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• Huang, S., Shen, L., Roelfsema, M.R.G., Becker, D., and Hedrich, R. (2023) ‘Light-gated channelrhodopsin sparks proton-induced calcium release in guard cells’, Science, 382(6676), 1314–1318, available: https://doi.org/10.1126/science.adj9696.
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  @article{Huang_2023, author = {Huang, Shouguang and Shen, Like and Roelfsema, M. Rob G. and Becker, Dirk and Hedrich, Rainer}, journal = {Science}, keywords = {ca2+-signals}, month = 12, number = 6676, pages = {1314–1318}, publisher = {American Association for the Advancement of Science (AAAS)}, title = {Light-gated channelrhodopsin sparks proton-induced calcium release in guard cells}, volume = 382, year = 2023 }

  %0 Journal Article %1 Huang_2023 %A Huang, Shouguang %A Shen, Like %A Roelfsema, M. Rob G. %A Becker, Dirk %A Hedrich, Rainer %D 2023 %I American Association for the Advancement of Science (AAAS) %J Science %N 6676 %P 1314–1318 %R 10.1126/science.adj9696 %T Light-gated channelrhodopsin sparks proton-induced calcium release in guard cells %U http://dx.doi.org/10.1126/science.adj9696 %V 382
• Hedrich, R. and Roelfsema, M. (2023) ‘Cracking the code of plant herbivore defense’, Cell, 186, 1300–1302, available: https://doi.org/10.1016/j.cell.2023.02.025.
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  In 1916, Ricca hypothesized that plant defense mediators are transported by xylem vessels. While it was discovered that electrical waves generated at plant wounds also transmit information over great distances, the molecular nature of the so-called Ricca factor remained unclear. In this issue of Cell, Gao et al. identify thioglucoside glucohydrolases as a Ricca factor in Arabidopsis.

  @article{hedrich2023cracking, abstract = {In 1916, Ricca hypothesized that plant defense mediators are transported by xylem vessels. While it was discovered that electrical waves generated at plant wounds also transmit information over great distances, the molecular nature of the so-called Ricca factor remained unclear. In this issue of Cell, Gao et al. identify thioglucoside glucohydrolases as a Ricca factor in Arabidopsis.}, author = {Hedrich, Rainer and Roelfsema, M.R.G.}, journal = {Cell}, keywords = {wounding}, pages = {1300-1302}, title = {Cracking the code of plant herbivore defense}, volume = 186, year = 2023 }

  %0 Journal Article %1 hedrich2023cracking %A Hedrich, Rainer %A Roelfsema, M.R.G. %D 2023 %J Cell %P 1300-1302 %R 10.1016/j.cell.2023.02.025 %T Cracking the code of plant herbivore defense %U https://www.sciencedirect.com/science/article/abs/pii/S009286742300168X?via%3Dihub %V 186 %X In 1916, Ricca hypothesized that plant defense mediators are transported by xylem vessels. While it was discovered that electrical waves generated at plant wounds also transmit information over great distances, the molecular nature of the so-called Ricca factor remained unclear. In this issue of Cell, Gao et al. identify thioglucoside glucohydrolases as a Ricca factor in Arabidopsis.
• Huang, S., Maierhofer, T., Hashimoto, K., Xu, X., Karimi, S.M., Müller, H., Geringer, M.A., Wang, Y., Kudla, J., Smet, I.D., Hedrich, R., Geiger, D., and Roelfsema, M.R.G. (2023) ‘The CIPK23 protein kinase represses SLAC1-type anion channels in Arabidopsis guard cells and stimulates stomatal opening’, New Phytologist, 238(1), 270–282, available: https://doi.org/10.1111/nph.18708.
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  @article{Huang_2023, author = {Huang, Shouguang and Maierhofer, Tobias and Hashimoto, Kenji and Xu, Xiangyu and Karimi, Sohail M. and Müller, Heike and Geringer, Michael A. and Wang, Yi and Kudla, Jörg and Smet, Ive De and Hedrich, Rainer and Geiger, Dietmar and Roelfsema, M. Rob G.}, journal = {New Phytologist}, keywords = {anion-channel}, month = {01}, number = 1, pages = {270–282}, publisher = {Wiley}, title = {The CIPK23 protein kinase represses SLAC1-type anion channels in Arabidopsis guard cells and stimulates stomatal opening}, volume = 238, year = 2023 }

  %0 Journal Article %1 Huang_2023 %A Huang, Shouguang %A Maierhofer, Tobias %A Hashimoto, Kenji %A Xu, Xiangyu %A Karimi, Sohail M. %A Müller, Heike %A Geringer, Michael A. %A Wang, Yi %A Kudla, Jörg %A Smet, Ive De %A Hedrich, Rainer %A Geiger, Dietmar %A Roelfsema, M. Rob G. %D 2023 %I Wiley %J New Phytologist %N 1 %P 270--282 %R 10.1111/nph.18708 %T The CIPK23 protein kinase represses SLAC1-type anion channels in Arabidopsis guard cells and stimulates stomatal opening %U https://doi.org/10.1111%2Fnph.18708 %V 238

• Jones, J.J., Huang, S., Hedrich, R., Geilfus, C.-M., and Roelfsema, M.R.G. (2022) ‘The green light gap: a window of opportunity for optogenetic control of stomatal movement’, New Phytologist, 236(4), 1237–1244, available: https://doi.org/10.1111/nph.18451.
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  @article{Jones_2022, author = {Jones, Jeffrey J. and Huang, Shouguang and Hedrich, Rainer and Geilfus, Christoph-Martin and Roelfsema, M. Rob G.}, journal = {New Phytologist}, keywords = {optogenetics}, number = 4, pages = {1237-1244}, publisher = {Wiley}, title = {The green light gap: a window of opportunity for optogenetic control of stomatal movement}, volume = 236, year = 2022 }

  %0 Journal Article %1 Jones_2022 %A Jones, Jeffrey J. %A Huang, Shouguang %A Hedrich, Rainer %A Geilfus, Christoph-Martin %A Roelfsema, M. Rob G. %D 2022 %I Wiley %J New Phytologist %N 4 %P 1237-1244 %R 10.1111/nph.18451 %T The green light gap: a window of opportunity for optogenetic control of stomatal movement %U https://doi.org/10.1111%2Fnph.18451 %V 236

• Nuhkat, M., Brosche, M., Stoelzle-Feix, S., Dietrich, P., Hedrich, R., Roelfsema, M.R.G., and Kollist, H. (2021) ‘Rapid depolarization and cytosolic calcium increase go hand-in-hand in mesophyll cells’ ozone response’, New Phytologist, 232(4), 1692–1702, available: https://doi.org/10.1111/nph.17711.
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  Plant stress signalling involves bursts of reactive oxygen species (ROS), which can be mimicked by the application of acute pulses of ozone. Such ozone-pulses inhibit photosynthesis and trigger stomatal closure in a few minutes, but the signalling that underlies these responses remains largely unknown. We measured changes in Arabidopsis thaliana gas exchange after treatment with acute pulses of ozone and set up a system for simultaneous measurement of membrane potential and cytosolic calcium with the fluorescent reporter R-GECO1. We show that within 1 min, prior to stomatal closure, O-3 triggered a drop in whole-plant CO2 uptake. Within this early phase, O-3 pulses (200-1000 ppb) elicited simultaneous membrane depolarization and cytosolic calcium increase, whereas these pulses had no long-term effect on either stomatal conductance or photosynthesis. In contrast, pulses of 5000 ppb O-3 induced cell death, systemic Ca2+ signals and an irreversible drop in stomatal conductance and photosynthetic capacity. We conclude that mesophyll cells respond to ozone in a few seconds by distinct pattern of plasma membrane depolarizations accompanied by an increase in the cytosolic calcium ion (Ca2+) level. These responses became systemic only at very high ozone concentrations. Thus, plants have rapid mechanism to sense and discriminate the strength of ozone signals.

