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PILOT PRODUCTION OF NOVEL RECOMBINANT BACTERIAL COLLAGEN FOR USE AS A TISSUE ENGINEERING SCAFFOLD

Peng Y.P., Stoichevska V.S., Howell L.H., Schacht K.S., Dumsday G.D., Werkmister J.W. and Ramshaw J.R. CSIRO Materials Science and Engineering, Clayton 3169, Australia.

Collagens have a characteristic triple-helical structure with a (Gly-Xaa-Yaa)n repeating sequence. Collagen has proven safe and effective in numerous medical products, and is frequently used in tissue engineering studies. Collagen is usually extracted from animals, but there is a concern of transmissible diseases. This introduces the potential use of recombinant collagens. However, recombinant mammalian collagens require the functional co-expression of prolyl 4-hydroxylase (P4H) to achieve collagen stability. Alternatively, collagen-like sequences have been characterised from several bacteria. These collagens lack hydroxyproline, yet form stable triple-helices at 35-38 C, and can be readily expressed in E. coli. In this study we have used the collagen-like protein Scl2 from S. pyogenes. We have previously shown that this collagen is non-immunogenic and non- cytotoxic, and can be fabricated into sponges suitable as tissue engineering scaffolds. This collagen has limited biological interactions so can be used as a blank slate into which selected functional domains, such as heparin or integrin binding, have been inserted. Also, multimeric constructs of the single Scl2 collagen-like domain have been constructed. The present study has shown that these various new constructs can be readily produced in 2L or larger fermentation and that initial yields of better than 6 g/L can be obtained in several cases. Functional studies confirm their suitability as tissue engineering scaffolds.

INVESTIGATIONS INTO THE EFFECT OF DIFFERENT EXPRESSION CONDITIONS ON THE LEVEL OF SOLUBLE TEV PROTEASE

Quinsey N.S. and Bottomley S.P. Protein Production Unit, Department of Biochemistry and Molecular Biology, Monash University.

The Tobacco etch virus (TEV) protease is a commonly used protease for the removal of the solubility tags attached to many recombinant proteins. It has a very sequence specific cleavage site, which makes it a very attractive protease to use for this cleavage event. However, the expression of a soluble form of TEV has slowed the development of this protease for many commercial applications. Major improvements in the solubility of this protein could be achieved with the introduction of a double mutation (L56V/S135G), without reducing the catalytic efficiency of the resulting mutant protein. Recently there have been a number of new medias and cell lines on the market that promise to either increase in the yield or the purity of the recombinant protein being produced. This work here will investigate the effects of these reagents on the crude soluble expression of TEV to determine if we can further increase the level of soluble protein expression.

HYBRID STRUCTURE-LIGAND-BASED DRUG DISCOVERY: CAN WE HAVE THE BEST OF BOTH WORLDS?

Roach M.J. and Menz R.I. Flinders University, Sturt Road, Bedford Park, South Australia.

Structure-based approaches to in silico drug discovery require knowledge of the 3D shape of a protein and involves ‘docking’ and scoring a chemical library to the receptor pocket. Ligand-based approaches require knowledge of the minimum necessary characteristics a molecule must possess for target recognition and searches a chemical library for ligands with these features. Both have their strengths and weaknesses. Structure-based approaches are incredibly more computationally expensive but are not limited to target proteins for which there are multiple known binders. They also have a greater potential for finding novel classes of inhibitors as they’re not limited by information derived from known binders. The aim is to combine both approaches in a way that utilises the speed of a ligand-based approach and the greater hit diversity and fewer requirements of a structure-based approach. A ligand-based model is built from structure-based ‘docking’ of a few dozen probe molecules and hence does not require knowledge of multiple known binders. The ligand-based model is used to pre-filter a chemical library, immensely reducing the total CPU time required. To date this approach has been tested on seven target enzymes that have previously been used in numerous validation studies. The ligand-based models have shown excellent results with an average enrichment of 65.37 at 0.5% and 40.5 at 1% when known actives were screened against the UCSF DUD decoy libraries. This new hybrid approach could theoretically screen a chemical library in the millions, on a regular PC in a matter of days.

CHARACTERIZATION OF VIRAL DNA REPLICATION, MRNA EXPRESSION LEVEL AND PROTEIN PRODUCTION OF BACULOVIRUS INFECTED SF9 CULTURES AT VARIOUS CELL DENSITIES

Huynh H.T., Tran T.T.B., Chan L.C.L., Nielsen L.K. and Reid S. Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, St Lucia, Qld 4072, Australia.

The drop in cell specific yield when infecting at high cell densities is the main limitation for enhancing the volumetric yield for baculovirus expression in insect cell systems. Despite this knowledge being available for many years, the evaluation of the cell density effect in terms of virus replication and mRNA expression in addition to foreign protein translation is still limited. In the current study, a wide range of 9 different cell densities at the time of infection (TOIs) from 0.5 to 8 x 106 cells/ml of Sf9 cells infected with a β-gal AcMNPV was investigated for β-gal yield, total viral DNA (vDNA) copies and β-gal mRNA expression. The results showed that a drop in cell specific β-gal yield appeared as early as the final cell density of 1.3 x 106 cells/ml with a gradual decline after that point. At the highest final cell density investigated (11.2 x 106 cells/ml); the specific β-gal yield was about 5% of that at 0.6 x 106 cells/ml. A similar rate of drop was observed for the β-gal mRNA expression levels, to about 10% of the highest value, while a drop in viral DNA copies to 50% of the peak value was also seen. It is suggested that the drop in β-gal yield with the increment of the final cell densities is largely a result of the drop in virus replication, leading to a reduction in β-gal mRNA expression.


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ESSENTIAL ROLE FOR THE 5β-SCYMNOL TRI-HYDROXY SIDE CHAIN IN HEPATOPROTECTION AGAINST PARACETAMOL TOXICITY IN SWISS MICE

Hodges L.1, Macrides T.1, Carter F.1, Glowacki L.1 and Wright P.2 1Natural Products Research Group, RMIT University, Australia. 2Toxicology Key Centre, RMIT University, Australia.

Paracetamol (APAP) overdose is a model of bioactivation, covalent binding and oxidative stress in hepatotoxicity. Sodium scymnol sulfate (3α,7α,12α,24,26-pentahydroxy-5β-cholestan-27-yl sodium sulfate) is the native salt of the shark bile alcohol 5β-scymnol (5β-cholestan-3α,7α,12α,24,26,27-hexol) and a major constituent of the Japanese liver tonic ‘deep sea liver oil’. Scymnol is protective of APAP-induced hepatotoxicity in mice, however the effectiveness of sodium scymnol sulfate is untested. In this study, exposure of male Swiss mice to a hepatotoxic dose of APAP (350 mg/kg, ip) increased serum hepatocellular enzyme activities, decreased hepatocellular glutathione (GSH) concentration and induced periacinar hepatocellular necrosis. Treatment by scymnol (single dose 70 mg/kg or multiple dose 5 mg/kg/7 days, ip) decreased significantly the extent of APAP-induced hepatotoxicity without inhibiting GSH depletion. Sodium scymnol sulfate, which lacks the tri-hydroxy side chain of scymnol, did not inhibit GSH depletion or protect against the toxicity. Scymnol reacted directly with NAPQI (the toxic metabolite of APAP), and inhibited cytochromes P450 (CYP1A2 and CYP2E1) and glutathione transferase (GST) activities in vitro, but at concentrations unlikely to be hepatoprotective in vivo. Scymnol, but not scymnol sulfate, was able to chelate iron ions when tested in monomeric concentration in solution. Octahedral complexing metal ions play a major role in exacerbating the mitochondrial oxidant stress that is invoked post APAP activation, and their chelation by scymnol may help explain its comparatively greater hepatoprotective effect in vivo. Together, these results provide further insight as to the hepatoprotective mechanism of scymnol, and in particular, the essential function of the tri-alcohol-substituted aliphatic side chain moiety in APAP-induced toxicity in male Swiss mice.

COMPREHENSIVE CELL HEALTH ANALYSIS ON A NOVEL MULTI-PARAMETRIC CELL ANALYSER

Gillis K.1, Khan A.1, Clor J.1, Tyagarajan K.1 and Noone C.2 1EMD Millipore, Hayward, CA. 2Merck Millipore, Kilsyth Victoria.

The study of anti-cancer compounds and cancerous cell lines requires access to cell health information that includes parameters such as cellular vitality, cell cycle and apoptosis and provides a complete picture of cell health. Traditionally, expensive and complicated instrumentation has been required for comprehensive cell health analysis. Here we present data from a novel, low-cost, easy to use, compact, high performance instrument, the Muse™ Cell Analyzer which provides multi-dimensional cell health information. The instrument utilizes a combination of highly simplified mix and read assay protocols with a touch-screen interface which greatly simplifies data acquisition and analysis. The objective of this study was to evaluate performance of Muse™ Count and Viability Assays when compared to traditional methods including hemacytometers and automated image based systems. Cytotoxicity effects of anti-cancer compounds such as on multiple cell types could be reliably obtained on the instrument platform. The combination of three critical assays for cell count and viability, apoptosis and cell cycle status on a simple low cost instrument makes the Muse™ Cell Analyzer a versatile and accurate solution for comprehensive cell health analysis.

OVER-EXPRESSION OF RCAN1 CAUSES DOWN SYNDROME-LIKE IMMUNE DYSFUNCTION

Martin K.R.1, Corlett A.1, Layton D.2, Seach N.2, Boyd R.L.2, Scott B.3 and Pritchard M.A.1 1Department of Biochemistry and Molecular Biology, Monash University. 2MISCL, Monash University. 3MIMR, Monash University.

Individuals with Down syndrome (DS) are more susceptible to infections, have a higher incidence of haemopoietic malignancies and are more likely to develop certain types of autoimmune disorders such as thyroiditis, coeliac disease and Type I diabetes. Thus DS appears to be a combination of immunodeficiency and immune dysfunction, but the underlying genetic mechanisms remain unresolved. RCAN1 (Regulator of calcineurin 1) is a gene located on human chromosome 21 that is over-expressed in DS, and has been shown to regulate calcineurin/NFAT signalling. We report that its up-regulation in mice replicates immunophenotypes observed in DS and that these are independent of calcineurin. RCAN1-TG mice display thymic and peripheral immune organ T cell developmental defects. Thymic cellularity is reduced by a substantial loss of mature CD4+ and CD8+ thymocytes and a concomitant loss of medullary epithelium. Mature T lymphocytes are reduced in number and exhibit reduced proliferative capacity and aberrant cytokine production. T cell defects are stem cell intrinsic as transfer of wild-type bone marrow into RCAN1-TG recipients restored medullary thymic epithelium and T cell numbers in the thymus, spleen and lymph nodes. However, bone marrow transplantation failed to improve T cell function, suggesting an additional role for RCAN1 in the non-haemopoietic stromal compartment. RCAN1 therefore plays an essential role in the processes that govern T cell development and function, and as such when over-expressed, may be a component of immune dysfunction in DS.

UNDERSTANDING THE GENETIC BASIS OF TOLERANCE TO LOW PH AND SO2 IN S. CEREVISIAE

Schmidt S.A.1, Li J.1, 2, Kolouchova R.1, Borneman A.R.1, Henschke P.A.1 and Chambers P.J.1 1The Australian Wine Research Institute, Adelaide, Australia. 2China Agricultural University, Bejing, China.

Saccharomyces cerevisiae is the transformative microorganism in the production of wine from grape juice. There is a multitude of stresses encountered by yeast upon inoculation into grape juice. Two of these are low juice pH and the presence of antioxidant/antimicrobial sulphur dioxide (SO2). Grape juice typically has a pH of between 3 and 4. Within this range SO2 exists in equilibrium between the active molecular form (SO2) and the bisulphite anion (HSO2

3-), tending increasingly to the molecular form as conditions become more acidic. Therefore these two stresses can interact to negatively affect yeast growth. As a consequence commercial yeast strains need to be robust in relation to these conditions. Through examination of grape juice composition and subsequent analysis of the contribution of individual elements to fermentation performance of different yeast strains we identified pH as a key determinant of strain dependent fermentation performance. In this work we are uncovering how juice SO2 concentrations can interact with juice pH to influence not only fermentation performance but dramatically impact on cell viability. The degree to which viability is affected is a yeast strain dependent phenomenon. We have used a wine-yeast gene-deletion collection, in continuous culture competitive fitness assays to determine the genetic basis for pH and SO2 tolerance.. The relative fitness of mutants within mixed populations was assessed using an Illumina multiplex barcoding strategy. In this way we have been able to map the contributions of different genes to yeast strain robustness and to better understand how pH and SO2 interact to exert their effects.


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LOSS OF CASPASE-2 RESULTS IN INCREASED OXIDATIVE STRESS AND EARLY ONSET OF AGEING IN MICE

Shalini S., Dorstyn L., Wilson C., Puccini J. and Kumar S. Centre for Cancer Biology, SA Pathology, Frome road, Adelaide, SA 5000, Australia.

Caspase-2 is one of the first mammalian caspases to be identified however its physiological role remains controversial. Our recent studies using caspase-2 knockout (Casp-2-/-) mice and cells implicate this caspase in DNA damage and genomic instability, tumour suppression and ageing1,2. We found that Casp-2-/- mice have a shorter lifespan, show early hair greying, reduced fat and increased bone loss that are all indicative of early ageing3. Aged Casp-2-/- mice show enhanced oxidative stress accompanied by reduced activity of antioxidant enzymes and increased DNA damage. Interestingly, in the aged Casp-2-/- mice expression of FoxO family members (FoxO1 and FoxO3a) was significantly reduced. Oxidative damage is one of the key factors that accounts for ageing. Our study clearly demonstrates that increased DNA damage and oxidative stress associated with caspase-2 deficiency are the causal factors leading to early onset of ageing related traits in Casp-2-/- animals. However the mechanism by which caspase-2 regulates oxidative stress remains unclear. Currently we are using proteomics approaches to identify global changes in the total proteome of young and aged wild-type and Casp-2-/- mice and potential caspase-2 targets. This will help us identify caspase-2 targets involved in its recently discovered roles in tumour suppression, oxidative stress response and ageing. 1. Kumar S (2009) Nat Rev Cancer 9, 897-903. 2. Dorstyn L, Puccini J, Wilson CH, Shalini S, Nicola M, Moore S, Kumar S (2012) Cell Death Differ. In Press. 3. Shalini S, Dorstyn L, Wilson C, Puccini J, Ho L, Kumar S (2012) Cell Death Differ. In Press.

REGULATION OF RAB11 AND EXOCYTOSIS OF IMMUNE RESPONSE MEDIATORS

Sorvina A., Brooks D.A., Bader C. and Shandala T. Sansom Institute for Health Research, University of South Australia, Adelaide SA5000, Australia.

The coordinated response of the innate immune system to the invasion of pathogenic microorganisms is mediated by secretion of immune-mediators, including cytokines (e.g. tumor necrosis factor alpha, interferon-gamma etc.) and antimicrobial peptides. Altered regulation of secretion has been implicated in autoimmune syndromes and immune-deficiency disorders, such as Griscelli, Hermansky-Pudlak and Chediak-Higashi syndromes. Within the cell, these immune-mediators are included into specialist exocytic vesicles for delivery to the plasma membrane. The plasma membrane tethering of exocytic vesicles is orchestrated by complex molecular machinery including, for example Rab11 small GTPase. The latter cycles between its active GTP-bound form, and inactive GDP-bound form, which is thought to be regulated by guanine nucleotide exchange factors and GTPase activating proteins, respectively. While for vertebrates, these guanine nucleotide exchange factors and GTPase activating proteins for Rab11 GTPase have been predicted by recent yeast two-hybrid and biochemical studies, their role in regulation of intracellular Rab11 function is yet to be proven. Many of these proteins are evolutionary conserved in Drosophila. Moreover, in Drosophila fat body cells, we showed that the intracellular localization of Rab11 depends on its GTP- or GDP-binding status, where a constitutively active form of Rab11 (YFP-Rab11Q70L) is preferentially found near the plasma membrane, and its inactive form (YFP-Rab11S25N) remains in the peri-nuclear zone. Using this Drosophila model, we are undertaking genetic and cell biological analysis to identify a role for these proteins in regulating Rab11 function in vivo. The regulation of Rab11 vesicles and their cargo has direct significance for innate immune function, particularly for the exocytosis of antimicrobial peptides.

AN ONCOGENIC ROLE OF CALCIUM AND INTEGRIN BINDING PROTEIN 1 VIA MODULATION OF SPHINGOSINE KINASE 1

Gliddon B.L., Jarman K.E., Moretti P.A.B. and Pitson S.M. Centre for Cancer Biology, SA Pathology, Frome Rd, Adelaide, SA, 5000.

Sphingosine kinase 1 (SK1) catalyzes the formation of the bioactive lipid sphingosine 1-phosphate, and has been implicated in a number of biological processes in mammalian cells, including enhanced proliferation, inhibition of apoptosis, and oncogenesis. We have previously examined the molecular mechanisms regulating SK1 and found that its re-localisation from the cytoplasm to the plasma membrane is essential for oncogenic signalling by this enzyme. We have now established the mechanism whereby this agonist-dependent translocation of SK1 occurs by identifying a calcium-myristoyl switch protein CIB1 (calcium and integrin binding protein 1) that directly mediates this process. Furthermore, we found that CIB1 is overexpressed in a range of human cancers, and its overexpression can enhance plasma membrane localisation of SK1, enhance cell proliferation, and induce neoplastic transformation of cells in a SK1-dependent manner. Thus, CIB1 represents a key regulator of SK1 and is a potential target for therapeutic intervention in cancer.

ALTERED EXPRESSION OF THE PROTEIN HANDLING COMPLEX AFTER ACUTE EXPOSURE TO LEPTIN

Grinfeld E., McAinch A.J. and Hryciw D.H. Victoria University.

In obese individuals, elevated leptin is filtered through the glomerulus, and leptin is processed in the proximal tubule cells by the scavenger receptor megalin. Megalin is part of an endocytic protein complex that is required for the uptake of a number of ligands including albumin. Research studies have indicated that the C-terminus of megalin can act as a signal transducer in renal tubules. As elevated albumin induces fibrosis via transforming growth factor-β1 (TGF-β1), elevated leptin levels seen in the obese may also bind to megalin to upregulate fibrotic pathways via this cytokine. We investigated the effect of acute leptin exposure on the level of expression of megalin and the endocytic protein complex; the Chloride channel 5 (ClC-5), Na+/H+ exchanger 3 (NHE3), NHE regulatory factor 1 (NHERF1) and NHERF2, in addition to TGF-β1, an indicator of fibrosis. Opossum kidney (OK) proximal tubule cells were exposed to physiological (0.05 and 0.1 μg/ml) and pathophysiological (0.25 and 0.5 μg/ml) of leptin for 2 hours. mRNA and Protein was isolated and investigated by Real time PCR and Western blot analysis. At all leptin concentrations, megalin, NHE3, ClC-5, NHERF1 and NHERF2 mRNA was unaltered. ClC-5, NHE3 and NHERF1 protein levels were also unchanged. At increasing leptin concentrations, NHERF2 protein significantly increased compared to control. Megalin protein significantly decreased at all leptin concentrations compared to control, however when comparing physiological and pathophysiological concentrations there were no changes in megalin protein expression. Finally, real time PCR analysis of TGF-β1 expression indicated that at pathophysiological concentrations of leptin, fibrotic pathways are activated. Thus, acute exposure to leptin acts on specific cellular targets in proximal tubule cells.


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DUAL NUCLEAR IMPORT MECHANISMS OF SEX DETERMINING FACTOR SRY; INTRACELLULAR CA2+ AS A SWITCH

Kaur G. and Jans D.A. Nuclear Signalling Laboratory, Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia.

The critical role of the chromatin remodelling factor SRY (Sex determining region on the Y chromosome) during foetal development is dependent on two nuclear localisation signals (NLSs) that flank the DNA binding high mobility group (HMG) domain; the β-NLS and the CaM-NLS which mediate nuclear transport through the conventional transport molecule Importin β1 (Impβ1) and the calcium-binding protein calmodulin (CaM), respectively. We reconstituted nuclear import mediated by the two NLSs in vitro, establishing Ran-independence of CaM-NLS-dependent transport. The β- and CaM-NLSs were found to be independently functional out of the context of the SRY HMG domain, dependent on Impβ1 and CaM binding, respectively. Intriguingly, direct protein binding assays also indicated competitive binding of Impβ1 and CaM to the SRY HMG domain. To assess the potential role of intracellular calcium in modulating SRY nuclear accumulation, Cos-7 cells expressing SRY and control constructs were treated with agents elevating or reducing intracellular Ca2+ levels. The in vivo results, supported by experiments in vitro where transport was assessed with/without 2μM Ca2+, indicate a Ca2+-dependent mode of nuclear transport via the CaM-NLS/CaM, with inhibition of β-NLS/Impβ1 mediated nuclear import by intracellular Ca2+. The results imply a novel mechanism of mutual exclusivity of nuclear transport via the two NLSs with intracellular Ca2+ as the switch between the two.

SEROTONIN 2B RECEPTOR ACTIVATES TRPC6 CHANNEL VIA GαQ-PLC-DEPENDENT PATHWAY IN PELVIC GANGLION NEURONS

Cha S.K.1, Kim J.H.1, Park K.S.1, Jeong S.W.1, Yeh B.Y.2 and Kong I.D.1 1Department of Physiology, Yonsei Univ. Wonju College of Medicine 162 Ilsan-Dong Wonju Republic of Korea. 2Department of Biochemistry, Yonsei Univ. Wonju College of Medicine 162 Ilsan-Dong Wonju Republic of Korea.

Serotonin 2B receptors (5-HT2BRs) expressed in pelvic ganglion neurons. However, the cellular mechanisms underlying the effect of 5-HT to regulate neuronal activity via 5-HT2BRs have not yet been identified. In this study, we examined modulation of intracellular Ca2+ ([Ca2+ ]i) by 5-HT2BRs in male rat major pelvic ganglion (MPG) neurons. RT-PCR analysis revealed that MPG neurons express several subtypes of serotonin receptors including Gαq-coupled 5-HT2BR. BW723C86, a specific 5-HT2BR agonist, increased intracellular [Ca2+ ]i, which was prevented by SB204742, a 5-HT2BR antagonist. 5-HT2BR-mediated increase of [Ca2+ ]i was inhibited by the phospholipase C (PLC) inhibitor U73122 but not by its inactive analogue, U73343. 5-HT2BR-mediated [Ca2+ ]i increase was blocked by canonical transient receptor potential (TRPC) channel blockers such as SKF96365, La3+ and Gd3+, indicating the involvement of TRPC channels. In addition, BW723C86 evoked an inward TRPC-like current which was blocked by a La3+. TRPC1 and TRPC6 channels were expressed in MPG neurons in RT-PCR analysis. We confirmed that 5-HT2BR activated TRPC6 but not TRPC1 using HEK293 cells coexpressing 5-HT2BR and TRPC6 or TRPC1 channel. The 5-HT2BR-mediated activation of TRPC6 current was inhibited by PLC inhibitors and RGS4, a protein that blocks signaling via Gαq. Taken together, our data suggest that 5-HT2BR activates TRPC6 channel via Gαq-PLC-dependent pathway and may, at least partly, contribute to regulation of in ganglionic transmission.

NON-CLASSICAL ACTIONS OF ANDROGEN RECEPTOR

Mann N.K1, Peet D.J.1, Ivell R.1, 2 and Anand-Ivell R.2 1University of Adelaide, Adelaide, Australia. 2Institute of Farm animal research, Dummerstorf, Germany.

The Androgen Receptor (AR), which belongs to the superfamily of nuclear receptors, mediates the actions of androgens in the cells. The genomic or “classical” pathway of AR signaling has been studied in extenisvely where it regulates target gene expression in response to androgens by translocating to the nucleus, binding Androgen Response Elements (AREs) in DNA and recruiting other transcription factors. But not all the actions of the AR can be explained by this pathway, and in the last two decades a lot of studies have been focussed on the non-genomic or “non-classical” actions of the AR. In non-classical AR-mediated signalling, either AR does not translocate to the nucleus and mediates cytoplasmic signalling, or it mediates nuclear signalling but does not bind to the DNA directly via AREs. This project has focussed on the androgen-dependent regulation of the oxytocin gene, which lacks a classical ARE. We show that the oxytocins gene is regulated by androgens in an AR-dependent manner, with a distinct dose response to classically regulated AR-regulated genes. The requirement for nuclear localised AR is demonstrated by the inability of an AR with a mutated NLS (ARΔNLS) to activate the oxytocins promoter. In contrast, an AR DNA binding domain mutant (ARΔDBD) activates the oxytocins promoter with similar activity to wild type AR, confirming regulation independent of direct DNA binding. Deletion analysis of the oxytocins promoter demonstrates AR-mediated regulation is mediated by a 20 bp fragment including putative sites for FoxA1 and Sp1 transcription factors, both of which have been implicated in non-classical AR signalling. Current experiments are directly addressing the role of FoxA1 and Sp1.

USING ZEBRAFISH TO IDENTIFY FACTORS INVOLVED IN NEUROGENESIS AND BRAIN REGENERATION

Kaslin J., Wikhe K., Tang J. and Colquhoun D. Australian Regenerative Medicine Institute, Level 1, Building 75,Monash University Clayton Campus, Wellington road, Clayton, Victoria 3800, Australia.

The mammalian brain has a limited regenerative capacity. In contrast, zebrafish display widespread life-long neurogenesis and can regenerate parts of its CNS. Yet, the cellular and molecular mechanisms that allow brain regeneration in zebrafish are poorly understood. We have studied the zebrafish regenerative capacity by using telencephalic and cerebellar lesions as models. We show that most cell types are restored following a traumatic lesion. However, the regenerative capacity is linked to adult neurogenesis i.e. only cell types that are homeostatically produced in the adult brain regenerates well. Interestingly, it seems like that the zebrafish brain provides a permissive environment for regeneration because we do not detect scarring or a persisting inflammation. Furthermore, we have identified genes directly involved in regulating the regenerative response in zebrafish by micro-array analysis. In summary, zebrafish provides exciting potential as a new powerful model for studying brain regeneration and neuronal stem cell diversity in the vertebrate brain.


POSTERS TUESDAY



DIFFERENTIATION OF MULTIPOTENT STEM CELLS DERIVED FROM THE EPIBLAST OF POSTIMPLANTATION MOUSE EMBRYOS ON 3D SCAFFOLDS

Kaufman-Francis K., Kojima Y., Studdert J.B., Steiner K.A., Power M.D., Loebel D.A.F. and Tam P.P.L. Embryology Unit, Children’s Medical Research Institute, Locked bag 23, Wentworthville, NSW 2145, Australia.

The generation of transplantable pancreatic β cells in vitro is a major goal of cell-based therapy for type 1 diabetes. This is currently hampered by our poor understanding of the necessary environment for pancreatic lineage differentiation. The hypothesis for this study is that three-dimensional ECM-enriched microenvironment will promote efficient growth and differentiation of stem cell-derived pancreatic tissue. To test our hypothesis we reconstructed the pancreatic niche in vitro, by utilizing biodegradable polymeric scaffolds to establish a three-dimensional culture system. These scaffolds may provide an appropriate 3D matrix architecture that mimics the nature tissue environment, for progenitor cells to undergo pancreas differentiation and organogenesis. Epiblast stem cells (EpiSCs), which were derived from epiblast of postimplantation mouse embryo at different stages of germ layer formation, were cultured on the scaffolds and compared with the outcome of cultures on a planar (2D) surface. Our results revealed significant differences in differentiation capacity of the EpiSCs in general and particularly in generating definitive endoderm progenitors between culture on 2D substrate and 3D polymer scaffolds. The 3D microenvironment further allows the generation of a more complex tissue organization and the differentiation of the EpiSCs as they are directed by factors that promote pancreatic differentiation. These experiments are the first steps towards the goal of engineering a defined synthetic environment that will facilitate the in vitro production of pancreatic β cells.

CHARACTERISTICS OF MULTIPOTENT STEM CELLS DERIVED FROM THE EPIBLAST AND ECTODERM OF POSTIMPLANTATION MOUSE EMBRYOS

Kojima Y., Studdert J.B., Steiner K.A., Kaufman-Francis K., Power M.D., Loebel D.A.F. and Tam P.P.L. Children’s Medical Research Institute, Locked bag 23, Wentworthville, NSW 2145, Australia.

Epiblast stem cells (EpiSC) are multiipotent cells that have been derived from epiblast of post-implantation mouse embryo at stages prior to and during germ layer formation. It is not known at which advanced developmental stage that EpiSC-like cells can still be derived from, and if the EpiSC derived from later stages have common characteristics with the conventional EpiSC. In this study, epiblast or ectoderm from a wide range of developmental stages defined by stringent morphological criteria were tested in an identical culture condition for the ability to generate EpiSC. Over 40 EpiSC lines were derived from epiblasts of pre-gastrula stage up to as advanced as from ectoderms of neural plate stage. All the lines displayed similar morphology and expressed pluripotency markers, and when injected to immunodeficiency mice, teratomas were formed. Transcriptome analysis revealed that these lines showed gene expression profiles comparable to established EpiSC, and by the comparison of transcriptome from dissected epiblasts/ectoderm of various stages, all EpiSC lines were most closely related to the epiblasts during germ layer formation. The lineage potential of the EpiSC was tested by in vitro differentiation as embryoid bodies (EB). When grown as EB in serum-containing medium, EpiSC could initiate differentiation into endoderm lineage earlier than embryonic stem cells (ESC) but their potency varied considerably. Ongoing analysis comparing the transcriptome of EpiSC to dissected epiblasts of corresponding stages and to their differentiation potency will reveal the relationship between EpiSC and the epiblast from which they were derived and shed further light on the mechanisms of pluripotency maintenance and lineage commitment.

NEURAL CREST CELL MIGRATION, A LITTLE LESS PER-PLEXIN

Lumb R., Kabbara S., Scherer M. and Schwarz Q. Centre for Cancer Biology.

The migration of neural crest cells is vital to the development of the sensory and sympathetic nervous system, cranial bones and tendons and pigment cells of the skin. Neuropilin / Sema3 signalling has recently been shown to be essential in the guidance of neural crest cells, ensuring they reach target destinations for differentiation within the vertebrate embryo. Due to the absence of a functional cytoplasmic domain Neuropilins are required to interact with a co-receptor to confer downstream signalling. However, the co-receptor(s) Neuropilins use to transmit downstream signalling in neural crest cell guidance is unknown. Here, A-type Plexins have been investigated as potential Neuropilin co-receptors in neural crest cell migration. Using in-situ hybridisation and immuno-fluorescence, we show for the first time that A-type Plexins are expressed in an overlapping pattern within the cranial neural crest cells and in independent patterns within trunk neural crest cells. To further elucidate the function of A-type Plexins, vectors that target and knock down of single or multiple A-type Plexins have been developed. These vectors pave the way for investigations into the role of A-type Plexins not only within NCC migration, but within neural development, angiogenesis, immune responses and within tumour development and proliferation.

THE HISTONE VARIANT H3.3 IS REQUIRED FOR FERTILITY IN THE MOUSE

Tang M.1, Jacobs S.A.1, O’Bryan M.K.2, Wong L.2 and Mann J.R.1 1Murdoch Childrens Research Institute. 2Monash University

Histones package DNA and regulate chromatin structure and function through post-translational modifications (PTMs) to residues in their N-terminal tails, these tails probably being the most conserved peptide sequences in all of life. For example, the most important histone in terms of PTMs is histone H3 in which the tri-methylation of lysine 9 (H3K9me3) is an essential waypoint in heterochromatin formation. Canonical histone H3 (H3.1 and H3.2) is incorporated into chromatin during S phase only. We thought that the variant H3.3, which can be incorporated into chromatin at any phase of the cell cycle, could be especially important in germ cell development. This is because the germ line undergoes large-scale epigenetic change when non-dividing, and at this stage can only incorporate H3.3. To investigate this possibility, we made null mutations in each of the two unlinked genes encoding histone H3.3—H3f3a and H3f3b. For H3f3a, homozygous mutants were essentially viable and fertile. This was surprising, as a hypomorphic gene-trap mutation of this gene was previously shown to cause high-level neonatal mortality. Possibly this was due to dysregulation of H3.3 induced by the gene trap sequences rather than H3.3 deficiency. For H3f3b, we found that heterozygous mutants were small in size. Females were fertile, but males were infertile due to breakdown in the formation of haploid spermatids. Due to this defect, it is not possible to breed homozygous mutants. These results establish a functional role for H3.3 in fertility in the mouse in which there is a stringent requirement for full H3f3b activity.


POSTERS TUESDAY



DO EXPANDED POLYALANINE TRACT MUTATIONS IN ARX ALTER INTERACTION WITH THE NOVEL PROTEIN PARTNER UBQLN4 AND DISRUPT DEGRADATION BY UBIQUITIN-PROTEASOME PATHWAY?

Mattiske T.1, Gecz J.1, 2 and Shoubridge C.1, 2 1Neurogenetics, SA-Pathology, SA 5006, Australia. 2Department of Paediatrics, University of Adelaide, SA 5005, Australia.

