Final Report Summary - FUNCPTOXTHELL (The role and functioning of the PTOX in stress tolerance in extremophile Thellungiella halophila)
In the coming years, climate change is predicted to have a significant impact on crop productivity globally. This involve not only a general warming worldwide but, more significantly for its impact on agriculture, an increase in the incidence of extreme events - extreme temperatures, droughts, salinity and flooding. Most crop species are, to a greater or lesser extent, stress sensitive. Work presented here, characterising the extremophile arabidopsis relative - thellungiella halophila - is a novel and powerful model system for studying adaptations of photosynthesis to stress. Our understanding of photosynthetic regulation is currently making great progress, in part through the identification of proteins, e.g. plastid terminal oxidase (PTOX), that are impaired in such regulation.
Previously, we provided evidence for plastid terminal oxidase (PTOX) to act in thellungiella as a significant electron sink from photosynthesis, with this pathway being absent in arabidopsis. This activity could substantially reduce reactive oxygen species (ROS) production under stress, protecting from photooxidative damage, and may play a crucial role in determining high stress tolerance of thellungiella. PTOX protein has been examined in various plant systems before, however, whilst some evidence for a low level of activity have been presented, no indication for a substantial flow of electrons from photosystem two (PSII) to oxygen under steady state photosynthesis has been observed, such as seen in thellungiella. The simplest hypothesis to explain this was that there are structural differences between thellungiella PTOX and that in other species. Alternatively, other factors, in addition to the core PTOX peptide, may be required.
The aim of the project was/is to investigate the role and functioning of the plastid terminal oxidase in thellungiella and in particular the factors that allow it to act effectively as a sink for electron transport from PSII, as well as, to investigate whether thellungiella PTOX activity can be transferred into another species and whether this has the potential to increase stress tolerance.
Under the aegis of this project, using an agrobacterium tumefaciens-mediated transfer of transfer deoxyribonucleic acid (T-DNA) and floral dipping, we successfully produced several lines of the thellungiella halophila and arabidopsis thaliana plants overexpressing thellungiella PTOX at various levels, in order to determine whether the thellungiella PTOX protein alone has substantial capacity to act as a terminal electron acceptor. In the course of our experiments, in planta alteration to PTOX through overexpression was demonstrated to have a certain potential for increasing an alternative oxidase activity and its functioning as significant sink for electrons from PSII. The fact, however, it was observed exclusively in thellungiella plants overexpressing native protein, with this overexpression having no major effect in arabidopsis, as well as, the fact the final electron flux diverted by PTOX in wild type and PTOX-overexpressing thellungiella under salinity is not significantly different, strongly suggested contribution of an additional factors or regulatory mechanisms in response of plastid terminal oxidase in thellungiella to stress.
The data obtained indicated either the structural and functional differences, or, different localisation of the PTOX protein in thellungiella in relative to the arabidopsis protein. Our experiment, in which we heterologously expressed thellungiella PTOX in e. coli, revealed however, the same specificity for plastoquinone and similar concentration-dependent effects of different inhibitors, as it was previously reported for arabidopsis. As also expected, iron proved in the current experiments with thellungiella to be essential for the catalytic function of the enzyme and not able to be substituted by other metal cations at the PTOX catalytic centre. Our results showed that the high in planta PTOX activity observed in thellungiella is unlikely to be a result of possible structural differences e.g. in surface charge or domain variation giving rise to differences in functioning, as compared to the arabidopsis protein.
Alternatively, we postulated possible interactions with further peptides being required for PTOX activity in thellungiella, as it was discussed previously for the mitochondrial alternative oxidase (AOX) protein. Our studies with isolated thylakoid membranes demonstrated substantial amounts of PTOX protein being co-isolated with other polypeptides, with this, however, being observed only when assayed in material derived from the light-adapted thellungiella plants - grown under both control and salt stress conditions. Our findings suggested the potential PTOX-PSII or/and PTOX-cyt b6f interaction, for which we will currently seek a direct evidence using an alternative methods, allowing in vitro confirmation of membrane and membrane-associated protein interactions.