  @article{Nuhkat_2021, abstract = {Plant stress signalling involves bursts of reactive oxygen species (ROS), which can be mimicked by the application of acute pulses of ozone. Such ozone-pulses inhibit photosynthesis and trigger stomatal closure in a few minutes, but the signalling that underlies these responses remains largely unknown. We measured changes in Arabidopsis thaliana gas exchange after treatment with acute pulses of ozone and set up a system for simultaneous measurement of membrane potential and cytosolic calcium with the fluorescent reporter R-GECO1. We show that within 1 min, prior to stomatal closure, O-3 triggered a drop in whole-plant CO2 uptake. Within this early phase, O-3 pulses (200-1000 ppb) elicited simultaneous membrane depolarization and cytosolic calcium increase, whereas these pulses had no long-term effect on either stomatal conductance or photosynthesis. In contrast, pulses of 5000 ppb O-3 induced cell death, systemic Ca2+ signals and an irreversible drop in stomatal conductance and photosynthetic capacity. We conclude that mesophyll cells respond to ozone in a few seconds by distinct pattern of plasma membrane depolarizations accompanied by an increase in the cytosolic calcium ion (Ca2+) level. These responses became systemic only at very high ozone concentrations. Thus, plants have rapid mechanism to sense and discriminate the strength of ozone signals.}, author = {Nuhkat, Maris and Brosche, Mikael and Stoelzle-Feix, Sonja and Dietrich, Petra and Hedrich, Rainer and Roelfsema, M. Rob G. and Kollist, Hannes}, journal = {New Phytologist}, keywords = {ca2+-signals}, month = {09}, number = 4, pages = {1692–1702}, publisher = {Wiley}, title = {Rapid depolarization and cytosolic calcium increase go hand-in-hand in mesophyll cells' ozone response}, volume = 232, year = 2021 }

  %0 Journal Article %1 Nuhkat_2021 %A Nuhkat, Maris %A Brosche, Mikael %A Stoelzle-Feix, Sonja %A Dietrich, Petra %A Hedrich, Rainer %A Roelfsema, M. Rob G. %A Kollist, Hannes %D 2021 %I Wiley %J New Phytologist %N 4 %P 1692--1702 %R 10.1111/nph.17711 %T Rapid depolarization and cytosolic calcium increase go hand-in-hand in mesophyll cells' ozone response %U https://doi.org/10.1111%2Fnph.17711 %V 232 %X Plant stress signalling involves bursts of reactive oxygen species (ROS), which can be mimicked by the application of acute pulses of ozone. Such ozone-pulses inhibit photosynthesis and trigger stomatal closure in a few minutes, but the signalling that underlies these responses remains largely unknown. We measured changes in Arabidopsis thaliana gas exchange after treatment with acute pulses of ozone and set up a system for simultaneous measurement of membrane potential and cytosolic calcium with the fluorescent reporter R-GECO1. We show that within 1 min, prior to stomatal closure, O-3 triggered a drop in whole-plant CO2 uptake. Within this early phase, O-3 pulses (200-1000 ppb) elicited simultaneous membrane depolarization and cytosolic calcium increase, whereas these pulses had no long-term effect on either stomatal conductance or photosynthesis. In contrast, pulses of 5000 ppb O-3 induced cell death, systemic Ca2+ signals and an irreversible drop in stomatal conductance and photosynthetic capacity. We conclude that mesophyll cells respond to ozone in a few seconds by distinct pattern of plasma membrane depolarizations accompanied by an increase in the cytosolic calcium ion (Ca2+) level. These responses became systemic only at very high ozone concentrations. Thus, plants have rapid mechanism to sense and discriminate the strength of ozone signals.
• Huang, S., Ding, M., Roelfsema, M.R.G., Dreyer, I., Scherzer, S., Al-Rasheid, K.A.S., Gao, S., Nagel, G., Hedrich, R., and Konrad, K.R. (2021) ‘Optogenetic control of the guard cell membrane potential and stomatal movement by the light-gated anion channel Gt {ACR}1’, Science Advances, 7(28), available: https://doi.org/10.1126/sciadv.abg4619.
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  Guard cells control the aperture of plant stomata, which are crucial for global fluxes of CO2 and water. In turn, guard cell anion channels are seen as key players for stomatal closure, but is activation of these channels sufficient to limit plant water loss? To answer this open question, we used an optogenetic approach based on the light-gated anion channelrhodopsin 1 (GtACR1). In tobacco guard cells that express GtACR1, blue- and green-light pulses elicit Cl- and NO3- currents of -1 to -2 nA. The anion currents depolarize the plasma membrane by 60 to 80 mV, which causes opening of voltage-gated K+ channels and the extrusion of K+. As a result, continuous stimulation with green light leads to loss of guard cell turgor and closure of stomata at conditions that provoke stomatal opening in wild type. GtACR1 optogenetics thus provides unequivocal evidence that opening of anion channels is sufficient to close stomata.

  @article{Huang_2021, abstract = {Guard cells control the aperture of plant stomata, which are crucial for global fluxes of CO2 and water. In turn, guard cell anion channels are seen as key players for stomatal closure, but is activation of these channels sufficient to limit plant water loss? To answer this open question, we used an optogenetic approach based on the light-gated anion channelrhodopsin 1 (GtACR1). In tobacco guard cells that express GtACR1, blue- and green-light pulses elicit Cl- and NO3- currents of -1 to -2 nA. The anion currents depolarize the plasma membrane by 60 to 80 mV, which causes opening of voltage-gated K+ channels and the extrusion of K+. As a result, continuous stimulation with green light leads to loss of guard cell turgor and closure of stomata at conditions that provoke stomatal opening in wild type. GtACR1 optogenetics thus provides unequivocal evidence that opening of anion channels is sufficient to close stomata.}, author = {Huang, Shouguang and Ding, Meiqi and Roelfsema, M. Rob G. and Dreyer, Ingo and Scherzer, Sönke and Al-Rasheid, Khaled A. S. and Gao, Shiqiang and Nagel, Georg and Hedrich, Rainer and Konrad, Kai R.}, journal = {Science Advances}, keywords = {Nicotiana}, month = {07}, number = 28, publisher = {American Association for the Advancement of Science ({AAAS})}, title = {Optogenetic control of the guard cell membrane potential and stomatal movement by the light-gated anion channel Gt {ACR}1}, volume = 7, year = 2021 }

  %0 Journal Article %1 Huang_2021 %A Huang, Shouguang %A Ding, Meiqi %A Roelfsema, M. Rob G. %A Dreyer, Ingo %A Scherzer, Sönke %A Al-Rasheid, Khaled A. S. %A Gao, Shiqiang %A Nagel, Georg %A Hedrich, Rainer %A Konrad, Kai R. %D 2021 %I American Association for the Advancement of Science ({AAAS}) %J Science Advances %N 28 %R 10.1126/sciadv.abg4619 %T Optogenetic control of the guard cell membrane potential and stomatal movement by the light-gated anion channel Gt {ACR}1 %U https://doi.org/10.1126%2Fsciadv.abg4619 %V 7 %X Guard cells control the aperture of plant stomata, which are crucial for global fluxes of CO2 and water. In turn, guard cell anion channels are seen as key players for stomatal closure, but is activation of these channels sufficient to limit plant water loss? To answer this open question, we used an optogenetic approach based on the light-gated anion channelrhodopsin 1 (GtACR1). In tobacco guard cells that express GtACR1, blue- and green-light pulses elicit Cl- and NO3- currents of -1 to -2 nA. The anion currents depolarize the plasma membrane by 60 to 80 mV, which causes opening of voltage-gated K+ channels and the extrusion of K+. As a result, continuous stimulation with green light leads to loss of guard cell turgor and closure of stomata at conditions that provoke stomatal opening in wild type. GtACR1 optogenetics thus provides unequivocal evidence that opening of anion channels is sufficient to close stomata.
• Dindas, J., Dreyer, I., Huang, S., Hedrich, R., and Roelfsema, M.R.G. (2021) ‘A voltage-dependent Ca2+-homeostat operates in the plant vacuolar membrane’, New Phytologist, 230(4), 1449–1460, available: https://doi.org/10.1111/nph.17272.
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  @article{Dindas_2021, author = {Dindas, Julian and Dreyer, Ingo and Huang, Shouguang and Hedrich, Rainer and Roelfsema, M. Rob G.}, journal = {New Phytologist}, keywords = {cell}, month = {03}, number = 4, pages = {1449–1460}, publisher = {Wiley}, title = {A voltage-dependent Ca2+-homeostat operates in the plant vacuolar membrane}, volume = 230, year = 2021 }

  %0 Journal Article %1 Dindas_2021 %A Dindas, Julian %A Dreyer, Ingo %A Huang, Shouguang %A Hedrich, Rainer %A Roelfsema, M. Rob G. %D 2021 %I Wiley %J New Phytologist %N 4 %P 1449--1460 %R 10.1111/nph.17272 %T A voltage-dependent Ca2+-homeostat operates in the plant vacuolar membrane %U https://doi.org/10.1111%2Fnph.17272 %V 230