Aristaless related homeobox (ARX) is a paired-type homeodomain transcription factor with critical roles in embryonic development. Mutations in ARX are frequent and give rise to non-syndromic intellectual disability, infantile spasms and brain malformation syndromes. ARX has four polyalanine tracts and mutations of the first two account for over half of all reported cases. To identify proteins interacting with ARX we used yeast-2 hybrid assay and identified ubiquilin 4 (UBQLN4) interacting with the region of the polyalanine tracts 1 and 2. UBQLN4 protein contains a Ubiquitin-like and Ubiquitin associated domains and acts as proteasome-substrate adaptor or carrier. We have investigated protein-protein interactions of UBQLN4 and ARX with expanded alanine tracts 1 and 2 and demonstrate that in HEK293T cells recombinant UBQLN4 localised to aggregates containing mutant ARX protein. Co-immunoprecipitation experiments show that while the interaction of mutant ARX and UBQLN4 is maintained, it is at a decreased level compared to the wild-type ARX. We are currently investigating if proteasomal degradation of mutant ARX is altered using a pulse chase assay approach. The ARX- UBQLN4 interaction may link the production of ARX to its efficient degradation by proteasome to ensure tight control of this transcription factor. We postulate that this fine regulation is affected as a consequence of at least polyalanine tract 1 and 2 expansion mutations. The resulting changes to ARX protein turnover are likely contributing if not driving the molecular pathogenesis of these mutations, which also present as clinically highly pleiotropic, ranging from mild intellectual impairment to severe epilepsy with intellectual disability.

SPECIES-DEPENDENT EFFECTS OF VITAMIN D METABOLITE 1, 25 (OH)2D3 DURING OSTEOGENIC DIFFERENTIATION OF MESENCHYMAL STEM CELLS IN VITRO

Pande V.1, Martin E.1, Croom S.1, Chousalkar K.2, Bhanugopan M.1 and Quinn J.1 1School of Animal & Veterinary Science Charles Sturt University, Wagga Wagga NSW, 2678, Australia. 2School of Animal and Veterinary Sciences, the University of Adelaide, Roseworthy, AUSTRALIA 5173.

Leg disorders in broilers are an important welfare and economic issue for the poultry industry. Vitamin D deficiency is a major cause of leg abnormalities in poultry. The use of supplementary dietary vitamin D3 metabolite 1, 25 (OH)2D3 to combat bone disorders in growing and adult birds is well documented. However, there is paucity of literature on how Vitamin D3 modulates osteogenesis in species other than rodents and humans and therefore how it is affecting bone growth in avian species. We have examined the effects of 1, 25 (OH)2D3 on osteoblast differentiation using rat, horse and chick mesenchymal stem cells (MSCs). MSCs were subjected to a standard osteogenic differentiation protocol in the absence of, or presence of low (2.4 nM) and high (24 nM) concentrations of 1, 25 (OH)2D3. We found that high doses of 1, 25 (OH)2D3 inhibited calcium deposition during osteogenic induction of MSCs in all species examined. However, mineralisation was evident in chick MSCs cultures in a dramatically shorter timeframe than in mammalian counterparts. To conclude, avian systems are highly sensitive to mineralisation indicating underlying developmental differences. Moreover, high concentrations of 1, 25 (OH)2D3 are detrimental to normal skeletal development in all species. Therefore, optimal species-specific concentrations need to be established in vivo for all species individually to determine critical levels of supplementary dietary Vitamin D3 for optimal bone health.

THE MIR-15 FAMILY REGULATES POSTNATAL CARDIAC REGENERATIVE CAPACITY IN MAMMALSPorrello E.R.1, 2, Mahmoud A.I.3, Simpson E.2, Kocabas F.3, Grinsfelder D.3, Canseco D.3, Mammen P.P.3, Rothermel B.A.2, 3, Olson E.N.2 and Sadek H.A.3 1School of Biomedical Sciences, The University of Queensland, Brisbane, Australia. 2Department of Molecular Biology, UT Southwestern Medical Center, Dallas, USA. 3Department of Internal Medicine, UT Southwestern Medical Center, Dallas, USA.

The inability of the adult mammalian heart to regenerate following injury represents a substantial barrier in cardiovascular medicine. We recently identified a brief window during post-natal development when the mammalian heart retains significant regenerative potential following amputation of the ventricular apex. However, one major unresolved question is whether the neonatal mouse heart can also regenerate in response to myocardial ischemia, the most common antecedent of heart failure in humans. Here, we induced ischemic myocardial infarction (MI) in 1-day-old mice by permanent ligation of the left anterior descending coronary artery, and found that this results in extensive myocardial necrosis and systolic dysfunction at day 3 post-MI. Remarkably, the neonatal heart mounted a robust regenerative response resulting in full functional recovery within 21 days. Neonatal heart regeneration was associated with a robust induction (6-fold) of cardiomyocyte proliferation. Genetic lineage tracing of cardiomyocytes from Myh6-MerCreMer transgenic mice demonstrated that the majority of regenerated cardiomyocytes following neonatal MI were derived from pre-existing cardiomyocytes rather than from a stem cell population. The molecular mechanisms that regulate neonatal cardiac regenerative capacity are not understood but we have recently identified a large family of microRNAs, known as the miR-15 family, which regulate postnatal mitotic arrest of cardiomyocytes. Transgenic over-expression of miR-195, a member of the miR-15 family, was sufficient to impair the neonatal cardiac regenerative response and was associated with increased fibrosis and reduced cardiomyocyte proliferation. Finally, pharmacological inhibition of the miR-15 family by administration of locked nucleic acid-modified antimiRs from an early post-natal age until adulthood induced myocyte proliferation in the adult heart and improved left ventricular systolic function following ischemia-reperfusion injury. These findings indicate that the neonatal mammalian heart can regenerate following myocardial infarction and that the miR-15 family contributes to postnatal loss of cardiac regenerative capacity in mammals.

ROLE OF ATCIPK16 IN PLANT SALINITY TOLERANCE

Huang W., Tester M. and Roy S. Australian Centre for Plant Functional Genomics.

Soil salinity is a significant environmental problem affecting agriculture around the world leading to reduced crop yield. High concentrations of Na+ affect cell metabolism and compete with K+ for the binding sites of enzymes which play important roles in cellular function. One mechanism for improving salinity tolerance of crop plants is to minimise the accumulation of Na+ in the shoot. AtCIPK16 (Calcineurin B-like-interacting protein kinase 16) has been identified as a novel candidate gene important in increasing salinity tolerance. Over-expression of AtCIPK16 has been shown to reduce shoot sodium in a number of species. In both hydroponic and soil culture, Arabidopsis with constitutive over-expression of AtCIPK16 show significant reductions in Na+ concentration in shoot, compared with wild type and nulls, while Arabidopsis with amiRNA knockdown of AtCIPK16 exhibit an increase of Na+ concentration in shoot. While it can be clearly seen that alterations in the expression of AtCIPK16 result in increased salinity tolerance, little is known, however, about the role the protein plays in tolerance mechanisms. Therefore, AtCIPK16’s downstream targets and its cellular location needs to be investigated to elucidate its role in plant salinity tolerance. Yeast two hybrid systems were used to confirm the potential upstream CBL partners of AtCIPK16. Split YFP was used in both transient expression in protoplasts and stable expression to further identify interacting partner of AtCIPK16 and cellular location of their complex. Progress on antibody pull-down assays, to identify the downstream target of AtCIPK16, will also be presented.


POSTERS TUESDAY



CAN SUCROSE TRANSPORTER OVEREXPRESSION INCREASE MICRONUTRIENT ACCUMULATION IN RICE GRAINS?

Huynh M., Jenkins C.L.D., Anderson P.A. and Stangoulis J.C.R. School of Biological Sciences, Flinders University, SA, 5042.

In cereals, the sucrose transporter SUT1 is highly expressed in the grain aleurone layer and its expression closely follows the pattern of assimilate uploading during grain filling. Zinc and iron form complexes with compounds in the phloem and follow a similar pathway as sucrose entering the grain. This study is examining a novel approach to increase micronutrients in cereals by manipulating grain uptake capacity to create a stronger sink for nutrients. Overexpression of barley HvSUT1 in wheat endosperm resulted in a higher capacity for sucrose uptake, leading to higher grain protein content due to altered storage product gene expression1. Increased sink strength and higher protein content in grains has been shown to correlate positively with micronutrient levels. We therefore hypothesize that plants that overexpress SUT1 in rice endosperm tissue will produce grains with higher iron and zinc levels. To test this hypothesis, rice plants have been transformed using Agrobacterium to integrate barley HvSUT1 driven by the rice globulin promoter, Glb-1, to target overexpression of a sucrose transporter specifically in the endosperm. Preliminary data on transformed rice lines will be presented. This work provides a platform for future detailed analysis of altered gene expression and storage product and micronutrient concentration and distribution in rice grains from selected GlbHvSUT-overexpressing transgenic rice lines.

1Weichert, N., Saalbach, I., Weichert, H., Kohl, S., Erban, A., Kopka, J., Hause, B., Varshney, A., Sreenivasulu, N.,

Strickert, M., Kumlehn, J., Weschke, W. and Weber, H. (2010) Increasing Sucrose Uptake Capacity of Wheat Grains

Stimulates Storage Protein Synthesis, Plant Physiology, 152(2):698-710..

CALCIUM ENHANCES THE GUANYLATE CYCLASE ACTIVITY OF THE MOONLIGHTING ENZYME PSKR1 BUT INHIBITS KINASE ACTIVITY IN VITRO

Muleya V.1, Wheeler J.I.1, Kwezi L.2, Ruzvidzo O.2, Gehring C.3 and Irving H.R.1 1Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, VIC 3052, Australia. 2Department of Biological Sciences, North-West University, Private Bag X2046, Mmabatho, 2735, South Africa. 3Division of Chemistry, Life Sciences and Engineering, 4700 King Abdullah University of Science and Technology, Thuwal 23955-6900, Kingdom of Saudi Arabia.

Calcium is a universal signalling molecule that interacts with the second messenger cGMP formed by guanylate cyclases (GCs) to mediate many physiological plant responses. Previously we identified a novel class of receptor-GCs containing the GC catalytic centre embedded within the kinase domain including the phytosulfokine receptor AtPSKR1. We showed that the recombinant cytoplasmic domain (amino acids 681 to 1008) of AtPSKR1 has both guanylate cyclase and kinase activity in vitro. We used site directed mutagenesis to show that G922K but not G922E in the GC catalytic centre, was essential for GC activity (Kwezi et al. JBC 286: 22580-8, 2011). Here we report that the GC activity of these recombinant proteins is enhanced at physiological levels of calcium whereas the same calcium treatments inhibit and abolish kinase activity. In addition PSKR1 kinase activity is reduced in the presence of the GC product cGMP and preliminary experiments indicate that this is correlated with decreased phosphorylation of serine residues. Our results suggest that the presence of free calcium ions is required for optimal GC activity. In addition, the products of the dual enzymatic functions of AtPSKR1 appear to directly affect the opposing enzymatic action indicative of mutual autoregulation. The challenge now lies in understanding interactions between the GC and kinase domains at the molecular level and how these receptors capitalize on their dual functionality in planta.

NITROGEN ISOTOPE DISCRIMINATION PREDICTABLY CHANGES AMONG HYDROPONICALLY GROWN KNOCKOUT LINES OF ARABIDOPSIS THALIANA

Kalcsits L. and Guy R.D. Department of Forest Sciences, University of British Columbia, 2424 Main Mall, Vancouver, BC, Canada V6T1Z4.

The nitrogen isotope composition of plants has potential to act as a time-integrated measure reflecting nitrogen uptake, assimilation and allocation in plants. Here, we report genotypic manipulation of nitrogen isotope discrimination in plants using T-DNA and gamma-ray mutant lines of Arabidopsis thaliana Col-0. Nitrate Reductase 2 (NR2), Nitrate Reductase 1 (NR1) and Nitrate Transporter 2.1-2 (NRT2.1-2) mutant lines and the Col-0 wild type were grown hydroponically under steady-state NO3

- conditions at either 100 or 1000 μM NO3- for 35 days. Using

a model explaining whole-plant and intraplant fractionation of nitrogen isotopes, calculated nitrogen uptake, assimilation and allocation dynamics in the mutant lines were compared to the wild type. NR2 showed an increased proportion of assimilation of NO3

- in leaves while NR1 showed an increased proportion of nitrogen assimilation in roots. These observations are consistent with expression of the NR1 gene predominantly in leaves and the NR2 gene predominantly in roots. NRT2.1-2 showed an overall reduction in NO3

- assimilation and an increased partitioning of NO3

- assimilation to the roots, presumably because of decreased symplastic intercellular movement of NO3

- in the root. This reduced symplastic intercellular movement also resulted in an increase in efflux or leakage across the root cell membranes. By restricting expression of genes critical for either nitrate assimilation or uptake, phenotypic changes in nitrogen dynamics within the plant were reflected by changes in whole-plant and intraplant nitrogen isotope discrimination.

TAMYB13 IS A MASTER REGULATOR IN CONTROLLING THE EXPRESSION OF GENES INVOLVED IN THE FRUCTAN SYNTHESIS PATHWAY IN WHEAT

Kooiker M., Drenth J., Glassop D., McIntyre C.L. and Xue G.P. CSIRO Plant Industry, 306 Carmody Rd, St Lucia, Qld 4067, Australia.

Fructans are the major component of temporary carbon reserve in the stem of temperate cereals, and serve as an important carbon source for grain filling. Three families of fructosyltransferases are known to be directly involved in the fructan synthetic pathway in wheat. However, the mechanism of regulation of the genes involved in fructan synthesis is still poorly understood. To date, only one transcription factor has been identified as a possible regulator of fructan synthesis. Using transient transactivation assays, TaMYB13 has been shown to be a regulator of sucrose:sucrose 1-fructosyltransferase (1-SST) and sucrose:fructan 6-fructosyltransferase (6-SFT). In this study we produced transgenic lines overexpressing TaMYB13 to investigate its target genes and its influence on the fructan synthetic pathway. We show that the fructan synthetic pathway is a major target of the TaMYB13 transcription factor, with all fructosyltransferases being upregulated in our transgenic lines. In vitro DNA-binding studies showed that TaMYB13 is able to bind the TaMYB13 DNA-binding motifs present in the promoters of several target genes. The expression of these targets is highly correlated with that of TaMYB13 in different tissues, developmental stages and genetic backgrounds, suggesting a direct regulation of these targets by TaMYB13. The increased fructosyltransferase expression resulted in a significant increase in fructan accumulation in TaMYB13-overexpressing transgenic lines, confirming the key role of TaMYB13 in the regulation of the fructan synthesis pathway.


POSTERS TUESDAY



CARBOXYLATE-EXUDING CLUSTER ROOTS IN NUTRIENT- RICH LANDSCAPESLambers H.1, Bishop J.G.2, Hopper S.D.1, 3, Laliberté E.1 and ZúñIga-Feest A.4 1School of Plant Biology, The University of Western Australia. 22School of Biological Sciences, Washington State University, USA. 3Royal Botanic Gardens, Kew, UK. 4Laboratorio de Ecofisiología Vegetal, Universidad Austral de Chile, Chile.

Australia and South America were once part of Gondw analand. About 180 million years ago, this ancient continent started to break up, and the geological fate of south-w estern Australia and Chile turned out to be vastly different. We compared Proteaceae in Chile w ith those in south-w estern Australia. The Proteaceae represent an ancient Gondwanan plant family. Ancestors of the present species either ‘rafted’ across, w hen the Gondw anan continent broke up or arrived via longdistance, transoceanic dispersal. In a recent review (Lambers et al. 2010), the mineral nutrition and grow th strategies of a w ide range of species in south-western Australia w ere compared w ith those elsew here in the w orld. Here we focus on a single family (Proteaceae) and on two vastly contrasting landscapes (south-w estern Australia and Chile). South-western Australia is an ancient landscape w here numerous Proteaceae are found, invariably on the most nutrient-impoverished soils. Chile, on the other hand is a typical young landscape, w here Proteaceae are typically found on rich, volcanic soils, but w ith an abundance of aluminium and iron and a low pH, rendering P poorly available for plants. The ecophysiological mechanisms of cluster roots in the contrasting landscapes are the same, but their role in P cycling is fundamentally different. In old landscapes, cluster roots of Pr oteaceae allow functioning under extremely P- impoverished soils, w hereas they act as ecosystem engineers in young landscapes. We speculate that cluster roots of other species in other young landscapes play a similar role in P cycling and facilitation (Lambers et al. 2012). Lambers H, Brundrett MC, Raven JA & Hopper SD 2010 Plant mineral nutrition in ancient landscapes: high plant species diversity on infertile soils is linked to functional diversity for nutritional strategies. Plant Soil 334:11-31. Lambers H, Bishop JG, Hopper SD, Laliberté E & Zúñiga-Feest A 2012. Phosphorus-mobilisation ecosystem engineering: the roles of cluster roots and carboxylate exudation in young P-limited ecosystems. Ann. Bot., in press.

FUNCTIONAL CHARACTERIZATION OF TWO PUTATIVE CHLORIDE TRANSPORTERS IN ARABIDOPSIS

Li B.1, 2, Jha D.1, 2, Johnson A.1,Tester M.1, 2, Gilliham M.2, and Roy S.1, 2 1Australian Centre for Plant Functional Genomics, PMB 1 Glen Osmond SA 5064, Australia. 2School of Agriculture, Food and Wine, The University of Adelaide. SA 5000, Australia.

Chloride (Cl-) is an essential plant micronutrient, but toxic when accumulated to high concentrations within the plant shoot cytoplasm. Exclusion of Cl- from the shoot has been shown to be important for the plant salinity tolerance, particularly in legumes and woody species. In these species, Cl- is considered more toxic than sodium (Na+). To increase the salinity tolerance of plants, it is necessary to understand the mechanisms of Cl- transport through the plant, however, not much is known about the molecular identity of plant Cl- transporters. Here we identified two root-expressed putative Cl- transporters, Proton dependent Oligo-peptide Transporters 1 & 2 (POT1 and POT2) in Arabidopsis thaliana using root stelar specific microarrays. Stable and transient subcellular localization using protein:GFP and Protein:YFP fusions suggest both POT1&2 are plasma membrane targeted proteins. Promoter:reporter gene fusions and quantitative real time PCR showed that POT1 is expressed in the cells associated with vascular system, and it is down regulated by salt and ABA. Electrophysiological functional characterisation of POT1 in Xenopus oocytes showed that POT1 can facilitate Cl- movements across the membrane. Artificial microRNA knockdown of POT1 in Arabidopsis decreases Cl- accumulation significantly in the shoot, suggesting POT1 has a role in Cl- xylem loading. In contrast, constitutive over expression of POT1 increases shoot Cl- accumulation. Contrary to POT1, POT2 is up regulated by salt, and is expressed in root epidermal and cortical cells, indicating participation of POT2 in Cl- excretion from the root when plant is salt stressed

THE MICRORNA394-REGULATED F-BOX PROTEIN IS INVOLVED IN PLANT DEVELOPMENT IN ARABIDOPSIS THALIANA

Litholdo Junior C.G.1, 2, Eamens A.3 and Waterhouse P.1, 3 1School of Biological Sciences, The University of Sydney. 2CAPES Foundation, Ministry of Education of Brazil. 3School of Molecular Bioscience, The University of Sydney.

The microRNA394 (miR394) family is highly conserved in plants, consisting of two members; MIR394A and MIR394B, both of which produce a 20 nucleotide mature small RNA. The mature miR394a/b miRNA regulates the expression of a single target gene. This gene encodes to an F-box protein, which generally targets other proteins for ubiquitination, however, the role of miR394-targeted F-Box protein remains unknown. Analysis of the MIR394A/B promoter-driven reporter vectors revealed GUS expression in the vasculature of young leaves, roots and floral tissue, including anthers and pollen sacks. The F-Box reporter gene vector showed an overlapping expression pattern. Loss-of-function plants of the miR394-targeted F-Box protein are wild-type in appearance. However, plants harboring a miR394-resistant F-Box transgene display alterations to both leaf polarity and flower development. In an attempt to identify the proteins regulated by the miR394-targeted F-Box protein, and to confirm their role in plant development, proteomic analysis was carried out by mass spectrometry. This approach identified candidate proteins that are differentially expressed in the F-Box loss-of-function mutant and miR394 resistant transgene expressing plant line. Further analysis to confirm that these candidate proteins are bona fide targets of the miR394-regulated F-Box protein are currently underway and it will give additional information on the biological function of miR394 in plant development.

GLOBAL EXPRESSION CHANGES OF TRANSPORTER GENES IN THE CHLOROPHYLL D-PRODUCING CYANOBACTERIUM ACARYOCHLORIS MARINA UNDER OXYGEN STRESS

Loughlin P.C., Lin Y. and Chen M. School of Biological Sciences, University of Sydney, Sydney, NSW 2006.

The cyanobacterium Acaryochloris marina is unique in its ability to use chlorophyll d as its major photosynthetic pigment. Chlorophyll d has a red shifted maximum when compared with the ubiquitous chlorophyll a, allowing A. marina to efficiently utilise photons in the far-red spectrum for oxygenic photosynthesis. As such, A. marina occupies ecological niches depleted in visible light such as under chlorophyll a -containing cyanobacteria. The fully sequenced genome of A. marina has revealed the unusually large genome of this organism and provided an insight into how it is able to exploit such niches. Using RNA-Seq we have built on this genomic data and examined global gene expression changes of cultured A. marina cells in response to oxygen enriched (>75%) or microaerobic (oxygen <0.02%) conditions. Changes in the expression levels of transporters are of particular interest as they are a major mechanism by which the cell regulates internal concentrations of particular solutes. We have observed expression changes of a wide range of transporters under the conditions studied, reflecting changes in the biochemical needs of A. marina under oxygen stresses. Some transporters, such as putative ferrous and ferric iron transporters are upregulated under high and low oxygen, whereas others, such as specific phosphate transport systems, are downregulated, suggesting a possible general stress response. Other transporters are more specifically up or downregulated under high or low oxygen conditions indicating an oxidative stress or hypoxic response. Our results demonstrate the plasticity in expression levels of transporters under different oxygen conditions in this unique chlorophyll d -containing organism.


POSTERS TUESDAY



GENETIC REGULATION OF PHLOEM PARENCHYMA TRANSFER CELL DEVELOPMENT IN ARABIDOPSIS

McCurdy D.W., Hou J., Wu Y., Arun Chinnappa K.S. and Sheahan M.B. The University of Newcastle.

Transfer cells (TCs) are specialized plant cells characterized by wall ingrowths that amplify plasma membrane surface area to achieve enhanced rates of nutrient transport. TCs trans-differentiate from differentiated cells at sites where enhanced rates of nutrient exchange across key apo/symplasmic boundaries is required to achieve optimum growth and development. Despite the importance of TC development to plant performance, little is known of the genetic mechanisms regulating construction of their intricate wall ingrowths. TC development involves large-scale transcriptional regulation, with changes in gene expression presumably co-ordinated within a hierarchical cascade regulated by key transcription factors. To identify such factors we investigated genetic control of wall ingrowth formation in phloem parenchyma (PP) TCs in Arabidopsis leaves. Wall ingrowth deposition in PP TCs is substantially enhanced upon exposing plants to various abiotic stresses. Using a targeted bioinformatics approach, we identified a list of ~30 MYB-related and ~20 NAC genes that were commonly up-regulated in leaf tissue under these stress conditions. Phenotypic screening of homozygous knockout lines for these genes using aniline blue staining of mature leaves, which detects callose as a component of these wall ingrowths, identified several novel MYB-related and NAC domain genes as potential regulators of PP TC development. Knockout of one MYB-related gene in particular, AT4G09450, showed more than a 20-fold reduction in the appearance of PP TCs, as revealed by aniline blue staining. Further studies investigating the functions of these genes in regulating PP TC development in Arabidopsis will be reported.

BUILDING A DYNAMIC ARCHITECTURAL AND BIOCHEMICAL PLANT MODEL FOR RICE (ORYZA SATIVA)

Meacham K.G.1, 2, Furbank R.1, Von Caemmerer S.2 and Sirault X.1 1High Resolution Plant Phenomics Centre, CSIRO Plant Industry, Canberra, ACT 2601, Australia. 2Plant Science Division, Research School of Biology, Australian National University, Canberra, ACT, Australia.

It is widely recognized that yield increases required to meet global demand in rice production, must be achieved through increases in Radiation and Light Use Efficiency (RUE and LUE). Traditional breeding techniques have hit a recognized plateau and potential yield in breeders trials at IRRI have not increased for 30 years. Defining LUE as kg biomass/PAR intercepted, and assuming that RUE is a product of LUE and light interception, improving RUE becomes a matter of understanding the relationship between plant and canopy architecture, and plant and canopy biochemistry. With a subset of sequenced rice lines from IRRI, we are working towards creating a dynamic biochemical and architectural plant model. Weekly 3D imaging with a novel digitization platform is used to characterize plant architecture and phenology, whilst photosynthesis is characterized with gas exchange measurements of CO2 response, light response curves and measurements of chlorophyll fluorescence. Nitrogen partitioning and distribution throughout the plant is derived from SPAD and chlorophyll extraction. We are presenting early results from the 3D imaging platform and the underlying models into which it will be incorporated.

OVERGROWTH MUTANTS OF BARLEY: PHENOTYPES OF SELECTED OVERGROWTH ALLELES IN A SLOOP GENETIC BACKGROUND

Miraghazadeh A.1, Harding C.A.1, Hossain S.1, Zwart A.2 and Chandler P.M.1 1CSIRO, Plant Industry, GPO Box 1600, ACT 2601. 2CSIRO Mathematics, Informatics and Statistics, GPO Box 664.

Overgrowth mutants have been isolated and characterised in a Himalaya genetic background. They involve mutation in one of two genes known to encode negative regulators of signalling by the plant hormone gibberellin (GA): the DELLA gene Slender (Sln1), and the Spindly (Spy1) gene. Their mutant phenotypes result from increased GA signalling, although the extent depends on both the allele involved, and the GA response being assessed. They represent novel germplasm for traits such as malting quality, early vigour, plant height at maturity, and grain size. To assess their potential use in breeding, eight of the thirteen overgrowth alleles have been back-crossed into a Sloop genetic background. Greenhouse studies on the BC2F2 generation indicated that particular alleles were associated with either enhanced production of α-amylase by endosperm, or with a 15% increase in grain size (p<0.001). After one further back-cross generation we identified BC3F2 sister lines with a Sloop (>90%) genetic background that are homozygous for either the WT or for the mutant overgrowth allele. The phenotypes of sister lines were studied in the greenhouse and the results showed that semidwarf and tall overgrowth alleles increased the lengths of heads and rachis internodes, as well as grain size. The most interesting effect was on grain size, with four alleles being associated with statistically significant increases ranging from 13-17%. In single rows in the field the differences were about 10%. Our results indicate that overgrowth alleles have the potential to increase grain size, and that this single gene trait could be useful in the appropriate breeding programs. We gratefully acknowledge support for this work from the GRDC.

MECHANISTIC MODULATION OF A BIFUNCTIONAL GC-LINKED RECEPTOR KINASE, PSKR1

Muleya V.1, Wheeler J.I.1, Ruzvidzo O.3, Kwezi L.3, Marondedze C.2, Thomas L.2, Gehring C.2 and Irving H.R.1 1Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkvile, VIC 3052, Australia. 2Division of Chemistry, Life Sciences and Engineering, 4700 King Abdullah University of Science and Technology, Thuwal 23955-6900, Kingdom of Saudi Arabia. 3Department of Biological Sciences, North-West University, Private Bag X2046, Mmabatho, 2735, South Africa.

The phytosulfokine receptor 1, PSKR1 is a membrane-localised leucine-rich repeat receptor-like kinase that possesses intrinsic guanylate cyclase (GC) activity which is conferred by a GC catalytic centre that is embedded within its kinase domain. This unusual domain architecture represents a novel class of GC-linked receptor kinases. This novel class of kinases was unearthed using sequence homology-guided bioinformatic data mining tools. Only four members of this new class of kinases have been shown to possess both kinase and GC activity. Since there is a paucity of information as to how this dual catalytic activity is regulated in these molecules, we set out to explore the regulatory factors that modulate the dual catalysis in this unusual family of receptor kinases. Western analysis demonstrated that specific anti-phospho antibodies to pSer, pThr and pTyr residues were present in the cytoplasmic domain of recombinant PSKR1. Using mass spectrometry, we have confirmed that recombinant PSKR1 has 14 phosphorylation sites in its cytoplasmic domain including 3 phospho-tyrosines. The kinase activity was further analysed under different calcium concentrations and revealed that calcium has an inhibitory role on the kinase activity of PSKR1. We have previously demonstrated that the cGMP inhibits the kinase activity of PSKR1 and that calcium enhances the GC function of PSKR1. Taken together, our observations indicate that calcium and cGMP act as molecular switches of PSKR1-mediated signalling.


POSTERS TUESDAY



SILENCING OF SELECTED HvCKX GENES DECREASE THE CYTOKININ DEHYDROGENASE ACTIVITY IN DEFINED ORGANS OF BARLEY PLANTS AND LEAD TO HIGHER PLANT PRODUCTIVITY

Nadolska-Orczyk A., Zalewski W. and Orczyk W. Plant Breeding and Acclimatization Institute - National Research Institute, Radzikow, 05-870 Blonie, Poland.

The HvCKX genes encode cytokinin dehydrogenase. The enzyme is involved in regulation of cytokinin level in different tissues/organs of barley plants, however the detailed functions of the genes are not known. We applied expression profiling and stable interference-based (RNAi) technology to characterize two of them, HvCKX1 and HvCKX2. The highest expression of HvCKX1 was detected in developing spikes and in the seedling roots of wild type barley. Our earlier results have already proved that silencing of this gene decreased the level of the cytokinin dehydrogenase in those parts of barley plants what correlated with the higher plant productivity (J. Exp. Bot. 61: 1839-1851). The expression of the second gene, HvCKX2 was high in the developing spikes as well as in the young and fully developed leaves. The mean productivity of 33 transgenic T0 plants (the yield and the number of seeds per plant) was significantly higher than in the control – plants transformed with the empty vector. The lowest relative levels of the HvCKX2 transcript in spikes 7 DAP of segregating T1 plants were from 0.4 to 0.6 and in spikes of segregating T2 plants ranged from 0.0 to 0.2. The lowest relative levels of CKX activity were below 0.2 in developing leaves, below 0.4 in developed leaves and below 0.6 in spikes 7 DAP. There was a positive correlation between the low level of transcript in spikes 7 DAP of T1 and T2 plants and the higher plant productivity. The data of silencing and phenotype characteristic in T0 to T2 generation will be presented and discussed.

IMPACTS OF THE EXPRESSION OF ALANINE AMINOTRANSFERASE ON THE NITROGEN USE EFFICIENCY OF BARLEY

Negrini A.C.A.1, 2, Gauthier P.1, Long B.1, Garnica D.1, Evans J.1, Millar H.3, Okamoto M.4, Kaiser B.4, Kridl J.C.5 and Atkin O.K.1 1Research School of Biology, Australian National University. 2Embrapa Vegetables, Brazil. 3ARC CoE in Plant Energy Biology, University of Western Australia. 4Waite Research Institute, University of Adelaide. 5Arcadia Biosciences Inc. Davis, CA USA.

Nitrogen is the most important nutrient for crop productivity and achieving high yields relies on the application of large amounts of N-based fertilizers. However, N incorporated into crops rarely exceeds 40% of the total applied, indicating an inefficiency in N-utilization. The unused N is a source of pollution. Furthermore the production of N fertilizers consumes large amounts of energy. Therefore, to increase productivity in a sustainable way, it is necessary to improve N-use efficiency (NUE), while maintaining high yields. Previous research has shown that the regulated expression of alanine aminotransferase (AlaAT) may improve the N use efficiency of crops, particularly when cultivated under low N. Technology that combines the AlaAT gene from barley and the promoter from the abiotic stress-inducible antiquitin gene from rice (OsAnt1) is currently being tested in barley. The purpose of our current research is to evaluate the impacts of the AlaAT overexpression on different metabolic parameters, using plants that are being evaluated for NUE under field conditions. Measurements of growth, photosynthesis, respiration, enzyme activity and gene and protein expression are being conducted under optimal and sub-optimal growth conditions, including under low N supply. The results will be discussed.

AUXIN, ABSCISIC ACID AND INVERTASE ACTIVITY DURING GRAIN-FILL IN RICE

Nonhebel H.M. and Bennett K. University of New England.

Grain fill in cereals is limited in varying degrees by both source availability and sink strength. We are investigating the roles of indole-3-acetic acid (IAA) and abscisic acid (ABA) in sink strength of developing rice grains. Most published measurements of IAA and ABA in developing grains have used ELISA based methods. Using combined liquid chromatography-tandem mass spectrometry in multiple reaction monitoring mode we have shown that the IAA concentration of grains increases 50-fold during grain fill. The major increase in IAA coincides with the start of the major starch deposition phase of grain fill from 7-10 days after pollination (DAP); IAA levels remain high during the maturation phase of grain development. ABA content also increases up to approximately 10 DAP after which it drops rapidly. The amounts of IAA and ABA detected as well as the timescale of production is very different from published ELISA data and suggests possible roles during the starch deposition phase of grain development. Work of other labs has linked the production of IAA in maize to activity of the cell wall invertase ZmCWI2. In addition the ZmCWI2 orthologue in rice, Grain Incomplete Filling 1 (GIF1) has been proposed to play a key role in grain fill and sink strength. Other published work has suggested a connection between ABA and invertase activity. We are investigating the link between IAA and ABA production and invertase activity in developing rice grains. We will present data suggesting that IAA may be involved in regulating activity of cell wall invertase.

EXTRACELLULAR REACTIVE OXYGEN SPECIES REGULATE INGROWTH WALL FORMATION DURING TRANSFER CELL TRANS-DIFFERENTIATION

Xia X., Zhang H.-M., Andriunas F.A., Offler C.E. and Patrick J.W. School of Environmental and Life Sciences, The University of Newcastle, Callaghan, NSW 2308, Australia.

