Evaluating the contribution of tonoplast and plasma membrane transporters in salinity tissue tolerance in barley

Salinity is one of the major abiotic stresses affecting the world food supply. Salt affected soil has area coverage of 950 million ha which accounts for 10% of the land surface of the globe. Fifty percent of the irrigated land (230 million ha) is affected by salinity costing the world food production $US27 billion per annum. In Australia alone, 67$AUD 1330 million per annum. In comparison to other strategies, the use of salt tolerant plants is cost effective and sustainable way of controlling salinity. While numerous attempts have been made to develop salt tolerant varieties over the last few decades, most of the efforts were focused on tackling individual components or genes contributing to overall salt tolerance. As a result, the progress in the field was much slower than expected and we still lack truly salt tolerant varieties in the farmers‚ÄövÑvº field. Salinity tolerance is a multi-faceted physiological trait, and all the beneficial effects of improving a function of one gene/mechanism may be lost or overturned by plethora of other contributing factors. This calls for a need of pyramiding‚ÄövÑvp several key traits in one ideotype. Before this can be implemented into the practice, the essentiality and a relative contribution of various traits should be quantified, for each particular species. The aim of this PhD study was to identifying the major contributing mechanisms to salt tolerance in barley. The major focus was on the following aspects: (1) essentiality of transcriptional vs post translational factors in mediating plant adaptive responses to salinity; (2) quantifying the relative contribution of osmo- and tissue-tolerance mechanisms in barley; (3) identifying components involved in Na+ sequestration in vacuole; and (4) revealing the role of H+-ATPase in vacuolar ion sequestration and overall salinity stress tolerance. In the first part of this PhD study, the relative contribution of ionic, osmotic and oxidative stresses to the overall salinity tolerance in barley was studied, both at the whole plant and cellular level. In addition, the gene expression profile of key genes in ionic and oxidative homeostasis (NHX, RBOH, SOD, AHA and GORK) as a way of comparing the contribution of transcriptional and post translational factors for salinity tolerance was also investigated. The major findings can be summarized to two main points. These are: (i) tissue tolerance is the dominant component in which root K+ retention and lower sensitivity to stress induced hydroxyl radical production are the main ones; (ii) responses at the post-translational level is more important than at the transcriptional level for understanding the mechanisms for salt tolerance. Overall, for better tissue tolerance, sodium sequestration, K+ retention and resistance to oxidative stress are important salt tolerance mechanisms. It is concluded that every crop improvement programs for salinity stress tolerance should take into consideration all this components. In the second part of this PhD study, we showed that unlike a number of reports for saltsensitive salt excluder‚ÄövÑvp species (such as Arabidopsis or rice), expressing AtNHX1 in barley had no beneficial effect on plant performance under saline conditions. AtNHX1 Arabidopsis tonoplast Na+/H+ exchanger was expressed in barley (Hordeum vulgare L., cv. Golden Promise) and the plants grown under saline growth condition. The transgenic plants were compared with null segregant for biomass, water content, gas exchange, and Na+ and K+ content of the leaf. The transgenic barley plants expressing AtNHX1 has not shown significant difference from the null segregant for any of the trait at least under our experimental conditions. The lack of phenotype in barley which adapts salt including‚ÄövÑvp strategy was explained by one or more of the following: (i) low level of activity of vacuolar H+-PPiase and vacuolar H+-ATPase causing poor proton gradient; (ii) the lack of controlling passive leak of sodium via Na+ permeable slow activating and fast activating channels in the vacuole; (iii) insufficient ATP pool to assist the H+ pumping activity; (iv) the AtNHX1 protein may not be folded properly, inactive or mis-targeted. In the last part of this PhD study, improvement in salinity stress tolerance was obtained in barley crops by expressing V-ATPase subunit C gene. Most of the reports until now have not shown improvement at the grain yield level and also have not explained the physiological mechanisms behind. Also, no previous attempt to express the V-ATPase subunit C was done in barley. Accordingly, we expressed AtVHA-C gene in barley and grown under 300mM NaCl. The barley plants expressing the gene were compared with wild type plants for dry biomass, leaf pigment content, stomatal conductance, grain yield, and leaf Na+ and K+ content. The transgenic barley plant expressing AtVHA-C have shown smaller reduction in biomass and grain yield compared to wild type plants. The beneficial effect in the AtVHA-C expressing plant was due to better maintenance of stomatal conductance resulted from the accumulation of Na+ and K+ in the leaf that lead to osmotic adjustment and less reliance on the de novo synthesis of organic osmolytes. To recap, salinity tolerance is a multigenic physiological trait involving various mechanisms that may differ between species. For barley, the dominant mechanism appears to be the tissue tolerance. This includes better K+ retention in the root; reduced tissue sensitivity to oxidative stress; and more efficient sodium sequestration in the leaf. We have shown that for a better sodium sequestration all the components such as proton pumping for generating proton gradient in the tonoplast membrane, control of the back leak of sodium through Na+ permeable SV and FV channels, and ensuring properly folded, active and correctly targeted NHX protein to the tonoplast all should be considered and modified as one set, to achieve a positive outcome and improve crop salinity tolerance. Finally, we have also shown the importance of vacuolar H+-ATPase towards salinity tolerance by the contribution of vacuolar H+-ATPase for the accumulation of Na+ and K+ in the leaf where they contribute towards osmotic adjustment, and hence, conservation of energy otherwise spent for organic osmolyte production