• Thor, K., Jiang, S., Michard, E., George, J., Scherzer, S., Huang, S., Dindas, J., Derbyshire, P., Leit{\~{a}}o, N., DeFalco, T.A., Köster, P., Hunter, K., Kimura, S., Gronnier, J., Stransfeld, L., Kadota, Y., Bücherl, C.A., Charpentier, M., Wrzaczek, M., MacLean, D., Oldroyd, G.E.D., Menke, F.L.H., Roelfsema, M.R.G., Hedrich, R., Feij{{ó}}, J., and Zipfel, C. (2020) ‘The calcium-permeable channel {OSCA}1.3 regulates plant stomatal immunity’, Nature, 585(7826), 569–573, available: https://doi.org/10.1038/s41586-020-2702-1.
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  @article{Thor_2020, author = {Thor, Kathrin and Jiang, Shushu and Michard, Erwan and George, Jeoffrey and Scherzer, Sönke and Huang, Shouguang and Dindas, Julian and Derbyshire, Paul and Leit{\ {a}}o, Nuno and DeFalco, Thomas A. and Köster, Philipp and Hunter, Kerri and Kimura, Sachie and Gronnier, Julien and Stransfeld, Lena and Kadota, Yasuhiro and Bücherl, Christoph A. and Charpentier, Myriam and Wrzaczek, Michael and MacLean, Daniel and Oldroyd, Giles E. D. and Menke, Frank L. H. and Roelfsema, M. Rob G. and Hedrich, Rainer and Feij{\'{o}}, Jos{\'{e}} and Zipfel, Cyril}, journal = {Nature}, keywords = {ca2+-signals}, month = {08}, number = 7826, pages = {569–573}, publisher = {Springer Science and Business Media {LLC}}, title = {The calcium-permeable channel {OSCA}1.3 regulates plant stomatal immunity}, volume = 585, year = 2020 }

  %0 Journal Article %1 Thor_2020 %A Thor, Kathrin %A Jiang, Shushu %A Michard, Erwan %A George, Jeoffrey %A Scherzer, Sönke %A Huang, Shouguang %A Dindas, Julian %A Derbyshire, Paul %A Leit{\~{a}}o, Nuno %A DeFalco, Thomas A. %A Köster, Philipp %A Hunter, Kerri %A Kimura, Sachie %A Gronnier, Julien %A Stransfeld, Lena %A Kadota, Yasuhiro %A Bücherl, Christoph A. %A Charpentier, Myriam %A Wrzaczek, Michael %A MacLean, Daniel %A Oldroyd, Giles E. D. %A Menke, Frank L. H. %A Roelfsema, M. Rob G. %A Hedrich, Rainer %A Feij{{ó}}, Jos{{é}} %A Zipfel, Cyril %D 2020 %I Springer Science and Business Media {LLC} %J Nature %N 7826 %P 569--573 %R 10.1038/s41586-020-2702-1 %T The calcium-permeable channel {OSCA}1.3 regulates plant stomatal immunity %U https://doi.org/10.1038%2Fs41586-020-2702-1 %V 585
• Han, B., Jiang, Y., Cui, G., Mi, J., Roelfsema, M.R.G., Mouille, G., Sechet, J., Al-Babili, S., Aranda, M., and Hirt, H. (2020) ‘CATION-CHLORIDE CO-TRANSPORTER 1 (CCC1) mediates plant resistance against Pseudomonas syringa’, Plant Physiology, 182, 1052–1065, available: https://doi.org/10.1104/pp.19.01279.
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  Plasma membrane (PM) depolarization functions as an initial step in plant defense signaling pathways. However, only a few ion channels/transporters have been characterized in the context of plant immunity. Here, we show that the Arabidopsis (Arabidopsis thaliana) Na1:K1:2Cl- (NKCC) cotransporter CCC1 has a dual function in plant immunity. CCC1 functions independently of PM depolarization and negatively regulates pathogen-associated molecular pattern-triggered immunity. However, CCC1 positively regulates plant basal and effector-triggered resistance to Pseudomonas syringae pv. tomato (Pst) DC3000. In line with the compromised immunity to Pst DC3000, ccc1 mutants show reduced expression of genes encoding enzymes involved in the biosynthesis of antimicrobial peptides, camalexin, and 4-OH-ICN, as well as pathogenesis-related proteins. Moreover, genes involved in cell wall and cuticle biosynthesis are constitutively down-regulated in ccc1 mutants, and the cell walls of these mutants exhibit major changes in monosaccharide composition. The role of CCC1 ion transporter activity in the regulation of plant immunity is corroborated by experiments using the specific NKCC inhibitor bumetanide. These results reveal a function for ion transporters in immunity-related cell wall fortification and antimicrobial biosynthesis.

  @article{han2020cationchloride, abstract = {Plasma membrane (PM) depolarization functions as an initial step in plant defense signaling pathways. However, only a few ion channels/transporters have been characterized in the context of plant immunity. Here, we show that the Arabidopsis (Arabidopsis thaliana) Na1:K1:2Cl- (NKCC) cotransporter CCC1 has a dual function in plant immunity. CCC1 functions independently of PM depolarization and negatively regulates pathogen-associated molecular pattern-triggered immunity. However, CCC1 positively regulates plant basal and effector-triggered resistance to Pseudomonas syringae pv. tomato (Pst) DC3000. In line with the compromised immunity to Pst DC3000, ccc1 mutants show reduced expression of genes encoding enzymes involved in the biosynthesis of antimicrobial peptides, camalexin, and 4-OH-ICN, as well as pathogenesis-related proteins. Moreover, genes involved in cell wall and cuticle biosynthesis are constitutively down-regulated in ccc1 mutants, and the cell walls of these mutants exhibit major changes in monosaccharide composition. The role of CCC1 ion transporter activity in the regulation of plant immunity is corroborated by experiments using the specific NKCC inhibitor bumetanide. These results reveal a function for ion transporters in immunity-related cell wall fortification and antimicrobial biosynthesis.}, author = {Han, Baoda and Jiang, Yunhe and Cui, Guoxin and Mi, Jianing and Roelfsema, M. Rob G. and Mouille, Grégory and Sechet, Julien and Al-Babili, Salim and Aranda, Manuel and Hirt, Heribert}, journal = {Plant Physiology}, keywords = {resistance}, pages = {1052–1065,}, title = {CATION-CHLORIDE CO-TRANSPORTER 1 (CCC1) mediates plant resistance against Pseudomonas syringa}, volume = 182, year = 2020 }

  %0 Journal Article %1 han2020cationchloride %A Han, Baoda %A Jiang, Yunhe %A Cui, Guoxin %A Mi, Jianing %A Roelfsema, M. Rob G. %A Mouille, Grégory %A Sechet, Julien %A Al-Babili, Salim %A Aranda, Manuel %A Hirt, Heribert %D 2020 %J Plant Physiology %P 1052–1065, %R 10.1104/pp.19.01279 %T CATION-CHLORIDE CO-TRANSPORTER 1 (CCC1) mediates plant resistance against Pseudomonas syringa %U http://www.plantphysiol.org/content/early/2019/12/05/pp.19.01279/tab-figures-data?versioned=true %V 182 %X Plasma membrane (PM) depolarization functions as an initial step in plant defense signaling pathways. However, only a few ion channels/transporters have been characterized in the context of plant immunity. Here, we show that the Arabidopsis (Arabidopsis thaliana) Na1:K1:2Cl- (NKCC) cotransporter CCC1 has a dual function in plant immunity. CCC1 functions independently of PM depolarization and negatively regulates pathogen-associated molecular pattern-triggered immunity. However, CCC1 positively regulates plant basal and effector-triggered resistance to Pseudomonas syringae pv. tomato (Pst) DC3000. In line with the compromised immunity to Pst DC3000, ccc1 mutants show reduced expression of genes encoding enzymes involved in the biosynthesis of antimicrobial peptides, camalexin, and 4-OH-ICN, as well as pathogenesis-related proteins. Moreover, genes involved in cell wall and cuticle biosynthesis are constitutively down-regulated in ccc1 mutants, and the cell walls of these mutants exhibit major changes in monosaccharide composition. The role of CCC1 ion transporter activity in the regulation of plant immunity is corroborated by experiments using the specific NKCC inhibitor bumetanide. These results reveal a function for ion transporters in immunity-related cell wall fortification and antimicrobial biosynthesis.
• Dindas, J., Becker, D., Roelfsema, M.R.G., Scherzer, S., Bennett, M., and Hedrich, R. (2020) ‘Pitfalls in auxin pharmacology’, New Phytologist, 227(2), 286–292, available: https://doi.org/10.1111/nph.16491.
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  @article{dindas2020pitfalls, author = {Dindas, Julian and Becker, Dirk and Roelfsema, M. Rob G. and Scherzer, Soenke and Bennett, Malcolm and Hedrich, Rainer}, journal = {New Phytologist}, keywords = {hair;}, number = 2, pages = {286-292}, title = {Pitfalls in auxin pharmacology}, volume = 227, year = 2020 }