The intricate, and often polarized, ingrowth walls of transfer cells (TCs) amplify their plasma membrane surface areas to confer a transport function of supporting high rates of nutrient exchange across apo-/symplasmic interfaces. The TC ingrowth wall comprises a uniform wall layer on which wall ingrowths are deposited. Signals and signal networks inducing trans-differentiation events leading to formation of TC ingrowth walls are poorly understood. Vicia faba cotyledons offer a robust experimental model to examine TC induction as, when placed into culture, their adaxial epidermal cells rapidly (h) and synchronously form polarized ingrowth walls accessible for experimental observations. Using this model, we recently reported findings consistent with extracellular hydrogen peroxide, produced through a respiratory burst oxidase homologue/superoxide dismutase pathway, initiating cell wall biosynthetic activity and providing directional information guiding deposition of the polarized uniform wall (Andriunas et al 2012). However, our conclusions rest on observations derived from pharmacological manipulations of hydrogen peroxide production and correlative gene expression data sets. We present a series of additional studies that verify that extra- and not intracellular hydrogen peroxide, as part of a sequential series of signals and signalling cascades, regulates ingrowth wall formation. Further, we established that the hydrogen peroxide is generated by a respiratory burst oxidase homolog/superoxide dismutase pathway and not by cell wall peroxidases. A model of signals and signalling pathways regulating ingrowth wall formation during TC trans-differentiation is presented. Andriunas et al. 2012. Reactive oxygen species form part of a regulatory pathway initiating trans-differentiation of epidermal transfer cells in Vicia faba cotyledons. J. Exp. Bot. PMID: 22442421; doi:10.1093/jxb/ers029.


POSTERS TUESDAY


POS-TUE-067 POS-TUE-068CRYOTHERAPY OF GRAPEVINE TO REMOVE LEAFROLL VIRUS FROM INFECTED PLANTS

Pathirana R. Plant & Food Research, Private Bag 11600, Palmerston North 4442, New Zealand.

Infections by viruses and other pathogens are a threat to the grapevine industry. A robust method for removing all microorganisms from infected tissue is important for cultivar imports, germplasm maintenance and to produce grafted material for the industry. The demonstration of virus and phytoplasma eradication by methods used in cryopreservation of plants has led to the establishment of cryotherapy: a new method for cleaning infected plant material of vegetatively propagated species. Vitrification-based cryopreservation techniques have been shown to be the most adaptable across species. In droplet vitrification, plant tissue pre-treated with vitrification solution is placed on aluminium foil in a droplet of vitrification solution and directly immersed in liquid nitrogen. We have cryopreserved shoot tips and axillary buds of 10 Vitis genotypes by this method and regenerated plants. Only highly cytoplasmic, non-vacuolar meristematic cells survive the freezing process because only such cells can tolerate the high dehydration caused by the vitrification solution; therefore, cryotherapy can be considered a precise method of meristem culture. To test the suitability of cryotherapy for virus eradication, we used Lakemont Seedless and Chardonnay infected with Grapevine leafroll-associated virus-3 (GLRaV-3, an Ampelovirus), Sauvignon blanc and Pinot gris infected with Grapevine leafroll-associated virus-2 (a Closterovirus), and another clone of Sauvignon blanc infected with both Grapevine leafroll-associated virus-1 (an Ampelovirus) and GLRaV-3. Plants regenerated after cryotherapy tested negative (DAS-ELISA) for all three viruses, whereas untreated control plants tested positive. Droplet vitrification has the potential to be a novel and precise tool for virus eradication and establishment of high-health grapevine germplasm collections. Acknowledgement: This work is funded by New Zealand Winegrowers (NZW 10-107).

OVULE CULTURE AND EMBRYO RESCUE FACILITATES INTERSPECIFIC HYBRIDISATION IN BLUEBERRY

Pathirana R.1, Scalzo J.2, Frew T.3, Pathirana S.1, Timmerman-Vaughan G.3 and Morgan E.1 1Plant & Food Research, Private Bag 11600, Palmerston North 4442, New Zealand. 2Plant & Food Research, Private Bag 1401, Havelock North 4157, New Zealand. 3Plant & Food Research, Private Bag 4704, Christchurch 8140, New Zealand.

About 80 blueberry (Vaccinium) accessions represent four species in New Zealand at tetraploid, pentaploid and hexaploid levels. In order to increase the variability available to breeders and to introgress characters from different species into parental breeding populations, twenty-eight interspecific, interploidy crosses were made. Ovules were cultured aseptically 28 and 35 days after pollination. Embryos were dissected from the enlarged ovules and cultured on regeneration media. From 8 of these crosses, more than 200 plants regenerated within 4 - 8 weeks. In ovulo embryo culture was more successful when the maternal parent had a lower ploidy than the male parent. Flow cytometry revealed that 4n x 6n crosses produced pentaploids, while the progeny of 4n x 5n and 5n x 6n crosses exhibited a ploidy similar to the maternal parent. RAPD marker analyses confirmed the hybrid nature of progeny from three of the four crosses examined to date. Hybrid plants were cloned in vitro and successfully acclimated in the greenhouse. Our study has demonstrated that in vitro ovule culture and embryo rescue is useful in overcoming post-zygotic barriers to interspecific hybridisation among Vaccinium species with differing ploidies.

TRANSGENIC MANIPULATION OF THE C4 CYCLE: ANTISENSE REDUCTION OF NADP-MALIC ENZYME IN FLAVERIA BIDENTIS

Pengelly J.J.L.1, Tan J.2, Furbank R.T.3 and Von Caemmerer S.1 1Research School of Biology, The Australian National University, Canberra, Australia. 2National University of Singapore, Singapore. 3High Resolution Plant Phenomics Centre, CSIRO Plant Industry, Canberra, Australia.

An antisense construct targeting the C4 isoform of NADP-malic enzyme (NADP-ME), the primary enzyme decarboxylating malate in bundle sheath cells to supply CO2 to Rubisco, was used to transform the dicot Flaveria bidentis. Transgenic plants (α-NADP-ME) generated, exhibited a 34-75% reduction in NADP-ME activity relative to wild-type with no visible growth phenotype. We characterised the effect of reducing NADP-ME on photosynthesis by measuring in vitro photosynthetic enzyme activity, gas exchange and real-time carbon isotope discrimination. In α-NADP-ME plants with less than 40% of wild-type NADP-ME activity, CO2 assimilation rates at high intercellular CO2 (Ci) were significantly reduced, whereas the in vitro activity of both PEP carboxylase and Rubisco were increased. Carbon isotope discrimination (Δ) measured concurrently with gas exchange in these plants showed a lower Δ and thus a lower calculated leakiness of CO2 (the ratio of CO2 leak rate from the bundle sheath to the rate of CO2 supply). Comparative measurements on antisense Rubisco small subunit (α-SSu) F. bidentis plants showed the opposite effect of increased Δ and leakiness. We use these measurements to estimate the C4 cycle rate, bundle sheath leak rate and bundle sheath CO2 concentration (Cs). The comparison of α-NADP-ME and α-SSu demonstrates that the coordination of the C3 and C4 cycle that exists during environmental perturbations by light and CO2 can be disrupted through transgenic manipulations. Furthermore our results suggest that the efficiency of the C4 pathway could potentially be improved through a reduction in C4 cycle activity or increased C3 cycle activity.

XYLEM REFILLING AND RECOVERY FROM DROUGHT: ARE THERE FUNDAMENTAL DIFFERENCES BETWEEN CONIFERS AND ANGIOSPERMS?

Choat B.1, Brodersen C.R.2 and McElrone A.J.3 1University of Western Sydney, Hawkesbury Institute for the Environment, Richmond, NSW, 2753, Australia. 2Citrus Research and Education Center, University of Florida, 700 Experiment Station Road, Lake Alfred, FL 33850, USA. 3Department of Viticulture & Enology, University of California, Davis, CA 95616, USA.

Plants are capable of lifting water to heights of over 100m and extracting water from dry and saline substrates. To achieve this, plants have evolved a transport system that relies on water sustaining a tensile force, such that the xylem sap is held below its vapour pressures. However, this transport mechanism comes with its own set of problems, most notably that water under tension is prone to cavitation, which results in the formation of a gas bubble (embolism). Embolism reduces the capacity of the xylem tissue to deliver water to sites of gas exchange. This can limit productivity and in extreme cases, reach lethal levels causing plant death. It has now been established that some plants can refill embolised xylem conduits, restoring them to a functional state. The ability of plants to refill embolised xylem conduits appears to be active process driven by parenchyma cells adjacent to xylem conduits (vessels or tracheids) although the details of the mechanism have not been elucidated. We compared refilling activity in one angiosperm species (Vitis vinifera) and one conifer (Sequoia sempervirens) using synchrotron based x-ray micro computed tomography (microCT). Evidence from microCT demonstrated that V. vinifera was capable of refilling embolised vessels on a time scales of hours. In contrast, S.sempervirens showed no capacity to refill embolised tracheids even after two weeks of well watered conditions. We hypothesize that that S. sempervirens is unable to refilled embolised tracheids because it lacks the xylem parenchyma and carbohydrates reserves necessary to drive the refilling process. This may represent a fundamental difference in the ability of angiosperms and conifers to recover from drought.


POSTERS TUESDAY



XYLANS IN PLANTAGO SPECIES

Phan J., Tucker M.R. and Burton R.A. ARC Centre of Excellence in Plant Cell Walls, University of Adelaide, Waite Campus, Urrbrae SA 5064, Australia.

Plant cell wall polysaccharides are an essential source of human dietary fibre (DF). Studies of diseases such as hypercholesterolemia, obesity, bowel-related disorders, type 2 diabetes and colon cancer show that a diet incorporating DF helps to positively impact and alleviate symptoms. Consequently, there are now a diverse range of commercially available DF supplements, all of which have varying therapeutic effects and benefits. One example is Metamucil®, which is derived from the husk of Plantago ovata seeds and predominantly comprised of xylan. The aim of this project is to characterise xylan in diverse Plantago species using chemical, microscopic and transcriptomic methods. Plantago is an ideal model for the study of xylan synthesis, since the ovule tissues that synthesise xylan are easy to harvest, candidate biosynthetic genes have been identified and the polysaccharide contents of its seed husk are easily extracted, manipulated and analysed. Initial results suggest that the amount and structure of xylan varies within the husk of different Plantago species. The possibility of identifying natural variants showing differential expression of biosynthetic enzymes will provide further insight into our understanding of polysaccharide biosynthesis.

USE OF HIGH THROUGHPUT PHENOTYPING OF BRACHYPODIUM DISTACHYON TO ENABLE NEW BREEDING STRATEGIES

Poiré R.1, Sirault X.1, Bragg J.2, Vogel J.2, Watt M.3 and Furbank R.1 1High Resolution Plant Phenomics Centre, CSIRO Plant Industry, Canberra, ACT 2601, Australia. 2USDA, ARS, WRRC Albany, California 94710, USA. 3CSIRO Plant Industry, Canberra, ACT 2601, Australia.

Increasing plant biomass productivity on marginal land is a challenge for improving grass crops for energy production. The genetic determinants underlying biomass production in crops are poorly understood because of the complexity of their genomes, their growth requirements and a lack of phenotyping tools. This work illustrates a practical use of phenomics tools to elucidate the mechanisms underlying plant performance using high-throughput imaging platforms and destructive analysis of the model grass species Brachypodium distachyon (L). We describe a range of single pot and tray based methodologies for non-invasive estimation of plant growth and function in controlled conditions on set of natural accessions and a population of homozygous T-DNA mutants generated from Bd21-3. Chlorophyll fluorescence and infrared thermography are used to obtain information on photochemical efficiency, electron transport rate and transpiration validated against physiological measures of gas exchange and leaf destructive sampling (biomass and total N concentration). A 10-fold variation in biomass accumulation was observed in the 160 natural accessions set as well as a 5-fold variation in their photosynthetic activity. Non-destructive imaging techniques used to estimate plant growth show a 2-fold difference in the rate of biomass accumulation from an early development stage. The T-DNA population is currently used to link performance traits underlying higher biomass accumulation to specific genomic regions. As Brachypodium distachyon shares a high degree of genetic homology with wheat and energy crops it will enable new breeding strategies.

INVESTIGATING THE MECHANISMS OF SHOOT CHLORIDE EXCLUSION IN ARABIDOPSIS

Qiu J.1, 2, Roy S.2 and Gilliham M.1 1School of Agriculture, Food, and Wine, University of Adelaide. 2Australian Centre for Plant Functional Genomics.

Salinity tolerance is correlated with shoot chloride (Cl-) exclusion in many horticultural and crop species (e.g. grapevine, soybean). The key regulatory step in root-to-shoot transfer of Cl- is conferred by plasma membrane-localised anion transporters associated with the root vasculature. Therefore, reducing long-distance Cl- transport by manipulating the regulation of anion transporters in the root vasculature is a strategy that promises to increase plant tolerance to salinity environment. Candidate anion transporters were identified from a preliminary microarray study. Quantitative PCR indicated transcriptional levels of candidate anion transporters decreased upon NaCl and ABA. Mutant lines of these candidate genes, including over-expression, knock-out and amiRNA knock-down lines are being generated to further phenotype the function of these proteins under salinity stress. In addition, heterologous expression in Xenopus laevis oocyte is being performed to characterize the electrophysiological properties of candidate chloride transporters, data on these results will be presented too. Furthermore, nitrate (NO3

-) and Cl- share similar transport pathways and NO3- shoot

content is reduced when Cl- is increased under salt stress, which is a component of the stress phenotype; this may also be an undesirable consequence of manipulating long-distance Cl- transport. Using an inhibitor of anion transport we have uncoupled the transfer of NO3

- and Cl- to the shoot under salt stress; Cl- content from root-to-shoot was significantly reduced under salt stress, whilst the NO3

- content was similar to that in unstressed conditions. This blocker is being used to understand further the mechanisms that control the activity of anion transporters.

WATER RELATIONS IN FIVE BREEDING LINES OF ALMOND (PRUNUS DULCIS)

Rahimi Eichi V.1, Wirthensohn M.1, Tyerman S.1 and Downey M.2 1School of Agriculture, Food and Wine, University of Adelaide, Waite Campus, Glen Osmond SA 5064, Australia. 2Department of Primary Industries, Victoria, Mildura.

The Almond (Prunus dulcis) is a nut tree in the family Rosaceae which compared to other nuts is relatively drought tolerant. Nevertheless, the production under irrigation is more than 10-fold higher compared to rainfed conditions. Intrinsic water use efficiency (WUEintr), the ratio of assimilation (A) to stomatal conductance (gs), can be used as a criterion for calculating the crop water use index in breeding programs. Here we examine WUEintr and the relationships between leaf transpiration (E), A and leaf hydraulic conductivity (kleaf) in five breeding lines of almonds comprised of Carmel x Tarraco, Johnston x Lauranne, Nonpareil x Tarraco, Nonpareil x Lauranne and Nonpareil x Vayro. After exposing the plants to the same environmental conditions for 1 week, A, E, and kleaf and the total leaf area (LA) were measured. There were significant differences in kleaf, E and A between different varieties, especially between Johnston x Lauranne and Nonpareil x Lauranne. For WUEintr, significant differences were only observed between Carmel x Tarraco and Nonpareil x Lauranne. According to numerous reports, any decrease in kleaf can lead to the reduction of gs and E. However, under well watered conditions, stomatal functioning cannot have a large effect on light-saturated carbon assimilation rates; hence, different values of A are probably controlled by biochemical processes within the leaf that can be affected by genotype variations.


POSTERS TUESDAY



TRANSACTIVATION OF ALMTS BY EXTERNAL ANIONS CAN CONFER SUSTAINED CELLULAR ANION EFFLUX

Ramesh S.A.1, Tyerman S.D.1, Ryan P.R.2 and Gilliham M.11ARC Centre of Excellence in Plant Energy Biology, School of Agriculture, Food and Wine, University of Adelaide, SA 5064. 2CSIRO Plant Industry, GPO Box 1600, Canberra, ACT 2601.

Aluminium-activated Malate Transporters (ALMTs) are named after TaALMT1 cloned from Al tolerant wheat, which encodes a plasma membrane localised protein that catalyses the efflux of malate from roots when exposed to micromolar concentrations of soil Al3+. Soil and cell wall malate then chelate the toxic Al3+, thus conferring Al tolerance. ALMTs are now known to form a large anion channel family found in all plant species – Arabidopsis (14), Vitis (13), soybean (33), and rice (9). However unlike TaALMT1, most ALMTs are not activated by Al3+, nor have any role in Al tolerance. Instead, the roles of ALMTs are diverse and include processes such as mineral nutrition, vacuolar malate accumulation and stomatal aperture control. In most cases, ALMTs facilitate a sustained efflux of anions out of cells down a diminishing electrochemical gradient. In wheat, TaALMT1 facilitates sustained malate efflux over many hours but the mechanism by which this occurs is poorly understood. Here, using ALMT expression within Xenopus oocytes we show that transactivation, the activation of the channel by permeating anions on the external (trans) side at low mM concentrations (i.e. Km for malate ≈ 1 mM), is a property that could help facilitate this process. We show transactivation is common in ALMTs from Arabidopsis, wheat, rice, barley and grapevine, and can be replicated in intact plants. To gain further insight into the physiological roles of ALMTs, we also examine ALMT selectivity and the sensitivity of transactivation to pH. * Authors Ramesh and Tyerman contributed equally to this work.

PREFERENTIAL ALLOCATION OF ENERGY TO PROTEIN SYNTHESIS IN RICE COLEOPTILES UNDER OXYGEN DEPRIVATION

Edwards J.M.1, Roberts T.H.1, 2 and Atwell B.J.1 1Macquarie University. 2University of Sydney.

The amount of energy available for cellular processes in plants is severely limited by oxygen deprivation, as occurs during waterlogging. While synthesis of ATP has been widely estimated in O2-deprived tissues, less is known about how this limited ATP is used. We studied 3-d-old coleoptiles of a flooding-tolerant variety of rice (Oryza sativa) grown in solution to determine (i) rates of ATP regeneration under normoxia (aerated), hypoxia (3% O2) and anoxia (N2) and (ii) steady-state rates of synthesis of proteins, lipids, nucleic acids and cell walls, as well as K+ transport. Using published bioenergetics data, we then compared the costs of synthesizing each class of biopolymer and the proportion of available ATP allocated to each process. Protein synthesis consumed the largest proportion of ATP synthesized under all three oxygen regimes. Importantly, the proportion of ATP allocated to protein synthesis in anoxia (52%) was more than double that in normoxic coleoptiles (19%). Rates of protein synthesis in coleoptiles from rice cultivars and mutants with contrasting tolerance to oxygen deficits confirmed that protein synthesis and turnover account for most of the energy consumed under anoxia. We conclude that establishment of rice seedlings under water is largely due to allocation of energy to vital processes, particularly protein synthesis. Reference: Edwards JM, Roberts TH and Atwell BJ (in press) Quantifying ATP turnover in anoxic coleoptiles of rice (Oryza sativa) demonstrates preferential allocation of energy to protein synthesis. J Exp Bot.

PROGRESS TOWARDS THE GENERATION OF SALINITY TOLERANT WHEAT AND BARLEY

Tilbrook J.1, 2, Schilling R.1, 2, Trittermann C.1, 2, Plett D.1, 2, Jacobs A.1, 2, Mcdonald G.2, Tester M.1, 2, 3 and Roy S.J.1, 2 1ACPFG, Waite Campus, PMB1, Glen Osmond, SA 5064, Australia. 2The University of Adelaide, Waite Campus, PMB1, Glen Osmond, SA 5064, Australia. 3The Plant Accelerator, Waite Campus, PMB1, Glen Osmond, SA 5064, Australia.

Saline soils have a severe impact on crops in Australia and around the world. To improve the salinity tolerance of wheat and barley we must first understand the mechanisms of salinity tolerance in plants then test under field conditions how manipulating such pathways can lead to yield improvements. Through a variety of approaches, we have identified a number of candidate genes involved in a variety of salt tolerance mechanisms from a variety different species. Numerous candidate genes have been identified for controlling the movement of both Na+ and Cl- in to and through a plant. Using using non-destructive imaging technology of salt stressed wheat and barley, we have also revealed a large variation in osmotic tolerance (sensu Munns & Tester, 2008), with great potential for future gene discovery and introgression in to crops. In addition to understanding the science behind these mechanisms, we have now undertaken both GM and non-GM field trials of enhanced crop plants to assess their performance in the field, away from the controlled conditions of the laboratory. Initial field trials of our most advanced GM lines indicate we have been able to increase yield on saline soils by over 10%. Work will be presented on the current progress of our research into ion transport and osmotic tolerance, as well as our results from field trials.

GENETIC AND BIOLOGICAL RESISTANCE TO ROOT LESION NEMATODE IN WHEAT

Linsell K.J.1, 2, Davies K.2, Riley I.1, 2, Wallwork H.1 and Oldach K.1, 2 1South Australian Research and Development Institute, GPO Box 397, Adelaide, SA, 5001. 2The University of Adelaide, Waite Campus, PMB 1, Glen Osmond, SA, 5064.

The root lesion nematode, Pratylenchus thornei, is a migratory nematode that feeds and reproduces within the wheat root cortex resulting in cell death (root lesions), causing severe reductions in yield. In this study, a doubled haploid wheat population from a cross between the synthetic derived cultivar Sokoll (resistant) and the cultivar Krichauff (susceptible) was used to identify QTL associated with P. thornei resistance and to gain a simple understanding of the resistance mechanisms and how they interfere with the nematode’s biology. A genetic map was constructed and two highly significant (P < 0.001) P. thornei resistance QTL were detected on the distal ends of the short arms of chromosomes 2B and 6D. The QTL QRlnt.sk-6D (LOD 18) and QRlnt.sk-2B.1 (LOD 8.7) explained 43% and 24% of the phenotypic variation in this population. Current fine mapping is being conducted on populations segregating for either of the two QTL. The study revealed through analysis of each invasive stage of the root, that resistance in the Sokoll x Krichauff population occurs post penetration. The motility and egg hatching of P. thornei was suppressed in un-inoculated resistant roots/exudates, suggesting resistant genotypes constitutively produce inhibiting compounds. QTL linked to hatching and motility suppression were identified and co-located to the P. thornei resistance QTL on chromosomes 2B and 6D implying that the resistance mechanism targets motility and juvenile hatching.


POSTERS TUESDAY



THE ROLE OF PLANT CELL WALL-DERIVED OLIGOSACCHARIDES IN DEFENCE SIGNALLING IN BARLEY

Jasim B.N., Little A., Tucker M.R., Lahnstein J., Shirley N.J., Burton R.A. and Fincher G.B. ARC Centre of Excellence in Plant Cell Walls, School of Agriculture, Food & Wine, The University of Adelaide.

Plants have evolved complex mechanisms to deal with biotic and abiotic stresses. These mechanisms involve endogenous and exogenous factors, including physical and chemical barriers. The components and structure of plant cell walls are believed to play an important role in these mechanisms owing to their heterogeneity and complexity. Degradation of cell wall polysaccharides by hydrolytic enzymes generates oligosaccharides that can act as signals triggering plant defence systems which act to restrict pathogen spread. To date, a number of oligosaccharide elicitors have been characterised, but research has been focused on elicitors derived from fungal or dicot cell walls. Due to the many differences observed between the cell walls of monocot and dicot species, it is predicted that there may be novel signalling molecules present within the barley cell wall. The aim of this study is to investigate the involvement of plant cell wall-derived oligosaccharides in defence signalling in barley. Identifying these oligosaccharides in plant tissues would be useful for understanding the defence response in both host and non-host plants.

EFFECT OF ELEVATED CARBON-DIOXIDE ON HOST-PATHOGEN INTERACTION OF WHEAT AND FUSARIUM SPP

Melloy P.1, 2, Chakraborty S.1, Aitken E.2 and Luck J.3 1CSIRO Plant Industries, 306 Carmody Road, St Lucia, Queensland 4067 Australia. 2School of Agriculture and Food Sciences, University of Queensland, St Lucia, Queensland 4072, Australia. 3CRC for Plant Biosecurity, PO Box 5012 Bruce ACT 2617.

The effect of climate change and rising atmospheric carbon dioxide on plant pathogens and subsequently their effect on agricultural productivity have raised food security concerns. Previous studies on the host and pathogen interactions between wheat and Fusarium pseudograminearum, the causal agent of crown rot (CR), has shown an increase in symptom expression and fungal biomass in host tissue. Here we report on two experiments which aim to determine if CR resistance of selected wheat genotypes are expressed consistently at ambient and elevated CO2. We have assessed CR symptom on a selection of 16 wheat lines; in three glasshouse environments ambient conditions, elevated CO2 with ambient temperature and elevated CO2 and temperature, and in a Free to Air Carbon-Dioxide Enrichment (FACE) facility at Horsham, Victoria under ambient and elevated CO2 treatments. In all glasshouse and field experiments the mean proportion of tillers per plant with CR symptoms, and the mean index of stem browning (ISB) were higher at elevated CO2. However, at the FACE trial the difference in CR symptoms between the two CO2 treatments was not significant (P>0.05). In the glasshouse ISB correlated negatively to plant dry weight at ambient and elevated CO2 but positively at elevated CO2 and temperature. Wheat genotypes differed in their performance at the two CO2 levels, but the differences in ISB were non-significant.

POSITIONAL CLONING OF THE GENE RRS1 MEDIATING RESISTANCE TO THE FUNGUS RHYNCHOSPORIUM SECALIS IN BARLEY

Oldach K.H.1, 2, Rongala J.1, Al-Ani I.1, 2 and Baumann U.3 1South Australian Research and Development Institute, Plant Genomics Centre, Waite Campus, Urrbrae, SA, 5064, Australia. 2University of Adelaide, Waite Campus, Urrbrae, SA, 5064, Australia. 3Australian Centre for Plant Functional Genomics, Plant Genomics Centre, Waite Campus, Urrbrae, SA, 5064, Australia.

Scald disease of barley caused by Rhynchosporium secalis is a major foliar disease in all barley-growing regions of the world. Crop surveys in Australia indicate average annual yield losses of 10% with extreme losses surpassing 45% due to reduced kernel weight, malt and feed grain quality. To date, 16 major genes have been identified that control resistance against strains of R. secalis, but no resistance gene to this pathogen has been cloned. Numerous of these 16 genes appear to coincide at a few loci suggesting resistance gene clusters at specific genome regions or the existence of numerous alleles of a few resistance genes. In either case, not knowing the DNA sequence of the resistance genes prevents the full utilisation of the available resistance genes/alleles in barley breeding programs. The gene Rrs1 is part of a resistance gene cluster on barley chromosome 3H. It is outstanding among the R. secalis resistance genes as the fungal peptide, NIP1, has previously been described as the effector mediating the Rrs1-dependent resistance in barley (Rohe et al., 1995). Here, we report on the progress of the approach to map-based clone the scald resistance gene Rrs1. In addition, we describe an objective quantification method using TaqMan probes to assess the level of resistance in different barley genotypes.

IDENTIFICATION OF WHEAT GENES DIFFERENTIALLY REGULATED IN TcLr9 LINE DURING AN INCOMPATIBLE WHEAT-BROWN RUST INTERACTION

Dmochowska-Boguta M.1, Alaba S.2, Lasota E.1, Nadolska-Orczyk A.1, Karlowski W.2 and Orczyk W.1 1Plant Breeding and Acclimatization Institute – National Research Institute, Radzikow, 05-870 Blonie, Poland. 2Laboratory of Computational Genomics, Institute of Molecular Biology and Biotechnology, Adam Mickiewicz University, Umultowska 89, 61-614 Poznan, Poland.

Brown rust caused by Puccinia triticina is one of the most devastating wheat diseases. Over sixty Lr genes conferring resistance against brown rust have been identified in wheat, rye and wild Triticeae. Despite the importance of the genes there is still a very limited knowledge about their function and the Lr-dependent processes leading to plant resistance. The goal of the work was to identify and to analyze wheat transcripts specifically regulated during an incompatible wheat-brown rust interaction in TcLr9 line. The cDNA clones (177 submitted to the NCBI dbEST database) of the pathogen-regulated transcripts were isolated by means of suppression-subtractive-hybridization from a pair of isogenic lines: susceptible Thatcher and resistant TcLr9 after inoculation with a single spore isolate of Puccinia triticina. The subtraction time-points were selected based on the earlier described steps of complex wheat-brown rust interaction (Orczyk et al., 2010; Dmochowska-Boguta et. al. 2012 accepted). The identified transcripts were analyzed bioinformatically, by extending their sequences with know cDNA and EST data, protein coding region prediction and functional domain annotation as well as comparison with available databases of coding and non-coding sequences. Subsequently, their relative expression was quantified by qRT-PCR. The potential role of the transcripts in wheat response for brown rust infection is discussed. Acknowledgements The research was financed by National Science Centre, grant UMO-2011/01/B/NZ9/02387.


POSTERS TUESDAY

POS-TUE-081 POS-TUE-082APPLICATION OF SILENCING SUPPRESSOR IN SINGLE CELL CO-EXPRESSION OF TARGET PROTEINS

Reis R.S.1, Litholdo Junior C.G.1, Mackay J.1 and Waterhouse P.1, 2 1University of Sydney. 2CSIR0 Plant Industry.

The majority of plant viruses encode silencing suppressor proteins (SSPs), which can impair the host s defence system. Polerovirus P0 (PLP0) protein is a nuclear SSP, which presents an F-box domain and was shown to interact with Skp1, a component of the ubiquitination complex. Moreover, P0 destabilizes siRNA-unloaded AGO1, preventing de novo formation of the RISC complex, possibly via ubiquitination and degradation of AGO1 in the proteasome. Here we demonstrate that the PLP0 protein can be separated into two distinct proteins that complement each other in planta resulting in silencing suppression similar to the full length protein. PLP0 protein structure prediction, using ab initio approach, presents two distinct domains or units linked together by, approximately, 20 amino acids long unstructured sequence. Alignment of the amino acid sequences of PLP0 available in the non-redundant database of NCBI showed high conservation throughout the whole sequence. Interestingly, it shows a variable sequence, 5 amino acids long, that matches with the predicted linker between the two domains of PLP0. Based on the structure prediction and alignment of PLP0, this protein was separated into two proteins, here called A and B, and each different set of PLP0-A/B was assayed for its silencing suppression activity. Two sets of proteins, PLP0-A2/B2 and PLP0-A3/B3, showed suppressor activity when co-infiltrated in N. benthamiana. PLP0-A2 and PLP0-B2 were placed into two different plasmids with each plasmid also encoding one of a pair of proteins of interest. When co-infiltrated, only cells that contain both constructs have silencing suppression activity and these cells have enhanced expression of the pair of target proteins. This approach was tested with different proteins of interest and showed potential application for single cell co-expression of partner proteins, in pull-down assay, as well as in repressible binary expression system.

INVESTIGATING FLAX RUST EFFECTOR UPTAKE INTO PLANT CELLS

Wu W.J.1, Koeck M.1, 2, Zhang A.3, Catanzariti A.-M.1, Dodds P.N.2 and Hardham A.R.1 1Plant Science Division, Research School of Biology, The Australian National University, Canberra ACT 0200. 2CSIRO Plant Industry, Canberra ACT 2601. 3School of Chemistry and Molecular Biosciences, University of Queensland, St Lucia, Queensland 4072.

The flax rust pathogen, Melampsora lini, is an obligate biotrophic fungus that relies on living plants for its growth and reproduction. During invasion, the rust fungus forms a specialized structure called a haustorium. In addition to helping the pathogen acquire nutrients from plant cells, haustoria play an important role in facilitating infection by secreting effector proteins into the plant cytoplasm. Fungal effector proteins have no clearly conserved host targeting motifs comparable to the RxLR motif of oomycete effectors. While the uptake domains of the flax rust fungal effectors AvrM and AvrL567 have been identified, the uptake domains of AvrP, AvrP4 and AvrP123 rust effectors have not yet been determined. In addition, little is known about the mechanism by which fungal effector proteins gain access into the plant cytoplasm during infection or their role in facilitating plant colonization. Our research aims to localise AvrP, AvrP4 and AvrP123 effectors during infection, to determine their uptake pathway into the plant cell cytoplasm and to investigate their role in establishing disease. Localization studies will use antibodies raised against purified AvrP, AvrP4 and AvrP123. Yeast two hybrid screen has identified another secreted flax rust protein, siA2 that interacts with a number of rust effectors including AvrP123, AvrM and AvrL567. Our research aims to determine the interacting domains of siA2 and the rust effectors using yeast two hybrid and luciferase assays.

EZH2 REGULATES HISTONE METHYLATION ALONG THE INK4A/ARF LOCUS IN TWIST-1 EXPRESSING MESENCHYMAL STEM CELLS AND MEDIATES LINEAGE DETERMINATION

Cakouros D.1, Hemming S.E.1, Isenmann S.1, Cooper L.1, Anderson P.J.2 and Gronthos S.1 1Mesenchymal Stem Cell Group, Department of Haematology, SA Pathology, Frome Road, Adelaide, SA, Australia/Hanson Institute/ Centre for Stem Cell Research, Robinson Institute, University of Adelaide. 2Australian Craniofacial Unit, Women’s and Childrens Hospital, 72 King William Road North Adelaide, SA 5006, Australia.

