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AtNOR1 gene confers salt tolerance on Arabidopsis
Abstracts / Comparative Biochemistry and Physiology, Part A 146 (2007) S255 – S266 1
suggest that ascorbate degradation also increases during elongation growth. The degradation of ascorbate to oxalate/Lthreonate operates extra-cellularly in cultured Rosa cells2. The study reported here compares the kinetics of the changes in cellular ascorbate, glutathione and sugars through the phases of cell division and cell expansion, relating the observed changes in antioxidant pools sizes to the relative effects on ascorbate and glutathione synthesis and degradation during the different phases of cell development in culture. In addition, we have also followed the changes in the activity of poly (ADP-ribose) polymerase (PARP) that occur through the periods of mitosis and cell expansion.
S259
P5.14 Role of nitric oxide in plants challenged by elicitors or cadmium: another paradigm for double-edged role of nitric oxide D. Wendehenne, A. Pugin, (CNRS/Université de Bourgogne); G. Dobrowolska, (Institute of Biochemistry and Biophysics, Warsaw)
P5.13 Functional analysis of H2O2 signalling in photorespiratory Arabidopsis mutants deficient in catalase
We previously reported that nitric oxide (NO) was rapidly produced in plant cells challenged by elicitors of defence responses and hyper-osmotic stresses. NO synthesis is required as a step in the pathways leading to the rise in cytosolic concentration ([Ca 2+ ]cyt). NO enhances [Ca 2+ ]cyt by promoting phosphorylation-dependent events. Accordingly, NO released by a NO donor or produced endogenously in response to hyperosmotic stresses and elicitors was shown to induce the activation of the protein kinase NtOSAK which belongs to the SnRK2 family. NO activates NtOSAK by promoting the phosphorylation of two serine residues. Taken together, these data highlight a role for NO as a Ca2+-mobilizing messenger in plant cells. On the other hands, we found that NO produced in response to cadmium (Cd) promotes Cd toxicity by favouring root growth inhibition and Cd assimilation. NO-dependent candidate genes that might contribute to this process have been identified. In summary, depending on the physiological context, NO acts as a signalling or a toxic molecule.
G. Noctor, (Université de Paris Sud)
doi:10.1016/j.cbpa.2007.01.654
1. Hidalgo, A., Garci´a-Herdugo, G., Gonza´lez-Reyes, J. A., Morre´, D. J. & Navas, P. (1991): Ascorbate free-radical stimulates onion root growth by increasing cell elongation. Bot. Gaz. 152, 282–288. 2. Green MA & Fry SC (2005) Vitamin C degradation in plant cells via enzymatic hydrolysis of 4-O-oxalyl-L-threonate. Nature 433, 83–87. doi:10.1016/j.cbpa.2007.01.652
Photorespiration is among the most important light-dependent sources of H2O2 in the leaves of C3 plants. Work in several species has shown that catalases are important in metabolizing photorespiratory H2O2 generated in the peroxisomes by the action of glycolate oxidase. We are using Arabidopsis T-DNA insertion mutants to examine the role of catalases in controlling leaf redox state and the functional consequences of decreased intracellular capacity to process H2O2. During growth in air, where photorespiration is rapid, the decreased capacity of catalase mutants to metabolize H2O2 causes severely decreased growth and, under some conditions, lesions on the leaves. These phenotypic effects are linked to oxidative stress as evidenced by profiling of marker transcripts and redox metabolites. In high CO2 growth conditions, however, where photorespiration is suppressed, the mutant has the same phenotype as wild type plants and no oxidative stress is apparent. Redox perturbation reappears when mutants grown at high CO2 are transferred to air. Thus, the knocked-out catalase has a specific role in processing H2O2 produced during photorespiration. This conditional mutant enables redox state to be readily and controllably perturbed by ambient CO2, and is being exploited to examine the roles of intracellular H2O2 availability in oxidative signalling, plant function and responses to the environment. doi:10.1016/j.cbpa.2007.01.653