  %0 Journal Article %1 dindas2020pitfalls %A Dindas, Julian %A Becker, Dirk %A Roelfsema, M. Rob G. %A Scherzer, Soenke %A Bennett, Malcolm %A Hedrich, Rainer %D 2020 %J New Phytologist %N 2 %P 286-292 %R 10.1111/nph.16491 %T Pitfalls in auxin pharmacology %V 227

• Sussmilch, F., Hedrich, R., and Roelfsema, M.R.G. (2019) ‘Acquiring control: The evolution of stomatal signalling pathways’, Trends in Plant Science, 24(4), 342–351, available: https://doi.org/DOI/10.1016/j.tplants.2019.01.002.
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  @article{noauthororeditor, author = {Sussmilch, Frances and Hedrich, Rainer and Roelfsema, M. Rob G.}, journal = {Trends in Plant Science}, keywords = {ABA}, number = 4, pages = {342-351}, title = {Acquiring control: The evolution of stomatal signalling pathways}, volume = 24, year = 2019 }

  %0 Journal Article %1 noauthororeditor %A Sussmilch, Frances %A Hedrich, Rainer %A Roelfsema, M. Rob G. %D 2019 %J Trends in Plant Science %N 4 %P 342-351 %R DOI/10.1016/j.tplants.2019.01.002 %T Acquiring control: The evolution of stomatal signalling pathways %V 24
• Huang, S., Waadt, R., Nuhkat, M., Kollist, H., Hedrich, R., and Roelfsema, M.R.G. (2019) ‘Calcium signals in guard cells enhance the efficiency by which abscisic acid triggers stomatal closure’, New Phytologist, available: https://doi.org/doi: 10.1111/nph.15985.
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  * During drought, abscisic acid (ABA) induces closure of stomata via a signaling pathway that involves the calcium (Ca2+)-independent protein kinase OST1, as well as Ca2+-dependent protein kinases. However, the interconnection between OST1 and Ca2+ signaling in ABA-induced stomatal closure has not been fully resolved. * ABA-induced Ca2+ signals were monitored in intact Arabidopsis leaves, which express the ratiometric Ca2+ reporter R-GECO1-mTurquoise and the Ca2+-dependent activation of S-type anion channels was recorded with intracellular double-barreled microelectrodes. * ABA triggered Ca2+ signals that occurred during the initiation period, as well as in the acceleration phase of stomatal closure. However, a subset of stomata closed in the absence of Ca2+ signals. On average, stomata closed faster if Ca2+ signals were elicited during the ABA response. Loss of OST1 prevented ABA-induced stomatal closure and repressed Ca2+ signals, whereas elevation of the cytosolic Ca2+ concentration caused a rapid activation of SLAC1 and SLAH3 anion channels. * Our data show that the majority of Ca2+ signals are evoked during the acceleration phase of stomatal closure, which is initiated by OST1. These Ca2+ signals are likely to activate Ca2+-dependent protein kinases, which enhance the activity of S-type anion channels and boost stomatal closure.

  @article{noauthororeditorcalcium, abstract = {* During drought, abscisic acid (ABA) induces closure of stomata via a signaling pathway that involves the calcium (Ca2+)-independent protein kinase OST1, as well as Ca2+-dependent protein kinases. However, the interconnection between OST1 and Ca2+ signaling in ABA-induced stomatal closure has not been fully resolved. * ABA-induced Ca2+ signals were monitored in intact Arabidopsis leaves, which express the ratiometric Ca2+ reporter R-GECO1-mTurquoise and the Ca2+-dependent activation of S-type anion channels was recorded with intracellular double-barreled microelectrodes. * ABA triggered Ca2+ signals that occurred during the initiation period, as well as in the acceleration phase of stomatal closure. However, a subset of stomata closed in the absence of Ca2+ signals. On average, stomata closed faster if Ca2+ signals were elicited during the ABA response. Loss of OST1 prevented ABA-induced stomatal closure and repressed Ca2+ signals, whereas elevation of the cytosolic Ca2+ concentration caused a rapid activation of SLAC1 and SLAH3 anion channels. * Our data show that the majority of Ca2+ signals are evoked during the acceleration phase of stomatal closure, which is initiated by OST1. These Ca2+ signals are likely to activate Ca2+-dependent protein kinases, which enhance the activity of S-type anion channels and boost stomatal closure.}, author = {Huang, Shouguang and Waadt, Rainer and Nuhkat, Maris and Kollist, Hannes and Hedrich, Rainer and Roelfsema, M. Rob G.}, journal = {New Phytologist}, keywords = {SLAH3,ABA,}, title = {Calcium signals in guard cells enhance the efficiency by which abscisic acid triggers stomatal closure}, year = 2019 }

  %0 Journal Article %1 noauthororeditorcalcium %A Huang, Shouguang %A Waadt, Rainer %A Nuhkat, Maris %A Kollist, Hannes %A Hedrich, Rainer %A Roelfsema, M. Rob G. %D 2019 %J New Phytologist %R doi: 10.1111/nph.15985 %T Calcium signals in guard cells enhance the efficiency by which abscisic acid triggers stomatal closure %X * During drought, abscisic acid (ABA) induces closure of stomata via a signaling pathway that involves the calcium (Ca2+)-independent protein kinase OST1, as well as Ca2+-dependent protein kinases. However, the interconnection between OST1 and Ca2+ signaling in ABA-induced stomatal closure has not been fully resolved. * ABA-induced Ca2+ signals were monitored in intact Arabidopsis leaves, which express the ratiometric Ca2+ reporter R-GECO1-mTurquoise and the Ca2+-dependent activation of S-type anion channels was recorded with intracellular double-barreled microelectrodes. * ABA triggered Ca2+ signals that occurred during the initiation period, as well as in the acceleration phase of stomatal closure. However, a subset of stomata closed in the absence of Ca2+ signals. On average, stomata closed faster if Ca2+ signals were elicited during the ABA response. Loss of OST1 prevented ABA-induced stomatal closure and repressed Ca2+ signals, whereas elevation of the cytosolic Ca2+ concentration caused a rapid activation of SLAC1 and SLAH3 anion channels. * Our data show that the majority of Ca2+ signals are evoked during the acceleration phase of stomatal closure, which is initiated by OST1. These Ca2+ signals are likely to activate Ca2+-dependent protein kinases, which enhance the activity of S-type anion channels and boost stomatal closure.
• Liu, Y., Maierhofer, T., Rybak, K., Sklenar, J., Breakspear, A., Johnston, M., Fliegmann, J., Huang, S., Roelfsema, M., Felix, G., Faulkner, C., Menke, F., Geiger, D., Hedrich, R., and Robatzek, S. (2019) ‘Anion channel SLAH3 is a regulatory target of chitin receptor-associated kinase PBL27 in microbial stomatal closure’, Elife, 8(e44474), available: https://doi.org/https://doi.org/10.7554/eLife.44474.
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  In plants, antimicrobial immune responses involve the cellular release of anions and are responsible for the closure of stomatal pores. Detection of microbe-associated molecular patterns (MAMPs) by pattern recognition receptors (PRRs) induces currents mediated via slow-type (S-type) anion channels by a yet not understood mechanism. Here, we show that stomatal closure to fungal chitin is conferred by the major PRRs for chitin recognition, LYK5 and CERK1, the receptor-like cytoplasmic kinase PBL27, and the SLAH3 anion channel. PBL27 has the capacity to phosphorylate SLAH3, of which S127 and S189 are required to activate SLAH3. Full activation of the channel entails CERK1, depending on PBL27. Importantly, both S127 and S189 residues of SLAH3 are required for chitin-induced stomatal closure and anti-fungal immunity at the whole leaf level. Our results demonstrate a short signal transduction module from MAMP recognition to anion channel activation, and independent of ABA-induced SLAH3 activation.