One of the main impairments to tissue maintenance during ageing is the reduced capacity for stem cell self-renewal over-time due to senescence, the irreversible block in proliferation. We have previously described that the basic HLH) transcription factor, Twist-1 can greatly enhance theβHelix-loop-Helix ( life span of human bone marrow derived mesenchymal stem/stromal cells (MSC). We now show that Twist-1 potently suppresses senescence and the Ink4A/Arf locus with a dramatic decreased expression of p16 and to some extent, p14. Furthermore, the polycomb group protein and histone methyl transferase, Ezh2 which suppresses the Ink4A/Arf locus was found to be induced by Twist-1 resulting in an increase in H3K27me3 along the Ink4A/Arf locus but not H3K4me3, repressing transcription of both p16/p14 and therefore inhibiting senescence of human MSCs. Reduced Twist-1 wild type expression and function in bone cells derived from Saethre Chotzen patients also revealed an increase in senescence. In addition we characterize the role of Ezh2 in mediating differentiation of MSCs into osteoblasts, adipocytes and chondrocytes using both functional and transcription based approaches. These studies for the first time link Twist-1 to histone methylation of the Ink4A/Arf locus by controlling the expression of histone methyltransferase Ezh2 and identify a role for Ezh2 in mediating differentiation of human MSCs.

POS-TUE-083 POS-TUE-084GENETIC VARIABILITY OF THE ALPHA-AMYLASE 1 GENE FAMILY IN BARLEY

Cu S.T., Roumeliotis S. and Eglinton J. University of Adelaide, School of Agriculture Food and Wine, Waite Campus, Glen Osmond, South Australia, 5064, Australia.

Barley α-amylase (EC 3.2.1.1) is an endohydrolase that catalyses the cleavage of internal α-(1,4)-glucosyl linkages of amylose and amylopectin and is a key enzyme in the degradation of starch during brewing. The α-amylase family consists of two groups differentiated by their isoelectric points (pIs); the low pI group AMY1 and the high pI group AMY2. The gene families coding for AMY1 and AMY2 are amy2 and amy1, located on chromosome 7H and 6H respectively. In this study, natural variation in α-amylase 1 genes was examined in 26 barley varieties and 14 single nucleotide polymorphisms (SNPs) were found in the ORFs. This variation was investigated and shown to have a significant impact on enzyme activity in a mapping population derived from Flagship and two wild-barley accessions.


POSTERS TUESDAY



IN VIVO SITE-SPECIFIC INCORPORATION OF UNNATURAL CROSS-LINKING AMINO ACIDS INTO IGF-II FOR THE STUDY OF THE IGF-II INTERACTION WITH THE INSULIN AND TYPE 1 IGF RECEPTORS

Alvino C.L., Evans M., Delaine C., Ong S., McCarthy P., Wallace J.C. and Forbes B.E. The University of Adelaide.

Insulin-like growth factor (IGF) II is a potent mitogen with structural homology to IGF-I and insulin. IGF-II binds both the type 1 IGF receptor (IGF-1R) and the exon 11- isoform of the insulin receptor (IR-A) with high affinity. IGF-II is frequently over-expressed in human cancers and numerous studies have demonstrated its ability to signal via the IR-A and IGF-1R to induce cancer cell proliferation and migration. Inhibition of receptor activation by IGF-II has therefore become a major target therapeutic intervention. Recently we have been characterising the molecular mechanism of interaction between IGF-II and the IGF-1R and IR-A, and have described two distinct receptor binding surfaces on IGF-II (site 1 and site 2)(Alvino, C.L. et al (2009). A Novel Approach to Identify Two Distinct Receptor Binding Surfaces of IGF-II. J Biol Chem 284, 7656-7664). Here we outline our approach to site-specifically introduce a cross linking amino acid (benzophenylalanine, Bpa) into IGF-II using an expression system developed by Prof. Schultz (Scripps)(Wang, L., and Schultz, P.G. (2004). Expanding the genetic code. Angew Chem Int Ed Engl 44, 34-66; Chin, J.W. et al. (2002). Addition of a photocrosslinking amino acid to the genetic code of E. coli. PNAS 99, 11020-11024 ), which allows selective incorporation of unnatural amino acids into recombinant proteins. Using this approach we have generated a series of novel cross-linking IGF-II analogues which we are using to probe specific contacts made by key residues within the binding surfaces with the receptors.

AUTOMATION OF THE BUCCAL CYTOME AND NEUTRAL LIPID ANALYSES IN HUMAN BUCCAL CELLS BY LASER SCANNING CYTOMETRY

Francois M.1, 2, Leifert W.R.1, Thomas P.1, Martins R.2 and Fenech M.1 1CSIRO Animal Food and Health Sciences, Adelaide, SA. 2Centre of Excellence in Alzheimer’s Disease Research and Care, Edith Cowan University, Joondalup, WA.

The buccal micronucleus cytome assay has previously been used and validated using visual scoring microscopy to measure distinct differences between the cytome profiles associated with normal ageing relative to that for premature ageing clinical outcomes such as Down’s syndrome and Alzheimer’s disease. The aim was to develop an automated laser scanning cytometry (LSC) protocol to study the buccal cytome assay including nuclear anomalies (aneuploidy) as well as the accumulation of neutral lipids in the different buccal cell types. Cells derived from the buccal mucosa were applied to a microscope slide, stained with Fast Green (cytoplasm), Oil Red O (neutral Lipid) and DAPI (nuclei). The frequency of the different buccal cell types was scored (basal cells 16%, transitional cells 35%, differentiated cells 24% and karyolytic cells 25%). The DNA content and neutral lipid content of basal cells was significantly higher compared with the other cell types. This automated method allows for the co-location of neutral lipids and DNA content within the specific cell types and can be combined with other cellular markers for high content automated analyses in population studies.

MONOMERIC STATUS OF 14-3-3ZETA IS CONTROLLED BY SALT BRIDGING INTERACTIONS WITHIN THE DIMER INTERFACE

Goodwin K.L.1, 2, Carver J.A.2 and Woodcock J.M.1 1Centre for Cancer Biology, SA Pathology, Adelaide, SA 5000, Australia. 2School of Chemistry and Physics, The University of Adelaide, Adelaide, SA 5005, Australia.

14-3-3 proteins are an important key in the cell cycle, playing a role in apoptosis, cell growth and the modulation of a target proteins catalytic activity, in a phosphorylation dependant manner. The dimeric structure of 14-3-3 is important for these in vivo functions. The monomeric state of 14-3-3, previously produced by the phosphorylation of the buried hydrophobic residue, Ser58, has been shown to be less stable and less active than its dimeric counterpart. This dimeric disruption does not prevent 14-3-3 from binding to phospho-peptide targets, but the monomers are unable to perform any structure alteration of target proteins. The regulation of 14-3-3 in vivo is thought to be regulated by this phosphorylation ofSer58. Three major salt bridges have been hypothesised to be present in the dimer interface of 14-3-3zeta, which enable it to homodimerise (Asp21-Lys85, Glu5-Lys74, Arg18-Glu89). Disruption of two of these sites was undertaken by site-directed mutagenesis and the monomeric nature of each mutant was determined by employing Bis-ANS, crosslinking with Native PAGE and SEC-MALLS techniques. These have shown that the disruption of one of these salt bridging sites causes sufficient dimer disruption to produce monomers of 14-3-3zeta. This indicates that these salt bridges are essential for the formation of dimeric 14-3-3 proteins.

PATCHED-DEFICIENT MICE EXHIBIT INCREASED LIVER INJURY FOLLOWING THIOACETAMIDE TREATMENT

Grzelak C.A.1, Patkunanathan B.1, Warner F.J.1, Shackel N.A.1, 2, 3 and McCaughan G.W.1, 2, 3 1Liver Injury and Cancer, Centenary Institute, Newtown, NSW. 2Faculty of Medicine, Unversity of Sydney, Sydney, NSW. 3A. W. Morrow Gastroenterology and Liver Centre, RPAH, Camperdown, NSW.

Reactivation of the Hedgehog (Hh) signalling pathway is associated with liver injury, fibrosis and hepatocellular carcinoma (HCC) development. Mice deficient in the Hh receptor/repressor Patched1 (Ptch1) have increased baseline Hh activation (Goodrich et al. Science 1997). This study aimed to characterize the degree of liver damage and Hh activation in Ptch1-lacZ mice, following hepatocellular liver damage. Methods: Wildtype (WT) and Ptch1-lacZ heterozygote (Ptch1+/-) C57BL/6 male mice (n=6-9/group) were treated with thioacetamide (TAA) (300mg/L; ad lib) for 8 weeks. Serum liver function tests, H+E, picrosirius red (PSR), WB and immunofluorescence (IF) determined the extent of liver injury. qRT-PCR and WB assessed Hh and target gene expression in whole liver. Results: ALT (p=0.0027) and AST (p=0.0012) enzyme levels were significantly increased in Ptch1+/- vs WT mice following TAA. Greater collagen-I deposition occurred in Ptch+/- littermates, combined with a 2.24-fold increase in α-SMA protein and increased vimentin+ cells. Progenitor cell expansion (EpCAM+) associated with the Ptch+/- phenotype. Hh ligand protein levels were equivalent in Ptch+/- and WT following TAA. However, Hh pathway activation significantly increased in Ptch+/- vs WT as indicated by Gli1 mRNA upregulation (p=0.0465). The degree of hepatocyte injury and Hh activity correlated significantly (ALT vs Gli1 mRNA; Spearman r=0.6907; p=0.0011). Conclusions: Patched-deficient mice exhibit an enhanced liver injury/repair and Hh response to TAA treatment, compared to WT littermates. Cirrhosis is considered a premalignant condition to HCC development, and Hh is a known pro-oncogenic pathway. This work provides further evidence that Hh is an integral pathway that drives liver injury progression, and potentially HCC development.


POSTERS TUESDAY

POS-TUE-089 POS-TUE-090BUILDING, EVALUATING AND REBUILDING BIOINFORMATICS PIPELINES

Lawrence D.M. SA Cancer Genomics Facility, Centre for Cancer Biology, SA Pathology, Adelaide, SA, Australia.

High throughput sequencing requires a significant amount of computer processing before data is ready for human analysis. Usually, many of the steps needed for an experiment can be performed by existing software, and these are combined together to form a pipeline. While fitting existing software into a pipeline is generally much more efficient than writing your own, 3rd party tools still need to be evaluated and tested. Other issues include deciding between the (often very large) numbers of competing tools, large differences in tool quality, not fully understanding internal algorithms and managing tool bugfixes and upgrades. As it’s hard to know what you’re not seeing, losing good data at each step is also a constant danger. We discuss strategies to try and identify and minimise this risk. With new techniques, tools and upgrades being released, even well understood, mature pipelines will have parts constantly upgraded or replaced, and for research work pushing the boundaries of existing tools, it is a given that large sections will be rewritten as a true understanding of the problem and data is only reached after much trial and error. While seemingly straightforward for small jobs, constant upgrades means that maintaining state of the art, reproducible pipelines for different experiments starts to require a similar level of discipline as large software projects. We discuss techniques for automated testing and evaluation of tools, managing upgrades, and a more iterative method of working with other researches.

GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE IS A KEY COMPONENT OF THE ANTIFUNGAL ACTIVITY OF THE PLANT DEFENSIN NAD1

Bleackley M.R.1, 2, Wiltshire J.L.1, 2, Van Der Weerden N.L.1, 2 and Anderson M.A.1, 2 1La Trobe Institute for Molecular Sciences. 2Hexima Ltd.

Plants lack the adaptive immune system found in higher animals to elicit pathogen specific immune responses and thus depend on a broad spectrum innate immune system for protection against microbial pathogens. A crucial element of this immune system is the expression of small proteins and peptides that have antimicrobial activity. Plant defensins are a family of small, basic proteins that contain 4-5 disulfide bonds, many of which are known to possess potent antifungal activity. The defensin from ornamental tobacco Nicotiana alata, NaD1, is active against plant pathogens. The mechanism appears to require the cell wall and is hypothesized to involve internalization of the defensin and killing of the fungal cell through an interaction with an intracellular target. More recently NaD1 was also shown to be active against human fungal pathogens including Candida albicans as well as the model yeast Saccharomyces cerevisiae. The activity of NaD1 against human pathogens demonstrates the potential for the use of this plant defensin for novel antifungal therapeutics. Understanding the mechanism by which NaD1 is killing fungi is essential for achieving the full potential of plant defensins in treatment of fungal infections. Using the model yeast S. cerevisiae we have shown that NaD1 interacts with Tdh3p using an NaD1 coupled sepharose column and analysis of fractions eluted with free NaD1 by MALDI TOF. Tdh3p is one of three isoforms of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) found in S. cerevisiae. A tdh3-delta strain was also shown to be resistant to NaD1 compared to wildtype and to have decreased membrane permeabilization kinetics. This has led to the working hypothesis that the antifungal activity of NaD1 is inherently linked to GAPDH, possibly through the induction of programmed cell death, a process in which GAPDH has a well defined role.


POSTERS WEDNESDAY

POS-WED-001 POS-WED-002


ISOMIRS - THE OVERLOOKED REPERTOIRE THAT CONTRIBUTES TO THE DYNAMIC MICRORNAOME

Neilsen C.T.1, 2, Bracken C.P.1, 3 and Goodall G.J.1, 2, 3 1Centre for Cancer Biology, SA Pathology, Frome Road, Adelaide, South Australia, 5000, Australia. 2School of Molecular and Biomedical Science, The University of Adelaide, Adelaide, South Australia, 5005, Australia. 3School of Medicine, The University of Adelaide, Adelaide, South Australia, 5005, Australia.

The emergence of deep sequencing has led to both the identification of novel microRNAs (miRNAs) and the growing appreciation of extensive sequence heterogeneity within individual miRNA loci. These variants, termed isomiRs, display length and/or sequence variations and are often expressed in a cell-specific manner. A number of studies have recently emerged to show the potential of isomiRs to impart differences in target selection, miRNA stability and loading into RNA-induced silencing complexes (RISCs), Thus necessitating caution be taken by researchers to consider which miRNA forms they are actually studying. However, despite their abundance, reports indicating the differential functionality of isomiRs have only begun emerging in the past few years and are largely confined to specific variants of specific miRNAs. As such, the widespread significance of isomiRs remains an important issue yet to be fully resolved.

ALTERATIONS IN RIBOSOMAL PROTEIN EXPRESSION; POTENTIAL ROLES IN CHILDHOOD ACUTE LYMPHOBLASTIC LEUKAEMIA

Nelmes G.1, Ho N.1, Henry M.2 and Catchpoole D.R.1 1The Tumour Bank/CCRU at the Children’s Hospital at Westmead, NSW, 2145. 2Phoenix Children’s Hospital, Phoenix, Arizona, USA.

Ribosome biogenesis is a tightly regulated cellular process essential for protein synthesis originally thought to be rigid with little or no alteration possible without causing cell death. Recent work has challenged this notion highlighting that the composition of the ribosome can dramatically influence translational control, disease and oncogenesis. One example of this phenomenon is Diamond-Blackfan Syndrome where mutations to RPS19 lead to anaemia. A growing number of cancers have been identified as having increased or altered ribosomal protein (RP) gene expression. This combined with ‘ribosomopathies’, a collection of genetic diseases with impaired ribosome function which often have higher incidence of cancer, highlights the need to examine the role of RPs in oncogenesis. Our work investigating RP gene expression has focused on childhood acute lymphoblastic leukaemia (ALL). RNA from diagnostic bone marrows (BM) from 39 pre-B ALL patients and 10 normal samples was used to generate gene expression data using the Affymetrix HU133A chip. The gene expression profile of RPs in ALL were chaotic and highly active compared with the healthy BM. In contrast the gene RPS6KA2, identified as a tumour suppressor in epithelial ovarian cancer, was significantly down regulated. Interrogation of our dataset using Biplots, GeneRaVE, Stepwise Diagonal Discriminant Analysis identified three RPs within the 20 most parsimonious genes which best discriminate between relapse and non-relapse. Examination of the regulatory networks involving RPs using MINER identified a lineage-specific relationship between RPL38 and CD34 gene expression. We hypothesise a role for RPL38 in haematopoietic progenitor cells. These results suggest a role of RPs within ALL requiring further investigation which will be discussed.

EXPRESSION AND FUNCTIONAL ANALYSIS OF DECAPENTAPLEGIC GENE ORIGINATED FROM THE BOMBYX MORI

Park S.W., Goo T.W., Choi G.H., Kim S.R. and Kang S.W. Sericultural & Apicultural Materials Division, National Academy of Agricultural Science, RDA, Suwon 441-100, Republic of Korea.

The bone morphogenetic and osteogenic proteins (BMPs/OPs) belong to the transforming growth factor-β (TGF-β) supergene family act as soluble signals for the de-novo initiation of bone formation. Based on the 75% sequence homology, these BMP were suggested to be the human homologue of the Drosophila Dpp protein. In this study, we focused on a B. mori Dpp homologue as a candidate molecule for bone morphogenetic protein. The molecular cloning of the Dpp gene and its expression in intact and regenerating B. mori are useful for artificial bone regeneration and may help us to understand the bone formation effect of silkworm as a food and drug application materials.

QUANTIFICATION OF RNA INTEGRITY BY STANDARD qPCR, FOR MEASURING TRANSCRIPT NUMBER

Brisco M.J. and Morley A.A. Dept Haematolgy & Genetic Pathology, Flinders University of S Australia, Bedford Park, SA 5042.

RNA, a labile molecule, needs careful handling, to avoid nucleases and damage. For expression profiling, RNA degradation needs assessing, and we report a straightforward PCR method, to quantify lesions/1,000 bases. Briefly, the method uses a Poisson model, to explore how lesions affect RNA integrity, reverse transcription, and qPCR. It uses efficient RT-qPCR to amplify 3-5 products from a reference gene; plots Ct vs. amplicon length; and estimates degradation from regression. It then corrects quantification data for amplicon length and degradation. RNA from fresh blood, reverse transcribed (Superscript III) and RT-qPCR performed, using N-ras, APC or GAPDH as reference genes, had a mean of 1.59 lesions/1000 bases (SD=0.61; n=33). RNA degraded by 30 min at 55C or 90C, gave 3 lesions/1000 or 90/1000 respectively, and lesions increased progressively with temperature. In 30 heat-treated RNA samples, degradation estimates from the new method, correlated strongly with estimates from ΔΔCt (y=0.90x+0.73; r=0.89, p<<0.0005). To mimic expression profiling, 4 GAPDH amplicons were quantified, relative to 4 APC amplicons, in 3 normal RNAs and 3 heat-treated RNAs. Results from conventional quantitation varied up to 18-fold, influenced by degradation, target amplicon’s length, and standard amplicon’s length. The new method corrected for these factors and removed their effect. It was compared to RIN as assessed on an Agilent 2100 BioAnalyzer. Untreated RNA (1 lesion/1000) and 75C-treated RNA (10 lesions/1000), yielded RIN scores of 7-8 and 2. For RNAs treated >75C, RIN score remained 2, but PCR measured lesions increasing progressively. The new method is simpler, cheaper and more informative than RIN. It requires <10pg RNA, and should be suitable for single-cell analysis.


POSTERS WEDNESDAY



DNA LOOP DOMAINS: TESTING THE THEORY OF ENHANCER BLOCKING INSULATORS IN E. COLI

Priest D.G., Shearwin K.E. and Dodd I.B. The University of Adelaide.

The genome is the prime reference for life’s instructions, yet life must ‘read itself’ in the dynamic environment of the cell, robustly executing replication and differentiation whilst constantly fending off disease. Evolution, has recruited diverse biophysical and spatiotemporal phenomena to develop complex regulatory hierarchies infiltrating every corner of the cell. A prime example is that of enhancers, genetic elements that regulate genes located hundreds of kilobases away on the genome. It is now established that enhancers activate their target genes through direct physical contact, with the intervening chromatin looping out, however, since enhancers function in a position and orientation-independent manner, the question arises as to how inappropriate enhancer-promoter cross-talk is prevented. Enhancer reach is proposed to be restricted by insulators, genetic elements dotted throughout the genome that loop to one another via DNA binding proteins, thereby subdividing chromatin into distinct topological domains. The ‘loop-domain’ model suggests that when enhancer and promoter are on separate chromatin loops they will be insulated from one another. The model’s fundamental tenet is that alternating DNA loops will ‘interfere’ with one another, a phenomenon that has yet to be directly measured in vivo. I have employed well-studied DNA looping proteins in a tractable prokaryotic system to quantify DNA looping interference, thereby providing support for the loop-domain model.

GENOME-WIDE ANALYSIS IN DEVELOPMENTAL EYE DISEASE TO DETERMINE UNDERLYING GENETIC FACTORS

Prokudin I.1, Storen R.1, Simons C.2 and Jamieson R.1, 3 1Eye Genetics Group, Children’s Medical Research Institute, Sydney, Australia. 2Queensland Facility of Advanced Bioinformatics, University of Queensland, Brisbane, Australia. 3Sydney Medical School, University of Sydney, Sydney, Australia.

Abnormalities in development of the lens, anterior segment and whole eye frequently lead to severe visual impairment. Disease genes have only been identified in a minority of cases, and in these cases marked genetic heterogeneity and overlap in developmental phenotypes is noted. In addition, affected families are frequently small or there are singleton cases rendering sequential sequencing or traditional linkage approaches difficult for disease gene identification. Advances in next-generation DNA sequencing technology now allow a massively parallel genome-wide approach in coding exon analysis. In this project we aim to identify candidate disease genes in patients with developmental eye diseases including cataract/microcornea, Peters anomaly and microphthalmia/coloboma. We undertook exome capture and sequencing in ten probands using paired-end next-generation sequencing. Captured single nucleotide polymorphisms (SNPs) and indels were subject to filtering based on the coverage, SNP quality scores, region (exonic), functional consequences of the variant and allele frequency. A candidate gene approach was utilized to examine for mutations in genes known to be affected in these disorders. Further analyses were undertaken to examine orthologues of genes known to cause developmental eye anomalies in mice and other model systems, as well as examination of signaling pathways and target genes with roles in eye development. In three patients we have identified known and novel mutations in genes known to function in eye development and novel candidates are under investigation from our further analyses. Our findings demonstrate the power of exome sequencing technology in identification of causative genetic factors in patients with highly heterogeneous developmental eye disorders.

DOES THE RED GENE IN GRAPES AFFECT THE FLAVOUR OF WINE?

Rinaldo A.R.1, 2, Boss P.K.1, Ford C.M.2 and Walker A.R.1 1CSIRO Plant Industry, Waite Campus, Adelaide. 2School of Agriculture, Food and Wine, The University of Adelaide.

Red and white wines, made from red and white grapes respectively, have very distinctive flavour and aroma profiles. While this could be partially attributed to the different methods used to produce these wines, we are testing the hypothesis that some of these distinguishing attributes are due to the presence of different flavour and aroma precursors in red and white grapes. A family of compounds known as anthocyanins are responsible for the red/purple colour in grapes and wine. The transcription factors VvMYBA1 and VvMYBA2 have been identified as important regulators of anthocyanin synthesis and the two genes encoding these factors are mutated in white grapevine cultivars. The present research has used both naturally occurring and transgenic mutant grapevines in which VvMYBA1 and VvMYBA2 gene expression has been altered to establish if there is a link between berry colour and wine flavour and aroma. Using controls and transgenic Chardonnay grapevines altered to produce anthocyanins, as well as Shiraz vines with partial (rose Shiraz) or complete silencing of the VvMYBA1 gene (white Shiraz), wines have been produced using micro-fermentation. Naturally occurring Cabernet Sauvignon mutant grapes possessing rose and white coloured phenotypes were also fermented. The volatile composition of each wine was analysed and transcriptome analysis of transgenic berries was also performed. The results of this research suggest that the MYBA transcription factors in grapevine may act to down-regulate monoterpene biosynthesis resulting in a reduced concentration of monoterpene flavour precursors in red grapes. Monoterpenes are important wine flavour compounds that attribute to tropical and floral aromas more commonly predominant in white wines.

CHARACTERISATION OF PEPTIDES ENCODED BY UPSTREAM OPEN READING FRAMESAndrews S.1, Friend L.1, Smith R.1 and Rothnagel J.A.1, 2 1School of Chemistry & Molecular Biosciences, University of Queensland, St. Lucia, Qld, 4072, Australia. 2The Institute for Molecular Bioscience, University of Queensland, St. Lucia, Qld, 4072, Australia. Upstream open reading frames (uORFs) generally attenuate the expression of the main coding sequence of mRNAs by intercepting scanning ribosomes during translation. However, we hypothesize that some uORFs are translated to yield bioactive microproteins. We use the term uPEPs to describe peptides encoded by uORFs. In order to improve the identification of uORFs and to allow rapid visual inspection of conserved uORFs (and their encoded uPEPs) we developed a program that presents the degree of homology as a ‘heat map’ (uPEPperoni). This program performs a blast sequence comparison between a human transcript with that from other species. We have used this program to identify ~500 uPEP sequences. We then cloned 11 of these uPEP sequences into a GFP expression vector (Clontech). After appropriate quality assurance, these plasmids were transfected into HeLa cells and GFP expression visualized by confocal scanning microscopy. We found that 9 of these exhibited distinct cellular localization patterns that could provide insight into their cellular roles. Four uPEPs showed largely nuclear localization, five showed cytoplasmic localization and two were indistinguishable from the GFP alone control. Importantly, 5 uPEPs exhibited organelle-specific localization including to mitochondria, endoplasmic reticulum, Golgi and the nucleolus. The distinct localization of these uPEPs was confirmed using alternative tags (SNAP & CLIP; New England BioLabs). In addition, we reversed the orientation of the tag and found that for some uPEPs this resulted in abrogation of the localization seen for the N-terminal tag suggesting that signal sequences present in the uPEP were masked by the tag. Additionally, we confirmed the localization of one uPEP using a synthetic peptide tagged with a FITC fluorescent label. The surprisingly high hit rate (82%) for specific sub-cellular localization for these peptides is suggestive of independent biological functions and supports our hypothesis that a subset of uPEPs have independent bioactivities.


POSTERS WEDNESDAY



ROLE OF ZEB1 AND CHROMATIN MODIFYING ENZYMES IN EPITHELIAL-TO-MESENCHYMAL TRANSITION

Sadras F.1, Attema J.L.1, Gregory P.A.1 and Goodall G.J.1, 2, 3 1Centre for Cancer Biology, SA Pathology, Adelaide. 2School of Molecular and Biomedical Science, University of Adelaide. 3Discipline of Medicine, The University of Adelaide, Adelaide, SA 5005, Australia.

Epithelial-to-mesenchymal transition (EMT) regulates cell plasticity during normal development and wound healing but also plays an important role in cancer. Breast, colon, prostate and other solid epithelial cancers are thought to metastasise after undergoing a variation of EMT. Metastasis enables tumour cells to separate from the main tumour mass and spread throughout the body, seeding other organs and is the main cause of cancer related death. Our laboratory has previously shown that the microRNA-200 family maintains the epithelial state and are strong negative regulators of EMT. A key target of the miR200 family is the transcriptional repressor Zinc finger E-box-binding homeobox 1 (ZEB1), specifically expressed in mesenchymal and metastatic cell lines. The expression of ZEB1 is sufficient for epithelial cells to undergo EMT, thereby increasing tumor invasiveness; conversely, the expression of miR200 family members triggers a mesenchymal-to-epithelial transition. The mode of action of ZEB1 in EMT is poorly understood. However, ZEB1 has been shown to directly interact with a number of chromatin associated factors including BRM/SWI2-related gene 1 and Histone acetyltransferase p300, and indirectly with euchromatic histone-lysine N-methyltransferase 2. ZEB1 has also been shown to be present on several epithelial gene promoters, including E-Cadherin and the miR-200c cluster. We aim to identify specific ZEB1 interacting proteins and determine their effects on the epigenetic state of epithelial breast cells during the course of an induced EMT. Our work will provide novel insights into the mechanisms by which ZEB1 controls the molecular processes of EMT in mestastic breast cancer.

HUMAN PLASMA INFUSED WITH JET PROPULSION-8 (JP-8) AVIATION FUEL DISPLAYS CYTOTOXICITY FOR HUMAN HAEMATOPOIETIC CELLS.Scott A.P.1,2, Jansen E.S.1, Warner R.3, Gallagher R.L.2, M Bowlay2, Crowley L.C.1, Christensen M.E.1, Wallis T.P.2, McWhinney A.C.4, Gardner I.R.3, Price G.R.2, Venter D.J.2, Bowling F.G.4,5 and Waterhouse N.J.1,5.

1Apoptosis and Cytotoxicity Laboratory, Mater Medical Research Institute, Brisbane, Australia. 2Department of Pathology, Mater Adults Hospital. Brisbane, Australia. 3Defence Centre for Occupational Health, Department of Defence, Canberra, Australia. 4Department of Metabolic Medicine, Mater Children’s Hospital, Brisbane, Australia. 5Department of Medicine, University of Queensland, Brisbane.Exposure to jet propulsion-8 (JP-8) aviation fuel, which is commonly used in military aircraft, has been linked to a variety of pathologies including immunological and neurological disorders. Understanding the effects of JP-8 exposure is therefore of significant interest to workers exposed to JP-8 through the four formal F-111 Deseal/Reseal Program, some of whom developed various disorders at a greater than expected rate. Previous research investigating the cellular effect of JP-8 has used ethanol as a diluent. However this is unlikely to mimic the in-vivo environment, where any toxic component that would cause a wide variety of pathologies is likely to be transported in the plasma. We have found that although JP-8 is insoluble in plasma, low concentrations of JP-8 infused plasma rapidly induced apoptosis of haematopoietic cells. Lower concentrations were sufficient to alter the gene expression profile of these cells. Further investigations are required to determine whether toxic and sub-toxic levels of JP-8 carried by human plasma can account for the pathology observed in personnel exposed to JP-8.

EFFECTS OF AQUEOUS EXTRACTS OF ROSEMARY AND GREEN COFFEE ON THE DEPRESSION-INDUCED BY FORCED SWIMMING TEST ON BRAIN OF RATS

Shati A., Alzylaey K. and Elsaid F. KING KHALID UNIVERSITY, BIOLOGY DEPARTMENT, ABHA, SAUDI ARABIA.

Forced swimming test (FST) is considered as an animal model of depression and used to induce a depressive-like behavior. Natural products have antidepressant effect and have lower side effect. Sprague dawely rats were used in this study and divided into 4 groups (5/group); Control group: daily received normal saline solution orally administered; Depression group: daily exposed to FST for 10 min for 15 days; Depression& rosemary group: daily exposed to FST for 10 min and orally administered with rosemary extract at 200 mg/kg b. wt. for 15 days; Depression& green coffee group: daily exposed to FST for 10 min and orally administered with green coffee extract at 600 mg/kg b. wt for 15 days. Activities of acetylcholinesterase (AchE) and enzymatic antioxidants in brain were decreased in depression group when compared to controls. Also, brains of depression group suffered from high oxidative stress, represented by the increment of lipid peroxidation and hydrogen peroxide levels, accompanied with FST exposure. Molecular results showed that expression of AchE gene was low but the expression of P53, Bcl-2 (B cell lymphoma 2) and interleukins (IL-4& IL-12) genes was high in the brain of depression group. Both aqueous rosemary and green coffee extracts could attenuate the imbalance in antioxidants/oxidants system and amend of the distorted expression of the tested genes in the brain tissue. Therefore, these natural products could be used as antidepressants.

NON-ADDITIVE GENE EXPRESSION IN ARABIDOPSIS HYBRIDS

Tanurdzic M.1, Finigan P.2, Auer P.3, Ernst E.2, Doerge R.W.3 and Martienssen R.2 1School of Biological Sciences, The University of Queensland, St. Lucia, QLD 4072, Australia. 2Cold Spring Harbor Laboratory, Cold Spring Harbor NY 11724, USA. 3Department of Statistics, Purdue University, West Lafayette IN 47907, USA.

We have employed transcriptome sequencing and ultra-high throughput SNP genotyping in Arabidopsis hybrids to determine the contribution of homeoallele bias to non-additive gene expression. We have found that homologous genes with a biased transcript accumulation in favor of one homeoallele are no more likely to be non-additively expressed than those genes without such a bias. The source of this non-additive gene expression is still unclear, but presumably stems from cis- and trans-regulatory divergence between the progenitor-derived chromosome sets. Although some parental bias responsible for non-additive gene expression is inherited in cis, we conclude that non-additive gene expression in Arabidopsis hybrids is largely a trans- effect consistent with the Bateson-Dobzhansky-Muller hypothesis of gene interaction in hybrids. In contrast, the transcriptome and small RNA sequence analysis showed that differences between paternal and maternal small interfering RNA (siRNA) are responsible for epigenetic instability in transposon sequences, which leads to epigenetic reprogramming in hybrids and transcriptional activity of sequences that are normally transcriptionally silent in the parents.


POSTERS WEDNESDAY



TRANSPOSON-MEDIATED RECRUITMENT OF THE TAMATE1B GENE OF WHEAT INTO ALUMINIUM TOLERANCETovkach A.1, Lewis D.1, Rathjen T.1, Ryan P.R.1, Richardson A.E.1, Ramesh S.2, Tyerman S.D.2 and Delhaize E.1 1CSIRO Plant Industry, GPO Box 1600, Canberra, ACT 2601. 2School of Agriculture, Food, and Wine, University of Adelaide, Glen Osmond, SA 5064.