P5.15 Mutation of the AtNOS1/AtNOR1 gene confers salt tolerance on Arabidopsis J. Price, R. Taylor, S. Ramsay, G. Kettles, P. Dominy, (University of Glasgow) A gain-of-function screen of the Arabidopsis activation tagged collections for mutants with improved tolerance of NaCl identified several lines. Further characterization showed that one of these lines carries a single T-DNA insertion in the last exon of At3g47450. At that time the sequence was reported to show low homology to a nitric oxide synthase from Helix pomatia; we have designated this lesion as AtNOS1–2. Subsequently, confirmation was provided that the gene does encode an authentic nitric oxide synthase (AtNOS1; Guo et al. 2005 Science 302:100–103), although recently this assertion has been questioned (Zemojtel et al. 2006 TIPS:524–525; Crawford et al. 2006 TIPS:526–527). We have now isolated homozygous lines carrying the AtNOS1–2 lesion and characterized the phenotype. Homozygous AtNOS1–2 lines produce a truncated AtNOS1 message that results in a stunted, chlorotic, fertile phenotype. Measurements with a NO-sensitive flurophor, and with NO-sensitive electrodes, have failed to detect any significant differences in the production of NO between wild type and homozygous
S260
Abstracts / Comparative Biochemistry and Physiology, Part A 146 (2007) S255 – S266
AtNOS1–2 lines, suggesting the phenotype of the latter does not arise from differences in endogenous NO concentration. Treatment of wild type seedlings with NO donor SNP does confers salt tolerance suggesting that plant responses to high salinity are in part mediated through a NO signalling network. The implications of these results will be discussed. doi:10.1016/j.cbpa.2007.01.655
P5.16 SUMO proteases regulate ROS production in Arabidopsis L. Conti, E. O Donnel, J. Price, A. Love, P. Dominy, A. Sadanandom, (University of Glasgow) Plants have developed multiple strategies to cope with biotic and abiotic stress. A major goal of plant biology is to find how these stimuli are recognised, what signals they trigger and how they are integrated. Covalent protein modification plays a major role in cell signalling. Small Ubiquitin-like protein Modifiers (SUMO) are attached to key protein targets to modulate their activity. SUMO proteases convert proSUMO in the active form and remove SUMO from protein targets, a process which requires SUMO protease–target protein interaction. In plants, SUMO is involved in multiple signalling including pathogen resistance. OBS1 is a nuclear localised SUMO protease from Arabidopsis. Loss-of-function obs1 plants show increased thermotolerance compared to wild type. Heat shock perception requires OBS1 SUMO protease activity, as the ectopic expression of a protease deficient version of OBS1 phenocopies obs1 mutants. In contrast, OBS2 (the closest OBS1 homologue) knockouts or obs2/obs1 double mutants are hyper heat-sensitive suggesting that OBS1 and OBS2 act in the same pathway, but have opposite roles. Salicylic acid (SA) has been linked to heat tolerance and OBS1 and OBS2 are SA inducible. When sprayed with SA, obs1 plants produce a reduced oxidative burst compared to wild type, while obs2 and obs1/obs2 double mutants show an enhanced oxidative burst. These results suggest a molecular model whereby OBS1 modulates OBS2 activity to trigger the oxidative burst. Given the key role of the oxidative burst in plant stress responses, our data indicate that OBS1/OBS2 may play a key role in biotic and abiotic signalling. doi:10.1016/j.cbpa.2007.01.656
P5.17 Spatial dependence for H2O2-directed signalling in Arabidopsis leaves P. Mullineaux, N. Baker, U. Bechtold, M. Fryer, (University of Essex); W. Davies, (Lancaster University)
H2O2 from the chloroplast initiates intracellular and systemic signalling inducing protective and acclimatory mechanisms to environmental stress. H2O2 has been suggested to diffuse from the chloroplast to initiate this signalling. However, this model does not explain how H2O2 can diffuse to other subcellular compartments when the same stresses trigger an enhancement of antioxidant capacity in the cytosol. Therefore, we have proposed a hypothesis that argues for a spatial component for H2O2 signalling. In this hypothesis, a H2O2 signal is transduced in the chloroplast to a non-reactive oxygen species factor which exits the chloroplast, traverses the reducing