  @article{liu2019anion, abstract = {In plants, antimicrobial immune responses involve the cellular release of anions and are responsible for the closure of stomatal pores. Detection of microbe-associated molecular patterns (MAMPs) by pattern recognition receptors (PRRs) induces currents mediated via slow-type (S-type) anion channels by a yet not understood mechanism. Here, we show that stomatal closure to fungal chitin is conferred by the major PRRs for chitin recognition, LYK5 and CERK1, the receptor-like cytoplasmic kinase PBL27, and the SLAH3 anion channel. PBL27 has the capacity to phosphorylate SLAH3, of which S127 and S189 are required to activate SLAH3. Full activation of the channel entails CERK1, depending on PBL27. Importantly, both S127 and S189 residues of SLAH3 are required for chitin-induced stomatal closure and anti-fungal immunity at the whole leaf level. Our results demonstrate a short signal transduction module from MAMP recognition to anion channel activation, and independent of ABA-induced SLAH3 activation.}, author = {Liu, Y and Maierhofer, T and Rybak, K and Sklenar, J and Breakspear, A and Johnston, MG and Fliegmann, J and Huang, S and Roelfsema, MRG and Felix, G and Faulkner, C and Menke, FLH and Geiger, D and Hedrich, R and Robatzek, S}, journal = {Elife}, keywords = {chitin}, number = {e44474}, title = {Anion channel SLAH3 is a regulatory target of chitin receptor-associated kinase PBL27 in microbial stomatal closure}, volume = 8, year = 2019 }

  %0 Journal Article %1 liu2019anion %A Liu, Y %A Maierhofer, T %A Rybak, K %A Sklenar, J %A Breakspear, A %A Johnston, MG %A Fliegmann, J %A Huang, S %A Roelfsema, MRG %A Felix, G %A Faulkner, C %A Menke, FLH %A Geiger, D %A Hedrich, R %A Robatzek, S %D 2019 %J Elife %N e44474 %R https://doi.org/10.7554/eLife.44474 %T Anion channel SLAH3 is a regulatory target of chitin receptor-associated kinase PBL27 in microbial stomatal closure %U https://elifesciences.org/articles/44474#abstract %V 8 %X In plants, antimicrobial immune responses involve the cellular release of anions and are responsible for the closure of stomatal pores. Detection of microbe-associated molecular patterns (MAMPs) by pattern recognition receptors (PRRs) induces currents mediated via slow-type (S-type) anion channels by a yet not understood mechanism. Here, we show that stomatal closure to fungal chitin is conferred by the major PRRs for chitin recognition, LYK5 and CERK1, the receptor-like cytoplasmic kinase PBL27, and the SLAH3 anion channel. PBL27 has the capacity to phosphorylate SLAH3, of which S127 and S189 are required to activate SLAH3. Full activation of the channel entails CERK1, depending on PBL27. Importantly, both S127 and S189 residues of SLAH3 are required for chitin-induced stomatal closure and anti-fungal immunity at the whole leaf level. Our results demonstrate a short signal transduction module from MAMP recognition to anion channel activation, and independent of ABA-induced SLAH3 activation.
• Sussmilch, F., Roelfsema, M.R.G., and Hedrich, R. (2019) ‘On the origins of osmotically driven stomatal movements’, New Phytologist, 222, 84–90, available: https://doi.org/10.1111/nph.15593.
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  Stomatal pores with apertures that can be adjusted by changes in guard cell turgor have facilitated plant success in dry environments. We explore their evolutionary origins, considering recent findings from bryophytes. Unlike vascular plant stomata, which close to prevent water loss, bryophyte stomata become locked open to promote spore desiccation. We find that the families of ion channels, known to control stomatal movements in angiosperms, are ancient and represented across extant land plants. However, although angiosperm guard cells express specific ion channel genes, none appear specifically expressed in stomata-bearing moss tissues. Given the evolutionary shift in stomatal function from promotion to prevention of water loss, we postulate that ion channels adopted guard cell-specific functions after the divergence of bryophytes.

  @article{sussmilch2018origins, abstract = {Stomatal pores with apertures that can be adjusted by changes in guard cell turgor have facilitated plant success in dry environments. We explore their evolutionary origins, considering recent findings from bryophytes. Unlike vascular plant stomata, which close to prevent water loss, bryophyte stomata become locked open to promote spore desiccation. We find that the families of ion channels, known to control stomatal movements in angiosperms, are ancient and represented across extant land plants. However, although angiosperm guard cells express specific ion channel genes, none appear specifically expressed in stomata-bearing moss tissues. Given the evolutionary shift in stomatal function from promotion to prevention of water loss, we postulate that ion channels adopted guard cell-specific functions after the divergence of bryophytes.}, author = {Sussmilch, Frances and Roelfsema, M. Rob G. and Hedrich, Rainer}, journal = {New Phytologist}, keywords = {ion-channel,}, pages = {84-90}, title = {On the origins of osmotically driven stomatal movements}, volume = 222, year = 2019 }

  %0 Journal Article %1 sussmilch2018origins %A Sussmilch, Frances %A Roelfsema, M. Rob G. %A Hedrich, Rainer %D 2019 %J New Phytologist %P 84-90 %R 10.1111/nph.15593 %T On the origins of osmotically driven stomatal movements %V 222 %X Stomatal pores with apertures that can be adjusted by changes in guard cell turgor have facilitated plant success in dry environments. We explore their evolutionary origins, considering recent findings from bryophytes. Unlike vascular plant stomata, which close to prevent water loss, bryophyte stomata become locked open to promote spore desiccation. We find that the families of ion channels, known to control stomatal movements in angiosperms, are ancient and represented across extant land plants. However, although angiosperm guard cells express specific ion channel genes, none appear specifically expressed in stomata-bearing moss tissues. Given the evolutionary shift in stomatal function from promotion to prevention of water loss, we postulate that ion channels adopted guard cell-specific functions after the divergence of bryophytes.

• Voss, L., McAdam, S., Knoblauch, M., Rathje, J., Brodribb, T., Hedrich, R., and Roelfsema, M. (2018) ‘Guard cells in fern stomata are connected by plasmodesmata, but control cytosolic Ca2+ levels autonomously’, New Phytologist, 219(1), 206–215, available: https://doi.org/doi: 10.1111/nph.15153.
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  Recent studies have revealed that some responses of fern stomata to environmental signals differ from those of their relatives in seed plants. However, it is unknown whether the biophysical properties of guard cells differ fundamentally between species of both clades. Intracellular micro‐electrodes and the fluorescent Ca2+ reporter FURA2 were used to study voltage‐dependent cation channels and Ca2+ signals in guard cells of the ferns Polypodium vulgare and Asplenium scolopendrium. Voltage clamp experiments with fern guard cells revealed similar properties of voltage‐dependent K+ channels as found in seed plants. However, fluorescent dyes moved within the fern stomata, from one guard cell to the other, which does not occur in most seed plants. Despite the presence of plasmodesmata, which interconnect fern guard cells, Ca2+ signals could be elicited in each of the cells individually. Based on the common properties of voltage‐dependent channels in ferns and seed plants, it is likely that these key transport proteins are conserved in vascular plants. However, the symplastic connections between fern guard cells in mature stomata indicate that the biophysical mechanisms that control stomatal movements differ between ferns and seed plants.

  @article{vossguard, abstract = {Recent studies have revealed that some responses of fern stomata to environmental signals differ from those of their relatives in seed plants. However, it is unknown whether the biophysical properties of guard cells differ fundamentally between species of both clades. Intracellular micro‐electrodes and the fluorescent Ca2+ reporter FURA2 were used to study voltage‐dependent cation channels and Ca2+ signals in guard cells of the ferns Polypodium vulgare and Asplenium scolopendrium. Voltage clamp experiments with fern guard cells revealed similar properties of voltage‐dependent K+ channels as found in seed plants. However, fluorescent dyes moved within the fern stomata, from one guard cell to the other, which does not occur in most seed plants. Despite the presence of plasmodesmata, which interconnect fern guard cells, Ca2+ signals could be elicited in each of the cells individually. Based on the common properties of voltage‐dependent channels in ferns and seed plants, it is likely that these key transport proteins are conserved in vascular plants. However, the symplastic connections between fern guard cells in mature stomata indicate that the biophysical mechanisms that control stomatal movements differ between ferns and seed plants.}, author = {Voss, L.J. and McAdam, S.A.M. and Knoblauch, M. and Rathje, J.M. and Brodribb, T. and Hedrich, R. and Roelfsema, M.R.G.}, journal = {New Phytologist}, keywords = {ca2+-signals}, number = 1, pages = {206–215}, title = {Guard cells in fern stomata are connected by plasmodesmata, but control cytosolic Ca2+ levels autonomously}, volume = 219, year = 2018 }

