Associate Professor Robyn L Overall
Associate Professor
Plant Cell and Developmental Biology
School Biological Sciences
Room 239c Macleay Building A12
University of Sydney
NSW, 2006
AUSTRALIA
Personal WebsitAddress: http://www.bio.usyd.edu.au/SOBS/ADMIN/STAFF/overall.htm
Email: roverall@mail.usyd.edu.au
The Big Question
The main aim of my research program is to understand the structure, composition, function and regulation of plasmodesmata, the channels responsible for intercellular transport of ions, metabolites, signals and viruses in plants.
A secondary aim is to understand the mechanisms that bring about the intercellular co-ordination of growth and development within plant tissues. This work aims to elucidate the mechanisms that bring about the co-ordination of the cytoskeleton between neighbouring plant cells and the development and maintenance of polarities such as displayed in polar auxin transport.
The Short-term goals
Identification of plasmodesmatal proteins: We aim to identify novel proteins within
plasmodesmata to increase our understanding of their mode of operation. We are exploiting the unique anatomy of the large celled green alga Chara corallina to isolate proteins from walls with and without plasmodesmata. We are using a proteomics approach to obtain sequence for putative plasmodesmatal proteins unique to walls with plasmodesmata. The short-term goal is to confirm that any such proteins are actually localised to plasmodesmata. This will lead to functional studies using Arabidopsis mutant lines or modifications of gene expression.
Intercellular transport via the endoplasmic reticulum: This work aims to prove that intercellular transport through plasmodesmata takes place both via the cytoplasm and the endoplasmic reticulum. Micro-injection studies suggest that there is a cell-to-cell transport pathway for small molecules through the ER and our short-term goal is to confirm this at an ultrastructural level, using antibodies to injected fluorescent tracers.
High resolution of microtubule dynamics in plant cells: We are using high resolution scanning electron microscopy to study the plant microtubule cytoskeleton at high resolution. We are now able to visualize entire arrays of microtubules at high resolution, without the use of microtubule stabilizing agents. Our short term goal is to analyse these high resolution images of microtubules following a variety of treatments to characterise the dynamics of single and bundled microtubules.
Role of auxin influx carriers in polar auxin transport: We aim to determine if the auxin influx carrier plays a role in the polarity of polar auxin transport. Our approach is to study the effects of influx inhibitors on polar auxin transport characteristics and eventually to localise the influx carrier in plant cells.
Chilling sensitivity in anther development of rice and wheat: This work aims to unravel the cell biological basis for the chilling-induced male sterility observed in rice and wheat using a variety of techniques from immunocytochemistry to micro-injection.
| Group (lab) Members and areas of research
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Post-doctoral Fellows:
Dr Louise Cole is studying the role of the endoplasmic reticulum in intercellular transport in plants. She has been micro-injecting fluorescent tracers into cells expressing GFP within the endoplasmic reticulum. She has then attempted to localise these injected tracers in the injected cells to confirm their localisation to the ER. Dr Cole has expertise in electron microscopy, particularly freeze-substitution, and secretion and vesicle trafficking in fungal and plant cells.
Dr Laurence Cantrill has had a long-term interest in the regulation of intercellular communication in plants. He is studying the cell biology of chilling induced male sterility in rice and wheat. He has expertise in micro-injection and confocal microscopy.
Dr Terena Holdaway-Clarke is collaborating on the regulation of plasmodesmatal permeability.
PhD students:
Ms Christine Faulkner is searching for plasmodesmatal proteins using a proteomic approach on the giant celled green alga, Chara corallina . She is using 2-D gel electrophoresis and protein sequencing.
Ms Errin Johnson is studying the role of the auxin influx carrier in the polar transport of auxin. She is using radio-active tracers to monitor polar auxin transport and inhibitors of the influx carriers to investigate their role in transport.
Ms Deborah Barton is studying plant microtubules at high resolution using field emission scanning electron microscopy in combination with immunocytochemistry and micro-injection of labelled tubulin.
Honours students:
Ms Victoria Howard is studying the role of the plant cell wall in regulation of transport through plasmodesmata.
