This project was expected to contribute to the understanding of abscisic acid (ABA) perception in plants through identification of new ABA-binding proteins. The novel, biotinylated ABA derivative PBI686 (of biological activity comparable to natural ABA) has served as an affinity probe for isolation of ABA-binding proteins. Photoaffinity labeling in conjunction with affinity chromatography (streptavidin-biotin
based) was used for specific identification of target proteins from complex mixtures of cytosolic and membrane-bound proteins. Proteins of interest were identified by Mass Spectrometry through peptide mass fingerprinting and MS/MS ion search.<p>
Ribulose bisphosphate carboxylase/oxygenase (Rubisco) was identified as an ABA binding partner, and its interaction with ABA was initially confirmed by its ability to block the photoaffinity labeling reaction with PBI686. In addition, Surface Plasmon Resonance (SPR) experiments with ABA and Rubisco were performed, which provided further evidence for selective interaction between the two binding partners, with a very small preference towards (+)-ABA over (-)-ABA. SPR has also yielded the value of
equilibrium dissociation constant (KD) being 5 nM for (+)-ABA and 7 nM for (-)-ABA.
This was further confirmed by [3H] (±)-ABA binding assays, which have also shown that
non-radiolabeled (+)-ABA and (-)-ABA (at concentration 1000 fold higher) were able to
displace [3H] (±)-ABA from binding to Rubisco. Compounds other than ABA such as PA
(phaseic acid) or trans-(+)-ABA were not able to displace [3H] (±)-ABA, which has
suggested the selectivity of binding.
Further, Rubisco enzymatic activity in the absence of ABA was compared to that in
the presence of ABA at various concentrations. The results have clearly indicated the
effect of ABA on Rubiscos enzymatic activity. This was reflected on the enzymes Km
values being increased by seven fold in the presence of 10 mM ABA and 1 mM substrate
(RuBP). The interpretation of changes in enzyme kinetics upon inhibition by ABA most
resembles allosteric inhibition.
The biological function of this newly discovered interaction is interpreted as
ABAs ability to regulate plant growth during abiotic stress by its direct action on the
photosynthetic machinery - hypothesis often suggested in the literature.
| Identifer | oai:union.ndltd.org:USASK/oai:usask.ca:etd-08172009-071639 |
| Date | 15 September 2009 |
| Creators | Galka, Marek Michal |
| Contributors | Johnston, Linda, Cutler, Adrian, Loewen, Michele, Abrams, Suzanne, Palmer, David, Page, Matthew, Sanders, David, Wilson, Lee |
| Publisher | University of Saskatchewan |
| Source Sets | University of Saskatchewan Library |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://library.usask.ca/theses/available/etd-08172009-071639/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to University of Saskatchewan or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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