environment of the cytosol to the plasma-membrane where a second extracellular production of H2O2 occurs. The increased antioxidant capacity of the cytosol confines H2O2 to the extracellular and chloroplast compartments. We are testing this hypothesis using high light-stressed Arabidopsis leaves which accumulate H2O2 in peri-veinal bundle sheath cells. These cells specifically express the APX2 gene which requires active photosynthetic electron transport, chloroplast-sourced H2O2 and a systemic induction of APX2 expression which requires extracellular H2O2. We propose that an abscisic acid (ABA) mediated signal exits the chloroplast stimulated by increased H2O2 production in the chloroplast. Results from transgenic lines and mutants altered in APX2 expression will be presented that support the role of ABA as a signal from the chloroplast, supply evidence for a spatial separation of increased antioxidant pools and H2O2 and how this hypothesis may be extended to plants' responses to biotic and abiotic stresses. doi:10.1016/j.cbpa.2007.01.657
P5.18 Regulation of Cytochrome b6f complex in response to NADP(H)-redox poise S. Hald, P. Gallois, G. Johnson, (University of Manchester) When plants experience an imbalance between the absorption of light energy and the use of that energy to drive metabolism, they are liable to suffer from oxidative stress. Such imbalances arise due to environmental conditions (e.g. heat, chilling or drought) and can result in the production of reactive oxygen species (ROS). Various mechanisms have been characterised which help to limit ROS production, including non-photochemical quenching and the accumulation of antioxidants. Here we demonstrate a novel protective process – feedback redox regulation via the NADP+/NADPH pool – and demonstrate that this is vital in avoiding oxidative stress. Photosynthetic electron transport was studied in two transgenic tobacco (Nicotiana tabacum) lines — one having reduced levels of ferredoxin NADP+-reductase (FNR), the enzyme responsible for reducing NADP+, and the other reduced levels of glyceraldehyde 3phosphate dehydrogenase (GAPDH), the principle consumer of
suggest that ascorbate degradation also increases during elongation growth. The degradation of ascorbate to oxalate/Lthreonate operates extra-cellularly in cultured Rosa cells2. The study reported here compares the kinetics of the changes in cellular ascorbate, glutathione and sugars through the phases of cell division and cell expansion, relating the observed changes in antioxidant pools sizes to the relative effects on ascorbate and glutathione synthesis and degradation during the different phases of cell development in culture. In addition, we have also followed the changes in the activity of poly (ADP-ribose) polymerase (PARP) that occur through the periods of mitosis and cell expansion.
S259
P5.14 Role of nitric oxide in plants challenged by elicitors or cadmium: another paradigm for double-edged role of nitric oxide D. Wendehenne, A. Pugin, (CNRS/Université de Bourgogne); G. Dobrowolska, (Institute of Biochemistry and Biophysics, Warsaw)
P5.13 Functional analysis of H2O2 signalling in photorespiratory Arabidopsis mutants deficient in catalase
We previously reported that nitric oxide (NO) was rapidly produced in plant cells challenged by elicitors of defence responses and hyper-osmotic stresses. NO synthesis is required as a step in the pathways leading to the rise in cytosolic concentration ([Ca 2+ ]cyt). NO enhances [Ca 2+ ]cyt by promoting phosphorylation-dependent events. Accordingly, NO released by a NO donor or produced endogenously in response to hyperosmotic stresses and elicitors was shown to induce the activation of the protein kinase NtOSAK which belongs to the SnRK2 family. NO activates NtOSAK by promoting the phosphorylation of two serine residues. Taken together, these data highlight a role for NO as a Ca2+-mobilizing messenger in plant cells. On the other hands, we found that NO produced in response to cadmium (Cd) promotes Cd toxicity by favouring root growth inhibition and Cd assimilation. NO-dependent candidate genes that might contribute to this process have been identified. In summary, depending on the physiological context, NO acts as a signalling or a toxic molecule.