Aluminium tolerance in wheat is controlled by two parallel mechanisms, the first is the aluminium-activated efflux of malate from root apices via the anion channel TaALMT1; the second relies on the constitutive efflux of citrate from root apices. Here we describe the isolation and characterization of TaMATE1B from wheat and show that it encodes a citrate transporter located on the plasma membrane. The coding regions of TaMATE1B were identical in a genotype showing citrate efflux (cv. Carazinho) and one lacking citrate efflux (cv. Egret). Flanking sequences upstream of the coding region showed two differences between Carazinho and Egret. The first was a single nucleotide polymorphism (SNP) ~2 kb upstream from the start codon in Egret. The second difference was an 11.1 kb insert 25 bp upstream of the TaMATE1B start codon in Carazinho which was absent from the corresponding region in Egret. The influence of these differences on TaMATE1B expression was investigated by using different regions of the sequence upstream of TaMATE1B to drive the GFP expression in transgenic rice lines. In the apical millimetre of root, promoters that incorporated 1.4 kb and 2.4 kb regions upstream of TaMATE1B in cv. Carazinho (incorporating the 3’ region of the transposable element) generated 20-fold greater fluorescence as compared to the native promoter in Egret, and the SNP alone consistently increased fluorescence two-fold compared to the native promoter. We demonstrate that the transposable element-like insertion upstream of TaMATE1B in cv. Carazinho extends its expression to root apices resulting in constitutive efflux of citrate from these tissues. We propose that this mutation extends the original function of TaMATE1B, perhaps from one associated with Fe nutrition, to include Al tolerance as well.

TRANSCRIPTIONAL REGULATION AND GENE FUNCTIONS OF BACILLUS SP. CDB3 ARS 1

Yu X.1, Zheng W.1,2, Aquilina A.1 and Zhang R.1 1University of Wollongong, NSW, Australia. 2Northeast Forestry University, Harbin, China.

Arsenic compounds are widely dispersed in the environment, commonly existing in the form of arsenite or arsenate. Most organisms which can survive in arsenic-containing environments have evolved arsenic resistance (ars) operons. Bacillus sp. CDB3 isolated from an arsenic-contaminated cattle dip site contains an uncommon ars operon 1 bearing eight genes in the order arsRYCDAT-orf7-orf8. In this study, we investigated how transcription of the ars 1 is regulated and putatively identified the mechanisms by which these genes are involved in arsenic detoxification. The results of sequence analysis inferred that a functional hair-pin structure potentially exists between arsC and arsD, suggesting transcriptional regulation complexity. Northern blot analysis confirmed that ars 1 is regulated under arsenic induction and generates two detectable transcripts: a full-length 8-gene transcript of arsRYCDATorf7orf8 and 3-gene transcript of arsRYC. After mutagenesis to abolish the inverted repeat between arsC and arsD, the short transcript became undetectable, demonstrating its role as a transcriptional terminator. In addition, the results of gene deletion assays indicated that ArsA may couple with YqcL to form an ArsAY complex, which may enhance the efficiency of arsenite extrusion. The involvement of the novel genes orf7 and orf8 in arsenic resistance was also investigated. Disruption of orf7 had little influence on arsenic resistance. In contrast, orf8, a putative phosphatase gene, was selectively involved in arsenate resistance but not arsenite resistance, and furthermore, transformation of orf8 into E. coli JM109 significantly increased arsenate resistance, compared cells expressing a control plasmid. Work is in progress to identify the anti-terminator involved in the ars operon, and characterize the subtle coordination and functional differences between the two RNA transcripts, in order to understand how these genes provide efficient resistance to the toxin arsenic in Bacillus sp. CDB3.

CHRACTERIZATION OF ANTIMICROBIAL PEPTIDES OVER-EXPRESSED IN DAMAGED GUT CELLS OF DROSOPHILA MELANOGASTER

Yun E.Y., Yoon Y.I., Hwang J.S., Ahn M.Y. and Goo T.W. Department of Agricultural Biology, National Academy of Agricultural Science.

Inflammatory bowel disease (IBD) is a group of chronic disorders of unknown etiology characterized by inflammation of the gastrointestinal tract. To investigate the cause of IBD using Drosophila melanogaster, we made D. melanogaster intestine damage model by oral feeding with variety IBD inducer such as pathogenic bacteria Serratia marcescens, dextran sulfate sodium (DSS) and bleomycin, and got the survival rate and 50% lethal dose (LD50). After feeding with IBD inducer of LD50, the number of enteroendocrine cells secreting AMPs, expression level of AMPs (antimicrobial peptides), and toll, pelle, dorsal and relish, upstream signal of AMPs were up-regulated in damaged gut cells. Also, we confirmed that cell death by apoptosis was very increased, and ISCs were greatly increased in damaged gut cells. Whereas, results using RNA interference (RNAi) lines for various AMPs genes knockdown showed that the survival rate was increased and cell death by apoptosis was decreased in RNAi lines as compared with wild-type flies. Anti-toll, anti-dorsal, and anti-pelle-fed flies survived longer than normal flies after feeding with DSS. As above results, we suppose if IBD inducer was entered into fly gut, various AMPs will be up-regulated for self-defense. Over-expressed AMPs play a role for not only intestinal protection by destroying and removing harmful microorganisms, but also intestinal destruction by attacking their own cells like cytokines. Accordingly, we suggest each AMP over-expressed by IBD inducer is a kind of cause for IBD.

BIOLOGICAL CHANGES IN THE HIPPOCAMPUS FOLLOWING CHRONIC METHAMPHETAMINE USE: A PROTEOMIC APPROACH

Sauer M., Mirzaei M., Wearne T., Haynes P.A., Goodchild A.K. and Cornish J.L. Departments of Psychology and Chemistry and Biomolecular Sciences Macquarie University, North Ryde, NSW, Australia.

Methamphetamine is a highly addictive and harmful drug. Methamphetamine is known to act on dopamine and noradrenaline brain systems with previous studies highlighting protein changes of the striatum and amygdala following repeated methamphetamine administration. The hippocampus is also innverated by dopamine, however its role in methamphetamine abuse has not been determined. Therefore, this study assessed the biological changes in the hippocampus following chronic methamphetamine use. It was hypothesised that differentially expressed proteins would demonstrate evidence of neuroplasticity, particularly cytoskeletal and synaptic alterations in methamphetamine-treated rats, as well as early signs of neurotoxicity, such as oxidative stress. Using locomotor sensitization to methamphetamine as an animal model of methamphetamine abuse and psychosis, 16 male Sprague Dawley rats were randomly assigned to either methamphetamine or saline groups. Two weeks following 7 days of daily treatment with methamphetamine or saline, rats were euthanised and hippocampi were dissected for protein analysis. A label-free shotgun proteomic analysis using mass spectrometry was used to detect differentially expressed proteins indicative of biological changes. Methamphetamine treatment produced significant changes in the proteome of the hippocampus. Biological triplicate analysis revealed 1035 reproducibly identified proteins in treated rats (protein FDR<0.27%), and 969 proteins in control rats (protein FDR<0.27%). A large number of differentially expressed proteins indicative of cytoskeletal alterations such as vimentin, and synaptic alterations such as GTPase KRas were found. Differentially expressed proteins indicative of oxidative stress such as electron-transferring-flavoprotein dehydrogenase were also found. These biological changes may have significant implications for the reversal of learning and memory deficits, and induced psychosis in chronic methamphetamine users.


POSTERS WEDNESDAY



INVESTIGATING LIPID UPTAKE, METABOLISM AND BIOSYNTHESIS IN A SOUTHERN BLUEFIN TUNA CELL LINE

Scholefield A.M. and Schuller K.A. Flinders University.

Southern bluefin tuna (SBT, Thunnus maccoyii) are highly prized predatory marine fish with an industry value in South Australia of approximately $102 million in 2009-10. Currently, these fish are captured as wild juveniles and then fattened in sea cages off the coast of Port Lincoln before on-selling, mainly to the Japanese sashimi market. Captive spawning and complete aquaculture of SBT is currently being trialled by Clean Seas Tuna (CST). However, one of the challenges facing both the current method of tuna culture and the method being trialled by CST is ensuring adequate nutrition for the fish without the reliance on wild-caught sardines, their main food source. A manufactured feed would address this problem, however, there is little known about the dietary requirements of SBT in terms of fish oil and protein in the diet. The critical components of fish oil are the omega-3 long-chain polyunsaturated fatty acids (n-3 LC-PUFA), docosahexaenoic acid and eicosapentaenoic acid, which are only found in the marine environment. Fish, and other vertebrates, can synthesise n-3 LC-PUFA from the precursor α-linolenic acid which is abundant in certain plant oils but the efficiency of this synthesis is low. In this study, we have used a recently developed SBT cell culture model to investigate the effects of various fatty acids on cell proliferation, fatty acid uptake and n-3 LC-PUFA synthesis in SBT. This work furthers our understanding of lipid and fatty acid metabolism in SBT, the tolerance of SBT to shorter-chain fatty acids and the biosynthesis of n-3 LC-PUFA.

C3 - ADDING STRUCTURE TO YOUR RESEARCH

Seabrook S.A., Fazio V.J. and Newman J. CSIRO Collaborative Crystallisation Centre - www.csiro.au/c3.

Despite decades of research, creating crystals from bio-macromolecular samples to enable structure determination is still a roadblock, due to a lack of a priori knowledge regarding crystal formation. The Collaborative Crystallisation Centre (C3) is a full service technology platform that helps overcome this roadblock by enabling high throughput nano-scale crystallisation screening; we do a lot more with a lot less. Along with screening, the facility can make custom combinatorial screens and help characterise your sample to increase experimental throughput. We utilise an array of state of the art robotics and IT combined with automated incubation and imaging technology, which allows us to achieve highly original crystallisation strategies. This infrastructure is readily accessed via bespoke web-tools that allow C3 users to book experiments, view results and create optimised strategies online. More recently we’ve started to explore the field of protein crystallisation in the cubic phase, have implemented robust formulation screening protocols to stabilise problematic samples, developed a unique screen condition comparison tool which reduces redundancy in any given screening strategy and upgraded our automated imaging system with a UV source for two reasons: to locate more protein crystals and to filter out salt crystals. The biggest advantage of C3 is that we are a team of scientists dedicated to improving protein crystallisation strategies and challenging current processes, and we achieve this by working in close collaboration with national and international experts. C3 is accessible to any researcher, and has ties with several aligned CSIRO research groups that can assist with structural biology projects, from protein production through to solving a structure.

IDENTIFICATION AND STRUCTURAL CHARACTERISATION OF A NOVEL GENE PRODUCT IN MYCOBACTERIUM SMEGMATIS IMPLEMENTED IN MACROPHAGE SURVIVABILITYShahine A.1, Pelosi A.2, Brammanth R.2, Crellin P.2, Coppell R.2, Rossjohn J.1 and Beddoe T.1 1Department of Biochemistry & Molecular Biology, Monash University, Clayton, 3800, Vic. 2Department of Microbiology, Monash University, Clayton, 3800, Vic.

The Corynebactereae suborder of bacteria includes significant human pathogens such as mycobacterium tuberculosis and mycobacterium leprae. The emergence of drug resistant mycobacterial strains has resulted in an urgent need for the development of new treatments. Mycobacteria proliferate within the phagosomes of host macrophages, requiring a vast array of essential proteins for survival. The understanding of mycobacterial macrophage survivability would pave the way for the development of new anti-mycobacterial therapeutics. We’ve recently discovered a novel gene in Mycobacterium smegmatis that, when inactivated, leads to accelerated cell death in host macrophages within the first eight hours post infection. It was found that accelerated cell death was not as a result of oxidative stresses or acidic pH’s typical of a macrophage phagosomal environment. The identified gene product, denoted as MSMEG_5817, encodes a 13.5kDa protein of unknown function, and is found within all mycobacterial species for which whole genome sequence data is available. To gain an understanding of its function, the crystal structure of MSMEG_5817 has been solved to 2.1Å, by 3-wavelength MAD data collected on a selenomethionine derivative at the Australian Synchrotron. The crystal structure revealed a highly hydrophobic binding pocket, which may be involved in the binding and transportation of host macrophage lipids for mycobacterial survivability. Structural homology searches have revealed that MSMEG_5817 is most similar to the sterol carrier protein (SCP-x) class of proteins, which play roles in non-specific lipid transfer. MSMEG_5817 is a previously uncharacterised gene vital for the survivability within host macrophages, with a potential role in host-mycobacteria lipid transport. Interference with the function of MSMEG_5817 may provide a novel therapeutic approach for control of mycobacterial pathogens.

NuRDy PROTEINS: STRUCTURE DETERMINATION OF THE UNCHARACTERIZED CHD4 DOMAINS

Silva A.P.G., Mansfield R.E. and Mackay J.P. School of Molecular Biosciences, University of Sydney, New South Wales 2006, Australia.

The nucleosome remodeling and deacetylase (NuRD) complex is a key player in controlling both normal development and cancer progression. It is a widely expressed and conserved co-regulator complex with essential roles in gene regulation, but like other such complexes it is poorly defined at the protein level. It is our hypothesis that a structural analysis of the NuRD complex will provide a mechanistic understanding of its function and the likely causes underlying the association of NuRD with cancer biology. CHD4 (chromodomain-helicase-DNA-binding protein 4) is one of the proteins that constitute the core of the NuRD complex. We will present structural and biochemical data on several domains from this 218 KDa multidomain protein, and discuss the insights that these data provide into NuRD function.


POSTERS WEDNESDAY



INVESTIGATION OF THE MODE OF ACTIVATION OF THE INSULIN AND INSULIN-LIKE GROWTH FACTOR RECEPTORS

Sit K.C., Croll T.I., Walsh T. and Van Lonkhuyzen D. Cells and Tissues Domain, Institute of Health and Biomedical Innovation, Queensland University of Technology, Brisbane, Queensland. The insulin and insulin-like growth factor-1 receptors (IR and IGF1R, respectively) are two closely related receptors that play highly important roles in development, growth and metabolism. These receptors are also implicated with various diseases such as diabetes and cancer and are therefore regarded as major targets for drug research. Hindering the further development of drug research is the poorly understood mechanisms of how insulin and the insulin-like growth factor-1 (IGF-I) bind and activate their cognate receptors. Previous mutagenesis studies identified residues that were critically important for receptor binding and were later speculated to form one of the ligand binding sites on the IR. However, the latest crystal structure of the IR extracellular domain shows that the previously identified residues are, in fact, not in contact with insulin but with another region of the receptor, the C-terminus of the receptor alpha domain (CT peptide). The CT peptide plays a critical role in growth factor binding: IR or IGF1R constructs without it have no affinity for insulin or IGF-I, and affinity is rescued by the addition of soluble CT peptide. Clearly there is a need to understand how this region interacts with the rest of the receptor and establish a detailed model of ligand binding to both receptors and build upon information in the current literature. Molecular modelling and dynamics simulation techniques were utilised to study a truncated IR structure in complex with the CT peptide and analysed using various techniques including principal component analysis. The importance of the flexibility of the CT peptide was also investigated using linear or cyclic peptide analogues in Biacore experiments.

PROTEASE RESISTANT IGFBP-2 AS A POTENTIAL TUMOUR GROWTH INHIBITOR

Soh C.L., Delaine C., McNeil K., Wallace J.C. and Forbes B.E. School of Molecular and Biomedical Science, The University of Adelaide, Adelaide, South Australia.

The insulin and insulin-like growth factor (IGF) family is responsible for the regulation of both metabolic and mitogenic functions for normal growth and development. However, there is substantial evidence that upregulation of IGF signaling via the type 1 IGF receptor (IGF-1R) and the insulin receptor isoform A (IR-A) leads to increased cancer cell proliferation and survival. This makes the IGF system a good target for the development of potentially novel cancer therapies. As such, we have been developing as an inhibitor of tumour cell growth, a protease resistant IGF binding protein 2 (IGFBP-2) that will sequester IGFs and is unable to release IGFs to target receptors. We recently demonstrated that the mutant IGFBP-2 inhibited growth of MCF-7 breast cancer xenografts. We are now using immunohistochemistry to assess tumour vascularisation, proliferation and IGF-1R expression with the aim to determine the mechanism of action of mutant IGFBP-2 in tumour growth inhibition. In addition we aim to improve the protease resistance of our original mutant IGFBP-2 as our data suggest there are still sites that are cleaved in response to cancer cell proteases, including plasmin and matrix metalloproteinases. Here we present some preliminary data to demonstrate our strategy to improve the protease resistance of our modified IGFBP-2 constructs.

BIOCHEMICAL MODELS TO INVESTIGATE FUNCTION OF SINGLE-MINDED PROTEINS

Sullivan A.E., Raimondo A., Schwab T. and Whitelaw M.L. School of Molecular and Biomedical Science (Biochemistry), University of Adelaide, SA.

Single-Minded 1 (SIM1) and Single-Minded 2 (SIM2) are basic Helix-Loop-Helix (bHLH) Per-ARNT-Sim (PAS) domain-containing transcription factors that are well-conserved in mammals. Both proteins form heterodimers with the common partner factors ARNT or ARNT2 and bind DNA to either enhance or repress transcription of target genes. The bHLH/PAS domain regions of SIM1 and SIM2, which bind DNA and constitute the dimerisation interface with ARNT/ARNT2, are highly homologous (74% identity). This is consistent with observations that SIM1 and SIM2 complexes recognise identical DNA sequences on model promoters. However, respective knockout mice display distinct phenotypes that result in post-natal lethality, indicating that Sim1 and Sim2 are both vital for development and crucially non-redundant. Altered SIM1/2 functionality is also linked to disease states in humans. SIM1 deficiency has been associated with juvenile onset obesity, which is consistent with the hyperphagic obesity phenotype observed in Sim1-/+ mice. A human obesity study identified single nucleotide variants of SIM1, which we have analysed for effects on SIM1/ARNT2 activities including DNA binding, heterodimerisation, protein localisation and stability. We are also investigating if reciprocal mutations in fully conserved regions of SIM2 confer identical alterations in SIM2/ARNT activity. This will indicate if distinct structural aspects of SIM1 or SIM2 help impart specificity to their function in vivo. Increased expression of a SIM2 splice variant, encoding the shortened protein SIM2s, is implicated in tumour progression and invasiveness in several tissues, including prostate. We are currently pursuing a prostate cancer model in mice whereby SIM2s is ectopically expressed under the control of a tissue specific promoter. These studies are contributing to our understanding of SIM1 and SIM2 function from biochemical and physiological perspectives.

HOW CALCIUM INDUCES PERFORIN MEMBRANE BINDING? CRYSTAL STRUCTURES OF PERFORIN-LIKE C2 DOMAINS

Traore D.A.1, 2, Rosado C.J.1, Porter C.J.H.2 and Whisstock J.C.1 1Department of Biochemistry and Molecular Biology, Monash University, Australia. 2Monash Institute of Pharmaceutical Sciences, Monash University, Australia.

Perforin is a multi-domain protein that belongs to the MACPF/CDC family of pore forming toxins. Perforin is secreted as soluble monomers that oligomerise to form to pores on the target membrane upon calcium binding via a C-terminal type 1 C2 domain. Although the crystal structure of Perforin revealed insight into the overall pore structure (Law et al., Nature 2011), the initial event, the molecular mechanism of the calcium dependent membrane binding is yet to be understood. The aim of this study is to understand this mechanism by isolating the C2 domain. We cloned, expressed and purified the perforin-like C2 domains of Scopthtamus maximus (Turbot) and Sparus aurata (gilt-head sea bream). The C2 domain protein of these two organisms are active single modules proteins involved in the host immune response by binding to bacterial infected cells. Their expression is also up regulated when the organisms are challenged with a pathogen. The full-length proteins are intact C2 domain by itself and therefore constitute excellent models for understanding the structural basis of calcium and membrane binding of Perforin C2 domain. Here we present the crystal structures of these proteins in their apo and fully occupied calcium bound forms. These structures highlight the re-organisation of the important loops upon calcium binding. We also generated mutations in the calcium binding loops and analysed the effect of these mutations. The calcium binding of the wild type proteins and the mutants was monitored by fluorescence. This work provides critical insight into how Perforin interacts with lipid membranes via the C2 domain and how pores formation is triggered.


POSTERS WEDNESDAY



MULTI-PARAMETRIC SURFACE PLASMON RESONANCE – THE NEW GENERATION OF SPR

Granqvist N., Sadowski J. and Tuppurainen J. Bionavis Ltd.

Multi-Parametric Surface Plasmon Resonance (MP-SPR) is a novel approach to the half-a-century old optical phenomenon Surface Plasmon Resonance (SPR). For the last 20 years SPR has been predominantly used for biomolecular interaction analysis. MP-SPR broadens the application range also to biophysical studies, such as measurements of absolute refractive indices and formed layer thicknesses, and biomaterials studies, such as ceramic and polymer coating characterizations and biocompatibility studies. The MP-SPR method is a sensitive approach to measurements of biochemical interactions with both self assembled and hydrogel-coated sensors. The added functionality of MP-SPR allows also labeled SPR, which provides superior signal-to-noise ratio compared to traditional SPR, as well as unprecedented selectivity in the detection. MP-SPR is based on an optical arrangement that enables measurements on a number of sensor coatings that cannot be measured with traditional SPR. Apart from traditional hydrogel based biosensor coatings, a wide range of different sensor surfaces are also possible to use, such as different metals (Au, Ag, Al, Cu, Pt), polymers (PS, PEO, PMMA, cellulose), and ceramics (Al2O3, SiO2). This opens up new areas of applications of optical biosensor research, like developing new cellulose based point-of-care devices, solving challenges in drug-container interactions and quick biocompatibility characterization of implant materials and coatings.

A PROTEOMIC ANALYSIS OF THE PREFRONTAL CORTEX IN AN ANIMAL MODEL OF METHAMPHETAMINE-INDUCED BEHAVIOURAL SENSITIZATION

Wearne T.A.1, Mirzaei M.2, Goodchild A.K.3, Haynes P.A.2 and Cornish J.L.1 1Department of Psychology, Macquarie University. 2Department of Chemistry and Biomolecular Sciences, Macquarie University. 3Australian School of Advanced Medicine, Macquarie University.

Repeat administration of drugs of abuse, such as methamphetamine, produce a progressive increase in locomotor response to drug administration (sensitization) that is believed to mimic both the behavioural and neurochemical changes seen in mental health disorders, such as addiction and psychoses. Previous research has suggested that alterations to the prefrontal cortex (PFC) may mediate the aetiology and maintenance of these behavioural changes. The aim of the present experiment was to investigate changes to protein expression in the PFC following behavioural sensitization to chronic methamphetamine exposure. Male Sprague Dawley rats (n = 16) underwent repeated methamphetamine (1ml/kg intraperitoneal (i.p.) days 1 & 7; 5mg/kg i.p. days 2 – 6) or saline (1ml/kg i.p.) injections for 7 days. Following 14 days of withdrawal, rats were challenged with acute methamphetamine (1mg/kg i.p.). Sixty minutes after drug challenge, brains were removed and the PFC dissected out for label-free shotgun proteomic analysis using mass spectrometry (n=6). Behaviourally, a methamphetamine challenge resulted in significant sensitized locomotor response in methamphetamine pre-treated animals when compared to saline controls. Proteomic triplicate analysis of prefrontal cortices revealed 115 proteins that were differentially altered in methamphetamine treated rats in comparison to controls. These changes predominately involved proteins associated with cellular organisation, maintenance and function, cell-to-cell signalling, apoptosis and nervous system development and disease. These changes may underlie the mechanism of methamphetamine-induced sensitization and may thus serve to inform how changes to the PFC could underpin the cognitive and behavioural dysfunction commonly seen in mental illness and drug addiction.

THE INS AND OUTS OF THE STREPTOCOCCUS PNEUMONIAE SOLUTE BINDING PROTEIN PSAA

Ween M.P.1, Counago R.L.M.2, Morey J.R.1, Paton J.C.1 and McDevitt C.A.1 1Research Centre for Infectious Diseases, University of Adelaide. 2School of Chemistry and Molecular Biosciences, University of Queensland.

Streptococcus pneumoniae is a major human pathogen responsible for approximately one million deaths annually, predominantly in children under 5 in developing countries. Our research recently identified that pneumococcal susceptibility to zinc toxicity occurred via competition of high concentrations of zinc for PsaA, the manganese binding protein. This results in starvation of the essential metal ion manganese in the pneumococcus, which impairs growth and heightens susceptibility to oxidative stress. Intriguingly, Zn(II) binding to PsaA, unlike Mn(II), was irreversible. However, as the structures of PsaA with Mn(II) or Zn(II) were virtually identical, with the both metal ions coordinated by His67, His139, Glu205, and Asp280, the molecular basis for the irreversible Zn(II)-binding was unclear. Analysis of PsaA using a thermodynamic stability assay showed that Zn(II)-PsaA was more thermostable than Mn(II)-PsaA (+17.0°C vs +9.7°C) relative to metal free apo-PsaA. To elucidate the interaction of PsaA with the metal ligands point mutations of the metal-coordinating residues Glu205 (to Gln205) and Asp280 (to Asn280) were generated. Both mutants were observed to be entirely competent for Zn(II) binding, but Mn(II) binding was reduced in PsaA-Gln205 (21%) whilst binding was essentially abrogated in PsaA-Asn280 (9%). This interaction with metal ligands was further supported by thermodynamic stability assays, which showed PsaA-Gln205 and PsaA-Asn280 were stabilised, albeit to a lesser extent, by Zn(II) (+9.3°C and +4.6°C) whereas Mn(II) could not induce significant stabilisation. Taken together, this data implicates that the metal coordinating carboxylate residues, Glu205 and Asp280, have key roles in facilitating Mn(II) binding to PsaA and reveals a unique target for future drug design studies.

FRAGMENT BASED SCREENING METHODS TARGETING THE E. COLI β-SLIDING-CLAMP

Whittell L.R., Yin Z., Beck J.L., Kelso M.J. and Oakley A.J. School of Chemistry, University of Wollongong, New South Wales 2522, Australia.

Multi-drug resistant bacteria are emerging as a major threat to society as it is becoming increasingly difficult to treat life threatening infections such as, for example, sepsis, pneumonia and endocarditis. The β-sliding-clamp, an accessory protein to the Escherichia coli DNA polymerase III holoenzyme, plays an essential role in bacterial chromosomal replication. The clamp is a toroidal dimer that tethers DNA at the replication fork to the advancing DNA polymerase. It also serves as a site for interaction with many other proteins involved in DNA replication and repair and is one of the most “trafficked” proteins in the cell. Moreover, bacterial β-sliding clamp bears little homology to its analogous human counterpart. It is thus considered an excellent target for novel antibiotic development as inhibition will result in disruption of a vast number of essential cellular processes thus minimising the potential for development of target-based drug resistance. In the current work, fragment based screening (FBS) is being used to begin the lead discovery process with small molecular compound (fragments) with molecular weight less than 300 Da. X-ray crystallography and fluorescence polarisation have been applied in our laboratories to assess a range of commercially available fragment libraries and a range of weak to medium binders have been identified. The most promising of the fragments will be modified to improve the binding characteristics of the original fragment. Currently, the effects of small changes to the scaffold on binding are being assessed by crystallography, fluorescence polarisation, surface plasmon resonance and noncovalent mass spectrometry. These studies will address essential structural features for binding, providing a basis for thorough structure activity relationship assessment and/or linking the scaffold with other promising fragments.


POSTERS WEDNESDAY



MULTIPLE-LABELLING (2H/15N/13C) OF PROTEINS AT THE NATIONAL DEUTERATION FACILITY ENABLING STRUCTURAL INVESTIGATIONS USING NMR

Wilde K.L.1, Morris V.2, Linser R.3, Kwan A.H.2, Sunde M.2, Duff A.P.1 and Holden P.J.1 1National Deuteration Facility, ANSTO, Lucas Heights, NSW 2234. 2Schools of Molecular Bioscience and Medical Sciences, University of Sydney, NSW 2006. 3School of Chemistry, University of New South Wales, Sydney, NSW 2052.

The National Deuteration Facility (NDF*) has developed capabilities in biological molecular deuteration for the in vivo deuteration of proteins, where all or a fraction of the hydrogen atoms in an expressed protein are replaced by the stable isotope deuterium (2H). Of particular interest to the Nuclear Magnetic Resonance (NMR) community for both solution and solid state NMR applications, is the expansion of NDF capabilities to include production of multiple-labelled proteins where along with deuteration, carbon and/or nitrogen atoms in the protein are replaced by the stable isotopes 13C and 15N. As with deuterium (2H), the isotopes 13C and/or 15N are introduced into the minimal growth medium utilised for biomass production and protein expression following the NDF techniques for high-yield recombinant expression of partial or per-deuterated protein in Escherichia coli. As well as an overview of the labelling methods of the NDF, a particular example of expression of the labelled fungal protein EASΔ15 will be highlighted. EASΔ15 is a hydrophobin that self-assembles into monolayers at surfaces and interfaces. These monolayers are composed of laterally-assembled fibrils that are a form of functional amyloid. EASΔ15 was triple-labelled (2H/13C/15N) with per-deuteration for solid state NMR analysis, in order to investigate the molecular structure of the fibrillar form. *The NDF offers facilities, staff and expertise to the neutron beam and NMR user communities through externally refereed proposal schemes, accessible at www.ansto.gov.au.

ANALYSIS OF TYROSINASE VARIANT ALLELES IN CULTURED HUMAN MELANOCYTES

Sturm R.A.1, Jagirdar K.1, Smit D.J.1 and Duffy D.L.2 1Institute for Molecular Bioscience, University of Queensland. 2Queensland Institute of Medical Research.

Tyrosinase (TYR) was the first human pigmentation gene identified with reports showing it to be mutated in TypeI oculocutaneous albinism. Recent attention has concentrated on two variant alleles of TYR, Ser192Tyr and Arg402Gln, appearing at high frequency in Europeans. Studies have reported TYR enzyme activity for the 129Tyr allele to be only 60% active, possibly due to steric hindrance effects in the TYR protein copper-A catalytic site. The Arg402Gln polymorphism also has reduced enzyme activity, with exogenous expression showing that it encodes a thermolabile variant protein with only 25% activity. The role of these polymorphisms in normal pigmentation variation remains controversial, as such we wished to analyse the biochemical effects of these variants using human primary melanocytes genotyped for these alleles and grown in tissue culture. Over the past decade we have performed a systematic screen for common polymorphisms within major human pigmentation genes establishing a collection of melanocytes cultured from individual neonatal foreskins to ascertain clonal cell strains. We have compared the melanogenic activities of melanocytes carrying these TYR alleles as homozygous wildtype, heterozygous and homozygous variant. This includes assays of TYR protein, DOPAoxidase activity, glycosylation and temperature sensitivity of DOPAoxidase for each strain. Melanocytes homozygous for 402Gln produce significantly less TYR protein displaying an altered glycosylation pattern, and much reduced DOPA oxidase reaction. The 402Arg/Gln strains are intermediate in their activity suggesting a genetic heterozygote effect in those that carry these alleles. The relationship to normal variation in pigmentation of these TYR alleles has been studied in a collection of adolescent twins and melanoma patients with associations apparent for lighter eye colour, freckling and melanoma.

ORAI1, THE PORE-FORMING POLYPEPTIDE OF PLASMA MEMBRANE STORE OPERATED CALCIUM CHANNELS, IS PREDOMINATELY LOCATED IN THE ENDOPLASMIC RETICULUM IN LIVER SUBCELLULAR FRACTIONS

Tam K.C. and Barritt G.J. Department of Medical Biochemistry, School of Medicine, Flinders University.

Orai1 is the pore-forming polypeptide of store-operated Ca2+ channels (SOCs). STIM1, which is distributed throughout ER, senses a decrease in the ER Ca2+ concentration, moves in the ER membrane, interacts with, and activates, Orai1. A decrease in ER Ca2+ in a small peripheral ER subregion enriched in type I IP3 receptors and devoid of ribosomes appears to be critical for Orai1 activation. The aim of this study is to investigate the role of subregions of the ER in SOCs activation by determining the distribution of Orai1and STIM1 in liver subcellular fractions. Differential and Percoll gradient centrifugation of homogenates of rat livers treated with vehicle or phenylephrine (to activate SOCs) were used to prepare heavy (HM) and light (LM) microsomes (derived from rough and smooth ER, respectively), and plasma membrane vesicles (PM). Quantitative western blot was performed to determine the amounts of protein in each fraction. Distribution of calreticulin, and plasma membrane (Ca2+ +Mg2+)ATPase confirmed the identity of the HM, LM and PM fractions. STIM1 was distributed in the HM (87%) and LM (13%) with little in the PM (0.09%). Surprisingly, the majority of Orai1 is found in the HM (47%) and LM (27%) with about 0.02% in the PM. No significant change in STIM1 and Orai1 distribution was observed after phenylephrine treatment. Potential STIM1 binding proteins Orai1, α- tubulin and SERCA2 were not detected in STIM1 immunoprecipitates of HM. It is concluded that (i) a large reservoir of Orai1is maintained in the rough and smooth ER, (ii) STIM1 ha<sup>s additional ER functions.

DEVELOPMENT OF NOVEL CONJUGATED-QUINOLINE-INDOLE COMPOUNDS AS POTENTIAL ANTIMALARIAL AGENTS

Teguh S.C.1, 2, Hutton C.A.3, Klonis N.1, Duffy S.4, Avery V.M.4, Baell J.B.2 and Tilley L.M.1 1Department of Biochemistry and Molecular Biology, Bio21 Institute, University of Melbourne, VIC 3010. 2Monash Institute of Pharmaceutical Science, VIC 3052. 3School of Chemistry, Bio21 Institute, University of Melbourne, VIC 3010. 4Discovery Biology, Eskitis Institute for Cell & Molecular Therapies, Griffith University, Nathan, QLD 4111.

