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Characterisation of putative metal transport proteins in the nickel hyperaccumulator Senecio coronatus: investigating candidate genes for nickel tolerance and accumulation
The accumulation of exceptionally high concentrations of heavy metals in plant tissues is an extreme phenotypic trait that has evolved independently in multiple plant taxa. The majority of research undertaken in this area has been performed on zinc/cadmium hyperaccumulators and comparatively little...
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| Format: | Thesis |
| Language: | English |
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Department of Molecular and Cell Biology
2018
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| _version_ | 1867611310543339520 |
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| access_status_str | Open Access |
| author | Cowlin, Ross Martin |
| author2 | Ingle, Robert A |
| author_browse | Cowlin, Ross Martin Ingle, Robert A |
| author_facet | Ingle, Robert A Cowlin, Ross Martin |
| author_sort | Cowlin, Ross Martin |
| collection | Thesis |
| description | The accumulation of exceptionally high concentrations of heavy metals in plant tissues is an extreme phenotypic trait that has evolved independently in multiple plant taxa. The majority of research undertaken in this area has been performed on zinc/cadmium hyperaccumulators and comparatively little is known about the molecular mechanisms behind nickel accumulation. This is despite the fact that nickel hyperaccumulators constitute more than 75% of all known hyperaccumulator species. One such species is Senecio coronatus (Asteraceae), which is a useful model to study nickel hyperaccumulation - as both hyperaccumulator and non-accumulator populations have been identified on nickel-rich serpentine soils in South Africa. The nickel-transporting abilities of three proteins (ScMATE, ScVIT and ScCOP), previously shown to be constitutively over-expressed in shoot tissues of hyperaccumulating populations of S. coronatus, were investigated in order to determine if they play a role in nickel hyperaccumulation. The RNA-Seq derived nucleotide sequences of these genes were confirmed by reverse transcriptase PCR, and computational analysis suggested that the proteins encoded by these genes display identical topology to their homologues in Arabidopsis thaliana. Heterologous expression of these proteins in a metal-sensitive yeast strain was performed to determine whether they are capable of transporting nickel. Although a minor reduction in nickel sensitivity was observed in yeast expressing ScMATE, and a minor increase in ScCOP-expressing yeast, no marked changes in sensitivity to nickel were observed. C-terminal EYFP-tagged MATE and VIT fusion proteins were transiently expressed in live onion cells to determine the subcellular localization of these proteins in planta. Fluorescence microscopy indicated that MATE localises to the nucleus and VIT to the tonoplast or plasma membrane. |
| format | Thesis |
| id | oai:open.uct.ac.za:11427/27373 |
| institution | University of Cape Town (South Africa) |
| language | eng |
| license_str | Not specified — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from UCTD — University of Cape Town Open Access Repository |
| publishDate | 2018 |
| publishDateRange | 2018 |
| publishDateSort | 2018 |
| publisher | Department of Molecular and Cell Biology |
| publisherStr | Department of Molecular and Cell Biology |
| record_format | dspace |
| source_str | UCTD — University of Cape Town Open Access Repository |
| spelling | oai:open.uct.ac.za:11427/27373 Characterisation of putative metal transport proteins in the nickel hyperaccumulator Senecio coronatus: investigating candidate genes for nickel tolerance and accumulation Cowlin, Ross Martin Ingle, Robert A Molecular and Cell Biology The accumulation of exceptionally high concentrations of heavy metals in plant tissues is an extreme phenotypic trait that has evolved independently in multiple plant taxa. The majority of research undertaken in this area has been performed on zinc/cadmium hyperaccumulators and comparatively little is known about the molecular mechanisms behind nickel accumulation. This is despite the fact that nickel hyperaccumulators constitute more than 75% of all known hyperaccumulator species. One such species is Senecio coronatus (Asteraceae), which is a useful model to study nickel hyperaccumulation - as both hyperaccumulator and non-accumulator populations have been identified on nickel-rich serpentine soils in South Africa. The nickel-transporting abilities of three proteins (ScMATE, ScVIT and ScCOP), previously shown to be constitutively over-expressed in shoot tissues of hyperaccumulating populations of S. coronatus, were investigated in order to determine if they play a role in nickel hyperaccumulation. The RNA-Seq derived nucleotide sequences of these genes were confirmed by reverse transcriptase PCR, and computational analysis suggested that the proteins encoded by these genes display identical topology to their homologues in Arabidopsis thaliana. Heterologous expression of these proteins in a metal-sensitive yeast strain was performed to determine whether they are capable of transporting nickel. Although a minor reduction in nickel sensitivity was observed in yeast expressing ScMATE, and a minor increase in ScCOP-expressing yeast, no marked changes in sensitivity to nickel were observed. C-terminal EYFP-tagged MATE and VIT fusion proteins were transiently expressed in live onion cells to determine the subcellular localization of these proteins in planta. Fluorescence microscopy indicated that MATE localises to the nucleus and VIT to the tonoplast or plasma membrane. 2018-02-07T09:09:30Z 2018-02-07T09:09:30Z 2017 Master Thesis Masters MSc http://hdl.handle.net/11427/27373 eng application/pdf Department of Molecular and Cell Biology Faculty of Science University of Cape Town |
| spellingShingle | Molecular and Cell Biology Cowlin, Ross Martin Characterisation of putative metal transport proteins in the nickel hyperaccumulator Senecio coronatus: investigating candidate genes for nickel tolerance and accumulation |
| thesis_degree_str | Master's |
| title | Characterisation of putative metal transport proteins in the nickel hyperaccumulator Senecio coronatus: investigating candidate genes for nickel tolerance and accumulation |
| title_full | Characterisation of putative metal transport proteins in the nickel hyperaccumulator Senecio coronatus: investigating candidate genes for nickel tolerance and accumulation |
| title_fullStr | Characterisation of putative metal transport proteins in the nickel hyperaccumulator Senecio coronatus: investigating candidate genes for nickel tolerance and accumulation |
| title_full_unstemmed | Characterisation of putative metal transport proteins in the nickel hyperaccumulator Senecio coronatus: investigating candidate genes for nickel tolerance and accumulation |
| title_short | Characterisation of putative metal transport proteins in the nickel hyperaccumulator Senecio coronatus: investigating candidate genes for nickel tolerance and accumulation |
| title_sort | characterisation of putative metal transport proteins in the nickel hyperaccumulator senecio coronatus investigating candidate genes for nickel tolerance and accumulation |
| topic | Molecular and Cell Biology |
| url | http://hdl.handle.net/11427/27373 |
| work_keys_str_mv | AT cowlinrossmartin characterisationofputativemetaltransportproteinsinthenickelhyperaccumulatorseneciocoronatusinvestigatingcandidategenesfornickeltoleranceandaccumulation |