G. Noctor, (Université de Paris Sud)
doi:10.1016/j.cbpa.2007.01.654
1. Hidalgo, A., Garci´a-Herdugo, G., Gonza´lez-Reyes, J. A., Morre´, D. J. & Navas, P. (1991): Ascorbate free-radical stimulates onion root growth by increasing cell elongation. Bot. Gaz. 152, 282–288. 2. Green MA & Fry SC (2005) Vitamin C degradation in plant cells via enzymatic hydrolysis of 4-O-oxalyl-L-threonate. Nature 433, 83–87. doi:10.1016/j.cbpa.2007.01.652
Photorespiration is among the most important light-dependent sources of H2O2 in the leaves of C3 plants. Work in several species has shown that catalases are important in metabolizing photorespiratory H2O2 generated in the peroxisomes by the action of glycolate oxidase. We are using Arabidopsis T-DNA insertion mutants to examine the role of catalases in controlling leaf redox state and the functional consequences of decreased intracellular capacity to process H2O2. During growth in air, where photorespiration is rapid, the decreased capacity of catalase mutants to metabolize H2O2 causes severely decreased growth and, under some conditions, lesions on the leaves. These phenotypic effects are linked to oxidative stress as evidenced by profiling of marker transcripts and redox metabolites. In high CO2 growth conditions, however, where photorespiration is suppressed, the mutant has the same phenotype as wild type plants and no oxidative stress is apparent. Redox perturbation reappears when mutants grown at high CO2 are transferred to air. Thus, the knocked-out catalase has a specific role in processing H2O2 produced during photorespiration. This conditional mutant enables redox state to be readily and controllably perturbed by ambient CO2, and is being exploited to examine the roles of intracellular H2O2 availability in oxidative signalling, plant function and responses to the environment. doi:10.1016/j.cbpa.2007.01.653
P5.15 Mutation of the AtNOS1/AtNOR1 gene confers salt tolerance on Arabidopsis J. Price, R. Taylor, S. Ramsay, G. Kettles, P. Dominy, (University of Glasgow) A gain-of-function screen of the Arabidopsis activation tagged collections for mutants with improved tolerance of NaCl identified several lines. Further characterization showed that one of these lines carries a single T-DNA insertion in the last exon of At3g47450. At that time the sequence was reported to show low homology to a nitric oxide synthase from Helix pomatia; we have designated this lesion as AtNOS1–2. Subsequently, confirmation was provided that the gene does encode an authentic nitric oxide synthase (AtNOS1; Guo et al. 2005 Science 302:100–103), although recently this assertion has been questioned (Zemojtel et al. 2006 TIPS:524–525; Crawford et al. 2006 TIPS:526–527). We have now isolated homozygous lines carrying the AtNOS1–2 lesion and characterized the phenotype. Homozygous AtNOS1–2 lines produce a truncated AtNOS1 message that results in a stunted, chlorotic, fertile phenotype. Measurements with a NO-sensitive flurophor, and with NO-sensitive electrodes, have failed to detect any significant differences in the production of NO between wild type and homozygous
S260
Abstracts / Comparative Biochemistry and Physiology, Part A 146 (2007) S255 – S266
AtNOS1–2 lines, suggesting the phenotype of the latter does not arise from differences in endogenous NO concentration. Treatment of wild type seedlings with NO donor SNP does confers salt tolerance suggesting that plant responses to high salinity are in part mediated through a NO signalling network. The implications of these results will be discussed. doi:10.1016/j.cbpa.2007.01.655
