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

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

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Contrôle de l'oxydation et de la dégradation du pool de vitamine C chez la tomate et impact sur la qualité du fruit et la tolérance au stress. Le métabolisme de l’ascorbate et plus principalement le statut redox du pool d’ascorbate sont impliqués dans la tolérance au stress et dans les processus primaires de croissance et de développement de la plante. La teneur et le statut redox de l’ascorbate chez les plantes sont régulés par (i) ses voies de biosynthèse, (ii) par le cycle ascorbate-glutathion permettant le recyclage des formes semi-oxydées et oxydées de l’ascorbate et (iii) par sa dégradation, l’ensemble de ces processus étant sous le contrôle de l’environnement. Au cours de ce travail de thèse, des méthodes de transgénèse nous ont permis d’identifier, chez différents génotypes de tomate à petit et gros fruits, les bouleversements physiologiques et métaboliques permettant de compenser des modifications de l’activité des enzymes monodéhydroascorbate réductase (impliqué dans le cycle ascorbate-glutathion) et ascorbate oxydase. Nous avons observé d’importantes modifications phénotypiques altérant le rendement en fruits de la plante sous conditions de culture pouvant générer un stress et également en condition normale de culture. Des liens entre l’activité des enzymes précités avec le métabolisme des sucres, la photosynthèse et la conductance stomatique sont révélés. Le déséquilibre entre les activités oxydantes et réductrices de ces enzymes constitue la première étape vers une dégradation de l’ascorbate. Le taux de dégradation se révèle très faible à la lumière, tandis qu’à l’obscurité une forte accumulation des produits de dégradation l’oxalate, le thréonate ainsi que l’oxalyl-thréonate est observé dans les feuilles de tomate. Enfin, l’activité de l’enzyme MDHAR est corrélée au taux de dégradation à l’obscurité. Les travaux de cette thèse mettent en avant l’importance du statut redox du couple ascorbate / monodéhydroascorbate dans les processus de croissance cellulaire et entre dans la régulation du rendement chez la tomate, et influe la dégradation de l’ascorbate. / Ascorbate metabolism and particularly ascorbate redox status are involved in stress tolerance and growth processes of plant cells. The concentration of ascorbate and its redox status are under control of (i) its biosynthetic pathways, (ii) the ascorbate-glutathione cycle allowing recycling of semi-oxidized and oxidized forms of ascorbate and (iii) its degradation rate. These processes are under environmental control. Transgenic lines modified for the activity of monodehydroascorbate reductase (involved in ascorbate-glutathione cycle) and ascorbate oxidase were generated in cherry and large-fruited genotypes of tomato. Physiological and metabolic modifications related to the modification of these enzyme activities were studied. We observed large phenotypic alterations that affected fruit yield under both stress conditions and normal growth conditions. Links between ascorbate recycling and sugar metabolism, photosynthesis and stomatal conductance were also revealed. An imbalance between the oxidizing and reducing activities of these enzymes is the first step leading to ascorbate degradation. We have shown that the degradation rate was very low under light, whereas under darkness the degradation compounds oxalate, threonate and oxalyl-threonate accumulated in tomato leaves. Also, the degradation rate is correlated with MDHAR activity. These results highlight the crucial role of the redox status of the ascorbate / monodehydroascorbate couple in growth processes and yield stability in tomato, and the impact on ascorbate degradation.