Evaluating the contribution of tonoplast and plasma membrane transporters in salinity tissue tolerance in barley

Salinity is one of the major abiotic stresses affecting the world food supply. Salt affected soil has area coverage of 950 million ha which accounts for 10% of the land surface of the globe. Fifty percent of the irrigated land (230 million ha) is affected by salinity costing the world food production $US27 billion per annum. In Australia alone, 67$AUD 1330 million per annum. In comparison to other strategies, the use of salt tolerant plants is cost effective and sustainable way of controlling salinity. While numerous attempts have been made to develop salt tolerant varieties over the last few decades, most of the efforts were focused on tackling individual components or genes contributing to overall salt tolerance. As a result, the progress in the field was much slower than expected and we still lack truly salt tolerant varieties in the farmers‚ÄövÑvº field. Salinity tolerance is a multi-faceted physiological trait, and all the beneficial effects of improving a function of one gene/mechanism may be lost or overturned by plethora of other contributing factors. This calls for a need of pyramiding‚ÄövÑvp several key traits in one ideotype. Before this can be implemented into the practice, the essentiality and a relative contribution of various traits should be quantified, for each particular species. The aim of this PhD study was to identifying the major contributing mechanisms to salt tolerance in barley. The major focus was on the following aspects: (1) essentiality of transcriptional vs post translational factors in mediating plant adaptive responses to salinity; (2) quantifying the relative contribution of osmo- and tissue-tolerance mechanisms in barley; (3) identifying components involved in Na+ sequestration in vacuole; and (4) revealing the role of H+-ATPase in vacuolar ion sequestration and overall salinity stress tolerance. In the first part of this PhD study, the relative contribution of ionic, osmotic and oxidative stresses to the overall salinity tolerance in barley was studied, both at the whole plant and cellular level. In addition, the gene expression profile of key genes in ionic and oxidative homeostasis (NHX, RBOH, SOD, AHA and GORK) as a way of comparing the contribution of transcriptional and post translational factors for salinity tolerance was also investigated. The major findings can be summarized to two main points. These are: (i) tissue tolerance is the dominant component in which root K+ retention and lower sensitivity to stress induced hydroxyl radical production are the main ones; (ii) responses at the post-translational level is more important than at the transcriptional level for understanding the mechanisms for salt tolerance. Overall, for better tissue tolerance, sodium sequestration, K+ retention and resistance to oxidative stress are important salt tolerance mechanisms. It is concluded that every crop improvement programs for salinity stress tolerance should take into consideration all this components. In the second part of this PhD study, we showed that unlike a number of reports for saltsensitive salt excluder‚ÄövÑvp species (such as Arabidopsis or rice), expressing AtNHX1 in barley had no beneficial effect on plant performance under saline conditions. AtNHX1 Arabidopsis tonoplast Na+/H+ exchanger was expressed in barley (Hordeum vulgare L., cv. Golden Promise) and the plants grown under saline growth condition. The transgenic plants were compared with null segregant for biomass, water content, gas exchange, and Na+ and K+ content of the