P5.16 SUMO proteases regulate ROS production in Arabidopsis L. Conti, E. O Donnel, J. Price, A. Love, P. Dominy, A. Sadanandom, (University of Glasgow) Plants have developed multiple strategies to cope with biotic and abiotic stress. A major goal of plant biology is to find how these stimuli are recognised, what signals they trigger and how they are integrated. Covalent protein modification plays a major role in cell signalling. Small Ubiquitin-like protein Modifiers (SUMO) are attached to key protein targets to modulate their activity. SUMO proteases convert proSUMO in the active form and remove SUMO from protein targets, a process which requires SUMO protease–target protein interaction. In plants, SUMO is involved in multiple signalling including pathogen resistance. OBS1 is a nuclear localised SUMO protease from Arabidopsis. Loss-of-function obs1 plants show increased thermotolerance compared to wild type. Heat shock perception requires OBS1 SUMO protease activity, as the ectopic expression of a protease deficient version of OBS1 phenocopies obs1 mutants. In contrast, OBS2 (the closest OBS1 homologue) knockouts or obs2/obs1 double mutants are hyper heat-sensitive suggesting that OBS1 and OBS2 act in the same pathway, but have opposite roles. Salicylic acid (SA) has been linked to heat tolerance and OBS1 and OBS2 are SA inducible. When sprayed with SA, obs1 plants produce a reduced oxidative burst compared to wild type, while obs2 and obs1/obs2 double mutants show an enhanced oxidative burst. These results suggest a molecular model whereby OBS1 modulates OBS2 activity to trigger the oxidative burst. Given the key role of the oxidative burst in plant stress responses, our data indicate that OBS1/OBS2 may play a key role in biotic and abiotic signalling. doi:10.1016/j.cbpa.2007.01.656
P5.17 Spatial dependence for H2O2-directed signalling in Arabidopsis leaves P. Mullineaux, N. Baker, U. Bechtold, M. Fryer, (University of Essex); W. Davies, (Lancaster University)
H2O2 from the chloroplast initiates intracellular and systemic signalling inducing protective and acclimatory mechanisms to environmental stress. H2O2 has been suggested to diffuse from the chloroplast to initiate this signalling. However, this model does not explain how H2O2 can diffuse to other subcellular compartments when the same stresses trigger an enhancement of antioxidant capacity in the cytosol. Therefore, we have proposed a hypothesis that argues for a spatial component for H2O2 signalling. In this hypothesis, a H2O2 signal is transduced in the chloroplast to a non-reactive oxygen species factor which exits the chloroplast, traverses the reducing environment of the cytosol to the plasma-membrane where a second extracellular production of H2O2 occurs. The increased antioxidant capacity of the cytosol confines H2O2 to the extracellular and chloroplast compartments. We are testing this hypothesis using high light-stressed Arabidopsis leaves which accumulate H2O2 in peri-veinal bundle sheath cells. These cells specifically express the APX2 gene which requires active photosynthetic electron transport, chloroplast-sourced H2O2 and a systemic induction of APX2 expression which requires extracellular H2O2. We propose that an abscisic acid (ABA) mediated signal exits the chloroplast stimulated by increased H2O2 production in the chloroplast. Results from transgenic lines and mutants altered in APX2 expression will be presented that support the role of ABA as a signal from the chloroplast, supply evidence for a spatial separation of increased antioxidant pools and H2O2 and how this hypothesis may be extended to plants' responses to biotic and abiotic stresses. doi:10.1016/j.cbpa.2007.01.657
P5.18 Regulation of Cytochrome b6f complex in response to NADP(H)-redox poise S. Hald, P. Gallois, G. Johnson, (University of Manchester) When plants experience an imbalance between the absorption of light energy and the use of that energy to drive metabolism, they are liable to suffer from oxidative stress. Such imbalances arise due to environmental conditions (e.g. heat, chilling or drought) and can result in the production of reactive oxygen species (ROS). Various mechanisms have been characterised which help to limit ROS production, including non-photochemical quenching and the accumulation of antioxidants. Here we demonstrate a novel protective process – feedback redox regulation via the NADP+/NADPH pool – and demonstrate that this is vital in avoiding oxidative stress. Photosynthetic electron transport was studied in two transgenic tobacco (Nicotiana tabacum) lines — one having reduced levels of ferredoxin NADP+-reductase (FNR), the enzyme responsible for reducing NADP+, and the other reduced levels of glyceraldehyde 3phosphate dehydrogenase (GAPDH), the principle consumer of