A novel type of antimalarial compound, consisting of an indole-linked quinoline, was identified to have promising activity against Plasmodium falciparum (IC50 of ~200nM on 3D7 strain) from a high through-put screening campaign, and chosen as a lead antimalarial agent. A synthetic route has been determined and SAR investigations have been undertaken to improve the potency of the compound class, as well as to explore the pharmacophores. The indole ring was found to be necessary for potency. The compound set (especially those with quaternary nitrogen on the quinoline) shows improved activity against the chloroquine (CQ)-resistant K1 strain. Addition of alkyl groups on the 4-aminoquinoline side chain preferentially improved the activity against the CQ-sensitive 3D7 strain. The compounds show low cytotoxicity against HEK mammalian cells. Although the exact mechanism of action is still unknown, the novel compound class exhibits some activity as inhibitors of the formation of β-hematin. This suggests that the mode of action of these compounds may be similar to the currently used 4-aminoqunoline antimalarials; yet they circumvent the resistance mechanism. Further optimisation of the antimalarial activity of this compound class, as well as the deeper understanding of its mechanism of action at cellular and molecular level, are currently under investigation.


POSTERS WEDNESDAY



EVALUATION OF AN INTRACELLULAR METABOLITE EXTRACTION PROTOCOL FOR BACULOVIRUS-INFECTED INSECT CELLS

Tran T.T.B., Huynh H.T., Chan L.C.L., Nielsen L.K. and Reid S. Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Brisbane 4072, Australia.

The baculovirus-insect cell system has been extensively studied for the production of recombinant proteins, biopesticides for pest control, as well as vaccines and vectors for gene therapy. Metabolomic studies, which involve the analysis of intracellular metabolite levels, is a potentially useful approach for understanding host cell and virus interactions, as well as the response of cells to their environment. This approach has been employed widely for yeast and bacteria, and more recently for mammalian cells. The application of such metabolomic studies for insect cells, especially for infected insect cells, has not been well reported to date. A quantitative and reproducible intracellular metabolite extraction protocol for infected insect cells has been developed previously in this group. Since baculovirus infection is lytic, accurate profiling of intracellular metabolite levels is likely to be restricted to a relatively short timeframe post infection, prior to significant cell lysis. The aim of this study is to investigate the extent of this timeframe, which is important for determining the dynamics of the endometabolome post infection. With this knowledge, subsequent studies can be designed to investigate whether limitations in the endometabolome are implicated in the decline in productivity when insect cells are infected at high cell densities. Sf9 insect cells infected with a recombinant baculovirus (βGal-AcMNPV) were used in this study. The intracellular metabolites of infected cultures were extracted every 12 hours, from 0 to 72 hours post infection (hpi). The results show that the extraction protocol was effective for infected Sf9 cells as late as 48 hpi, as a good recovery of intracellular metabolites was still obtained at that time.

THE PRO-METASTASIS PROTEIN NEDD9 MEDIATES TRAFFICKING OF THE EPIDERMAL GROWTH FACTOR RECEPTOR AND DOWN-STREAM SIGNALLING

Zhong J.1, 2, Turner K.1, Ching Y.W.1 and O’Neill G.1, 2 1Children’s Cancer Research Unit, Kids Research Institute, Children’s Hospital at Westmead. 2Discipline of Paediatrics and Child Health, University of Sydney.

Poor survival rates from glioblastoma brain tumours are due to extensive infiltration of healthy brain tissue that is a cardinal feature of these tumours. The docking protein NEDD9 has been established to play a role in glioblastoma invasion, however the partner proteins mediating this have not been elucidated. GST-pull-down from lysates of glioblastoma cells cultured in 3D collagen gels followed by MALDI TOF mass spectrometry identified the adaptor protein CIN85/Ruk/SETA/SH3KBP1 as a novel NEDD9 N-terminal SH3 domain interactor. Importantly, elevated CIN85 expression has been reported in glioblastoma. Moreover, the Epidermal Growth Factor Receptor (EGFR) is the most frequently amplified gene in glioblastoma brain tumours and CIN85 is involved in EGFR trafficking. We show that siRNA depletion of NEDD9 from glioblastoma cells blocked EGFR degradation, similar to CIN85 depletion and indicating a potential role for NEDD9 in EGFR trafficking. Kinetic analysis of fluorescently-tagged EGF ligand following NEDD9 depletion revealed sustained accumulation of internalized ligand in punctate vesicles, suggesting that NEDD9 may alter EGFR trafficking from the late endosome to the lysosome. Recently, the EGFR target gene, EGF induced early growth response-1 (EGR1), has been identified in a signalling axis that drives growth-factor induced migration in mammary cells. We find that NEDD9 depletion and EGF ligand accumulation in endosomes lead to a truncated time-course of EGR-1 expression in response to EGF, while activation of c-Fos and ERK were unaffected. Together, our data suggest that NEDD9 may play a role in EGFR-mediated signalling to regulate cell migration in glioblastoma.

THE NOVEL SECRETION SYSTEM OF P. GINGIVALIS AND OTHER BACTEROIDETES ATTACHES COPIOUS QUANTITIES OF ITS SUBSTRATE PROTEINS TO THE CELL SURFACE TO FORM DISTINCT CELL-SURFACE STRUCTURESVeith P.D.1, 2, Nor Muhammad N.A.1, 2, Chen Y.Y.1, 2, Dashper S.G.1, 2, Likic V.2, Gorasia D.G.1, 2, Hanssen E.G.2 and Reynolds E.C.1, 2 1Cooperative Research Centre for Oral Health Science, Melbourne Dental School, The University of Melbourne, Melbourne, Vic. 3010 Australia. 2Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Melbourne, Vic. 3010, Australia. A novel protein secretion system has been recently discovered in Porphyromonas gingivalis, one of the bacterial pathogens associated with periodontitis (severe gum disease). The substrates of this secretion system contain a conserved C-terminal domain (CTD) that directs their secretion across the outer membrane. After cleavage of the CTD by the C-terminal signal peptidase the secreted proteins are attached to the cell surface via a proposed covalent linkage to lipopolysaccharide to form an electron dense surface layer. The aim of this study was to predict the presence of CTDs in other species and determine which features of the secretion system are conserved. Using a hidden markov model we predicted 682 CTDs from 87 organisms, all within the bacteroidetes phylum. A more sensitive analysis predicted an average of over 30 CTD-containing proteins per organism. Proteomic analysis of P. gingivalis and three other species revealed the presence of highly abundant CTD-containing proteins in each species, that were substantially increased in molecular mass relative to that calculated from their predicted amino acid sequences, consistent with their modification with lipopolysaccharides. Inspection of the peptides identified from each protein revealed that the CTD was not present in the proteins identified from the membrane fractions, however peptides from the CTD were identified from low mass SDS-PAGE bands from culture fluid samples. The data indicated that cleavage of the CTD signal was conserved across P. gingivalis, Tannerella forsythia, Prevotella intermedia, Parabacteroides distasonis and Cytophaga hutchinsonii. Imaging of the whole bacteria by cryo-electron microscopy demonstrated the presence of distinct cell surface structures in each of these species, whereas no cell surface structures beyond the outer membrane were observed in Bacteroides thetaiotaomicron, which contains no predicted CTDs. We propose that CTD cleavage and cell-surface attachment via covalent linkage to a glycolipid are conserved features that make this secretion system unique.

DEFINING THE MECHANISM(S) OF ACTION OF A NOVEL CLASS OF ANTI-OBESITY DRUG

Ng C.P.1, Vath J.2, Huges T.2 and Whitehead J.1 1Mater Medical Research Institute, Brisbane, QLD, Australia. 2Zafgen Inc, Cambridge, MA, USA.

Current anti-obesity strategies have failed to arrest the global obesity pandemic due to limited efficacy and or adverse side effects. There is an unmet need for effective therapies. Inhibitors of Methionine Aminopeptidase-2 (MetAP2) were originally developed as anti-cancer agents but have recently been repositioned as anti-obesity therapeutics. Studies in obese preclinical models demonstrate that MetAP2 inhibitors promote weight loss via reduced food intake and enhanced metabolism. Phase I clinical trials in obese adult women demonstrate that Beloranib is well-tolerated and promotes significant weight loss at effective doses (1.9-6.0 mg; -4.3.8 kg vs. +0.6 kg with placebo; n=17 & 11). We hypothesised that MetAP2 inhibitors mediate these effects, at least in part, via modulation of multiple signalling pathways to reduce inflammation and ER stress. The activity of c-Src, recently implicated in the aetiology of obesity-related inflammation, is dependent on MetAP2 activity for efficient cleavage and subsequent N-terminal myristoylation. Moreover, MetAP2 inhibitors are known to increase MetAP2 binding to ERK and eIF2α thereby reducing phosphorylation/activation. We have used Western blot, Alphascreen and immunofluorescence microscopy to investigate the effects of MetAP2 inhibitor treatment in vivo and in vitro. In obese mice, treatment with a MetAP2 inhibitor decreased liver phospho-ERK (p<0.001), as well as phospho-P38MAPK (p<0.05), and showed a trend towards reduced phospho-eIF2α. These effects were not observed in subcutaneous or mesenteric adipose tissue. In vitro studies performed in HUVECs demonstrated that MetAP2 inhibitor treatment reduced EGF-stimulated phosphorylation of ERK and c-Src but not TNFα-stimulated phosphorylation of p38MAPK. These results suggest that MetAP2 inhibitors represent a novel class of potentially effective anti-obesity agents. Further work is required to tease out the mechanisms that underpin the beneficial effects with the former likely to reflect pleiotropic, tissue/cell-type specific effects.


POSTERS WEDNESDAY



SPHINGOSINE KINASE 1, A THERAPEUTIC TARGET FOR ACUTE MYELOID LEUKAEMIA

Zhu W., Powell J.A., Coolen C., Pitman M.R. and Pitson S.M. Centre for Cancer Biology, SA Pathology, Frome Rd, Adelaide, South Australia.

Sphingosine kinase 1 (SK1) catalyses the formation of the bioactive lipid, sphingosine-1-phosphate (S1P). Dysregulation of SK1 has been shown to play crucial roles in solid tumour initiation, development, progression and chemotherapeutic resistance. Recent studies have also provided further evidence for the involvement of SK1 in a range of leukaemias. In our research, bone marrow biopsies collected from 53 acute myeloid leukaemia (AML) patients were examined for SK1 expression levels by qPCR. A median split of the 32 patients that received induction chemotherapy indicated that high SK1 expression was associated with shorter overall survival compared to patients with low SK1 expression (p=0.02). We have also shown that SK1 is constitutively activated in AML blasts, but not normal mononuclear cells. In addition, we have shown that SK1 inhibitors induce apoptosis in AML blasts as well as leukaemia stem progenitor cells (LSPC) (CD34+/CD38-/CD123+). AML is a stem cell disease and this LSPC population contributes to chemotherapy resistance and disease relapse. In summary, our studies suggest that high SK1 expression levels confer a poor prognosis for AML, SK1 is constitutively activated in AML and targeting SK1 may offer a new therapeutic avenue to treat AML.

THE MOLECULAR MECHANISM OF THE FUNCTIONAL CHANGES OF TRPV6 BY INTERNAL AND EXTERNAL PROTONS

Namkung J.1, Choi J.W.1, Yeh B.I.1 and Kong I.D.2 1Department of Biochemistry, Yonsei Univ. Wonju College of Medicine 162 Ilsan-Dong Wonju Republic of Korea. 2Department of Physiology, Yonsei Univ. Wonju College of Medicine 162 Ilsan-Dong Wonju Republic of Korea.

The transient receptor potential channel TRPV6 is located in the polarized epithelia of many tissues including the small intestine, placenta, prostate and pancreas. It was known that TRPV5 is very sensitive to pH which is very important for the function of it but the pH sensitivity of TRPV6 did not attract much interest since pH variations in the organs of the human body are rare. However, it has been recently reported that the calcium levels in skin epithelium play an important role in wound healing and aging. This suggests that TRPV6 in the skin epithelium could be the main regulator of Ca2+ homeostasis. We now report that the function of TRPV6 is inhibited by internal and external protons. Based on amino acid sequence analysis, we predicted that glutamate-521 of the extracellular domain plays a critical role as an extracellular proton sensor. The pKa of TRPV6 is about 7.5. To confirm intracellular proton sensor activity, we used inside-out patches. Intracellular acidification decreased the channel activity and the kinetics was greater than extracellular acidification. We demonstrated that intracellular and extracellular protons cross-regulate TRPV6 and this regulation through acidification underlies the pathogenesis of Ca2+ transport disruption in many diseased states.

STRESS-STIMULATED PHOSPHORYLATION OF THE MICROTUBULE DESTABILISER STATHMIN: A ROLE FOR PROTEIN KINASE A

Yip Y.Y., Bogoyevitch M.A. and Ng D.C.H. Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, VIC 3010, Australia.

Cellular responses towards detrimental stimuli is an important component of the cell survival mechanism. The cell cytoskeleton is often implicated in such events, where microtubule (MT) dynamics and organisation may be altered to preserve normal cellular architecture and function. However, the mechanisms that regulate these stress-induced modifications have yet to be elucidated in detail. In order to dissect the biochemical pathways involved, we focus on a key MT-regulatory protein: Stathmin (STMN). STMN functions as an MT destabiliser by sequestering free α/β tubulin heterodimers and its activity is tightly regulated via phosphorylation by a variety of kinases. Previously, we have shown STMN to be phosphorylated in response to hyperosmotic stress stimulation by the stress-activated c-Jun N-terminal kinase (JNK) at S38-STMN. In this study, Phos-tagTM gels and 2D-PAGE analyses revealed that phosphorylation profiles of STMN under hyperosmotic stress and growth factor stimulation are different. This suggests distinct regulatory mechanisms are involved in STMN stress signalling. Further investigation with site-specific phospho-antibodies showed S63-STMN is also phosphorylated under stress conditions. This site has been reported to play a critical role in tubulin association. Our inhibitor studies have successfully identified Protein Kinase A (PKA) as the kinase responsible for phosphorylation of this residue. In conclusion, our results indicate a novel, stress-stimulated STMN-mediated signalling pathway whereby S63-STMN is a target of PKA. These findings not only provide another important piece of evidence that STMN is a critical regulator of stress-induced MT remodelling but also further contribute to our understanding of global signalling events involved in microtubule reorganisation under stress.

ETHYLENE AND ROS REGULATE CA2+ CHANNEL ACTIVITY TO DIRECT WALL INGROWTH FORMATION OF TRANSFER CELLS

Zhang H.M., McCurdy D.W., Offler C.E. and Patrick J.W. School of Environmental and Life Sciences, The University of Newcastle, Callaghan NSW 2308, Australia.

Transfer cells (TCs) are specialized plant cells that trans-differentiate to facilitate membrane transport of nutrients. Their transport function is achieved by developing characteristic ingrowth walls comprised of a uniform wall layer on which wall ingrowths (WIs) are deposited. This wall structure provides a scaffold to amplify plasma membrane surface areas that are enriched in suites of nutrient transporters. Employing developing Vicia faba cotyledons as an experimental system to investigate TC trans-differentiation, we have identified ethylene, reactive oxygen species (ROS) and Ca2+ as key components of a signaling cascade that leads to ingrowth wall formation. During TC trans-differentiation, we have found that ROS determines polarity of uniform wall deposition while cytosolic Ca2+ plumes act as positioning signals directing loci at which WIs are formed. The cytosolic Ca2+ signal increased in intensity for the first 6 h of cotyledon culture and thereafter remained stable. Generating the Ca2+ signal was shown to be regulated by ethylene through two separate routes that controlled Ca2+ channel activity. One route is direct action of the phytohormone and the other is via an ethylene-regulated production of extracellular ROS as the active agent. At what level these signals exert their effects on Ca2+ channel activity will be addressed. A model describing interactions between Ca2+ and ethylene/ROS leading to TC trans-differentiation will be presented.


POSTERS WEDNESDAY



RNA POL II PAUSING: NOVEL MECHANISMS FOR PATTERNING MYC EXPRESSION DURING DEVELOPMENTCranna N.J.1, Lee Jue Er A.1, Mitchell N.C.1, Nie Z.2, Levens D.L.2, Hannan R.D.3 and Quinn L.M.1 1University of Melbourne. 2National Cancer Institute. 3Peter MacCallum Cancer Centre.

The prominent role of MYC in cancer has sparked extensive research into understanding how this factor functions and is regulated during normal development and is dysregulated in malignancy. In Drosophila dMyc is functionally homologous to c-MYC (MYC), and both are necessary and sufficient for cell growth and cell cycle progression. Although we are learning much about dMyc/MYC’s many targets and as a consequence, cellular function, we still do not understand the precise molecular mechanisms controlling the abundance of the MYC transcript, particularly during organ and tissue growth, where extracellular signaling pathways are required to determine distinct patterns of MYC transcription throughout development. RNA Pol II pausing refers to a group of post-initiation transcriptional events (eg. attenuation, stalling, poising and abortive elongation), which allow gene expression to be regulated at the level of Pol II release rather than by preinitiation. Poised Pol II has potential to facilitate; 1) rapid response to growth signals, 2) tight repression of transcription in the absence of appropriate signals and 3) synchronous activation of transcription among similarly fated cells within a tissue. Our studies have implicated the activator FBP (Psi in Drosophila) and repressor FIR (Hfp in Drosophila) in control of RNA Pol II pausing on MYC/dMyc (Liu et al. 2006 EMBO J 25: 2119-2130; Mitchell et al., 2010 Development 137, 2875-2884). More recently, from a genetic screen in Drosophila we have identified a distinct set of developmental signaling pathways that regulate the activity of Hfp and Psi, which in turn modulate RNA Pol II activity within the dmyc promoter to influence transcription of dmyc. Specifically, our data suggests the developmental signalling pathways (including TGFB, Hedgehog and Ras/PI3K) pattern dmyc transcription by either activating dmyc expression via Psi dependent release of Pol II or, conversely, tightly repress promoter activity by Hfp induced maintenance of RNA Pol II pausing.

A NEW MODEL OF DEVELOPMENT OF THE MAMMALIAN OVARY AND FOLLICLES

Hummitzsch K.1, Irving-Rodgers H.F.1, 2, Hatzirodos N.1, Bonner W.M.1, Wilhelm D.3 and Rodgers R.J.1 1Robinson Institute, University of Adelaide, SA. 2Institute of Health and Biomedical Innovation, Queensland University of Technology, QLD. 3Institute for Molecular Biosciences, The University of Queensland, QLD.

In mammalian gonads the somatic cells are pivotal for nurturing the germ cells, for determining if an ovary or testis develops from the indifferent gonad and for producing sex steroid hormones. Using bovine fetal ovaries (n = 80) we identified a novel cell type, termed GREL for Gonadal Ridge Epithelial-Like cells. To identify markers, we cultured and clonally isolated GREL cells and conducted microarray analysis. Using these and other cell markers (n = 26) we conducted immunohistochemistry and electron microscopy and chronologically tracked all somatic cells during development. We showed that the gonadal ridge/ovarian primordium is initially formed by proliferation of GREL cells at the surface epithelium of the mesonephros. At this stage the primoridum is not covered by a classical surface epithelium. Primordial germ cells (PGCs) migrate into the ovarian primordium. Stroma from the underlying mesonephros penetrates the primordium partitioning the developing ovary into irregularly-shaped ovigerous cords composed of GREL cells and PGCs/oogonia; the cords are separated from the stroma by a basal lamina. As the stroma penetrates further to just below the outer layers of GREL cells located at the ovarian surface, the stroma spreads laterally. GREL cells at the ovarian surface then form a mature epithelium with an underlying basal lamina and an epithelial-stromal interface. The stroma continues to partition the ovigerous cords into follicles containing an oogonium/oocyte surrounded by GREL cells. In contrast to prevailing theories we conclude that both granulosa cells and surface epithelium are derived from a common precursor GREL cell.

GENERATION OF PLURIPOTENT STEM CELL LINES FROM NUCLEAR TRANSFER BOVINE EMBRYOS

Kim D., Park S. and Roh S. Seoul National University School of Dentistry.

Autologous stem cells derived from somatic cell nuclear transfer embryos (SCNT-ESC) are valuable in animals for conservation of endangered species or production of transgenic animals. Here, we established SCNT-ESC lines of Korean cattle (Bos Taurus coreanae). Reconstructed embryos by SCNT were cultured in vitro for 7 days, and SCNT blastocyst were placed individually on the feeder layer with 3i stem cell culture medium consisting of an equal volume of DMEM/F12 glutamax and Neurobasal medium with 1% (v/v) N2 and 2% (v/v) B27 supplements, plus three inhibitors (3i), 0.8 mM PD184352 (MEK 1/2 inhibitor), 2 mM SU5402 (FGF receptor inhibitor) and 3 mM CHIR99021 (GSK3 inhibitor). Primary colonies formed after 2-3 days of culture and the cell colonies were moved onto new fresh feeder layer after 8-9 days of incubation. The intact colonies were routinely passaged every 5-6 days. Eight bovine SCNT-ESC lines have been established from eight different SCNT bovine blastocysts. The cells show ESC-like colonies were positive to alkaline phosphatase staining. Immunofluorescence and RT-PCR analyses also confirm their characteristic of pluripotency as Oct4, Sox2, Nanog and stage-specific embryonic antigen-4 is positive. All bovine SCNT-ESC lines are being maintained in culture over 50 passages for more than 270 days. The cells show normal karyotypes consisting of 60 chromosomes (58XX) at passage 50. Embryoid bodies (EBs) were formed by suspension culture to analyze in vitro differentiation capability. The marker genes representing the differentiation into three germ layers such as ectodermal: beta-3-Tubulin, Nestin and Vimentin, endodermal: Somatostatin and Gata6, mesodermal: Connexin40 and BMP4 are expressed in EBs derived from bovine SCNT-ESC. This work was supported by Technology Development Program for Life Industry, MIFAFF (Grant numbers 109020-3 and 111160-4).

14-3-3ZETA MAINTAINS PHOPHORYLATION OF NDEL1 TO CONTROL NEURAL MIGRATION AND AXON GUIDANCE

Saleh E.O.1, 2, Ramshaw H.1, Xu X.1, Lopez A.F.1 and Schwarz Q.1 1Centre for Cancer Biology, SA Pathology, Adelaide, South Australia. 2Adelaide University of South Australia, Adelaide, South Australia.

Neuropsychiatric disorders such as schizophrenia are likely caused by a large number of genes with a small effect. Primarily they have complex traits believed to arise from multiple deficiencies within connected biological networks controlling neuronal migration, axonal pathfinding and synapse formation. We have recently shown that 14-3-3zeta mouse mutants display deficits reminiscent to schizophrenic patients such as severe capacity to learn and remember, hyperactivity and disrupted sensorimotor gating. Developmental abnormalities of the hippocampus were also observed which rose from aberrant neuronal migration and synapse formation. I have shown that phosphorylated Ndel1 levels are reduced in the 14-3-3zeta mouse mutants and thus propose that 14-3-3zeta binds CDK5 phosphorylated Ndel1 to promote interaction with LIS1 and thereby promote neuronal migration. To test this model I have developed an in vitro migration assay with neural stem cells that recapitulates the in vivo finding. This provides an ideal model system to dissect the molecular mechanism in the neural migration pathway. Consistent with my finding that Ndel1 is aberrantly phosophorylated in 14-3-3zeta mutants, I have also found that the nuclear-centrosome coupling during migration is perturbed in vitro. This suggests that defects in coupling may contribute to migration defects in the 14-3-3zeta mutants. Furthermore, we have also found that 14-3-3zeta interacts with DISC1, a schizophrenia risk factor, in an isoform specific manner and acts as a central risk factor in the schizophrenia protein interaction network. Thus we also propose that 14-3-3zeta forms a LIS1/ NUDEL/DISC1 complex to control axonal growth dynamics.


POSTERS WEDNESDAY



DEFINING THE ROLE OF THE NEDD4 UBIQUITIN LIGASE DURING LYMPHATIC VASCULAR MORPHOGENESIS IN THE MOUSE EMBRYO

Secker G.A.1, Kazenwadel J.S.1, Betterman K.L.1, Boase N.1, Sutton D.L.1, Kumar S.1, 2 and Harvey N.L.1, 1 1Division of Haematology, Centre for Cancer Biology, SA Pathology, Adelaide, Australia. 2School of Medicine, University of Adelaide, Adelaide, Australia.

Lymphatic vessels are vital for tissue fluid homeostasis, the absorption of dietary fats and immune cell trafficking. Despite the integral role that lymphatic vessels play in homeostasis and human disease, little is known about the signals that direct construction of the lymphatic vasculature during development. Our work is focused on the identification and characterisation of genes and signalling pathways involved in morphogenesis of the lymphatic vasculature in mice. Here, we present recent work dissecting the role of the ubiquitin ligase Nedd4 in embryonic vascular development. Protein ubiquitination is a highly conserved process that can lead to the degradation, stabilisation or change in subcellular localisation of target proteins. Our work has revealed that Nedd4 is crucial for morphogenesis of the blood and lymphatic vascular networks during mouse embryogenesis. Nedd4-/- embryos exhibit sparse, ruptured blood vessels and strikingly mispatterned lymphatic vessels. Current work aims to dissect the endothelial cell autonomous versus non-autonomous roles of Nedd4 during vascular development and to identify the protein targets of Nedd4 important for morphogenesis of the lymphatic vasculature in the mouse embryo.

SPATIAL MAPPING AND QUANTITATION OF DEVELOPMENTAL BRANCHING MORPHOGENESIS

Short K.M.1, Hodson M.J.2 and Smyth I.M.1, 3 1Monash University, Department of Biochemistry & Molecular Biology, Clayton, Melbourne, 3800. 2Block Software, Klemzig, Adelaide, 5087. 3Monash University, Department of Anatomy & Developmental Biology, Clayton, Melbourne, 3800.

Branching morphogenesis underlies the development of many organs, but its analysis is hindered by the absence of methods to quantify the complex spatial datasets which describe it. As a consequence, while many developmentally important genes are proposed to influence branching morphogenesis, we have no way of objectively assessing their individual contributions to this process. We report the development of a method to accurately quantify many aspects of branching morphogenesis and we demonstrate its application to studying organ development. We show its applicability in the embryonic mouse kidney, and lung. This advance will enable the rigorous characterization of the genetic and environmental factors that regulate this critical developmental mechanism.

REGULATION OF ARX TRANSCRIPTIONAL ACTIVITY BY PROTEIN KINASE C PHOSPHORYLATION: IMPLICATIONS FOR INTELLECTUAL DISABILITY

Tan M.H.1, 2, Hii C.S.3, Gecz J.1, 2 and Shoubridge C.1, 2 1Neurogenetics, SA-Pathology, SA 5006, Australia. 2Department of Paediatrics, University of Adelaide, SA 5005, Australia. 3Department of Immunopathology, SA-Pathology, SA 5006, Australia.

Aristaless-related homeobox (ARX) gene is a homeodomain transcription factor important for the development of forebrain, pancreas and testes. Mutations in ARX result in intellectual disability with or without other comorbidity such as epilepsy and lissencephaly. Here, we report phosphorylation of ARX at multiple sites, supporting the role for post-translational modification in regulating ARX function. Using in vitro kinase assays we identified ARX as a novel substrate of protein kinase C (PKC) and serine 174 as a putative PKC phosphorylation site. To investigate the functional relationship between PKC and ARX, HEK293T cells were stimulated with phorbol 12-myristate 13-acetate (PMA) and transcriptional activity of recombinant ARX was measured using dual-luciferase assay. Activation of PKC abolished transcriptional repression activity of ARX and this PMA-induced effect is reversed by the addition of a PKC inhibitor. To date, we have shown that at least two PKC isozymes, gamma and delta, phosphorylate ARX in vitro. Furthermore, co-expression of ARX with PKC delta enhances the transcriptional repression activity of ARX. We are currently investigating the impact of isozyme-specific PKC phosphorylation of ARX on the expression of ARX transcriptional targets by RT-qPCR. Our data demonstrates that ARX is a phosphoprotein and its activity is regulated by specific PKC isozymes. The precise physiological function of ARX phosphorylation remains to be determined, this modification is potentially important for normal brain development as the identified pS174 site is conserved down to zebrafish. The pS174 site might also be relevant for the majority of ARX mutations given its close proximity to the hypermutable polyalanine tracts.

MOLECULAR FUNCTION OF A NOVEL RNA BINDING PROTEIN IN EMBRYONIC AND MATURE ENDODERM CELLS

Fossat N., Tourle K., Radziewic T., Jones V., Studdert J.B., Power M., Steiner K., Loebel D.A.F. and Tam P.P.L. Children’s Medical Research Institute, 214 Hawkesbury Rd, Westmead, NSW 2145, Australia.

Through transcriptome-wide screening of foregut endodermal specific genes in E8.5 mouse embryos, Rbm47 was identified. Rbm47 encodes a novel 64kDa putative RNA binding protein with three N-terminal RNA recognition motifs. We demonstrated in vitro and in vivo RNA binding by RBM47. We confirmed expression in mouse embyronic endoderm as well as adult mouse endoderm derived tissues, including pancreas, liver and intestine, and in the visceral endoderm of the yolk sac, an extraembryonic structure important for trophic support of the embryo. To study Rbm47 gene function, FLP and Cre recombinases were used combinatorially to conditionally switch Rbm47 activity “On” or “Off” in transgenic mice. The majority of mice lacking Rbm47 died at midgestation with additional deaths after birth before weaning. Surviving mice were born smaller than their wild type siblings and remained so during postnatal development, displaying gastro-intestinal tract defects. Re-activating the recombinase-silenced Rbm47 gene in the visceral endoderm rescued the embryonic lethal phenotype in Rbm47 mutant mice. We hypothesize that RBM47 function is important for trophic activity in both the visceral endoderm and the mucosal epithelium of the gut. Using a RBM47-specific antibody, we successfully immunoprecipitated and identified a potential RNA target of Rbm47, loss of function of which could partly account for the phenotype of Rbm47 mutant mice. Current studies focus on investigating the interaction of RBM47 with this RNA target, as well as identifying further RNA targets by combining immunoprecipitation and next generation sequencing.


POSTERS WEDNESDAY



REGULATION OF FRUIT SET BY CELL WALL INVERTASE: PHLOEM-SPECIFIC LOCALIZATION AND ACTIVITY BURST DURING OVARY-TO-FRUIT TRANSITION

Palmer W., Jin J., Ru L., Wang L., Patrick J.W. and Ruan Y.-L. School of Environmental & Life Sciences, University of Newcastle, NSW 2308, Australia.

Fruit- and seed-set determines yield potential of most crop species. These processes occur immediately following fertilization and are highly vulnerable to abiotic stresses (e.g., drought and heat) that often leads to fruit- and seed-abortion and hence yield loss (1, 2). Unraveling the molecular basis underpinning fruit- and seed-set is essential for developing innovative solutions to increase crop yield that needs to be doubled by 2050. We have previously demonstrated that cell wall invertase (CWIN) and its inhibitor (INH) play a critical role in seed and fruit development (2; 3). However, the mechanisms by which CWIN regulates fruit- and seed- set remain unknown (1). Here we show that spatial expression of CWIN and INH shifted from a dispersed cellular pattern in ovaries to a phloem-specific one in young fruit. Remarkably, CWIN and INH proteins were specifically co-expressed in cell walls of sieve elements located in ovaries and young fruit. Moreover CWIN activities dramatically increased in young fruit compared to those of ovaries. Proteomics analyses revealed potential biochemical pathways by which CWIN regulates fruit-set. The findings demonstrate a multi-level regulation of CWIN expression both spatially and temporally during the reproductive stage and provide new insights into the roles that CWIN plays in optimizing phloem unloading and fruit- and seed-set. REFERENCES (1) Ruan Y-L, Patrick JW, Bouzayen M, Osorio S & Fernie AR (2012) Trends in Plant Sciences (in press). (2) Ruan Y-L, Jin Y, Yang Y-J, Li G-J, Boyer JS (2010) Molecular Plant 3, 942–955. (3) Jin Y, N DA & Ruan Y-L (2009) Plant Cell 21, 2072–2089.

QUANTITATIVE TRAIT LOCI FOR TEMPERATURE AND PHOTOPERIOD RESPONSE IN SORGHUM

Sangma H.B.1, Van Oosterom E.J.1, Mace E.S.2, Jordan D.R.2 and Godwin I.D.1 1University of Queensland, Brisbane, QLD 4072, Australia. 2DPI&F, Warwick, QLD 4370, Australia.

Flowering time is a complex trait that determines the reproductive success of plants. Plants use various cues from the environment in order to regulate the timing of flowering. Temperature and photoperiod play a crucial role in determining flowering time in sorghum. Knowledge of genetic factors that underlie temperature and photoperiod control of flowering in sorghum is limited. The objective of this study was to identify the QTL and the underlying genes linked to temperature and photoperiod utilizing the phenotypic and genotypic resources available for two mapping populations which were developed by crossing African tropical (IS 8525) and wild sorghum (S.arundinaceum) lines with mildly photoperiod-sensitive elite line (R31945-2-2). Parameters for temperature and photoperiod were derived from thermal time (TT) and photoperiod sensitivity (PPS) respectively. TT and PPS QTL were identified through statistical analysis and were projected onto a consensus map to uncover the putative candidate genes for flowering time present underneath the QTL. Six TT QTL linked to chromosomes SBI-01, SBI-02, SBI-06 and SBI-10 and seven PPS QTL on chromosomes SBI-01, SBI-02, SBI-03, SBI-04, SBI-09 and SBI-10 were detected at different locations indicating the genetic control of temperature related flowering to be independent from that controlling the photoperiod. Twenty TT and nineteen PPS putative candidate genes were identified. Assigning clear function, especially temperature sensitivity to the candidate genes identified in this study will facilitate the prediction of possible changes that may occur in the flowering behavior of plants and therefore their reproductive success with respect to climate change.