  %0 Journal Article %1 vossguard %A Voss, L.J. %A McAdam, S.A.M. %A Knoblauch, M. %A Rathje, J.M. %A Brodribb, T. %A Hedrich, R. %A Roelfsema, M.R.G. %D 2018 %J New Phytologist %N 1 %P 206–215 %R doi: 10.1111/nph.15153 %T Guard cells in fern stomata are connected by plasmodesmata, but control cytosolic Ca2+ levels autonomously %U https://nph.onlinelibrary.wiley.com/doi/10.1111/nph.15153 %V 219 %X Recent studies have revealed that some responses of fern stomata to environmental signals differ from those of their relatives in seed plants. However, it is unknown whether the biophysical properties of guard cells differ fundamentally between species of both clades. Intracellular micro‐electrodes and the fluorescent Ca2+ reporter FURA2 were used to study voltage‐dependent cation channels and Ca2+ signals in guard cells of the ferns Polypodium vulgare and Asplenium scolopendrium. Voltage clamp experiments with fern guard cells revealed similar properties of voltage‐dependent K+ channels as found in seed plants. However, fluorescent dyes moved within the fern stomata, from one guard cell to the other, which does not occur in most seed plants. Despite the presence of plasmodesmata, which interconnect fern guard cells, Ca2+ signals could be elicited in each of the cells individually. Based on the common properties of voltage‐dependent channels in ferns and seed plants, it is likely that these key transport proteins are conserved in vascular plants. However, the symplastic connections between fern guard cells in mature stomata indicate that the biophysical mechanisms that control stomatal movements differ between ferns and seed plants.
• Dindas, J., Scherzer, S., Roelfsema, M., von Meyer, K., Müller H., Al-Rasheid, K., Palme, K., Dietrich, P., Becker, D., Bennett, M., and Hedrich, R. (2018) ‘AUX1-mediated root hair auxin influx governs SCFTIR1/AFB-type Ca2+ signaling’, Nature Communications, 9, 1174, available: https://doi.org/doi:10.1038/s41467-018-03582-5.
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  Auxin is a key regulator of plant growth and development, but the causal relationship between hormone transport and root responses remains unresolved. Here we describe auxin uptake, together with early steps in signaling, in Arabidopsis root hairs. Using intracellular microelectrodes we show membrane depolarization, in response to IAA in a concentration- and pH-dependent manner. This depolarization is strongly impaired in aux1 mutants, indicating that AUX1 is the major transporter for auxin uptake in root hairs. Local intracellular auxin application triggers Ca2+ signals that propagate as long-distance waves between root cells and modulate their auxin responses. AUX1-mediated IAA transport, as well as IAA- triggered calcium signals, are blocked by treatment with the SCFTIR1/AFB - inhibitor auxinole. Further, they are strongly reduced in the tir1afb2afb3 and the cngc14 mutant. Our study reveals that the AUX1 transporter, the SCFTIR1/AFB receptor and the CNGC14 Ca2+ channel, mediate fast auxin signaling in roots.

  @article{dindas2018aux1mediated, abstract = {Auxin is a key regulator of plant growth and development, but the causal relationship between hormone transport and root responses remains unresolved. Here we describe auxin uptake, together with early steps in signaling, in Arabidopsis root hairs. Using intracellular microelectrodes we show membrane depolarization, in response to IAA in a concentration- and pH-dependent manner. This depolarization is strongly impaired in aux1 mutants, indicating that AUX1 is the major transporter for auxin uptake in root hairs. Local intracellular auxin application triggers Ca2+ signals that propagate as long-distance waves between root cells and modulate their auxin responses. AUX1-mediated IAA transport, as well as IAA- triggered calcium signals, are blocked by treatment with the SCFTIR1/AFB - inhibitor auxinole. Further, they are strongly reduced in the tir1afb2afb3 and the cngc14 mutant. Our study reveals that the AUX1 transporter, the SCFTIR1/AFB receptor and the CNGC14 Ca2+ channel, mediate fast auxin signaling in roots.}, author = {Dindas, J. and Scherzer, S. and Roelfsema, M.R.G. and von Meyer, K. and Müller, H.M. and Al-Rasheid, K.A.S. and Palme, K. and Dietrich, P. and Becker, D. and Bennett, M.J. and Hedrich, R.}, journal = {Nature Communications}, keywords = {Ca2+-signals}, pages = 1174, title = {AUX1-mediated root hair auxin influx governs SCFTIR1/AFB-type Ca2+ signaling}, volume = 9, year = 2018 }

  %0 Journal Article %1 dindas2018aux1mediated %A Dindas, J. %A Scherzer, S. %A Roelfsema, M.R.G. %A von Meyer, K. %A Müller, H.M. %A Al-Rasheid, K.A.S. %A Palme, K. %A Dietrich, P. %A Becker, D. %A Bennett, M.J. %A Hedrich, R. %D 2018 %J Nature Communications %P 1174 %R doi:10.1038/s41467-018-03582-5 %T AUX1-mediated root hair auxin influx governs SCFTIR1/AFB-type Ca2+ signaling %U https://www.nature.com/articles/s41467-018-03582-5 %V 9 %X Auxin is a key regulator of plant growth and development, but the causal relationship between hormone transport and root responses remains unresolved. Here we describe auxin uptake, together with early steps in signaling, in Arabidopsis root hairs. Using intracellular microelectrodes we show membrane depolarization, in response to IAA in a concentration- and pH-dependent manner. This depolarization is strongly impaired in aux1 mutants, indicating that AUX1 is the major transporter for auxin uptake in root hairs. Local intracellular auxin application triggers Ca2+ signals that propagate as long-distance waves between root cells and modulate their auxin responses. AUX1-mediated IAA transport, as well as IAA- triggered calcium signals, are blocked by treatment with the SCFTIR1/AFB - inhibitor auxinole. Further, they are strongly reduced in the tir1afb2afb3 and the cngc14 mutant. Our study reveals that the AUX1 transporter, the SCFTIR1/AFB receptor and the CNGC14 Ca2+ channel, mediate fast auxin signaling in roots.
• Graus, D., Konrad, K.R., Bemm, F., Nebioglu, M.G.P., Lorey, C., Duscha, K., Guthoff, T., Herrmann, J., Ferjani, A., Cuin, T.A., Roelfsema, M.R.G., Schumacher, K., Neuhaus, E., Marten, I., and Hedrich, R. (2018) ‘High V-PPase activity is beneficial under high salt loads, but detrimental without salinity’, New Phytologist, 219, 1421–1432, available: https://doi.org/10.1111/nph.15280.
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  * The membrane-bound proton-pumping pyrophosphatase (V-PPase), together with the V-type H+-ATPase, generates the proton motive force that drives vacuolar membrane solute transport. Transgenic plants constitutively overexpressing V-PPases were shown to have improved salinity tolerance, but the relative impact of increasing PPi hydrolysis and protonpumping functions has yet to be dissected. * For a better understanding of the molecular processes underlying V-PPase-dependent salt tolerance, we transiently overexpressed the pyrophosphate-driven proton pump (NbVHP) in Nicotiana benthamiana leaves and studied its functional properties in relation to salt treatment by primarily using patch-clamp, impalement electrodes and pH imaging. * NbVHP overexpression led to higher vacuolar proton currents and vacuolar acidification. After 3 d in salt-untreated conditions, V-PPase-overexpressing leaves showed a drop in photosynthetic capacity, plasma membrane depolarization and eventual leaf necrosis. Salt, however, rescued NbVHP-hyperactive cells from cell death. Furthermore, a salt-induced rise in V-PPase but not of V-ATPase pump currents was detected in nontransformed plants. * The results indicate that under normal growth conditions, plants need to regulate the V-PPase pump activity to avoid hyperactivity and its negative feedback on cell viability. Nonetheless, V-PPase proton pump function becomes increasingly important under salt stress for generating the pH gradient necessary for vacuolar proton-coupled Na+ sequestration.

  @article{graus2018vppase, abstract = {* The membrane-bound proton-pumping pyrophosphatase (V-PPase), together with the V-type H+-ATPase, generates the proton motive force that drives vacuolar membrane solute transport. Transgenic plants constitutively overexpressing V-PPases were shown to have improved salinity tolerance, but the relative impact of increasing PPi hydrolysis and protonpumping functions has yet to be dissected. * For a better understanding of the molecular processes underlying V-PPase-dependent salt tolerance, we transiently overexpressed the pyrophosphate-driven proton pump (NbVHP) in Nicotiana benthamiana leaves and studied its functional properties in relation to salt treatment by primarily using patch-clamp, impalement electrodes and pH imaging. * NbVHP overexpression led to higher vacuolar proton currents and vacuolar acidification. After 3 d in salt-untreated conditions, V-PPase-overexpressing leaves showed a drop in photosynthetic capacity, plasma membrane depolarization and eventual leaf necrosis. Salt, however, rescued NbVHP-hyperactive cells from cell death. Furthermore, a salt-induced rise in V-PPase but not of V-ATPase pump currents was detected in nontransformed plants. * The results indicate that under normal growth conditions, plants need to regulate the V-PPase pump activity to avoid hyperactivity and its negative feedback on cell viability. Nonetheless, V-PPase proton pump function becomes increasingly important under salt stress for generating the pH gradient necessary for vacuolar proton-coupled Na+ sequestration.}, author = {Graus, Dorothea and Konrad, Kai R. and Bemm, Felix and Nebioglu, Meliha G. P. and Lorey, Christian and Duscha, Kerstin and Guthoff, Tilman and Herrmann, Johannes and Ferjani, Ali and Cuin, Tracey A. and Roelfsema, M. Rob G. and Schumacher, Karin and Neuhaus, Ekkehard and Marten, Irene and Hedrich, Rainer}, journal = {New Phytologist}, keywords = {leaf}, pages = {1421–1432}, title = {High V-PPase activity is beneficial under high salt loads, but detrimental without salinity}, volume = 219, year = 2018 }

