- Email: [email protected]
Coordinated decreases in cellular ascorbate and glutathione during the growth of Arabidopsis cell cultures
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Abstracts / Comparative Biochemistry and Physiology, Part A 146 (2007) S255 – S266
P5.9 Aquaporin gating in maize roots has large impacts on integrated physiological processes C. Ehlert, F. Tardieu, T. Simonneau, (INRA-Montpellier) The adjustment of water transport is fundamental to plant adaptation to many challenging environmental conditions. The discovery of aquaporins as membrane channels facilitating water transfer and recent insights into their gating properties have opened new avenues for elucidating regulation mechanisms at a molecular level. But their significance for whole plant adaptation remains poorly documented. This work aims to integrate advances in molecular biology of aquaporin gating with whole plant hydraulics and hydraulically controlled processes. Particularly, the significance of aquaporin gating in roots for stomatal functioning and growth of leaves was investigated. We induced aquaporin gating in maize roots by acid load, H2O2 and anoxia. Under all these conditions, aquaporin gating decreased cell turgor in the leaf elongation zone and dramatically reduced leaf growth. However these effects could only be observed when the plant transpired substantially attributing a critical role for aquaporins under high evaporative demand. Negligible effects of aquaporin gating on transpiration were observed indicating that aquaporin gating did not induce stomatal closure. Overall, we show that aquaporin gating modifies the hydraulic architecture of the whole plant with consequences for plant growth but not for transpiration. Turgor measurements demonstrate that leaf growth responses to aquaporin gating in roots are hydraulically controlled. Remarkably, plant growth responses to anoxia are also mediated by changes in cell turgor suggesting a common hydraulic signalling mechanism. We demonstrate that conditions affecting aquaporin activity in plant roots are determinant for whole plant tolerance to environmental stresses. doi:10.1016/j.cbpa.2007.01.649
P5.10 The interplay between redox and hormone signalling in Arabidopsis guard cells R. Desikan, (Imperial College London); J. Hancock, S. Neill, (University of the West of England, Bristol); R. Hooley, (University of Bath); K. Harter, (University of Tubingen) There is now much evidence that the reactive oxygen species (ROS) hydrogen peroxide (H2O2) plays an important role in a number of physiological responses, including stomatal closure. H2O2 is generated in stomatal guard cells in response to drought stress, increased concentrations of abscisic acid (ABA), ozone and also in response to elicitor/pathogen challenge. Generation of H2O2 occurs via the AtrbohD/F subunits of the NADPH oxidase complex. In recent work we have shown that the plant hormone ethylene, known to influence numerous processes such as fruit ripening, senescence, defence responses and seedling growth, also mediates stomatal closure in intact Arabidopsis
leaves. Using a combination of genetics and physiological tools, we have demonstrated that ethylene-induced stomatal closure is dependent on H2O2 synthesis via AtrbohF. Guard cells of the ethylene receptor mutants etr1–1 and etr1–3 are insensitive to ethylene, but not to ABA. Moreover stomata of the ethylene signalling ein2-1 and arr2 mutants do not close in response to ethylene, but do generate H2O2 following ethylene treatment. In addition, we have also observed that whilst ethylene alone can cause closure, ethylene in the presence of ABA does not cause stomatal closure in leaves. These data demonstrate that plant hormones mediate their effects in guard cells via complex interplay with redox signalling pathways. doi:10.1016/j.cbpa.2007.01.650
P5.11 Relationships between mitochondrial respiratory pathways, redox metabolism, nitrate sensing and regulation of plant architecture in Nicotiana sylvestris C. Foyer, (University of Newcastle) Recent studies suggest that redox status is an important modulator of carbon/nitrogen interactions and related signalling pathways in plants. We have analyzed this question by studying the N. sylvestris CMSII mutant, which lacks mitochondrial complex I and which respires through alternative pathways involving increases in NAD pools. This presentation will consider how these redox adjustments affect C/N metabolism and associated signalling. Transcript profiling of CMSII and WT leaves grown at low or high N availability provided little evidence for large pleiotropic effects of the loss of complex I on the CMSII transcriptome, while metabolite profiling identified marked increases in amino acids in CMSII, revealing that the mutant is enriched in N compounds. The consequences of this N-rich phenotype for plant architecture and resource allocation were examined by analysis of lateral root development and shoot/root ratios in plants grown at different conditions of external carbon and nitrogen availability. Changes in root and shoot N compounds in mutant and WT grown in the different conditions provide evidence that a root nitrate/amine ratio below 1 is characteristic of a high N signature that drives shoot biomass production and high shoot/root ratios. Measurements of gibberellic acid (GA) and abscisic acid status suggest that enhanced GA plays a part in signalling mechanisms that link N enrichment to shoot investment. doi:10.1016/j.cbpa.2007.01.651
P5.12 Coordinated decreases in cellular ascorbate and glutathione during the growth of Arabidopsis cell cultures V. Locato, T. Pellny, C. Foyer, (Rothamsted Research); L. De Gara, (University of Bari)
