Identifying a genetic basis for the tolerance to salinity exhibited by the resilient moss, Physcomitrella patens, could provide valuable information for use in the selection or modification of salinity tolerance in crop plants. The overall aim of the work described in this thesis was to identify, express and functionally characterise the protein products of two putative salinity tolerance genes from Physcomitrella, namely PpMdhar and PpENA1. The characterisation of PpMdhar and PpENA1 represents a two-pronged approach into investigating the salinity tolerance of Physcomitrella at the biochemical and transport level, respectively. The enzymes encoded by PpMdhars, monodehydroascorbate reductases (MDHARs), are central to the ascorbate-glutathione cycle, and recycle monodehydroascorbate molecules into the antioxidant, ascorbate. Hence, MDHARs play a part in maintaining the capacity of plant cells to remove toxic reactive oxygen species. Given that the production of reactive oxygen species is greatly increased in plants under salt stress, and that Physcomitrella is tolerant of high salt, MDHAR enzymes were expressed to determine whether they exhibit increased enzymic activity when compared with MDHARs from higher plants. The protein encoded by PpENA1 is Na⁺ transporting ATPase, which actively transports toxic Na⁺ ions across the cell membranes, and thereby minimizes the level of Na⁺ that accumulates in the cytoplasm. Thus, in contrast to the mechanism by which MDHARs may help Physcomitrella deal with the secondary effects of high salt, the PpENA1 protein could enable the moss to actively exclude Na⁺ ions, and thereby avoid cellular toxicity. A link between salinity and the transcription of PpMdhar and PpENA1 is reported here, and the function of each gene is investigated. A comprehensive characterisation of the enzymic action of expressed PpMDHAR enzymes is described, demonstrating that the biochemical mechanisms used by Physcomitrella in dealing with salt-induced reactive oxygen species are likely to be conserved with vascular plants. The physiological effects of the expression of PpENA1 are investigated via complementation experiments in yeast, and the membrane location of the protein is determined. The Na⁺ binding-sites of PpENA1 are predicted using homology modelling and amino acid residues crucial for Na⁺ transport are tested experimentally via site-directed mutagenesis. Finally, the introduction of a new, functional Na⁺ binding-site into an inactivated form of the PpENA1 protein demonstrates that a degree of control is possible over the Na⁺ binding-sites in PpENA1. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1337385 / Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 2008
| Identifer | oai:union.ndltd.org:ADTP/285347 |
| Date | January 2008 |
| Creators | Drew, Damian Paul |
| Source Sets | Australiasian Digital Theses Program |
| Detected Language | English |
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