It was unclear how a high rate of electron transport could be maintained between PSII and PTOX. Electron transport from PSII to PTOX requires the long-distance diffusion of plastoquinol through the thylakoid membrane from one protein to the other. The thylakoid membrane, however, is highly protein-rich which has been shown to limit quinone diffusion. We proposed that in salt-treated thellungiella there might be a different protein distribution. The results obtained from the control plants indicate clearly that PTOX is located mainly within the stromal lamellae, with only minor amounts being found in grana fraction, as it was concluded based on the location studies of the arabidopsis thaliana PTOX, as well as, with PTOX over-expressed in tobacco. In the thellungiella plants grown under high salinity, however, we observed significant increase in PTOX abundance within the thylakoid membrane originating from the grana, with PTOX being thus co-localised with a substantial proportion of PSII complexes. Importantly, this could explain functioning of the thellungiella PTOX as an efficient sink for electrons from PSII. The question arose, however, as to the factor/regulatory mechanism triggering this modified PTOX distribution within the thellungiella thylakoid membranes due to salt effect, as well as, to what extent it may assist PTOX targeting. We proposed that modification of the lipid composition within thellungiella chloroplast - influencing the membranes fluidity, protein transport and targeting - might be essential for altered PTOX distribution in thylakoid membranes under salinity. Significant increase in unsaturation of fatty acids in phospatidylglycerol (PG) and digalactosyldiacylglycerol (DGDG) fraction of chloroplast envelope, as well as, increase in unsaturation level in monogalactosyldiacylglycerol (MGDG) fraction from thellungiella thylakoid membranes due to salinity was observed in our experiments. In line with these findings, we are planning to generate currently, beyond the life-time of this project, thellungiella halophila plants in which the expression of the enzymes responsible for fatty acid processing in chloroplast will be suppressed to verify whether it is likely to restrict in planta PTOX activity in thellungiella. If these studies indicate that PTOX activity in thellungiella has been restricted due to suppression of the selected chloroplast desaturases, we will make an attempt to produce arabidopsis plant overexpressing both thellungiella PTOX and chloroplast enzymes responsible for fatty acids desaturation, and, verify whether this has the potential to increase arabidopsis stress tolerance.
Thellungiella halophila can survive high salt concentrations. Importantly however, thellungiella is not only tolerant of salt but has cross tolerance of other stresses, including drought and extreme temperatures. Thus, in addition to specific adaptations to salt stress, it must possess stress tolerance mechanisms that are of more general importance. We exposed thellungiella plants to a range of abiotic stresses - salinity, extreme temperatures and high light. We demonstrated PTOX expression to be rather a common feature in thellungiella response to stress, functioning however with different capacity under various stress conditions. We also successfully generated thellungiella plants in which the PTOX protein expression has been suppressed using ribonucleic acid (RNA) interference, corresponding to reduction in PTOX protein expression. We showed the alterations to PTOX through suppression to have a little effect on PSII photochemistry under the control conditions. By contrast, with greater level of PTOX suppression PSII electron transport was shown to be slightly inhibited with increasing stress and with this effect being deteriorated under high-light conditions. The higher PTOX suppression in thellungiella was also demonstrated to have important impacts on the extent of secondary oxidative stress - it was shown that inhibition of plastid terminal activity in thellungiella in fact results in higher accumulation of damage to membrane components. Thus we proved that down-regulation of naturally high stress-inducible PTOX activity in thellungiella was demonstrated to result at least partially in suppressing the stress-tolerant phenotype.
Climate is the great factor that mankind has not yet managed to control. Climate change is one of the most severe environmental factors limiting the productivity of crops globally. The annual cost of this is estimated in billions of Euro, with this being expected to rise in the coming decades. This, combined with the growing world population, makes this vital that we increase our understanding of how plants respond to stress. The need to ensure on-going food security in the face of changing climates is widely recognised as a major concern and is prominent amongst the recently defined European Union (EU) priorities. A better understanding of how photosynthesis is regulated in response to stress is therefore essential in meeting future demands for stress tolerant crops. Most crop species are, to a greater or lesser extent, stress sensitive. Studies on such species provide rather information on how and when protective mechanisms can fail but are of less use in allowing us to identify mechanisms to increase stress tolerance. It certainly is studies of extremophiles that have the greatest potential to provide novel stress tolerance mechanisms. Work presented here, characterising the extremophile arabidopsis relative - thellungiella halophila - is a novel and powerful model system for studying adaptations of photosynthesis to stress. With this project we made a direct and significant contribution towards meeting those priorities. By improving the current understanding of an extremophile we generated knowledge and resources that will certainly inform work into enhancing crop stress tolerance and we may even be able to directly show the transfer of a stress tolerance trait to another species. These current findings from this project place us in an ideal position to move towards producing crop plants with enhanced PTOX activity as a means to increase stress tolerance.