REGULATION OF ATHKT1;1 EXPRESSION, A GENE ENCODING FOR A SODIUM TRANSPORTER IN THE PLANT ARABIDOPSIS THALIANA

Schmoeckel S.M.1, 2, Sundstrom J.F.1, Tester M.1, 2, 3, Berger B.1, 2, 3 and Roy S.J.1, 2 1Australian Centre for Plant Functional Genomics, PMB 1, Glen Osmond, SA 5064, Australia. 2The University of Adelaide. 3The Plant Accelerator.

Salinity is a major threat to agriculture, resulting in reduced crop yields and endangering food security. With salt affected areas increasing, understanding the molecular mechanisms of salinity stress is of heightened importance. Elevated Na+ levels lead to osmotic and ionic stress, both having significant effects on plant performance and yield. Particularly, the accumulation of Na+ in the shoot has been shown to be detrimental. The Na+ transporter AtHKT1;1 is reported to be located in the xylem parenchyma of A. thaliana where it is involved in retrieving Na+ from the xylem, thereby reducing the amount of Na+ transported to the shoots. A. thaliana ecotype C24 was found to lack AtHKT1;1 expression in the roots and accumulated significantly higher amounts of Na+ in the shoot compared to ecotype Col-0. A tandem repeat 4 kb upstream of the start codon has been reported to enhance AtHKT1;1 gene expression. We have observed significant polymorphisms within this region between C24 and Col-0 ecotypes. In addition, we have discovered significant sequence polymorphisms in other areas of the promoter and within the gene itself. Here we show that despite a disrupted CAAT motive, a highly polymorphic region 50 bp upstream of the start codon has only minor influence on the expression of AtHKT1;1. Furthermore, we investigate the role of a 1.6 kb insertion in the second intron of C24 on the expression of the gene.

COMPARISON OF FUNGAL GENE EXPRESSION IN ARUM- AND PARIS-TYPE MYCORRHIZAL COLONISATION

Schultz C.J. University of Adelaide, School of Agriculture, Food and Wine, Waite Campus, PMB1, Glen Osmond 5024 Australia.

Arbuscular mycorrhizal (AM) fungi colonise plant roots establishing symbiotic interfaces across which nutrients are exchanged. The AM fungus Glomus intraradices forms Arum-type colonisation with many plant species but Paris-type with others [1]. Arum-type is characterised by an intercellular hyphal phase with terminal intracellular arbuscules, whereas Paris-type has entirely intracellular hyphae and coils and arbusculate coils in cortical cells. Genes controlling these differences are not known. Three potential candidate genes are GiAGL1-3 encoding cell wall proteins [2]. In vitro studies using recombinant proteins and synthetic peptides suggest that GiAGL1 & GiAGL3 can self-assemble and form polyproline II (PPII) helices. PPII helices are important motifs in structural proteins and molecular recognition. This study aims to determine if GiAGL genes are differentially regulated in roots with different fungal morphology. Three plant species were grown; two with Arum-type structures (Medicago truncatula and Allium porrum) and Asphodelus fistulosus with Paris-type. Gene expression was determined by quantitative PCR (QPCR). Plants inoculated with G. intraradices were well colonised (50-75%) and A. porrum plants showed a positive growth response. QPCR data revealed that GiAGL3 was expressed at 3-fold higher levels in As. fistulosus (Paris-type) than it was in the two plants with Arum-type morphology. The AM fungal phosphate transporter (GiPT1) was expressed in all colonised roots. These studies suggest that GiAGL3 could have a specific role in producing the coiled hyphae observed in Paris-type AM and this possibility will be explored in the future with AGL3-specific antibodies. [1] van Aarle et al 2005 New Phytol 166: 611; [2] Schultz et al 2008 Mycorrhiza 18: 403; [3] Creasey et al 2012 BBA Protein Proteomics 1824: 711.


POSTERS WEDNESDAY



ACTIVATION AND REPRESSION OF ANTHOCYANIN PIGMENTATION – INSIGHTS FROM THE MODEL PETUNIA

Schwinn K.1, Albert N.2, Montefiori M.3, Brendolise C.3, Boase M.1, Zhang H.1, Lewis D.1 and Davies K.1 1The New Zealand Institute for Plant & Food Research Limited, Private Bag 11600, Palmerston North 4442 New Zealand. 2Agresearch, Private Bag 11008 Palmerston North 4442, New Zealand. 3The New Zealand Institute for Plant & Food Research Limited, Private Bag 92169, Auckland 1142, New Zealand.

Plants produce anthocyanin pigment in response to a variety of developmental and stress signals. Complexes containing R2R3MYB and bHLH transcription factors (TFs) and a WD-repeat protein turn on the biosynthetic genes of the pathway. While many aspects of the activation of the pathway have been elucidated, the role of repression and repressor TFs in the control of this pathway is relatively poorly understood. Using Petunia, the main pigmentation model for the asterid clade of angiosperms, we are elucidating the control of developmental and light-responsive pigmentation in floral and vegetative tissues. Our focus has been on defining the roles and modes of function of the repressors, MYB27, a R2R3MYB containing a putative EAR domain, and MYBX, a R3MYB. We have also been further characterising DEEP PURPLE and PURPLE HAZE, novel members of the family of MYB activators of the pathway that we previously identified. Our progress will be presented as well as a model of the control of pigmentation that includes feedback inhibition by the repressor TFs and de-repression.

QTL STUDY FOR SALINITY TOLERANCE IN AUSTRALIAN BREAD WHEAT

Shamaya N.1, Rabie H.3, Berger B.2, Langridge P.1 and Tester M.1, 2 1ACPFG, University of Adelaide. 2The Plant Accelerator, University of Adelaide. 3ACPFG, Uni SA.

A major limitation to wheat production is soil salinity. Increasing salinity tolerance of wheat is a target for many wheat breeding programmes. This requires an understanding of the inheritance of salinity tolerance traits. In the present study, a population of recombinant inbred lines (RILs) derived from a cross between bread wheat (Triticum aestivum L.) cultivars Gladius and Drysdale was used to study Osmotic Stress Tolerance (OST) as a major trait associated with salinity tolerance. 162 RILs and their parents were grown in pots with soil. 0 mM and 75 mM NaCl were applied at the time of emergence of the fourth leaf. An imaging system (LemnaTec) was used to estimate the plant growth rate for 10 days after salt application and used to calculate the OST. Other traits such as Salt Tolerance Index (STI) (relative shoot biomass), and Na+ and K+ concentration in leaf four were estimated. The results of phenotyping were used with a genetic map for QTL analysis. A strong correlation was observed between OST and STI, whereas Na+ accumulation was only slightly correlated with STI. The QTL analysis detected four QTLs for OST; two significant QTLs on chromosomes 2B and 5D and two suggestive QTLs on chromosomes 1B and 7B. Two QTLs for OST overlapped with two significant QTLs for STI on 1B and 2B chromosome, whereas Na+ accumulation QTLs did not overlap with STI QTLs. These results imply that variation in salinity tolerance in this population is due primarily to OST. Further screening with more members of the Gladius/Drysdale RIL population will be conducted in order to validate QTLs for OST.

GENETIC VARIATION IN THE ROOT GROWTH RESPONSE OF BARLEY CULTIVARS TO SALINITY STRESS

Shelden M.C.1, Roessner U.1, Tester M.2 and Bacic A.3 1Australian Centre for Plant Functional Genomics, School of Botany, University of Melbourne, Parkville, VIC. 2Australian Centre for Plant Functional Genomics, University of Adelaide, Glen Osmond, SA. 3ARC Centre of Excellence in Plant Cell Walls, School of Botany, University of Melbourne, Parkville VIC.

Barley (Hordeum vulgare L.) is the most salt tolerant cereal crop and therefore is a good model to study salt tolerance mechanisms in cereals. In order to identify genetic variation in root growth traits of barley to the osmotic component of salinity stress, we examined seminal root growth at various concentrations of salinity. A high throughput, non-destructive, screening method was developed using nutrient agar media to accurately measure the seminal root elongation rate of eight barley cultivars consisting of a landrace, wild barley and a number of domesticated barley lines. Salinity inhibited root elongation in all cultivars, however changes in other root architectural traits such as total root length and seminal root number were also observed. Considerable variation between cultivars was observed in the root growth response with relative root elongation rates ranging between 60 – 90 % and 30-70 % at 100 and 150 mM NaCl, respectively. The screen identified the wild barley cultivar CPI71284-48 as the most tolerant, maintaining root growth and biomass in response to salinity. Seminal root elongation was most significantly inhibited in the landrace Sahara. Root and shoot Na+ concentrations increased and K+ concentrations decreased in all cultivars in response to salinity. However the root and shoot ion concentration did not correlate with root elongation rates suggesting that the Na+ and K+ concentration is not directly influencing root growth, at least during the osmotic phase of salt stress. The identification of genetic diversity in root growth responses in barley for osmotic stress tolerance provides important information for future genetic characterization. This study highlights the need for utilizing wild cultivars and landraces in looking for genetic traits for osmotic stress tolerance. We will now apply spatially resolved ‘omics approaches to elucidate the molecular pathway(s) involved in osmotic stress tolerance.

HIGH-THROUGHPUT SCREENING FOR PHOTOSYNTHETIC EFFICIENCY AND CAPACITY IN WHEAT

Silva-Perez V.1, 2, 3, Reynolds M.2, Condon T.3, Furbank R.4 and Evans J.1 1Science Division, Research School of Biology, Australian National University, Canberra, ACT, Australia. 2International Maize and Wheat Improvement Center. El Batan, Texcoco 56130, Mexico. 3CSIRO Plant Industry, Canberra, ACT 2601, Australia. 4High Resolution Plant Phenomics Centre, CSIRO Plant Industry, Canberra ACT 2601, Australia.

A worldwide challenge is to increase yield potential in wheat to address global food demand. One way to improve yield is to increase crop biomass by improving photosynthesis, the main process whereby plants obtain the energy and CO2 required for biomass production. An important technique that helps to understand plant photosynthetic performance is the analysis of gas exchange with a portable photosynthesis system (for example LICOR LI-6400XT). Using a modelling approach and gas exchange measurements we can extract valuable information such as Rubisco amount and kinetics and electron transport rate to analyse photosynthetic efficiency and capacity in plants. However, this method is not very useful in high-throughput screening as measurements take considerable time. This research seeks to develop and validate new tools to screen for superior photosynthetic performance among a broad range of wheat germplasm. One approach has been to compare gas exchange measurements and destructive analysis with leaf spectral reflectance and absorbance, measured using SPAD and the ASD Field Spec 3 spectro-radiometer. Here we report on the potential of spectral reflectance to rapidly screen for variation in photosynthetic parameters in a set of 16 wheat genotypes grown in the glasshouse in Canberra, Australia. In addition, we are analysing the mechanisms underlying the variability in photosynthesis among these genotypes.


POSTERS WEDNESDAY



A NOVEL STABLE ISOTOPE TECHNIQUE FOR EVALUATING THE HYDRAULIC ARCHITECTURE OF LEAVES

Simonin K.A., Hu J. and Barbour M.M. University of Sydney,, Private Bag 4011, Narellan, NSW 2567.

The structure and function of the water transport pathway in leaves has direct biophysical ties to leaf rigidity, longevity, photosynthetic metabolism and ultimately the life time return on dry mass investment in leaf surface area. The vast majority of studies characterizing within- and between-species variation in leaf hydraulic architecture have treated leaves like woody stems by focusing on the dead xylem conduits that make up the leaf venation network. However, some studies have suggested that living leaf tissues (e.g. palisade, spongy mesophyll, bundle sheath cells and epidermis) may be a major component of the leaf transpiration stream, but the hydraulic connections between the metabolically active tissues of the leaf and the dead xylem cells are not well understood. Recent work suggests the metabolically active tissues and the dead xylem cells do not contribute equally to the leaf transpiration stream such that leaves do not behave like a single water pool. Here we present a novel stable isotope technique for evaluating the extent to which leaves behave like a single pool of water and how variation in transpiration rate influences the behavior of the leaf water transport pathway. A single pool model was unable to accurately predict the change in the stable oxygen isotope composition of transpired water in response to a step change in source water isotope composition suggesting that leaf water is comprised of at least two pools of water.

UNDERSTANDING SUGARCANE SEED COMPOSITION TO ENABLE STUDY OF SEED LONGEVITY

Siqueira G.F.1, 2, Pierre J.S.1, Rae A.L.1, Crusciol C.A.C.2 and Bonnett G.D.1 1CSIRO, Plant Industry, 306 Carmody Road, St. Lucia, QLD, 4072. 2Sao Paulo State University - UNESP, Jose Barbosa de Barros, 1780, Botucatu, SP, Brazil, 18610-307.

The aim of the project is to understand the composition of sugarcane seeds. Sugarcane is planted from vegetative stalk and this is also the harvested product, so consequently little is known about sugarcane seeds. However knowledge about sugarcane seeds is important to assist research and development of GM sugarcane cultivars as part of the regulatory process for evaluating GM cultivars. As part of this work, we are following the fate of sugarcane seeds in the environment. The research reported here is aimed at determining the composition of sugarcane seeds so that this can be related to the longevity of seeds. We have observed oil bodies in the seeds and will report the quantification and composition of lipid in the seed. We will also report the composition of carbohydrate reserves present in the seed. Identification of the components of the seeds and the methods for the analysis will enable us to understand the deterioration of the seed over time.

COMPARATIVE STUDIES ON MEDICAGO TRUNCATULA AND MEDICAGO ORBICUALRIS: TOWARDS REGULATORY CONTROL OF SEED STORAGE PARTITIONING

Song Y.S.1, Sheahan M.B.S.1, Liao S.L.1, Wang X.D.W.1, Garg M.G.2 and Rose R.J.R.1 1Australian Research Council Centre of Excellence for Integrative Legume Research, School of Environmental and Life Sciences, The University of Newcastle, Newcastle, 2308, Australia. 2School of Biomedical Sciences and Pharmacy, The University of Newcastle, Newcastle, 2308, Australia.

Legume seed accumulates considerable starch, protein and oil, but the storage composition largely varies with species. The mechanism of regulating storage partitioning is still unclear. In this study, two Medicago species i.e. M. truncatula (a legume model) and M. orbicularis with contrasting oil content were used to compare the cellular and molecular basis underlying the differences in seed storage accumulation. M. orbicularis accumulates much less protein and oil than that in M. trunctula, with similar fatty acid composition in the two Medicago species. A TEM study consistently showed more abundant protein and oil bodies in M. truncatula. Seed protein and oil accumulation is developmentally regulated by master transcription factors (TFs) including LEC1/L1L, LEC2, FUS3 and ABI3 in Arabidopsis. The homologs of those genes in M. truncatula were found by phylogenetic analysis. The FUS3 expression was similar in both temporal patterns and levels between both Medicago species. LEC1 level was very low and hardly detected by qPCR in both Medicago species; L1L and ABI3 expression was lower in M. orbicualris than in M. truncatula. Oleosins are unique proteins in forming the monolayer membrane enclosing oil bodies, there being four oleosin isoforms in M. truncatula. Oleosin3 expression was of very low in M. truncatula, which suggested that this gene may not be involved in oil body storage and oleosin3-GFP data confirmed that. Oleosin1 expression level was high and similar in both M. truncatula and M. orbicualris; Oleosin2 and Oleosin4 were high in M. truncatula but lower in M. orbicularis. In conclusion, this study indicated that L1L and ABI3 were most likely responsible for regulating protein and oil production, and Oleosin2 and Oleosin4 were most closely linked to oil body packaging in Medicago. The findings can be used for directing molecular breeding or genetic transformation in improving protein or oil production in legumes.

ORGANIC ACID METABOLISM OF VITIS VINIFERA FRUIT

Jia Y.1, Higginson E.G.2, Contreras-Pezoa D.A.1, Sweetman C.1, Thomas M.R.2, Soole K.L.3 and Ford C.M.1 1School of Agriculture, Food and Wine, The University of Adelaide, South Australia. 2CSIRO Plant Industry, Adelaide Laboratory, Glen Osmond, South Australia. 3School of Biological Sciences, Flinders University, South Australia.

Tartaric and malic acids are the predominant organic acids of grape berries, and are important for fruit and wine quality. Tartaric acid, despite having been found in several plant species, accumulates to a significant level uniquely in the grape berry. It arises through the metabolism of ascorbic acid (Vitamin C), and while many of the intermediates of the tartaric acid biosynthesis pathway have been determined, the enzymes responsible are still largely unknown. Malic acid is integrated in complex primary metabolism, and displays developmental changes in net accumulation and degradation through transport and metabolism of the acid. The sensitivity of malic acid metabolism to environmental conditions also causes seasonal variation in grape berry acidity. We are investigating four aspects of organic acid metabolism in grapes to advance the current knowledge of organic acid metabolism and fruit acidity. Firstly, to uncover novel enzymes involved in tartaric acid synthesis, we identified a candidate grapevine gene that can be used for recombinant expression and functional characterisation. To determine how the metabolism of tartaric and malic acids are controlled in grape, we screened the concentration of these acids from numerous grapevine populations, to assist the discovery of genes linked to acidity. To identify transporters and transcription factors that regulate malate release from the vacuole during berry ripening, we have cloned candidate genes for functional characterisation. And fourthly, to determine the effect of elevated temperature on organic acid metabolism, we have conducted controlled atmosphere experiments using potted grapevines.


POSTERS WEDNESDAY



WARMING EFFECTS ON MALIC ACID METABOLISM IN FIELD-GROWN VITIS VINIFERA WINEGRAPES

Sweetman C.1, Jenkins C.L.D.2, Sadras V.O.3, Ford C.M.1 and Soole K.L.2 1School of Agriculture, Food and Wine, The University of Adelaide, Glen Osmond, South Australia. 2School of Biological Sciences, Flinders University, Bedford Park, South Australia. 3South Australian Research and Development Institute, Glen Osmond, South Australia.

Malic acid is a common metabolite involved in complex biochemical networks. In grapes, it is also an important determinant of juice acidity at harvest, and particularly influential for viticultural and winemaking decisions. Malic acid levels in grape berries are dictated by the genotype, environmental factors and their interaction during development. The acid seems to play a role in carbon sequestration during early development, in preparation for fruit ripening. A switch to malate mobilisation and utilisation coincides with the onset of ripening (véraison). Malic acid is sensitive to elevated temperature, which accelerates acid loss and thus affects pH of the grape juice at harvest; a costly issue for winemakers especially in warming regions of Australia. Field-based investigations using Vitis vinifera cv. Shiraz grapevines from the Barossa Valley indicated a developmentally dependent effect of warming on the metabolism of malic acid, which was also altered by selective day and night heating. Short-term treatments (3 days) effected little change on malic acid levels. Moderate term (10-day) and long term (21-day) treatments resulted in decreased malic acid levels with elevated daytime temperature during early development, véraison and ripening. These changes were linked to decreased activity of the malate synthesis enzyme, phosphoenolpyruvate carboxylase, although this could not always explain a temporary increase in levels of malate seen with night heating. Trials with continuous heating over two growing seasons advanced fruit development and attenuated thermal effects on malic acid metabolism.

EFFECTS OF ALTERING THE EXPRESSION OF CELLULOSE SYNTHASE (CESA) GENES IN BARLEY

Tan H.-T., Burton R.A., Shirley N.J., Henderson M. and Fincher G.B. ARC Centre of Excellence in Plant Cell Walls, Waite Campus, University of Adelaide, Urrbrae, SA 5064.

Over-expression of cellulose synthase (CesA) genes in barley may increase plant cellulose content and stem strength. CesA1, CesA2 and CesA6 have higher transcript levels in tissues more likely to be synthesising primary cell walls, while CesA4, CesA7 and CesA8 are involved in the synthesis of secondary cell walls (Burton et al., 2004). Growing and expanding cells mainly consist of primary walls whilst secondary cell walls are deposited upon cessation of cell growth (McNeil et al., 1984). In this study, five sets of transformed barley plants transgenic for the CesA1, CesA2, CesA4, CesA6 or CesA8 genes driven by the CaMV 35S promoter, were analysed for transcript levels, phenotype, tissue morphology, cellulose content and crystalline cellulose distribution in cell walls. Results showed that transcript levels of the primary CesA transgenes were lower than expected even though they were driven by a strong constitutive promoter. The lines transgenic for the secondary cell wall genes exhibited a brittle node phenotype with 35S:HvCesA4 exhibiting the most severe dwarfing phenotype where xylem cells in the vascular tissue are irregular in shape and partially collapsed, resembling the irx mutants described in Arabidopsis (Turner and Somerville, 1997). Transcript silencing together with cellulose content reduction are also observed in many lines. Where over-expression in transcript levels does occur this does not translate into a change in crystalline cellulose content but TEM images show a significant decrease in cell wall thickness. These results are somewhat counter-intuitive and indicate that over-expression of a single CesA gene cannot successfully increase cellulose content in the cell wall and further demonstrate that cellulose synthesis is a fundamental process that is extremely sensitive to perturbation.

CHARACTERIZATION OF ALTERNATIVE DEHYDROGENASES IN ORYZA SATIVA AND THEIR TRANSCRIPTION PROFILES IN RESPONSE TO STRESS

Taylor D.C.1, Foo C.Z.1, Smith C.A.1, Shavrukov Y.2, Day D.A.1 and Soole K.L.1 1School of Biological Sciences, Flinders University, Adelaide, SA. 2Australian Centre for Plant Functional Genomics, Urrbrae, SA.

Plants, fungi, protists and some primitive animals have a branched respiratory electron transport chain containing alternative enzymes which do not pump protons across the inner mitochondrial membrane, and hence are not coupled to ATP synthesis. These enzymes include alternative oxidase (AOX), as well as several Type II NAD(P)H dehydrogenases (NDHs). The type II NDH’s are located on both sides of the inner mitochondrial membrane and are hence able to oxidize both internal and external NAD(P)H. Bioinformatics was used to identify and classify these Type II NDHs from rice. It was found that homologues of all three Type II NDH families in A. thaliana were also present in rice. Quantitative real-time PCR reactions were performed on shoot and root tissue, and have confirmed the presence of OsNda1, OsNdb1, OsNdb3 and OsNdc1 on a transcript level. Crude mitochondrial isolations and oxygen electrode assays were done and these confirmed the existence of enzyme activity for the complete alternative respiratory pathway in rice. Two cultivars of rice were subjected to salinity stress in hydroponics, and it was observed that O. sativa japonica var. Nipponbare exhibited a higher salinity tolerance than O. sativa indica var. Langi. As expected, sodium levels in both the root and shoot samples increased drastically under salt stress, with higher levels of sodium in the shoot tissue. Quantitative real-time PCR will be performed to determine transcript levels of the alternative NDH’s and alternative oxidase under salt stress.

INVESTIGATION OF ULTRA SHORT TERM GROWTH BEHAVIOUR OF TWO CROPS SOYBEAN AND RADISH UNDER OZONE STRESS USING STATISTICAL INTERFEROMETRY TECHNIQUE(SIT)

Thilakarathne B.L.S.1, Kadono H.1 and Yonekura T.2 1Graduate School of Science and Engineering, Saitama University, Japan. 2Center for Environmental Science in Saitama, Kazo, Saitama, Japan.

SIT permits real time growth measurements of plant leaves (1,2), with very high sensitivity of sub-nanometer order. Nanometric growth rate fluctuations (NGRF) in very short periods of 5.5 seconds were found using SIT. The standard deviation (S.D) of NGRF was found as the indicator of the plant healthiness. In this study, SIT was applied to measure the effect of ozone on two crops of Soybean and Radish. Two crops were fumigated in three levels of ozone; Charcoal filtered air (CF), Non filtered air (NF) and 1.5 times of ambient ozone (NF*1.5) for 30 days. Both crops Soybean and Radish, showed statistically significant (p<0.05) reductions in S.D of NGRF for NF and NF*1.5 compared with CF. As a percentage Soybean showed 57 % and 73 % and Radish 42 % and 63 % reduction respectively. Furthermore, photosynthetic rate (A380) in Radish and Soybean showed significant reductions only for NF*1.5. However significant reductions for CF to NF air in Radish and NF to NF*1.5 air in Soybean were not seen in A380. SPAD showed significant reduction only for NF*1.5 compared to CF in both plants. Therefore the method based on the reduction of NGRF under ozone stress is more sensitive for smaller concentration of ozone where the conventional methods are invalid. Further, this study addresses fast assessment of environment pollution for plants. References: 1. Kobayashi K. et.al, Appl. Opt. 49, 6333-6339 (2010) 2. Rathnayake A.P. et.al, Environ. Exp. Bot. 64, 314-321 (2008).


POSTERS WEDNESDAY



INFLUENCE OF ABIOTIC STRESSES ON PEROXISOME DYNAMICS IN DIFFERENT PLANT TISSUES OF ARABIDOPSIS

Tiew T.W.Y., Sheahan M.B. and Rose R.J. ARC CILR, School of Environmental and Life Sciences, The University of Newcastle, Callaghan, NSW 2308.

Plants being sessile organisms cannot escape abiotic stresses and therefore have to adapt to the environment by inducing biochemical and/or morphogenic adaptation in response to these stresses, in order to maintain normal plant growth and development. We have shown that peroxisomes proliferate in response to reactive oxygen species (ROS) due to wounding which enables re-entry to the cell cycle in cultured cells. Here we investigate the role and response of peroxisomes, organelles central to ROS metabolism and homeostasis, during adverse environmental stimuli in various organs and tissues of Arabidopsis seedlings. Confocal microscopy was used to visualise peroxisome-GFP in various tissue and organs to assess the distribution and dynamics of both control and abiotic stressed arabidopsis seedlings. Intriguingly, quantitative analysis showed peroxisomes proliferate massively in aerial regions, predominantly in cotyledons, however in root regions of the plant peroxisomes reduce in number compared to control plants. The observations in the seedlings show peroxisomes respond to abiotic stresses in a organ and tissue specific way as they adapt to their changing environment.

FUNCTIONAL ANALYSIS OF PLANT CELL WALL POLYSACCHARIDES DURING REPRODUCTIVE DEVELOPMENT

Tucker M.R., Phan J., Aditya J., Fincher G.B. and Burton R.A. ARC Centre of Excellence in Plant Cell Walls, University of Adelaide, Waite Campus, Urrbrae SA 5064, Australia.

The plant cell wall is a dynamic structure that mediates interactions with the environment and provides mechanical support for plant growth. Alterations in the amount and structure of polysaccharides that reside within the wall also influence transitions between key developmental stages, and this is particularly evident during reproduction. Despite this, little is known about the biosynthetic and hydrolytic enzymes that act on a tissue and cell-specific level to influence cell identity, function and fate. Using monocot and dicot models, we are generating histochemical, chromatographic and transcriptional profiles of developing tissues to identify dynamic changes in polysaccharide composition and cell wall-related gene expression. This has led to the identification of specific cell wall-related genes for which the function of the corresponding polysaccharides in development is unknown. Interestingly, inter- and intra-specific variation in polysaccharide composition correlates with changes in the biosynthetic machinery. The functional significance of these differences is currently being investigated.

SHOOT-TO-ROOT SIGNALS CONTROLLING ROOT HYDRAULIC CONDUCTIVITY VIA AQUAPORINS

Vandeleur R.K., Sullivan W., Athman A., Gilliham M., Kaiser B.N. and Tyerman S.D. ARC Centre of Excellence in Plant Energy Biology, School of Agriculture, Food and Wine, University of Adelaide, Glen Osmond, SA 5064, Australia.

We examined the effect of shoot treatments on root hydraulic conductance (Lo, normalised to root dry weight) and expression of aquaporin genes, in order to investigate the mechanism(s) underlying the positive correlation between leaf transpiration and Lo. Shoot topping (approx. 30% reduction in leaf area) reduced Lo of grapevine (Vitis vinifera L.), soybean (Glycine max L.) and maize (Zea mays L.) by about 60%. More detailed investigations were carried out on soybean and grapevine where the reduction in Lo was not correlated with the degree of leaf area reduction. The response was present an hour after shoot topping and was still evident at least 5 days later. The half time of reduction in Lo was approximately 5 min after total shoot decapitation. Hydraulic (pressure) mediated signals are a possibility since we recorded a relatively rapid decrease in turgor within root cortex cells in soybean after shoot topping. However, there was no change in the cortical cell hydraulic conductivity (Lpcell). There was a significant reduction in the expression of several aquaporins in the PIP family of both grapevine and soybean. In soybean the signal acts upon aquaporin expression within one hour of shoot topping (5 fold reduction in a PIP1 (Glyma08g01860) expression) which is sustained over the period of reduced Lo, and which may contribute to the reduction in Lo. We are performing Q-PCR and insitu-PCR to determine where this gene is predominantly expressed in roots. There were also significant reductions in the expression level of PIP genes within 30 min following decapitation of soybean plants.

USING TRANSGENIC GRAPEVINES TO UNDERSTAND ANTHOCYANIN AND TANNIN SYNTHESIS

Walker A.R. and Robinson S.P. CSIRO Plant Industry.

Flavonoids are important secondary metabolites in grapes comprising the coloured anthocyanins, flavonols and tannins. The anthocyanins and tannins form pigmented polymers in red wine, providing stable colour and contributing to the texture and structure of the wine. While the synthesis of these compounds have been studied in many plants species including Arabidopsis, there are still questions around tannin synthesis and compositional contribution to mouth feel that have not been answered by studying model plants. We have identified genes encoding enzymes responsible for various steps in the flavonoid pathways, together with genes that regulate these steps. To clarify the functions of these genes we have generated transgenic grapevines with alterations in the expression in some of these genes. Compositional analysis of fruit and leaves from the transgenic vines is providing valuable information about how flavonoids are made in grapes.


POSTERS WEDNESDAY



POLLINIA REMOVAL IN CYMBIDIUM ORCHID FLOWERS INDUCES ETHYLENE-MEDIATED ANTHOCYANIN BIOSYNTHESIS

Wang L., Lewis D.H., Arathoon H.S., Ngo H., Boase M.R., Zhang H., Schwinn K.E. and Davies K.M. The New Zealand Institute for Plant & Food Research Limited, Private Bag 11600, Palmerston North, NewZealand.

Cymbidium orchid flowers have a complex form including a highly modified, distinctive petal, called the lip. The lip plays a key role in floral attraction of pollinators. Pollination in Cymbidium flowers involves the pollinia (pollen sac) being dislodged, which triggers a rapid induction of anthocyanin pigments in the lip and subsequent flower senescence. Ethylene has been linked as a signal during pollination. Induction of anthocyanin pigments after pollinia removal can be inhibited/delayed when ethylene inhibitors are applied to flowers to delay senescence. In this study we investigated whether ethylene influences anthocyanin production in the lips of Cymbidium cut flowers. After pollinia removal, changes in RNA transcripts of key anthocyanin biosynthetic (DFR) and regulatory (MYB) genes and ethylene biosynthetic genes (ACO1 and ACS1) were examined over a 48 hour period, in flowers of Cymbidium treated with or without the ethylene receptor inhibitor 1-Methylcyclopropene (1-MCP). The expression of the MYB, DFR, ACO1 and ACS1 were reduced in 1-MCP treated flowers where the pollinia had been removed, consistent with an absence of anthocyanin production in the lips of flowers in the same treatment. Ethylene does appear to act as a signal, initiating anthocyanin biosynthesis in Cymbidium orchid flowers after pollinia removal.

NEW INSIGHTS INTO ROLES OF CELL WALL INVERTASE IN EARLY SEED DEVELOPMENT

Wang L. and Ruan Y.-L. School of Environmental and Life Sciences, the University of Newcastle, Callaghan, NSW 2308, Australia.

Despite well-documented evidence on the essential roles of cell wall invertase (CWIN) in seed filling, it remains largely unknown as to how CWIN exerts its regulation early in seed development, a critical stage that sets yield potential. As the first step towards filling this knowledge gap, we systematically examined the spatial and temporal expression patterns of a cell wall invertase, GhCWIN1, in cotton seeds from pre-fertilization to pre-storage phase. GhCWIN1 mRNA was abundant at the innermost cell layer of seed coat at 5 DAA but became hardly detectable at 10 DAA, prior to the differentiation of this cell layer into transfer cells (TC) characterized with wall ingrowths. The observation suggests CWIN may negatively regulate TC differentiation. Within the filial tissues, GhCWIN1 transcript was evidently detected in endosperm cells undergoing nuclear division but not in these cells at the cellularization stage. Parallel experiments in Arabidopsis revealed strong expression of cell wall invertase AtCWIN4 in chalazal endosperm under endoreduplication. Thses foundings indicate a function of CWIN in chlorosomal replication during nuclear division but not cell wall biosynthesis in endosperm, contrasting to the role previously proposed for sucrose synthase. Analyses further revealed a ‘V’ shape localization pattern of GhCWIN1 in early torpedo stage embryo, spatially matching the embryonic provascular tissue. Moreover, AtCWIN4 also shown a preferential expression in embryonic provascular region, indicating a role of CWIN in vascular development. Together, these novel observations provided insights into the roles of CWIN in regulating early seed development both spatially and temporally. By comparing with previous studies on Sus expression in cotton seed, we propose models of CWIN- and Sus-mediated regulation of early seed development.

SELECTION OF THIN CELL-WALL BARLEY FOR REDUCED STEEPING DURING MALT PRODUCTION

Washington J.M.1, Burton R.A.1, Little A.1, Stewart D.2 and Fincher G.B.1 1ARC Centre of Excellence in Plant Cell Walls, School of Agriculture, Food and Wine, University of Adelaide. 2Viterra.

