No description available.

Soil salinity is a major abiotic stress for land plants, and multiple mechanisms of salt tolerance have evolved. Tissue tolerance is one of these mechanisms, which involves the sequestration of sodium into the vacuole to retain low cytosolic sodium concentrations. This enables the plant to maintain cellular functions, and ultimately maintain growth and yield. However, the molecular components involved in tissue tolerance remain elusive. Several candidate genes for vacuolar sodium sequestration have recently been identified by proteome analysis of vacuolar membranes purified from the salt-tolerant cereal Hordeum vulgare (barley). In this study, I aimed to characterize these candidates in more detail. I successfully cloned coding sequences for the majority of candidate genes with primers designed based on the barley reference genome sequence. During the course of this study a newer genome sequence with improved annotations was published, to which I also compared my observations. To study the candidate genes, I used the heterologous expression system Saccharomyces cerevisiae (yeast). I used several salt sensitive yeast strains (deficient in intrinsic sodium transporters) to test whether the candidate genes would affect their salt tolerance by mediating the sequestration of sodium into the yeast vacuole. I observed a reduction in growth upon expression for several of the gene candidate under salt-stress conditions. However, confocal microscopy suggests that most gene products are subject to degradation, and did not localize to the vacuolar membrane (tonoplast). Therefore, growth effects cannot be linked to protein function without further evidence. Various potential causes are discussed, including inaccuracies in the genome resource used as reference for primer design and issues inherent to the model system. Finally, I make suggestions on how to proceed to further characterize the candidate genes and hopefully identify novel sodium transporters from barley.

No description available.

Soil salinity is a serious environmental stress threatening productivity of major crops worldwide. Among the various biotic and abiotic strategies that exist, transgenic technologies provide a promising avenue to reduce yield losses in crops under saline environments. Recently, transgenic technology involving the TMT gene encoding thiol methyltransferase enzyme has been suggested as an effective solution for engineering a chloride detoxification capability into a high value crops to improve tolerance against chloride ion toxicity under saline environments. This proposed mechanism, however, results in the emission of methyl chloride (CH3Cl) from plants, which has deleterious effects on stratospheric ozone. This study was performed to examine the relationship between salt tolerance and chloride volatilizing capacity of transgenic plants containing TMT gene as well as to explore the possibility of generating transgenic rice crop containing TMT gene for salinity tolerance. To achieve these objectives, transgenic tobacco plants containing TMT gene were grown in comparison with wild type tobacco plants under three levels of sodium chloride (NaCl) salinity (0, 100 and 200 mM), three levels of soil water content (40%, 60% and 80% of the field capacity) and their tolerance to NaCl and water stress was studied. Plant growth parameters recorded included plant height, number of leaves, leaf area, stem dry weight, leaf dry weight, root dry weight, plant dry biomass and root/shoot ratio. Similarly, both types of plants were exposed to five levels of NaCl concentrations (0, 50, 100, 150 and 200 mM) and three levels of soil water content (40%, 60% and 80% of the field capacity), and the quantity of CH3Cl emitted was recorded. Significant decrease in plants growth parameters of both types of plants were recorded upon exposure to salinity and water stress. Under 100 mM NaCl, however, transgenic plants showed better tolerance to salinity by suffering less reduction in growth parameters compared to wild type plants. Under 200 mM NaCl, growth of both types of plants was completely inhibited. The interactive effects of salinity and water stress were more pronounced in wild type plants than in transgenic plants. Results also showed that all engineered plants acquired an ability to efficiently transform chloride ion to CH3Cl, and the rate of such transformation was higher under greater NaCl and soil water content compared to lower NaCl concentrations and soil water content. In order to explore the possibility of generating a transgenic food crop using TMT gene, a hypothetical transgenic rice crop was grown over 27 million hectares of the saline coastal areas of south and southeast Asia and the possible emission of CH3Cl from such ecosystem was inferred based on the CH3Cl emission data obtained from transgenic tobacco plants. The estimates showed that the possible CH3Cl emission from such ecosystem would be 219.21 Gg which is equivalent to 5.36 % of the global atmospheric emissions of CH3Cl.

