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Centre of Excellence Developments in Platform Technologies
Cell suspension cultures of Lolium multiflorum, Setaria viridis and Brachypodium distachyon have been generated and will be used in conjunction with previously established barley, rice, wheat and Arabidopsis cell culture and transformation systems. The sequenced genomes and genetic resources for rice and Arabidopsis have been supplemented by our access in 2011 to the barley genome scaffold sequence through our colleagues PI Waugh and Dr David Marshall from the James Hutton Institute in Dundee. The genome sequence will greatly accelerate our progress in reverse genetics approaches to the identification of candidate genes. A particular advantage of the Lolium multiflorum cultures, in which cell walls commonly consist of 20-30% (1,3;1,4)-ß-D-glucan, is that we can now generate and rapidly harvest hundreds of grams of biological material, including the cell walls themselves or membrane fractions and proteins that are involved in ß-glucan synthesis and cell wall synthesis more generally.
Although there is no published genome sequence for Lolium multiflorum, we initiated a de novo RNA-Seq sequencing of transcripts from the suspension-cultured cells using resources at the Australian Genome Research Facility that are funded through the financial support provided to the ARC CoE through BioPlatforms Australia. The resulting data can now be used as a reference set for future experiments in which cell wall biosynthesis will be perturbed by specific polysaccharide synthesis inhibitors or other chemicals that, for example, disrupt cytoskeleton function or trafficking. After obtaining a grant from the Victorian Life Sciences Computational Initiative (VLSCI) for supercomputer access in 2011-2012, we have constructed an assembly method for the raw sequencing data using open-source components that address quality assurance, sequence assembly, gene expression and gene prediction. The dataset consists of more than 24 GB of sequence, which in draft form comprises over 300,000 transcript sequences. Applying computation gene prediction methods results in over 80,000 coding sequence predictions, which are currently being validated using an established proteomics pipeline.
In collaboration with Professor Bill Willats of the University of Copenhagen, we have established and refined the Comprehensive Microarray Polymer Profiling (CoMPP) analysis method, which is a high-throughput screening technique for characterising the polysaccharide composition of cell walls in a wide range of biological contexts. Various plant cell wall samples from Arabidopsis thaliana, Nicotiana alata (ornamental tobacco), Eucalyptus globulus (bluegum) and Hordeum vulgare (barley) were analysed using CoMPP in 2011. Our original, published method was modified to reduce variability between biological replicates and a series of internal standards was employed to provide quantitative information on the glycan of interest. This approach is well suited for comparing the polysaccharide composition in different tissue or plant types, and tracking cell wall alterations that occur in response to mutation, development or environmental cues.
Several important bioinformatics resources have been obtained by the Centre during 2011. Through our Partner Investigator Robbie Waugh at the James Hutton Institute, we now have full access to the barley genome scaffold sequence, together with extensive re-sequencing and SNP data. In Australia, we have consolidated our Q-PCR data into a single database and have access to a number of transcript and co-expression databases that are relevant in our cell wall programs. As noted above, we are building a transcript database for Lolium multiflorum and our local bioinformatics capabilities have been greatly enhanced by visits of staff and students to the James Hutton Institute in the UK.
Glossary of Terms in Platform Technologies
A range of techniques, resources and expertise are applied across all three Research Programs of the ARC Centre. The following is a brief glossary of these technologies.
One of the challenges of the ARC Centre of Excellence in Plant Cell Walls is to integrate proteomic, transcriptomic, and metabolomic information to give a more complete picture of plant cell wall biology.