The cell walls of the barley starchy endosperm are an impediment to efficient malting, requiring enzymic degradation before hydrolytic enzymes can gain access to the starch and protein substrates contained within the cells. Penetration of water through starchy endosperm cell walls has been identified as a potentially limiting factor in the rate of grain hydration to initiate endosperm modification during steeping and subsequent germination stages. In this project we are investigating the roles of cell wall thickness, composition and modification efficiency in water movement across the endosperm during steeping, with a view to reducing steep times and hence water use in the malthouse. Barley starchy endosperm cell walls contain approximately 70% (1,3-1,4)-beta-glucan. Theoretically, barley with thin cell walls or reduced (1,3-1,4)-beta-glucan content may allow more efficient water penetration and reduced water usage during steeping. High through-put assays have been developed for screening a large mutant population for reduced cell wall content. Transgenic grain with reduced (1,3-1,4)-beta-glucan content have also been developed for proof of concept assays. Transgenic barley with reduced grain (1,3-1,4)-beta-glucan was found to have thinner cells walls in the starchy endosperm, however grain hydration rates during steeping were not increased in either mutant or transgenic barley with reduced (1,3-1,4)-beta-glucan. Pure mutant and transgenic grain will be used for steeping experiments and malt analysis in order to determine if reduced cell wall thickness allows faster modification at reduced steep-out moisture. Such lines may provide a greater understanding of the underlying mechanisms of cell wall modification during malting in order to provide breeders with information to improve the selection of high quality malting varieties that use less water in the malt-house.

CELL-TYPE SPECIFIC EXPRESSION OF MULTIPLE SALT TOLERANCE GENES TO IMPROVE PLANT SALINITY TOLERANCE

Wellman G., Roy S.J. and Jacobs A. Australian Centre for Plant Functional Genomics, Waite Campus, PMB1, Glen Osmond, SA 5064.

Salinisation of agricultural land is of growing concern world-wide, as excessive soil salinity has a detrimental effect on growth and yield of many plant species of agricultural importance. In cereals, the accumulation of sodium ions (Na+) in the shoot has a negative effect on shoot growth. In recent years many genes involved in Na+ transport have been identified from different plant species. Genetic modification utilising these genes has been used to manipulate Na+ transport with the aim of reducing Na+ accumulation in the shoot. Both constitutive and tissue-specific overexpression of such genes in transgenic plants has proved beneficial in reducing Na+ shoot accumulation. However, further reductions would be possible by fine tuning Na+ transport processes throughout the plant through co-expressing multiple salinity tolerance genes in specific cell-types. In this study we have developed dual enhancer trap lines in the model species Arabidopsis thaliana allowing the expression of two different reporter genes in different root cell types. These lines are being used to drive cell-specific expression of multiple genes involved in Na+ transport in different combinations to allow more accurate manipulation of Na+ transport throughout plants. Successful gene combinations that reduce total shoot Na+ would then be used in agriculturally important crop species such as wheat, and rice. As a proof of concept, transgenic barley lines expressing individual Na+ transporters under the control of cell-specific promoters have been combined through traditional crossing techniques. Current progress towards these goals will be presented.


POSTERS WEDNESDAY



EXPLORING GENE CO-EXPRESSION NETWORKS IN VITIS VINIFERA

Wong D.C.J., Sweetman C. and Ford C.M. School of Agriculture, Food and Wine, University of Adelaide, South Australia.

Large-scale gene expression profiling studies in model plants such as Arabidopsis have contributed to our understanding of how genes function and how an underlying biological process is governed by cooperation of a set of genes. Accumulation of these data has offered additional capabilities such as gene co-expression analysis (GCA). GCA is based on the understanding that genes encoding similar and/or related functions may exhibit comparable expression patterns over a range of experimental conditions. Datasets amenable to such analyses exist for non-model species including the cultivated grapevine, Vitis vinifera. We have constructed a grape co-expression network using publicly available microarray data encompassing a wide range of biological and environmental conditions. Topological analysis revealed that the network manifested “small-world” properties with a high modular structure, common to many biological networks. To identify functional modules within the co-expression network, graph clustering coupled with gene ontology enrichment analysis was applied. Together, this approach has led to the identification of modules of high biological relevance, reflecting well-defined biological processes such as photosynthesis, ribosome biogenesis and secondary cell wall formation in plants, offering a validation of this experimental approach. A meta-network containing modules and significant connected inter-module edges also provides another level of interpretation, as these inter-connected modules participate in biologically-related functions. We are currently developing a simple database resource for grapes which includes tools to perform GCA, expression meta-profiling and annotation searches. This resource will aid grape researchers in their prioritization of gene candidates for further study towards the understanding of biological processes related to many aspects of grapevine development and metabolism (eg. fruit ripening, pathogen defense, hormone and stress responses).

DO AQUAPORINS FACILITATE CO2 DIFFUSION? Zhao, M.1, Evans, J.2, McCaffery, S.2 and Tyerman, S.1 1School of Agriculture, Food and Wine, University of Adelaide, Glen Osmond, SA, 5064, Australia. 2Plant Science Division, Research School of Biology, The Australian National University, Canberra ACT 0200 Australia.

Aquaporins may facilitate CO2 diffusion across leaf plasma membranes reducing the effect of CO2 diffusion in photosynthesis (Evans et al. 2009). Using plasma membrane vesicles isolated from pea leaves, we examined links between CO2 permeability and water permeability. We used a pH dependant dye to detect changes in intravesicular pH as a result of the reaction of CO2 with water to produce carbonic acid. This reaction is catalysed by carbonic anhydrase (CA), which is entrapped within vesicles. Using stopped-flow spectrofluorimetry, a sudden change in external CO2 concentration is imposed and kinetics of intravesicular pH is used to calculate CO2 permeability. Temperature dependencies of both water and CO2 permeability can be used to infer diffusion/flow pathways across the membrane. Although previous research has assumed that CA is not rate limiting for this technique, we have observed that CA activity varies with temperature and influences the estimate of CO2 permeability. Taking into account the effect of CA activity, we examined inhibition by silver-sulfadiazine, an aquaporin inhibitor (Niemietz and Tyerman, 2002). This did not block CO2 uptake but strongly inhibited water flow. We also obtained vesicles from plants grown under different treatments that caused large differences in water permeability. There was no significant correlation between water permeability and CO2 permeability indicating that aquaporins do not directly affect CO2 diffusion. It remains to be tested how aquaporins may affect CO2 diffusion in leaves. Some possibilities will be discussed. Evans JR, et al. (2009) J Exp Bot 60: 2235-2248. Niemietz CM, Tyerman SD (2002) FEBS Letters 531: 443-447.

CAROTENOID BIOSYNTHETIC PATHWAY IN PETALS OF ORANGE FLOWER ESCHSCHOLZIA CALIFORNICA IS NOT FEEDBACK REGULATED BY ITS METABOLITES

Zhou J.1, 2, Mcmanus M.2, Hunter D.1, Lewis D.1 and Zhang H.1 1Plant and Food Research, Private Bag 11 600, Palmerston North 4442, New Zealand. 2Massey University, PO Box 11 222, Palmerston North 4442, New Zealand.

The carotenoid biosynthetic pathway has been studied extensively in the last century in many fruits and flowers. Although all the enzymes and the metabolites have been identified, very little is known about the regulation of the pathway. This is because studies have been limited to the few natural occurring mutants that happen to survive and produce an observable phenotype due to the fundamental role carotenoids played in the living system. Many questions have been asked regarding to its regulation mechanism. The one gathered most research attention was whether the carotenoid biosynthetic pathway is feedback regulated by its metabolites. To answer this question, viral induced gene silencing (VIGS) was utilised to generate transit gene knock down lines of the orange E. californica. The carotenoid profiles and gene expression patterns were studied in the carotenoid gene silenced flower petals. The accumulation of the intermediary carotenoid metabolites and the disappearance of the end products of the wt flower were observed. The gene expression patterns have also displayed dramatic decrease in the transcript abundance of the silenced genes. However, none of the metabolite accumulation brings a change to transcript level of the non-silenced genes. The data suggests the carotenoid biosynthetic pathway in the petals of orange E. californica is unlikely to be feedback regulated by its metabolites.

TRANSCRIPTIONAL PROFILING OF ZEA MAYS ROOTS REVEALS RAPID UP-REGULATION OF DEFENCE-RELATED GENES FOLLOWING INOCULATION WITH PHYTOPHTHORA CINNAMOMI

Allardyce J.A., Rookes J.E., Hussain H.I. and Cahill D.M. School of Life & Environmental Sciences, Deakin Univeristy, Waurn Ponds, Victoria, Australia.

Since the identification of Phytophthora cinnamomi as a causal agent of plant disease in Australia in the early 20th century, this soil-borne plant pathogen has caused widespread damage to vulnerable native ecosystems and agriculture systems across the country and shows no sign of abating. Management of the pathogen in the natural environment is difficult and the options are limited. In order to discover more about how resistant plants are able to defend themselves against this generalist pathogen, a microarray study of plant gene expression following root inoculation with P. cinnamomi was undertaken. Zea mays was used as a resistant model plant and microarray analysis was conducted using the Affymetrix GeneChip Maize Genome Array on root samples collected at 6 and 24 hours post inoculation. Over 300 genes were differentially expressed in inoculated roots compared to controls. Of the up-regulated genes which were functionally annotated, greater than 60% were implicated in plant defence responses to biotic stress including those involved in phytoalexin biosynthesis, glutathione S-transferases, serine-protease inhibitors, jasmonic acid/ethylene biosynthesis and other defence-related genes. Of particular interest was the identification of the two most highly up-regulated genes; terpene synthase11 (tps11) and kaurene synthase2 (an2), which are both involved in production of terpenoid phytoalexins. This is the first example of a study to investigate plant gene expression levels in response to P. cinnamomi at a global level and provides valuable insights into the mechanisms involved in defence.


POSTERS WEDNESDAY



THE ROLE OF CELL WALL DEGRADING ENZYMES IN PHYTOPHTHORA PATHOGENICITY

Torrena P.S., Blackman L.M. and Hardham A.R. Australian National University.

Plant cell walls are highly complex structures that are composed of a diverse range of complex polysaccharides and proteins. Wall components are highly cross-linked and stable, and plant cell walls constitute a strong and effective barrier that impedes the ingress of potential pathogens. In order to penetrate the plant cell wall, pathogens secrete a diverse array of cell wall degrading enzymes (CWDEs) that cleave the diversity of linkages within cell wall polysaccharides. Species of highly destructive plant pathogens in the genus Phytophthora contain especially large CWDE multigene families. It is believed that the large multigene families of CWDEs in Phytophthora reflect the need for a range of substrate specificities within an overall enzyme class. It is also believed that pathogens secrete cascades of CWDEs during the infection process but details of this process are limited. The aim of this research is to identify the full complement of genes encoding CWDEs in P. nicotianae with a particular focus on pectin methyl esterases (PMEs), polygalacturonases (PGs), cellulases (Cel), and xylanases (Xyl). Using lupin (Lupinus angusfolius) seedlings as a model system for studies of early infection by P. nicotianae, temporal patterns of gene expression will be determined using quantitative real-time PCR (qPCR) and transcriptome analysis. Results to date revealed that mRNA transcripts from a range of PME, PG, Cel and Xyl genes can be detected at 24 hour post inoculation (hpi) while expression of other PGs and Xyl genes is not detectable until 27 hpi and 36 hpi, respectively. The site of CWDE secretion will be determined by immunolabelling.

SITE-DIRECTED MUTAGENESIS OF AN AVIRULENCE PROTEIN, AVRLM6, OF THE PLANT PATHOGENIC FUNGUS, LEPTOSPHAERIA MACULANS

Van De Wouw A.P., Dubois D.J. and Howlett B.J. School of Botany, the University of Melbourne, Vic. 3010, Australia.

Blackleg disease, caused by the fungus, Leptosphaeria maculans, is the major disease of canola (Brassica napus) worldwide. Leptosphaeria maculans interacts with B. napus in a typical ‘gene-for-gene’ manner whereby for each seedling resistance gene in the plant there is a corresponding avirulence gene in the fungus. The avirulence gene, AvrLm6 is responsible for avirulence towards resistance gene Rlm6 in B. napus. Alignments of the AvrLm6 protein with similar proteins from three other fungal species identified some potential conserved regions. Using site-directed mutagenesis within a potential conserved motif of the AvrLm6 protein, we have identified at least two amino acids essential for conferring avirulence towards Rlm6.

A SINGLE PROTEIN PRODUCED BY MYCOSPHAERELLA GRAMINICOLA CAUSES NECROSIS AND CHLOROSIS IN WHEAT

Wagner M.1, 2, 3, Ash G.1, 3, Milgate A.3, 4 and Solomon P.2 1Charles Sturt University, Wagga Wagga, Australia. 2Australian National University, Canberra, Australia. 3EH Graham Centre, Wagga Wagga. 4NSW Department of Primary Industries, Wagga Wagga, Australia.

The dothideomycete Mycosphaerella graminicola is a major wheat pathogen, inflicting significant yield loss worldwide. Little is known about the biochemical mechanisms underlying its pathogenicity despite intensive efforts over several decades. Our laboratory has been investigating substances secreted by Mycosphaerella graminicola which induce wheat cell death. These substances are produced under specific in vitro growth conditions and induce disease like symptoms in planta. Purification by Anion-Exchange FPLC followed by LC-MS/MS analysis and gel filtration of the fraction strongly suggest that a small protein (effector) secreted by the pathogen is the causative agent. Current data does not suggest a gene-for-gene action as a wide range of wheat cultivars, both susceptible and resistant to the pathogen, as well as the close wheat-relative barley develop similar levels of symptoms. The putative effector is relatively small, its activity is light dependent and appears to be threshold dependent.

SOILBORNE DISEASE POTENTIAL IN POTATO FIELDS USING DNA TECHNOLOGY

Wiechel T.J.1, Crump N.S.2, De Boer R.F.1, McKay A.3 and Ophel Keller K.3 1Department of Primary Industries Biosciences Research Division Knoxfield Victoria 3180. 2ViCSPA Private Mail Bag 1 Healesville Victoria 3777. 3South Australian Research and Development Institute (SARDI) Waite Campus Urrbrae SA.

Biotic stress caused by soilborne pathogens is a major threat to agriculture resulting in reduced crop yields and endangering food security. With a continuously increasing land area affected by soilborne pathogens the ability to assess risk of disease development is of increased importance. DNA technology has been used to detect and quantify potato pathogens Spongospora subterranea (powdery scab) and pathogenic Streptomyces species (common scab). The test for S subterranea is based on the ITS region while the test for pathogenic Streptomyces sp is based on a single copy gene responsible for toxin production that is essential for disease development. This study aims to validate quantitative tests for two major soilborne pathogens of potato to determine disease potential in potato fields. Over 3 growing seasons, soil samples were collected from potato fields prior to planting and qPCR assays for S subterranea and pathogenic Streptomyces applied to determine the level of pathogen DNA present. At harvest, potato tubers were collected from tested fields and assessed for powdery and common scab. Levels of pathogen DNA in soil prior to planting was correlated with disease development at harvest. For correlation analysis DNA values of 0 were excluded. Correlation between planting DNA level of S subterranea and the incidence of powdery scab disease was 0.54. Correlation between planting DNA level of pathogenic Streptomyces and the incidence of common scab disease was 0.39. Further studies to understand the interaction between pathogen inoculum and environmental factors are continuing.


POSTERS WEDNESDAY



INVESTIGATING THE ERYTHROID ROLES OF THE TRANSCRIPTION FACTORS KLF3 AND KLF8

Funnell A.P.W.1, Mak K.S.1, Norton L.J.1, Burdach J.G.1, Twine N.A.1, Pelka G.J.2, Tam P.P.2, Pearson R.C.M.1 and Crossley M.1 1School of Biotechnology and Biomolecular Sciences, University of NSW, Australia. 2Childrens Medical Research Institute, NSW, Australia.

Kruppel-like factors (KLFs) are transcription factors that bind to CACCC-boxes in the control regions of their target genes. KLF1 is a master regulator of erythropoiesis and drives the expression of genes required for red blood cell development, such as adult β-globin. The related family member KLF3 is abundant in erythroid tissue and its expression is highly dependent on KLF1 activity. KLF3 is a transcriptional repressor and hence we hypothesised that during erythropoiesis a negative feedback loop is generated, such that KLF3 accumulates and represses KLF1-driven target genes to fine-tune their expression. KLF8, also a transcriptional repressor, is one such target gene. Disrupting this network, results in KLF8 overexpression in erythroid cells. We have further explored the biological roles of KLF3 and KLF8 using mouse models. KLF3 null mice, while viable, exhibit a compensated anaemia and defects in both foetal and adult erythropoiesis. Microarrays have revealed a cohort of genes that are derepressed in KLF3 null Ter119+ foetal liver cells, consistent with the view that KLF3 acts primarily as a transcriptional repressor. Mice homozygous for a mutant KLF8 allele display no overt erythroid phenotype. Crossing the KLF3 and KLF8 mutant lines, however, results in further deregulation of gene expression in erythroid cells and embryonic lethality. These transcription factors thus appear to be at least partially redundant and share a subset of target genes in common. Taken together, our results reveal an erythroid KLF network and suggest that the interplay between KLF1, KLF3 and KLF8 is important in orchestrating proper gene regulation during erythroid development.

MITOGENIC AND METABOLIC SIGNALLING VIA THE INSULIN RECEPTOR ISOFORM-A

Rajapaksha H.1, McCarthy P.1, 2, Wallace J.C.1, Hoffmann P.2, Booker G.1 and Forbes B.E.1 1Department of Biochemistry, University of Adelaide. 2Adelaide Proteomics Centre, University of Adelaide.

The insulin receptor (IR) is a receptor tyrosine kinase involved in cellular metabolism, growth and differentiation. In mammals, the IR exists as 2 isoforms; IR-A and IR-B. The IR-A isoform lacks 12 amino acids encoded by the exon 11 whereas IR-B contains those residues. Interestingly this subtle difference in sequence composition results in differences in ligand selectivity and signalling. At physiological concentrations insulin binds to both isoforms of the IR. Insulin-like growth factor II (IGF-II), which binds to the type 1 insulin like growth factor receptor with a high affinity, also binds to IR-A isoform with a high affinity. The interaction of insulin with both isoforms results predominantly in metabolic signalling, whereas the IGF-II interaction with IR-A gives rise to predominantly mitogenic signalling. Upregulation of IR-A and IGF-II promotes growth, survival and migration of cancer cells. However the mechanism of this differential signalling via IR-A upon binding insulin and IGF-II is very poorly understood. In this work, we hypothesised that different ligands are capable of inducing different phosphorylation patterns on the same receptor, leading to differences in recruitment of downstream signalling molecules. With this in mind, using a predominantly metabolic ligand S597, the predominantly mitogenic ligand qIGF-I (His4, Tyr15, Thr49, Ile51 IGF-I), insulin and IGF-II we characterised the phosphorylation of IR. Insulin, IGF-II and qIGF-I promoted rapid and sustained activation of the IR-A, whereas S597 was slower at promoting activation. A marked difference between S597 and the other ligands was seen when analysing Y960 phosphorylation (the docking site for insulin receptor substrates 1 and 2). This correlates with its poor mitogenic activity and inability to promote receptor internalization. These results indicate that differences in phosphorylation and internalisation could be the mechanism behind differential signalling by IR-A. Currently we are in the process of barcoding this phosphorylation patterns using quantitative proteomics.

THE ROLE OF AMYLOID FIBRIL PROTEINS IN HIV INFECTION

Regoeng K. University of Adelaide.

The research project aims to explore the broader roles of amyloid fibrils in Human Immunodeficiency Virus (HIV) infections. The mechanistic aspects of Semen enhancer of viral infection (SEVI), its fragments and other amyloid fibrils proteins (α-synuclein, α-lactalbumin and reduced carboxymethalated kappa casein) in HIV infection will be determined. SEVI was named after the fibrils formed by fragment of Prostate acid phosphate (PAP), PAP 248-286 with sequence; GIHKQKEKSRLQGGVLVNEILNHMKRATQIPSYKKLIMY. Amyloidogenic regions of SEVI were identified using different computer algorithms such as Zygregattor, Zipper DB and Tango and synthesized as independent fragments and given the names fragment 1-24 (F1-24) and fragment 10-20(F10-20). A wide range of biophysical and spectroscopic techniques including Thioflavin T assay, Transmission electron microscopy, Circular Dichroism spectroscopy and X-ray diffraction were used to monitor their fibril formation. The interaction of amyloid fibrils to glycoprotein 120(GP 120) found on the surface of HIV will be investigated. The project investigated toxicity of SEVI and its fragments to pheochromocytoma-12 cells, PC 12, (neurological cells) and colorectal cancer 2 cells, COCA2, (epithelial cells). The research also aims to achieve a better understanding of how anti-fibrils agents, clusterin and Epigallocatechin-3-Gallate (EGCG) affect fibril formation of SEVI and its fragments 10-20 and 1-24 and their enhancement HIV infection. HIV infection of Tzmbl cells will be performed with and without fibrils in the presence and absence of fibril inhibitors. In the longer term, it is hoped that the agents identified in this study that reduce HIV infection could be incorporated into tropical microbicides to prevent HIV sexual transmission.

INVESTIGATING THE FUNCTION OF AUTOPHAGY RELATED PROTEINS IN DROSOPHILA

Bader C.1, Brooks D.A.1 and Shandala T.1, 2 1School of Pharmacy and Medical Sciences, University of South Australia, Adelaide. 2School of Molecular and Biomedical Sciences, University of Adelaide, Adelaide.

Autophagy has been implicated in a number of high profile human diseases, including cancer, neurodegenerative disorders, and chronic inflammatory disease. Autophagy is essential for the recycling and degradation of cytoplasmic content, and functions in a range of cell processes including adaption to starvation, energy balance, organelle clearance, and ultimately cell homeostasis. Autophagy involves the formation of a double membrane structure, which envelops cytoplasmic contents to form an autophagosome structure. Autophagosome maturation proceeds by sequential fusion of this newly formed organelle with endosomes and lysosomes compartments, which provide hydrolytic enzymes that are utilised for the degradation of the autophagic vesicle contents. While the core autophagic molecular machinery has been identified, the step wise regulation of the processes of autophagosome initiation and maturation are not well understood, especially in vivo, in higher eukaryotes. Drosophila provides a genetically tractable in vivo model to investigate the regulation of autophagy, and due to the functional conservation of the core autophagic machinery, the findings can be extrapolated to mammalian systems. Using a rich collection of RNAi silencing transgenes, we are performing a genetic screen for possible regulators of the autophagic machinery. We aim to identify a regulatory mechanism directing the core machinery to the site of autophagosome nucleation, as well as controlling the steps of autophagosome maturation. This project will develop a greater understanding of this important cellular process and will have direct relevance for major human diseases involving autophagy.


POSTERS WEDNESDAY



DNA WRAPPING AND LOOPING BY A CIRCULAR 14-MER WHEEL OF THE BACTERIOPHAGE 186 CI REPRESSOR

Shearwin K.E.1, Wang H.2, Finzi L.2, Dunlap D.2, Murchland I.1 and Dodd I.B.1 1School of Molecular and Biomedical Science, The University of Adelaide, Australia. 2Department of Physics, Emory University, Atlanta, GA, USA.

As the field of synthetic biology grows, the requirements for synthetic gene control systems will become more subtle and complex, and higher order DNA:protein regulatory structures will be needed. However, not enough is yet known about the energetics and kinetics of DNA wrapping and looping to be able to predict or reliably manipulate the behaviour of these structures. The bacteriophage 186 CI system of DNA wrapping and looping is simple enough to reveal fundamental principles, yet displays complex behaviour. The effect of the CI repressor protein of bacteriophage 186 on lytic and lysogenic transcription is similar to other well-studied phages but is achieved in a unique way. 186 CI is proposed to form a circular wheel-like complex of seven dimers, with its DNA-binding domains on the outside, around which large single DNA segments or combinations of separate DNA segments can be ‘wrapped’ to provide the cooperative and competitive interactions needed for regulation. Although consistent with structural, biochemical and gene expression data, many aspects of this model are based on inference and have not been directly observed. Here, we use a range of biophysical approaches to provide direct evidence for a number features of the model. Multi-angle laser light scattering was used to confirm formation of a CI 14-mer in solution. Atomic force microscopy (AFM) analysis showed wheel-shaped and -sized CI particles both on and off DNA, with multimerization stimulated by DNA binding. AFM also revealed the various predicted wrapped and looped species for CI binding at the promoter region and the two distal sites. Tethered particle motion (TPM) experiments were consistent with wrapping and looping of DNA by CI in solution, where in contrast to lambda repressor, the looped species were exceptionally stable.

GAMMA-H2AX IS A BIOMARKER OF IONIZING RADIATION INDUCED DNA DAMAGE IN THE FRUIT FLY, BACTROCERA TRYONI

Siddiqui S.1, Filomeni E.1, Francois M.1, Collins S.R.2, Glatz R.V.3, Taylor P.W.2, Fenech M.1 and Leifert W.R.1 1CSIRO Food and Nutritional Sciences, Adelaide, SA, 5000,Australia. 2Department of Biological Sciences, Macquarie University, NSW, 2109, Australia. 3SARDI Sustainable Systems, Entomology, Waite Building, SA, 5064, Australia.

The response of eukaryotic cells to ionizing radiation (IR)-induced double strand DNA breaks is highly conserved and involves a DNA repair mechanism characterized by the early phosphorylation of histone protein H2AX (producing the active form γH2AX). The expression of an induced, radiation responsive γH2AX has not been reported in fruit flies of commercial relevance. We use Bactrocera tryoni (Queensland fruit fly or “Q-fly”) to investigate this response with a view to develop assays to quantify prior exposure of fruit flies to IR. A linear dose-response of γH2AX (0-400 Gy IR) was observed in whole Q-fly pupal lysates 24 hr post-IR and was detected at doses as low as 20 Gy. γH2AX signal was not routinely detectable in whole Q-fly pupal lysates at five days post IR (compared with one day post-IR). However, sensitivity of γH2AX detection was significantly improved by extraction of the histone proteins (containing γH2AX) from five days post-IR pupae. Additionally, using both Western blot and laser scanning cytometry techniques on isolated Q-fly nuclei we detected γH2AX signal in adult flies that had been irradiated 17 days earlier as pupae. We conclude that IR exposure in Q-fly leads to γH2AX signals that are persistent over a period of days and can easily be detected by Western blot or quantitative immunocytochemistry techniques. These approaches have potential as the basis for assays for analysis of prior IR exposure of pest fruit fly species.

INVESTIGATION OF A NOVEL BINDING PARTNER OF THE TUMOUR SUPPRESSOR p53

Su S.-P., Hahn M.A., Dickson K.-A. and Marsh D.J. Hormones and Cancer Division, Kolling Institute of Medical Research, Royal North Shore Hospital and University of Sydney, NSW, Australia.

Cell division cycle 73 (CDC73), also known as parafibromin, is a ubiquitously expressed tumour suppressor protein initially discovered in yeast. Mutation and/or downregulation or loss of nuclear CDC73 has been implicated in parathyroid, renal, breast, gastric, uterine and colorectal cancer. A limited number of CDC73 interacting partners have been reported, including β-catenin, actin-binding proteins actinin-2 and actinin-3, and from our own work, interaction with the ring finger protein E3 ubiquitin ligases RNF20 and RNF401. In the present study, we show that CDC73 also interacts with p53 - the most studied tumour suppressor known as the “guardian of the genome”. We treated HEK293 cells with the transcription inhibitor actinomycin D in order to activate p53, and were able to co-immunoprecipitate endogenous CDC73 and p53. Co-immunoprecipitation of CDC73 and p53 was not seen in untreated cells. p53 is upregulated in response to DNA damage and transcriptionally regulates a number of target genes including MDM2, p21 and BAX, whose primary functions are to either inhibit the cell cycle or induce apoptosis, thereby inhibiting tumour development. Experiments are ongoing to down-regulate CDC73 using siRNA in the p53-wild-type cell lines MCF7 and HEY (breast and ovarian cancer cell lines respectively) to determine the effect on p53 target genes. Preliminary data in MCF7 cells suggests that down-regulation of CDC73 decreases both transcript and protein levels of p53 gene targets. These findings suggest that the tumour suppressor function of CDC73 may derive, at least in part, from its association with p53. 1Hahn et al. Hum Mol Genet (2012) 21(3): 559-568.

THE STRUCTURAL AND FUNCTIONAL CHARACTERISATION OF THIOESTERASES WITHIN YERSINIA PSEUDOTUBERCULOSIS, MYCOBACTERIUM PARATUBERCULOSIS, AND LEGIONELLA PNEUMOPHILA

Swarbrick C.M.D.1, 2 and Forwood J.K.1, 2 1Charles Sturt University. 2EH Graham Centre for Agricultural Innovation.

Mycobacterium paratuberculosis is the causative agent of Ovine Johne’s Disease (OJD), an important disease within the Australian sheep industry. Since the discovery of OJD in Australia in 1980 it has been a costly disease for the Australian sheep industry, reducing weight gain, wool production and the potential to kill 10% of adult sheep if left untreated. Currently there is no cure for OJD however Animal Health Australia currently recommends use of vaccination, grazing management and biosecurity planning to prevent the spread. Legionella pneumophila, the causative agent of Legionnaires disease, has the ability to inhabit and replicate within alveolar macrophages when present in aerosols as a consequence of water cooling towers. Yersinia psuedotuberculosis causes the disease Yersinia, a food borne disease characterised by gastrointestinal symptoms. Research within bacterial species has demonstrated the presence of thioesterases crucial for the virulence of these pathogens. The characterisation of such proteins by x-ray crystallography and a range of complementary molecular techniques allows researchers to develop targeted drugs against these pathogenic organisms. In 2011 a number of potential inhibitors for a related human thioesterase were investigated with the potential to reduce inflammation and form the basis for an anti-inflammatory drug (Forwood et al., 2007, Kirkby et al., 2010, Swarbrick et al., 2011, Serek et al., 2006, Kirkby et al., 2011). Building on our expertise in this area, we will characterise a number of thioesterase proteins within these pathogenic bacteria as a platform for developing in vitro inhibitors and potential drug molecules. To date we have successfully cloned, expressed and purified a number of these proteins, with a number of crystal trials yielding the structure of the tesB thioesterase within Y. psuedotuberculosis, with functional data still to be collected.


POSTERS WEDNESDAY

POS-WED-089 POS-WED-090MAST CELLS ARE KEY NEGATIVE REGULATORS OF UVB-INDUCED SKIN TUMOURIGENESIS

Taing H.H.1, 2, Gordon E.J.1, Yip K.H.1, Betterman K.L.1, Pejler G.3, Galli S.J.4, Lopez A.F.1, Harvey N.L.1 and Grimbaldeston M.A.1, 2 1Centre for Cancer Biology, SA Pathology, Adelaide, South Australia. 2University of Adelaide, Adelaide, South Australia. 3Swedish University of Agricultural Sciences, Uppsala, Sweden. 4Stanford University School of Medicine, Stanford, USA.

Ultraviolet-B (UVB) radiation, a component of sunlight is considered the major etiologic factor in the pathogenesis of “non-melanoma skin cancers”. One of the hallmark features for the development and progression of these skin malignancies is the accumulation of inflammatory infiltrate and mast cells (MCs) in the peri-tumoural stroma. Recently, we reported that MCs can negatively regulate inflammatory responses caused by chronic low-dose UVB irradiation of the skin, via a pathway involving Vitamin D3 and MC-derived IL-10. In the current study we explored whether MCs maintain a protective regulatory ability in response to more excessive UVB exposures that cause tumour progression and aberrant lymphangiogenesis. In addition to MC-derived IL-10, our findings suggest that the preformed chymotrypsin-like serine protease, mouse mast cell protease 4 (mMCP4), a modulator of the extracellular matrix, provides protection against the progression of in situ squamous cell carcinoma. We demonstrate that MC-deficient c-kit mutant KitW/W-v mice engrafted with bone marrow-derived cultured mast cells (BMCMCs) from mMCP4-/- or IL-10-/- mice, yield higher rates of UVB-induced ear ulceration and skin neoplasia than wild-type BMCMC-engrafted KitW/W-v mice. Interestingly, in response to extensive chronic UVB irradiation mMCP4-/- BMCMC->KitW/W-v mice exhibit enlarged lymphatic vessels in their ears, suggesting a potential role of mMCP4 and its substrates in governing protection against pathological lymph vessel dysfunction. Taken together, our data indicate that MCs contribute towards homeostatic kinetics and processes that regulate and protect against UVB-induced skin carcinogenesis and aberrant lymphangiogenesis.

MECHANISTIC INSIGHT INTO THE PATHOLOGY OF POLYALANINE EXPANSION DISORDERS REVEALED BY A MOUSE MODEL FOR X LINKED HYPOPITUITARISM

Hughes J., Piltz S., Rogers N., McAninch D. and Thomas P. The University of Adelaide, Nth Tce Adelaide, 5005.

Polyalanine expansions in transcription factors have been associated with eight distinct congenital human diseases. It is thought that in each case the polyalanine expansion causes misfolding of the protein that abrogates protein function. Misfolded proteins form aggregates when expressed in vitro, however it is less clear whether aggregation is of relevance to these diseases in vivo. To investigate this issue, we used targeted mutagenesis of embryonic stem cells to generate mice with a polyalanine expansion mutation in Sox3 (Sox3-26ala) that is associated with X-linked Hypopituitarism (XH) in humans. By investigating both ES cells and chimeric mice we show that endogenous polyalanine expanded Sox3 does not form protein aggregates in vivo, but rather is present at dramatically reduced levels within the nucleus of mutant cells. Importantly, the residual mutant protein of chimeric embryos is able to rescue a block in gastrulation but is not sufficient for normal development of the hypothalamus, a region that is functionally compromised in Sox3 null embryos and individuals with XH. Together, these data provide the first definitive example of a disease-relevant PA mutant protein that is both nuclear and functional, thereby manifesting as a partial loss-of-function allele.

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