Soil salinity is a serious environmental stress threatening productivity of major crops worldwide. Among the various biotic and abiotic strategies that exist, transgenic technologies provide a promising avenue to reduce yield losses in crops under saline environments. Recently, transgenic technology involving the TMT gene encoding thiol methyltransferase enzyme has been suggested as an effective solution for engineering a chloride detoxification capability into a high value crops to improve tolerance against chloride ion toxicity under saline environments. This proposed mechanism, however, results in the emission of methyl chloride (CH3Cl) from plants, which has deleterious effects on stratospheric ozone. This study was performed to examine the relationship between salt tolerance and chloride volatilizing capacity of transgenic plants containing TMT gene as well as to explore the possibility of generating transgenic rice crop containing TMT gene for salinity tolerance. To achieve these objectives, transgenic tobacco plants containing TMT gene were grown in comparison with wild type tobacco plants under three levels of sodium chloride (NaCl) salinity (0, 100 and 200 mM), three levels of soil water content (40%, 60% and 80% of the field capacity) and their tolerance to NaCl and water stress was studied. Plant growth parameters recorded included plant height, number of leaves, leaf area, stem dry weight, leaf dry weight, root dry weight, plant dry biomass and root/shoot ratio. Similarly, both types of plants were exposed to five levels of NaCl concentrations (0, 50, 100, 150 and 200 mM) and three levels of soil water content (40%, 60% and 80% of the field capacity), and the quantity of CH3Cl emitted was recorded. Significant decrease in plants growth parameters of both types of plants were recorded upon exposure to salinity and water stress. Under 100 mM NaCl, however, transgenic plants showed better tolerance to salinity by suffering less reduction in growth parameters compared to wild type plants. Under 200 mM NaCl, growth of both types of plants was completely inhibited. The interactive effects of salinity and water stress were more pronounced in wild type plants than in transgenic plants. Results also showed that all engineered plants acquired an ability to efficiently transform chloride ion to CH3Cl, and the rate of such transformation was higher under greater NaCl and soil water content compared to lower NaCl concentrations and soil water content. In order to explore the possibility of generating a transgenic food crop using TMT gene, a hypothetical transgenic rice crop was grown over 27 million hectares of the saline coastal areas of south and southeast Asia and the possible emission of CH3Cl from such ecosystem was inferred based on the CH3Cl emission data obtained from transgenic tobacco plants. The estimates showed that the possible CH3Cl emission from such ecosystem would be 219.21 Gg which is equivalent to 5.36 % of the global atmospheric emissions of CH3Cl.