leaf. The transgenic barley plants expressing AtNHX1 has not shown significant difference from the null segregant for any of the trait at least under our experimental conditions. The lack of phenotype in barley which adapts salt including‚ÄövÑvp strategy was explained by one or more of the following: (i) low level of activity of vacuolar H+-PPiase and vacuolar H+-ATPase causing poor proton gradient; (ii) the lack of controlling passive leak of sodium via Na+ permeable slow activating and fast activating channels in the vacuole; (iii) insufficient ATP pool to assist the H+ pumping activity; (iv) the AtNHX1 protein may not be folded properly, inactive or mis-targeted. In the last part of this PhD study, improvement in salinity stress tolerance was obtained in barley crops by expressing V-ATPase subunit C gene. Most of the reports until now have not shown improvement at the grain yield level and also have not explained the physiological mechanisms behind. Also, no previous attempt to express the V-ATPase subunit C was done in barley. Accordingly, we expressed AtVHA-C gene in barley and grown under 300mM NaCl. The barley plants expressing the gene were compared with wild type plants for dry biomass, leaf pigment content, stomatal conductance, grain yield, and leaf Na+ and K+ content. The transgenic barley plant expressing AtVHA-C have shown smaller reduction in biomass and grain yield compared to wild type plants. The beneficial effect in the AtVHA-C expressing plant was due to better maintenance of stomatal conductance resulted from the accumulation of Na+ and K+ in the leaf that lead to osmotic adjustment and less reliance on the de novo synthesis of organic osmolytes. To recap, salinity tolerance is a multigenic physiological trait involving various mechanisms that may differ between species. For barley, the dominant mechanism appears to be the tissue tolerance. This includes better K+ retention in the root; reduced tissue sensitivity to oxidative stress; and more efficient sodium sequestration in the leaf. We have shown that for a better sodium sequestration all the components such as proton pumping for generating proton gradient in the tonoplast membrane, control of the back leak of sodium through Na+ permeable SV and FV channels, and ensuring properly folded, active and correctly targeted NHX protein to the tonoplast all should be considered and modified as one set, to achieve a positive outcome and improve crop salinity tolerance. Finally, we have also shown the importance of vacuolar H+-ATPase towards salinity tolerance by the contribution of vacuolar H+-ATPase for the accumulation of Na+ and K+ in the leaf where they contribute towards osmotic adjustment, and hence, conservation of energy otherwise spent for organic osmolyte production

GORK channel: a master switch of plant metabolism?

Potassium regulates a plethora of metabolic and developmental response in plants, and upon exposure to biotic and abiotic stresses a substantial K+ loss occurs from plant cells. The outward-rectifying potassium efflux GORK channels are central to this stress-induced K+ loss from the cytosol. In the mammalian systems, signaling molecules such as gamma-aminobutyric acid, G-proteins, ATP, inositol, and protein phosphatases were shown to operate as ligands controlling many K+ efflux channels. Here we present the evidence that the same molecules may also regulate GORK channels in plants. This mechanism enables operation of the GORK channels as a master switch of the cell metabolism, thus adjusting intracellular K+ homeostasis to altered environmental conditions, to maximize plant adaptive potential