Previously, we provided evidence for plastid terminal oxidase (PTOX) to act in thellungiella as a significant electron sink from photosynthesis, with this pathway being absent in arabidopsis. This activity could substantially reduce reactive oxygen species (ROS) production under stress, protecting from photooxidative damage, and may play a crucial role in determining high stress tolerance of thellungiella. PTOX protein has been examined in various plant systems before, however, whilst some evidence for a low level of activity have been presented, no indication for a substantial flow of electrons from photosystem two (PSII) to oxygen under steady state photosynthesis has been observed, such as seen in thellungiella. The simplest hypothesis to explain this was that there are structural differences between thellungiella PTOX and that in other species. Alternatively, other factors, in addition to the core PTOX peptide, may be required.
The aim of the project was/is to investigate the role and functioning of the plastid terminal oxidase in thellungiella and in particular the factors that allow it to act effectively as a sink for electron transport from PSII, as well as, to investigate whether thellungiella PTOX activity can be transferred into another species and whether this has the potential to increase stress tolerance.
Under the aegis of this project, using an agrobacterium tumefaciens-mediated transfer of transfer deoxyribonucleic acid (T-DNA) and floral dipping, we successfully produced several lines of the thellungiella halophila and arabidopsis thaliana plants overexpressing thellungiella PTOX at various levels, in order to determine whether the thellungiella PTOX protein alone has substantial capacity to act as a terminal electron acceptor. In the course of our experiments, in planta alteration to PTOX through overexpression was demonstrated to have a certain potential for increasing an alternative oxidase activity and its functioning as significant sink for electrons from PSII. The fact, however, it was observed exclusively in thellungiella plants overexpressing native protein, with this overexpression having no major effect in arabidopsis, as well as, the fact the final electron flux diverted by PTOX in wild type and PTOX-overexpressing thellungiella under salinity is not significantly different, strongly suggested contribution of an additional factors or regulatory mechanisms in response of plastid terminal oxidase in thellungiella to stress.
The data obtained indicated either the structural and functional differences, or, different localisation of the PTOX protein in thellungiella in relative to the arabidopsis protein. Our experiment, in which we heterologously expressed thellungiella PTOX in e. coli, revealed however, the same specificity for plastoquinone and similar concentration-dependent effects of different inhibitors, as it was previously reported for arabidopsis. As also expected, iron proved in the current experiments with thellungiella to be essential for the catalytic function of the enzyme and not able to be substituted by other metal cations at the PTOX catalytic centre. Our results showed that the high in planta PTOX activity observed in thellungiella is unlikely to be a result of possible structural differences e.g. in surface charge or domain variation giving rise to differences in functioning, as compared to the arabidopsis protein.
Alternatively, we postulated possible interactions with further peptides being required for PTOX activity in thellungiella, as it was discussed previously for the mitochondrial alternative oxidase (AOX) protein. Our studies with isolated thylakoid membranes demonstrated substantial amounts of PTOX protein being co-isolated with other polypeptides, with this, however, being observed only when assayed in material derived from the light-adapted thellungiella plants - grown under both control and salt stress conditions. Our findings suggested the potential PTOX-PSII or/and PTOX-cyt b6f interaction, for which we will currently seek a direct evidence using an alternative methods, allowing in vitro confirmation of membrane and membrane-associated protein interactions.