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

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / O objetivo deste trabalho foi avaliar parÃmetros fisiolÃgicos e bioquÃmicos em trÃs espÃcies vegetais com graus diferenciados de tolerÃncia ao estresse salino, a fim de melhor entender suas diferenÃas na tolerÃncia à salinidade. Para isto, sementes de algodÃo, feijÃo-de-corda e sorgo foram semeadas em copos plÃsticos contendo vermiculita umedecida com soluÃÃo nutritiva de Hoagland  forÃa (SNH Â), sendo o experimento conduzido em casa de vegetaÃÃo. PlÃntulas de cinco dias de idade foram transferidas para meio hidropÃnico (SNH Â), onde permaneceram por um perÃodo de seis dias para aclimataÃÃo. ApÃs esse perÃodo, as plantas foram submetidas a trÃs tratamentos salinos com valores de condutividade elÃtrica (CE) de 0,9 dS m-1 (baixa salinidade), 4,0 dS m-1 (mÃdia salinidade) e 8,0 dS m-1 (alta salinidade). A coleta foi realizada aos 25 dias apÃs o inÃcio do estresse. A salinidade reduziu significativamente a Ãrea foliar e a massa seca da parte aÃrea das trÃs espÃcies estudadas, especialmente as das plantas de feijÃo-de-corda e em menor proporÃÃo as do algodÃo. O potencial osmÃtico de folhas e raÃzes das trÃs espÃcies foram significativamente reduzidos nos tratamentos a 4,0 e 8,0 dS m-1 em comparaÃÃo com o de 0,9 dS m-1, exceto nas raÃzes de sorgo. Jà o teor relativo de Ãgua foliar nÃo apresentou alteraÃÃes com o aumento da CE do meio de crescimento. Os Ãons Na+ e Cl- aumentaram nas folhas e raÃzes das trÃs espÃcies, sendo que o algodÃo foi a espÃcie que mais reteve esses Ãons nos tratamentos a 4,0 e 8,0 dS m-1. As concentraÃÃes de K+ nas folhas de algodÃo e feijÃo-de-corda foram aumentadas pelos nÃveis crescentes de salinidade, enquanto nas plantas de sorgo foram diminuÃdas. Jà nas raÃzes as concentraÃÃes desse Ãon foram significativamente reduzidas nas trÃs espÃcies. De maneira geral, nos tratamentos de mÃdia e alta salinidade comparados com o de baixa salinidade, as concentraÃÃes de NO3- foram reduzidas em folhas e raÃzes das trÃs espÃcies. Os tratamentos a mÃdia e alta salinidade reduziram as concentraÃÃes de carboidratos solÃveis no algodÃo, enquanto aumentaram no feijÃo-de-corda e no sorgo. A concentraÃÃo de proteÃna solÃvel nÃo se alterou no feijÃo-de-corda em funÃÃo da salinidade, enquanto foi reduzida nas outras duas espÃcies. Os N-aminossÃluveis foram aumentados nas trÃs espÃcies, enquanto para a prolina, esses aumentos sà foram observados a 8,0 dS m-1. De modo geral, os parÃmetros de emissÃo de fluorescÃncia da clorofila a e a leitura SPAD nÃo foram alterados pela salinidade. Os nÃveis de peroxidaÃÃo lipÃdica foram significativamente aumentados nos tratamentos de mÃdia e alta salinidade no feijÃo-de-corda, nÃo sofreram alteraÃÃo no sorgo, enquanto foram reduzidos no algodÃo, quando comparados com o de baixa salinidade. A atividade das enzimas dismutase do superÃxido (SOD), catalase (CAT), peroxidases do ascorbato (APX) e do guaiacol (GPX) em folhas, nÃo foi alterada pelos tratamentos salinos a 4,0 e 8,0 dS m-1, com exceÃÃo de reduÃÃes nas atividades da SOD e GPX no algodÃo e da CAT no feijÃo-corda e, aumentos para a GPX no sorgo. Nas raÃzes, foram observados aumentos para a SOD no algodÃo e aumentos para a GPX no sorgo e feijÃo-de-corda, enquanto houve reduÃÃes da APX e GPX para o algodÃo. Os dados de crescimento aqui apresentados confirmam a maior tolerÃncia do algodÃo e a maior sensibilidade do feijÃo-de-corda ao estresse salino, enquanto que as alteraÃÃes na peroxidaÃÃo dos lipÃdios e nas enzimas antioxidativas nos levam a sugerir que o sistema enzimÃtico antioxidativo do algodÃo parece ser mais eficiente do que o das outras duas espÃcies estudadas, na eliminaÃÃo dos danos oxidativos ocasionados pela salinidade. à possÃvel, tambÃm, que a maior capacidade do algodÃo de acumular Ãons tÃxicos (Na+ e Cl-) nos tecidos fotossintetizantes contribua, pelo menos em parte, para sua maior tolerÃncia à salinidade. / The objective of this study was to evaluate physiological and biochemical parameters in three plant species with different degrees of salt tolerance in order to better understand their differences in salinity tolerance. For this, cotton seed, bean-to-string and sorghum were sown in plastic cups containing vermiculite moistened with  Hoagland solution strength ( SNH), the experiment being conducted in a greenhouse. Seedlings of five days of age were transferred to hydroponic medium (SNH Â), where they remained for a period of six days for acclimatization. After this period, the plants were subjected to three saline treatments with values ​​of electrical conductivity (EC) of 0.9 dS m-1 (low salinity), 4.0 dS m -1 (mean salinity) and 8.0 dS m 1 (high salt). Data were collected at 25 days after the onset of stress. Salinity significantly reduced leaf area and shoot dry mass of all species, especially, bean-to-string and to a lesser extent those of cotton. The osmotic potential of leaves and roots of the three species were significantly reduced in the treatments at 4.0 and 8.0 dS m-1 compared to 0.9 dS m-1 except the root sorghum. Since the leaf relative water content did not change with the increase in the EC medium. The Na + and Cl-increased in leaves and roots of three species, and cotton was the species that most of these ions retained in treatments 4.0 and 8.0 dS m-1. The concentrations of K + in leaves of cotton and bean-to-string were increased by increasing salinity levels, while in sorghum plants were decreased. Since the roots of this ion concentrations were significantly reduced in all three species. In general, the treatment of medium and high salinity compared with the low salinity, the concentrations of NO3-were reduced in leaves and roots of three species. Treatments at medium and high salinity reduced concentrations of soluble carbohydrates in cotton, while increased in the-string-beans and sorghum. The soluble protein concentration did not change the jack bean-string a function of salinity was reduced while the other two species. The N-aminossÃluveis were increased in all three species while for proline, the increases were only observed at 8.0 dS m-1. In general, the parameters of emission of fluorescence of chlorophyll SPAD readings were not affected by the salinity. Levels were significantly increased lipid peroxidation in the treatment of medium and high salinity of the bean-string, the sorghum did not change while the cotton were reduced compared with that of low salinity. The activity of the enzymes superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and guaiacol (GPX) in leaves was not affected by saline treatments at 4.0 and 8.0 dS m-1, except for reductions in the activities of SOD and GPX in cotton and string beans in CAT and GPX to increases in sorghum. In roots, increases were observed for SOD in cotton and increases for sorghum and beans in GPX-of-string while there were reductions of APX and GPX for cotton. The growth data presented here confirm the increased tolerance of cotton and the higher sensitivity of the jack bean-string to salt stress, whereas changes in lipid peroxidation and antioxidant enzymes lead us to suggest that the antioxidant enzyme system appears to be cotton more efficient than the other two species, the removal of oxidative damage caused by salinity. It is also possible that the greater ability of cotton to accumulate toxic ions (Na + and Cl-) in photosynthetic tissues contributes at least in part to its greater tolerance to salinity.