Ms Miranda Lee is searching for actin/myosin binding proteins in plasmodesmata using immunogold cytochemisry and western blotting on 2-D gels of plant cell wall protein extracts.
Visitors
Dr John Gardiner is searching for novel microtubule associated proteins in plant cells.
Dr Ingeborg Lang has worked on the nature of the connection between the protoplast and the cell wall in plasmolysed plant cells.
Current
- GRDC : The Cell Biology of cold-induced male sterility in wheat $314.446 (2004-2006)
Applications submitted or planned
- ARC: Proteomics of plasmodesmata. (planned)
| Techniques in the Laboratory
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Currently active
- Confocal microscopy (Zeiss LSM 5 Pascal )
- Micro-injection (pressure and iontophoresis) of fluorescent tracers and proteins
- Protein localisation
- Transient transformation by micro-projectile bombardment with GFP fusion proteins (with Jan Marc)
- Immunofluorescence
- Immuno-gold cytochemistry
- High resolution scanning electron microscopy (at EM unit)
- Protein identification
- Protein extraction
- 2-D gel electrophoresis
- Protein sequencing (at APAF)
- Western blotting
- Molecular biology
- PCR
- Cloning
- Gene Library screening
- Radio-active tracer transport experiments
In development
- Plant cell tissue culture
- Analysis Arabidopsis t-DNA mutant lines
- Protein expression for production of antibodies
Top three areas of knowledge
- Intercellular communication in plants via plasmodesmata.
- Plant cytoskeleton.
- Co-ordination in growth and development between adjacent plant cells
Top three technical skills
- Micro-injection of plant cells
- Protein localisation - immuno-cytochemistry and GFP fusion protein expression
- Proteomics
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Collings DA, Harper JDI, Marc J, Overall RL , Mullen RT (2002) Life in the fast lane: actin-based motility of plant peroxisomes. Canad. J. Bot. 80(4): 430-441.
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Whiffen LK, Dibbayawan TP, Overall RL (2002) High resolution microscopy of cell wall formation in regenerating Mougeotia (Chlorophyceae) protoplasts. Eur. J. Phycol 37 (3):339-347.
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Blackman LM, Overall RL (2001) Structure and function of plasmodesmata. Aust. J. Plant Physiol. 28:709-727.
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Cantrill LC, Overall RL , Goodwin PB (2001) Changes in symplastic permeability during adventitious shoot regeneration in tobacco thin cell layers. Planta 214:206-214.
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Holdaway-Clarke TL, Walker NAW, Reid RJ, Hepler PK , Overall RL (2001) Cytoplasmic acidification with butyric acid does not alter the ionic conductivity of plasmodesmata. Protoplasma 215:184-190.
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Overall RL , Dibbayawan TP, Blackman LM (2001) Intercellular alignments of the plant cytoskeleton. J. Plant Growth Reg. 20:162-169.
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Harper JDI, Fowke LC, Gilmer S, Overall RL , Marc J (2000) A centrin homologue is localised across the developing cell plate in gymnosperms and angiosperms. Protoplasma. 211: 207-209.
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Holdaway-Clarke TL, Walker NAW, Hepler PK, Overall RL (2000) Physiological elevations in cytoplasmic free calcium by cold or ion injection result in transient closure of higher plant plasmodesmata.Planta 210, 329-335.
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Blackman LM, Harper JDI, Overall RL (1999) Centrin localisation in higher plant plasmodesmata. Eur J Cell Biol. 78,297-304.
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Cantrill LC, Overall RL , Goodwin PB (1999) Cell-to-cell communication via plant endomembranes. Cell Biol. Int. 23, 653-651.
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Fowke L, Dibbayawan T, Schwartz O, Harper J, Overall RL (1999) Combined immunofluorescence and field emission scanning electron microscope study of plasma membrane-associated organelles in highly vacuolated suspensor cells of white spruce somatic embryos. Cell Bio. Int. 23,389-397.
Book Chapters
- Overall RL , White RG, Blackman LM, Radford JE (2000) Actin and Myosin in Plasmodesmata.. In: Actin: A Dynamic Framework for Multiple Plant Cell Functions , Staiger CJ, Baluska, F, Volkman D and Barlow PW (Eds) Kluwer, Netherlands, 497-515