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
Abstracts / Comparative Biochemistry and Physiology, Part A 146 (2007) S255 – S266
P5.9 Aquaporin gating in maize roots has large impacts on integrated physiological processes C. Ehlert, F. Tardieu, T. Simonneau, (INRA-Montpellier) The adjustment of water transport is fundamental to plant adaptation to many challenging environmental conditions. The discovery of aquaporins as membrane channels facilitating water transfer and recent insights into their gating properties have opened new avenues for elucidating regulation mechanisms at a molecular level. But their significance for whole plant adaptation remains poorly documented. This work aims to integrate advances in molecular biology of aquaporin gating with whole plant hydraulics and hydraulically controlled processes. Particularly, the significance of aquaporin gating in roots for stomatal functioning and growth of leaves was investigated. We induced aquaporin gating in maize roots by acid load, H2O2 and anoxia. Under all these conditions, aquaporin gating decreased cell turgor in the leaf elongation zone and dramatically reduced leaf growth. However these effects could only be observed when the plant transpired substantially attributing a critical role for aquaporins under high evaporative demand. Negligible effects of aquaporin gating on transpiration were observed indicating that aquaporin gating did not induce stomatal closure. Overall, we show that aquaporin gating modifies the hydraulic architecture of the whole plant with consequences for plant growth but not for transpiration. Turgor measurements demonstrate that leaf growth responses to aquaporin gating in roots are hydraulically controlled. Remarkably, plant growth responses to anoxia are also mediated by changes in cell turgor suggesting a common hydraulic signalling mechanism. We demonstrate that conditions affecting aquaporin activity in plant roots are determinant for whole plant tolerance to environmental stresses. doi:10.1016/j.cbpa.2007.01.649
P5.10 The interplay between redox and hormone signalling in Arabidopsis guard cells R. Desikan, (Imperial College London); J. Hancock, S. Neill, (University of the West of England, Bristol); R. Hooley, (University of Bath); K. Harter, (University of Tubingen) There is now much evidence that the reactive oxygen species (ROS) hydrogen peroxide (H2O2) plays an important role in a number of physiological responses, including stomatal closure. H2O2 is generated in stomatal guard cells in response to drought stress, increased concentrations of abscisic acid (ABA), ozone and also in response to elicitor/pathogen challenge. Generation of H2O2 occurs via the AtrbohD/F subunits of the NADPH oxidase complex. In recent work we have shown that the plant hormone ethylene, known to influence numerous processes such as fruit ripening, senescence, defence responses and seedling growth, also mediates stomatal closure in intact Arabidopsis
leaves. Using a combination of genetics and physiological tools, we have demonstrated that ethylene-induced stomatal closure is dependent on H2O2 synthesis via AtrbohF. Guard cells of the ethylene receptor mutants etr1–1 and etr1–3 are insensitive to ethylene, but not to ABA. Moreover stomata of the ethylene signalling ein2-1 and arr2 mutants do not close in response to ethylene, but do generate H2O2 following ethylene treatment. In addition, we have also observed that whilst ethylene alone can cause closure, ethylene in the presence of ABA does not cause stomatal closure in leaves. These data demonstrate that plant hormones mediate their effects in guard cells via complex interplay with redox signalling pathways. doi:10.1016/j.cbpa.2007.01.650
P5.11 Relationships between mitochondrial respiratory pathways, redox metabolism, nitrate sensing and regulation of plant architecture in Nicotiana sylvestris C. Foyer, (University of Newcastle) Recent studies suggest that redox status is an important modulator of carbon/nitrogen interactions and related signalling pathways in plants. We have analyzed this question by studying the N. sylvestris CMSII mutant, which lacks mitochondrial complex I and which respires through alternative pathways involving increases in NAD pools. This presentation will consider how these redox adjustments affect C/N metabolism and associated signalling. Transcript profiling of CMSII and WT leaves grown at low or high N availability provided little evidence for large pleiotropic effects of the loss of complex I on the CMSII transcriptome, while metabolite profiling identified marked increases in amino acids in CMSII, revealing that the mutant is enriched in N compounds. The consequences of this N-rich phenotype for plant architecture and resource allocation were examined by analysis of lateral root development and shoot/root ratios in plants grown at different conditions of external carbon and nitrogen availability. Changes in root and shoot N compounds in mutant and WT grown in the different conditions provide evidence that a root nitrate/amine ratio below 1 is characteristic of a high N signature that drives shoot biomass production and high shoot/root ratios. Measurements of gibberellic acid (GA) and abscisic acid status suggest that enhanced GA plays a part in signalling mechanisms that link N enrichment to shoot investment. doi:10.1016/j.cbpa.2007.01.651
P5.12 Coordinated decreases in cellular ascorbate and glutathione during the growth of Arabidopsis cell cultures V. Locato, T. Pellny, C. Foyer, (Rothamsted Research); L. De Gara, (University of Bari)
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