It was unclear how a high rate of electron transport could be maintained between PSII and PTOX. Electron transport from PSII to PTOX requires the long-distance diffusion of plastoquinol through the thylakoid membrane from one protein to the other. The thylakoid membrane, however, is highly protein-rich which has been shown to limit quinone diffusion. We proposed that in salt-treated thellungiella there might be a different protein distribution. The results obtained from the control plants indicate clearly that PTOX is located mainly within the stromal lamellae, with only minor amounts being found in grana fraction, as it was concluded based on the location studies of the arabidopsis thaliana PTOX, as well as, with PTOX over-expressed in tobacco. In the thellungiella plants grown under high salinity, however, we observed significant increase in PTOX abundance within the thylakoid membrane originating from the grana, with PTOX being thus co-localised with a substantial proportion of PSII complexes. Importantly, this could explain functioning of the thellungiella PTOX as an efficient sink for electrons from PSII. The question arose, however, as to the factor/regulatory mechanism triggering this modified PTOX distribution within the thellungiella thylakoid membranes due to salt effect, as well as, to what extent it may assist PTOX targeting. We proposed that modification of the lipid composition within thellungiella chloroplast - influencing the membranes fluidity, protein transport and targeting - might be essential for altered PTOX distribution in thylakoid membranes under salinity. Significant increase in unsaturation of fatty acids in phospatidylglycerol (PG) and digalactosyldiacylglycerol (DGDG) fraction of chloroplast envelope, as well as, increase in unsaturation level in monogalactosyldiacylglycerol (MGDG) fraction from thellungiella thylakoid membranes due to salinity was observed in our experiments. In line with these findings, we are planning to generate currently, beyond the life-time of this project, thellungiella halophila plants in which the expression of the enzymes responsible for fatty acid processing in chloroplast will be suppressed to verify whether it is likely to restrict in planta PTOX activity in thellungiella. If these studies indicate that PTOX activity in thellungiella has been restricted due to suppression of the selected chloroplast desaturases, we will make an attempt to produce arabidopsis plant overexpressing both thellungiella PTOX and chloroplast enzymes responsible for fatty acids desaturation, and, verify whether this has the potential to increase arabidopsis stress tolerance.
Thellungiella halophila can survive high salt concentrations. Importantly however, thellungiella is not only tolerant of salt but has cross tolerance of other stresses, including drought and extreme temperatures. Thus, in addition to specific adaptations to salt stress, it must possess stress tolerance mechanisms that are of more general importance. We exposed thellungiella plants to a range of abiotic stresses - salinity, extreme temperatures and high light. We demonstrated PTOX expression to be rather a common feature in thellungiella response to stress, functioning however with different capacity under various stress conditions. We also successfully generated thellungiella plants in which the PTOX protein expression has been suppressed using ribonucleic acid (RNA) interference, corresponding to reduction in PTOX protein expression. We showed the alterations to PTOX through suppression to have a little effect on PSII photochemistry under the control conditions. By contrast, with greater level of PTOX suppression PSII electron transport was shown to be slightly inhibited with increasing stress and with this effect being deteriorated under high-light conditions. The higher PTOX suppression in thellungiella was also demonstrated to have important impacts on the extent of secondary oxidative stress - it was shown that inhibition of plastid terminal activity in thellungiella in fact results in higher accumulation of damage to membrane components. Thus we proved that down-regulation of naturally high stress-inducible PTOX activity in thellungiella was demonstrated to result at least partially in suppressing the stress-tolerant phenotype.
Climate is the great factor that mankind has not yet managed to control. Climate change is one of the most severe environmental factors limiting the productivity of crops globally. The annual cost of this is estimated in billions of Euro, with this being expected to rise in the coming decades. This, combined with the growing world population, makes this vital that we increase our understanding of how plants respond to stress. The need to ensure on-going food security in the face of changing climates is widely recognised as a major concern and is prominent amongst the recently defined European Union (EU) priorities. A better understanding of how photosynthesis is regulated in response to stress is therefore essential in meeting future demands for stress tolerant crops. Most crop species are, to a greater or lesser extent, stress sensitive. Studies on such species provide rather information on how and when protective mechanisms can fail but are of less use in allowing us to identify mechanisms to increase stress tolerance. It certainly is studies of extremophiles that have the greatest potential to provide novel stress tolerance mechanisms. Work presented here, characterising the extremophile arabidopsis relative - thellungiella halophila - is a novel and powerful model system for studying adaptations of photosynthesis to stress. With this project we made a direct and significant contribution towards meeting those priorities. By improving the current understanding of an extremophile we generated knowledge and resources that will certainly inform work into enhancing crop stress tolerance and we may even be able to directly show the transfer of a stress tolerance trait to another species. These current findings from this project place us in an ideal position to move towards producing crop plants with enhanced PTOX activity as a means to increase stress tolerance.