Equisetum giganteum L., a giant horsetail, is one of the largest living members of an ancient group of non-flowering plants with a history extending back 377 million years. Its hollow upright stems grow to over 5 m in height. Equisetum giganteum occupies a wide range of habitats in southern South America. Colonies of this horsetail occupy large areas of the Atacama river valleys, including those with sufficiently high groundwater salinity to significantly reduce floristic diversity. The purpose of this research was to study the ecophysiological and biomechanical properties that allow E. giganteum to successfully colonize a range of habitats, varying in salinity and exposure. Stem ecophysiological behavior was measured via steady state porometry (stomatal conductance), thermocouple psychrometry (water potential), chlorophyll fluorescence, and ion specific electrodes (xylem fluid solutes). Stem biomechanical properties were measured via a 3-point bending apparatus and cross sectional imaging. Equisetum giganteum stems exhibit mechanical characteristics of semi-self-supporting plants, requiring mutual support or support of other vegetation when they grow tall. The mean elastic moduli (4.3 Chile, 4.0 Argentina) of E. giganteum in South America is by far the largest measured in any living horsetail. Stomatal behavior of E. giganteum is consistent with that of typical C3 vascular plants, although absolute values of maximum late morning stomatal conductance are very low in comparison to typical plants from mesic habitats. The internode stomata exhibit strong light response. However, the environmental sensitivity of stomatal conductance appeared less in young developing stems, possibly due to higher cuticular conductance. Exclusion of sodium (Na) and preferential accumulation of potassium (K) at the root level appears to be the key mechanism of salinity tolerance in E. giganteum. Overall stomatal conductance and chlorophyll fluorescence were little affected by salinity, ranging from very low levels up to half strength seawater. This suggests a high degree of salinity stress tolerance. The capacity of E. giganteum to adapt to a wide variety of environments in southern South America has allowed it to thrive despite tremendous environmental changes during their long tenure on Earth.

The Pocket-book mussel, Lampsilis ovata, is a native freshwater bivalve species that is endemic to North America. The salinity tolerance of this species is of interest because anthropogenic salinization events and climate change factors threaten their natural freshwater habitats. Furthermore, the invasive freshwater bivalve species Corbicula fluminea has been shown to display significant salinity tolerance, which may lead to negative competitive interactions with native freshwater bivalve species if the salinization of freshwater habitats exceeds thresholds beyond which native species can effectively cope. It was hypothesized that L. ovata would be sensitive to salinity conditions above 1 g/L and respond by closing their valves. To investigate this, juvenile pocket-book mussels were subjected to three experiments which measured tissue-water content, hemolymph osmolality, and oxygen consumption after salinity exposure to 0, 2.5, 5, and 10 g/L. The 96-hour exposure study showed that the 2.5 g/L and 5 g/L treatment groups had significantly lower average percent tissue-water content than the control group. The average percent tissue-water content for mussels exposed to 2.5 g/L and 5 g/L dropped 2.4% and 2.2%, respectively. In the 24-hour time-course study, it was observed that changes in the average percent tissue-water content for all treatment groups primarily occurred after four hours of exposure. In the same study, the osmolality of the control group maintained an average of 31.2 mOsm/kg over the 24-hour period, despite the osmolality of the treatment water being 2 mOsm/kg. The hemolymph osmolality concentration of mussels exposed to the 2.5 g/L and 5 g/L treatments increased to osmotically conform to their treatment waters. After 24 hours, the hemolymph osmolality of the 2.5 g/L and 5 g/L treatment groups was 79 mOsm/kg and 163 mOsm/kg, respectively. Contrastingly, the osmolality of mussels exposed to the 10 g/L treatment maintained an average hemolymph osmolality of approximately 132 mOsm/kg, while the osmolality of the treatment water was 320 mOsm/kg. Lastly, the oxygen-consumption study showed that mussels exposed to the 5 g/L treatment consumed a significantly lower amount of dissolved oxygen than that of the control and the 2.5 g/L treatment by an average of 1.6 mg O2/mg/h. The control group consumed an average of 4.66 mg O2/mg/h, while the 2.5 g/L treatment group consumed the highest amount of dissolved oxygen with an average of 5.05 mg O2/mg/h. The data collected from these studies suggest that juvenile L. ovata might not be able to tolerate salinities greater than 2.5 g/L for an extended amount of time. Mussels exposed to the 5 g/L treatment and the 10 g/L treatment demonstrated varying degrees of behavioral avoidance and much higher morbidity rates. In contrast, the 2.5 g/L treatment group showed minimal behavioral avoidance and an elevated oxygen consumption rate. When compared to similar studies performed on C. fluminea, these results support the hypothesis that L. ovata is more sensitive to saline conditions than the invasive species and could be replaced by the invasive species if habitat conditions exceeded 2.5 g/L salinity.