Honours Project Opportunities in Plant Cellular Molecular Biology

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Ass Prof Jan Marc

Proposed Honours projects 2011

Honours research projects are available in my lab to participate in the main focus of our research, namely, the role of phospholipase-cytoskeleton interactions in the transduction of hormonal and environmental stress signals to the cell interior. Phospholipases have been established as key enzymes in plant signal transduction, and the cytoskeleton is known to respond to both hormonal and environmental signals. Research in my laboratory has pioneered the role of the cytoskeleton as a downstream target, as well as transducer, of phospholipase signals. This discovery has attracted keen interest among plant biologists worldwide as a fundamental mechanism of plant responses to environmental stress signals, especially considering its importance in plant tolerance to adverse situations such as drought and salinity. Our research focuses on the cellular and molecular level of the signalling mechanism, using contemporary cellular and molecular tools and the model plant Arabidopsis. Our publications on this topic have attracted high citation rates, and the proposed Honours projects are at the cutting edge of this research area internationally. My colleagues Associate Professor Neville Firth and Dr. Penny Smith who specialize in cellular and molecular research approaches will be happy to co-supervise the projects.

1. Characterization of tubulin- and actin-binding domains in Arabidopsis PLDδ

We have found that silencing individual phospholipase D (PLD) isoforms using synthetic siRNAs produces distinct effects on the orientation of cortical microtubule arrays depending on the PLD isoform (Andreeva et al. 2009). AtPLDδ is unique in that its silencing promotes longitudinal orientation of cortical microtubules whereas silencing AtPLDβ or γ promotes transverse orientation. A simple explanation may be differences in their binding of either actin or tubulin, in combination with differences in their subcellular localization. Predicted tubulin- and actin-binding domains have been defined by alignment with a highly homologous mammalian protein. As a first step toward elucidating this mechanism, a previous Honours research project produced cytoskeletal deletion mutants in which either the tubulin- or the actin-binding domains, or both, were deleted from the AtPLDδ gene. These deletion mutants have been inserted into Gateway ‘entry’ vectors. The proposed Honours project will involve inserting these ‘entry’ clones into Gateway expression vectors harboring GFP tag, followed by Agrobacterium-mediated transformation into Arabidopsis cell suspension culture. The GFP fluorescent tag on individual cytoskeletal mutants will also facilitate subcellular localization using fluorescence microscopy. The GFP-AtPLDδ deletions will serve as ‘bait’ in affinity pull-down assays using anti-GFP magnetic beads to isolate complexes of interacting proteins from tissue homogenates. This will be followed by 2-D-gel electrophoresis, in-gel digestion with trypsin, and finally protein identification using nano LC-MS/MS mass spectrometry similarly to our previous experiments (Ho et al. 2009).

2. Characterization of tubulin- and actin-binding domains in AtPLDβ and γ isoforms

Because of the contrasting response of the AtPLDβ/γisoforms to siRNA silencing compared with silencing AtPLDδ in terms of microtubule orientation, we need to analyze their tubulin- and actin-binding properties as well as their sub-cellular localization as with the AtPLDδ isoform. In this project, cytoskeletal deletion mutants will be prepared as for the AtPLDδ above, using PCR to copy the desired segments of the AtPLDβ and γ genes, and then joining selected segments using ‘recombinant’ PCR. The cytoskeletal deletion mutants will then be inserted into Gateway ‘entry’ clones, followed by sequencing to confirm fidelity. The ‘entry’ clones will be inserted into Gateway expression vectors harboring RedFP or YellowFP tag, followed by Agrobacterium-mediated transformation into Arabidopsis cell suspension cultures, and pull-down assays and protein analysis as above. Again, the fluorescent tag on individual cytoskeletal mutants will allow subcellular localization using fluorescence microscopy. Multiple transformations of the different-coloured PLD isoforms into Arabidopsis cells will allow tracking individual isoforms during their subcellular re-localization in response to environmental stress signals such as salinity or the fungal elicitor xylanase.

Ideally, the first two projects would be carried out simultaneously by two Honours candidates because many of the experiments will be similar and thus any expertise could be shared for mutual benefit.

3. Association of AtPLDδ with actin, tubulin and flotillin in membrane microdomains.

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Our detection of Band-7 flotillin homologue in the GFP-AtPLDδ pull-down assay (Ho et al. 2009) is of interest because flotillin is a marker protein for lipid rafts, which are regarded as signalling platforms. Numerous Band-7 flotillin homologues have been reported in independent studies in plants, and many plant flotillin-like genes are annotated as nodulins. In this project, existing GFP-flotillin constructs will be transformed into Arabidopsis or tobacco BY-2 cell suspensions using particle bombardment. In addition, existing antibodies to flotillin will be used to immunolabel plasma membrane patches, or ‘ghost’, made by lysing protoplasts on a charged glass surface. The next step will be choosing flotillin homologues from the Arabidopsis database and constructing GFP-flotillin reporter genes for transformation into Arabidopsis cell suspension or making transgenic seedlings. Transformed cells or tissues will be used for tracking studies in combination with treatments using anti-microtubule or anti-actin drugs, or sterol depletion using methyl-β-cyclodextrin. GFP-flotillin reporters may be also used for transformation into Arabidopsis sterol-deficient mutant, smt1crc, or for treatments with fungal or hormonal signalling ligands. An alternative to standard confocal microscopy will be Total Internal Reflection Fluorescence (TIRF) optics or Fluorescence Lifetime Imaging microscopy (FLIM) for higher resolution close to the cell surface.

I would be most happy to discuss the details of the projects with potential Honours candidates.

References

  • Andreeva Z, Ho AYY, Barthet MM, Potocký M, Bezvoda R, Žárský V, Marc J (2009) Phospholipase D family interactions with the cytoskeleton: isoform  promotes plasma membrane anchoring of cortical microtubules. Functional Plant Biology 36, 600-612
  • Ho AYY, Day DD, Brown MH, Marc J (2009) Arabidopsis phospholipase D as an initiator of cytoskeleton-mediated signalling to fundamental cellular processes. Functional Plant Biology 36, 190-198