  %0 Journal Article %1 graus2018vppase %A Graus, Dorothea %A Konrad, Kai R. %A Bemm, Felix %A Nebioglu, Meliha G. P. %A Lorey, Christian %A Duscha, Kerstin %A Guthoff, Tilman %A Herrmann, Johannes %A Ferjani, Ali %A Cuin, Tracey A. %A Roelfsema, M. Rob G. %A Schumacher, Karin %A Neuhaus, Ekkehard %A Marten, Irene %A Hedrich, Rainer %D 2018 %J New Phytologist %P 1421–1432 %R 10.1111/nph.15280 %T High V-PPase activity is beneficial under high salt loads, but detrimental without salinity %V 219 %X * The membrane-bound proton-pumping pyrophosphatase (V-PPase), together with the V-type H+-ATPase, generates the proton motive force that drives vacuolar membrane solute transport. Transgenic plants constitutively overexpressing V-PPases were shown to have improved salinity tolerance, but the relative impact of increasing PPi hydrolysis and protonpumping functions has yet to be dissected. * For a better understanding of the molecular processes underlying V-PPase-dependent salt tolerance, we transiently overexpressed the pyrophosphate-driven proton pump (NbVHP) in Nicotiana benthamiana leaves and studied its functional properties in relation to salt treatment by primarily using patch-clamp, impalement electrodes and pH imaging. * NbVHP overexpression led to higher vacuolar proton currents and vacuolar acidification. After 3 d in salt-untreated conditions, V-PPase-overexpressing leaves showed a drop in photosynthetic capacity, plasma membrane depolarization and eventual leaf necrosis. Salt, however, rescued NbVHP-hyperactive cells from cell death. Furthermore, a salt-induced rise in V-PPase but not of V-ATPase pump currents was detected in nontransformed plants. * The results indicate that under normal growth conditions, plants need to regulate the V-PPase pump activity to avoid hyperactivity and its negative feedback on cell viability. Nonetheless, V-PPase proton pump function becomes increasingly important under salt stress for generating the pH gradient necessary for vacuolar proton-coupled Na+ sequestration.
• Hürter, A.-L., Fort, S., Cottaz, S., Hedrich, R., Geiger, D., and Roelfsema, M. (2018) ‘Mycorrhizal lipochitinoligosaccharides (LCOs) depolarize root hairs of Medicago truncatula’, PLoS One, 13(5), e0198126., available: https://doi.org/https://doi.org/10.1371/journal.pone.0198126.
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  Arbuscular Mycorrhiza and Root Nodule Symbiosis are symbiotic interactions with a high benefit for plant growth and crop production. Thus, it is of great interest to understand the developmental process of these symbioses in detail. We analysed very early symbiotic responses of Medicago truncatula root hair cells, by stimulation with lipochitinoligosaccharides specific for the induction of nodules (Nod-LCOs), or the interaction with mycorrhiza (Myc-LCOs). Intracellular micro electrodes were used, in combination with Ca2+ sensitive reporter dyes, to study the relations between cytosolic Ca2+ signals and membrane potential changes. We found that sulfated Myc- as well as Nod-LCOs initiate a membrane depolarization, which depends on the chemical composition of these signaling molecules, as well as the genotype of the plants that were studied. A successive application of sulfated Myc-LCOs and Nod-LCOs resulted only in a single transient depolarization, indicating that Myc-LCOs can repress plasma membrane responses to Nod-LCOs. In contrast to current models, the Nod-LCO-induced depolarization precedes changes in the cytosolic Ca2+ level of root hair cells. The Nod-LCO induced membrane depolarization thus is most likely independent of cytosolic Ca2+ signals and nuclear Ca2+ spiking.

  @article{hurter2018mycorrhizal, abstract = {Arbuscular Mycorrhiza and Root Nodule Symbiosis are symbiotic interactions with a high benefit for plant growth and crop production. Thus, it is of great interest to understand the developmental process of these symbioses in detail. We analysed very early symbiotic responses of Medicago truncatula root hair cells, by stimulation with lipochitinoligosaccharides specific for the induction of nodules (Nod-LCOs), or the interaction with mycorrhiza (Myc-LCOs). Intracellular micro electrodes were used, in combination with Ca2+ sensitive reporter dyes, to study the relations between cytosolic Ca2+ signals and membrane potential changes. We found that sulfated Myc- as well as Nod-LCOs initiate a membrane depolarization, which depends on the chemical composition of these signaling molecules, as well as the genotype of the plants that were studied. A successive application of sulfated Myc-LCOs and Nod-LCOs resulted only in a single transient depolarization, indicating that Myc-LCOs can repress plasma membrane responses to Nod-LCOs. In contrast to current models, the Nod-LCO-induced depolarization precedes changes in the cytosolic Ca2+ level of root hair cells. The Nod-LCO induced membrane depolarization thus is most likely independent of cytosolic Ca2+ signals and nuclear Ca2+ spiking.}, author = {Hürter, A-L. and Fort, S. and Cottaz, S. and Hedrich, R. and Geiger, D. and Roelfsema, M.R.G.}, journal = {PLoS One}, keywords = {membran-potential}, number = 5, pages = {e0198126.}, title = {Mycorrhizal lipochitinoligosaccharides (LCOs) depolarize root hairs of Medicago truncatula}, volume = 13, year = 2018 }

  %0 Journal Article %1 hurter2018mycorrhizal %A Hürter, A-L. %A Fort, S. %A Cottaz, S. %A Hedrich, R. %A Geiger, D. %A Roelfsema, M.R.G. %D 2018 %J PLoS One %N 5 %P e0198126. %R https://doi.org/10.1371/journal.pone.0198126 %T Mycorrhizal lipochitinoligosaccharides (LCOs) depolarize root hairs of Medicago truncatula %U http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0198126 %V 13 %X Arbuscular Mycorrhiza and Root Nodule Symbiosis are symbiotic interactions with a high benefit for plant growth and crop production. Thus, it is of great interest to understand the developmental process of these symbioses in detail. We analysed very early symbiotic responses of Medicago truncatula root hair cells, by stimulation with lipochitinoligosaccharides specific for the induction of nodules (Nod-LCOs), or the interaction with mycorrhiza (Myc-LCOs). Intracellular micro electrodes were used, in combination with Ca2+ sensitive reporter dyes, to study the relations between cytosolic Ca2+ signals and membrane potential changes. We found that sulfated Myc- as well as Nod-LCOs initiate a membrane depolarization, which depends on the chemical composition of these signaling molecules, as well as the genotype of the plants that were studied. A successive application of sulfated Myc-LCOs and Nod-LCOs resulted only in a single transient depolarization, indicating that Myc-LCOs can repress plasma membrane responses to Nod-LCOs. In contrast to current models, the Nod-LCO-induced depolarization precedes changes in the cytosolic Ca2+ level of root hair cells. The Nod-LCO induced membrane depolarization thus is most likely independent of cytosolic Ca2+ signals and nuclear Ca2+ spiking.

• Nguyen, T., Huang, S., Meynard, D., Chaine, C., Michel, R., Roelfsema, M., Guiderdoni, E., Sentenac, H., and Véry, A.-A. (2017) ‘A dual role for the OSK5.2 ion channel in stomatal movements and K+ loading into xylem sap’, Plant Physiology, 174(4), 2409–2418, available: https://doi.org/DOI: https://doi.org/10.1104/pp.17.00691.
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  The roles of potassium channels from the Shaker family in stomatal movements have been investigated by reverse genetics analyses in Arabidopsis (Arabidopsis thaliana), but corresponding information is lacking outside this model species. Rice (Oryza sativa) and other cereals possess stomata that are more complex than those of Arabidopsis. We examined the role of the outward Shaker K+ channel gene OsK5.2. Expression of the OsK5.2 gene (GUS reporter strategy) was observed in the whole stomatal complex (guard cells and subsidiary cells), root vasculature, and root cortex. In stomata, loss of OsK5.2 functional expression resulted in lack of time-dependent outward potassium currents in guard cells, higher rates of water loss through transpiration, and severe slowdown of stomatal closure. In line with the expression of OsK5.2 in the plant vasculature, mutant plants displayed a reduced K+ translocation from the root system toward the leaves via the xylem. The comparison between rice and Arabidopsis show that despite the strong conservation of Shaker family in plants, substantial differences can exist between the physiological roles of seemingly orthologous genes, as xylem loading depends on SKOR and stomatal closure on GORK in Arabidopsis, whereas both functions are executed by the single OsK5.2 Shaker in rice.