Targeting vacuolar sodium sequestration in plant breeding for salinity tolerance

Salinity is a major environmental issue affecting crop production around the globe, and creating a salt-tolerant germplasm is absolutely essential meeting the 2050 challenge of feeding a 9.3 billion population. While most efforts of plant breeders were focused around genes and mechanisms responsible for exclusion of cytotoxic Na+ from uptake, this is not the strategy naturally salt-tolerant halophyte species use. One of the hallmarks of halophytes is their ability to safely deposit large volumes of salt in their vacuoles, in the process termed vacuolar sodium sequestration. This chapter reviews molecular and physiological mechanisms mediating this process and prospects of their targeting in breeding programs

GORK channel: a master switch of plant metabolism?

Potassium regulates a plethora of metabolic and developmental response in plants, and upon exposure to biotic and abiotic stresses a substantial K+ loss occurs from plant cells. The outward-rectifying potassium efflux GORK channels are central to this stress-induced K+ loss from the cytosol. In the mammalian systems, signaling molecules such as gamma-aminobutyric acid, G-proteins, ATP, inositol, and protein phosphatases were shown to operate as ligands controlling many K+ efflux channels. Here we present the evidence that the same molecules may also regulate GORK channels in plants. This mechanism enables operation of the GORK channels as a master switch of the cell metabolism, thus adjusting intracellular K+ homeostasis to altered environmental conditions, to maximize plant adaptive potential

Expressing Arabidopsis thaliana V-ATPase subunit C in barley (Hordeum vulgare) improves plant performance under saline condition by enabling better osmotic adjustment

Salinity is a global problem affecting agriculture that results in an estimated US$27 billion loss in revenue per year. Overexpression of vacuolar ATPase subunits has been shown to be beneficial in improving plant performance under saline conditions. Most studies, however, have not shown whether overexpression of genes encoding ATPase subunits results in improvements in grain yield, and have not investigated the physiological mechanisms behind the improvement in plant growth. In this study, we constitutively expressed Arabidopsis Vacuolar ATPase subunit C (AtVHA-C) in barley. Transgenic plants were assessed for agronomical and physiological characteristics, such as fresh and dry biomass, leaf pigment content, stomatal conductance, grain yield, and leaf Na+ and K+ concentration, when grown in either 0 or 300 mM NaCl. When compared with non-transformed barley, AtVHA-C expressing barley lines had a smaller reduction in both biomass and grain yield under salinity stress. The transgenic lines accumulated Na+ and K+ in leaves for osmotic adjustment. This in turn saves energy consumed in the synthesis of organic osmolytes that otherwise would be needed for osmotic adjustment

Is the Addition of Dexmedetomidine to a Ketamine–Propofol Combination in Pediatric Cardiac Catheterization Sedation Useful?

Pediatric patients undergoing cardiac catheterization usually need deep sedation. In this study, 60 children were randomly allocated to receive sedation with either a ketamine-propofol combination (KP group, n = 30) or a ketamine-propofol-dexmedetomidine combination (KPD group, n = 30). Both groups received 1 mg/kg of ketamine and 1 mg/kg of propofol for induction of sedation, and the KPD group received an additional 1 mu g/kg of dexmedetomidine infusion during 5 min for induction of sedation and a maintenance infusion of 0.5 mu g/kg/h. In both groups, 0.2 mg/kg of propofol was administered as a bolus to maintain a Ramsey sedation score (RSS) greater than 4 throughout the procedure. None of the patients in either group required intubation. In the KP group, one patient required mask ventilation. The chin-lift maneuver needed to be performed for eight patients in the KP group and one patient in the KPD group (p 0.05). The mean recovery time was longer in the KP group (5.86 vs 3.13 min; p < 0.05). Adding dexmedetomidine to a ketamine-propofol combination led to a reduced need for airway intervention and to decreased movement during local anesthetic infiltration and throughout the procedure. The recovery time was shorter and hemodynamic stability good in the KPD group