  @article{nguyen2017osk52, abstract = {The roles of potassium channels from the Shaker family in stomatal movements have been investigated by reverse genetics analyses in Arabidopsis (Arabidopsis thaliana), but corresponding information is lacking outside this model species. Rice (Oryza sativa) and other cereals possess stomata that are more complex than those of Arabidopsis. We examined the role of the outward Shaker K+ channel gene OsK5.2. Expression of the OsK5.2 gene (GUS reporter strategy) was observed in the whole stomatal complex (guard cells and subsidiary cells), root vasculature, and root cortex. In stomata, loss of OsK5.2 functional expression resulted in lack of time-dependent outward potassium currents in guard cells, higher rates of water loss through transpiration, and severe slowdown of stomatal closure. In line with the expression of OsK5.2 in the plant vasculature, mutant plants displayed a reduced K+ translocation from the root system toward the leaves via the xylem. The comparison between rice and Arabidopsis show that despite the strong conservation of Shaker family in plants, substantial differences can exist between the physiological roles of seemingly orthologous genes, as xylem loading depends on SKOR and stomatal closure on GORK in Arabidopsis, whereas both functions are executed by the single OsK5.2 Shaker in rice.}, author = {Nguyen, T.H. and Huang, S. and Meynard, D. and Chaine, C. and Michel, R. and Roelfsema, M.R.G. and Guiderdoni, E. and Sentenac, H. and Véry, A-A.}, journal = {Plant Physiology}, keywords = {savity}, number = 4, pages = {2409–2418}, title = {A dual role for the OSK5.2 ion channel in stomatal movements and K+ loading into xylem sap}, volume = 174, year = 2017 }

  %0 Journal Article %1 nguyen2017osk52 %A Nguyen, T.H. %A Huang, S. %A Meynard, D. %A Chaine, C. %A Michel, R. %A Roelfsema, M.R.G. %A Guiderdoni, E. %A Sentenac, H. %A Véry, A-A. %D 2017 %J Plant Physiology %N 4 %P 2409–2418 %R DOI: https://doi.org/10.1104/pp.17.00691 %T A dual role for the OSK5.2 ion channel in stomatal movements and K+ loading into xylem sap %U http://www.plantphysiol.org/content/174/4/2409 %V 174 %X The roles of potassium channels from the Shaker family in stomatal movements have been investigated by reverse genetics analyses in Arabidopsis (Arabidopsis thaliana), but corresponding information is lacking outside this model species. Rice (Oryza sativa) and other cereals possess stomata that are more complex than those of Arabidopsis. We examined the role of the outward Shaker K+ channel gene OsK5.2. Expression of the OsK5.2 gene (GUS reporter strategy) was observed in the whole stomatal complex (guard cells and subsidiary cells), root vasculature, and root cortex. In stomata, loss of OsK5.2 functional expression resulted in lack of time-dependent outward potassium currents in guard cells, higher rates of water loss through transpiration, and severe slowdown of stomatal closure. In line with the expression of OsK5.2 in the plant vasculature, mutant plants displayed a reduced K+ translocation from the root system toward the leaves via the xylem. The comparison between rice and Arabidopsis show that despite the strong conservation of Shaker family in plants, substantial differences can exist between the physiological roles of seemingly orthologous genes, as xylem loading depends on SKOR and stomatal closure on GORK in Arabidopsis, whereas both functions are executed by the single OsK5.2 Shaker in rice.

• Jakobson, L., Vaahtera, L., Toldsepp, K., Nuhkat, M., Wang, C., Wang, Y.S., Horak, H., Valk, E., Pechter, P., Sindarovska, Y., Tang, J., Xiao, C.L., Xu, Y., Talas, U.G., Garcia-Sosa, A.T., Kangasjarvi, S., Maran, U., Remm, M., Roelfsema, M.R.G., Hu, H.H., Kangasjarvi, J., Loog, M., Schroeder, J.I., Kollist, H., and Brosche, M. (2016) ‘Natural variation in Arabidopsis Cvi-0 accession reveals an important role of MPK12 in guard cell CO2 signaling’, Plos Biology, 14(12), 25-, available: https://doi.org/10.1371/journal.pbio.2000322.
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  Plant gas exchange is regulated by guard cells that form stomatal pores. Stomatal adjustments are crucial for plant survival; they regulate uptake of CO2 for photosynthesis, loss of water, and entrance of air pollutants such as ozone. We mapped ozone hypersensitivity, more open stomata, and stomatal CO2-insensitivity phenotypes of the Arabidopsis thaliana accession Cvi-0 to a single amino acid substitution in MITOGEN-ACTIVATED PROTEIN (MAP) KINASE 12 (MPK12). In parallel, we showed that stomatal CO2-insensitivity phenotypes of a mutant cis (CO2-insensitive) were caused by a deletion of MPK12. Lack of MPK12 impaired bicarbonate-induced activation of S-type anion channels. We demonstrated that MPK12 interacted with the protein kinase HIGH LEAF TEMPERATURE 1 (HT1)—a central node in guard cell CO2 signaling—and that MPK12 functions as an inhibitor of HT1. These data provide a new function for plant MPKs as protein kinase inhibitors and suggest a mechanism through which guard cell CO2 signaling controls plant water management.

  @article{jakobson2016natural, abstract = {Plant gas exchange is regulated by guard cells that form stomatal pores. Stomatal adjustments are crucial for plant survival; they regulate uptake of CO2 for photosynthesis, loss of water, and entrance of air pollutants such as ozone. We mapped ozone hypersensitivity, more open stomata, and stomatal CO2-insensitivity phenotypes of the Arabidopsis thaliana accession Cvi-0 to a single amino acid substitution in MITOGEN-ACTIVATED PROTEIN (MAP) KINASE 12 (MPK12). In parallel, we showed that stomatal CO2-insensitivity phenotypes of a mutant cis (CO2-insensitive) were caused by a deletion of MPK12. Lack of MPK12 impaired bicarbonate-induced activation of S-type anion channels. We demonstrated that MPK12 interacted with the protein kinase HIGH LEAF TEMPERATURE 1 (HT1)—a central node in guard cell CO2 signaling—and that MPK12 functions as an inhibitor of HT1. These data provide a new function for plant MPKs as protein kinase inhibitors and suggest a mechanism through which guard cell CO2 signaling controls plant water management.}, author = {Jakobson, L. and Vaahtera, L. and Toldsepp, K. and Nuhkat, M. and Wang, C. and Wang, Y. S. and Horak, H. and Valk, E. and Pechter, P. and Sindarovska, Y. and Tang, J. and Xiao, C. L. and Xu, Y. and Talas, U. G. and Garcia-Sosa, A. T. and Kangasjarvi, S. and Maran, U. and Remm, M. and Roelfsema, M. R. G. and Hu, H. H. and Kangasjarvi, J. and Loog, M. and Schroeder, J. I. and Kollist, H. and Brosche, M.}, booktitle = {Plos Biology}, keywords = {MAP-kinase12}, number = 12, pages = {25–}, title = {Natural variation in Arabidopsis Cvi-0 accession reveals an important role of MPK12 in guard cell CO2 signaling}, volume = 14, year = 2016 }

  %0 Journal Article %1 jakobson2016natural %A Jakobson, L. %A Vaahtera, L. %A Toldsepp, K. %A Nuhkat, M. %A Wang, C. %A Wang, Y. S. %A Horak, H. %A Valk, E. %A Pechter, P. %A Sindarovska, Y. %A Tang, J. %A Xiao, C. L. %A Xu, Y. %A Talas, U. G. %A Garcia-Sosa, A. T. %A Kangasjarvi, S. %A Maran, U. %A Remm, M. %A Roelfsema, M. R. G. %A Hu, H. H. %A Kangasjarvi, J. %A Loog, M. %A Schroeder, J. I. %A Kollist, H. %A Brosche, M. %B Plos Biology %D 2016 %N 12 %P 25-- %R 10.1371/journal.pbio.2000322 %T Natural variation in Arabidopsis Cvi-0 accession reveals an important role of MPK12 in guard cell CO2 signaling %U http://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.2000322 %V 14 %X Plant gas exchange is regulated by guard cells that form stomatal pores. Stomatal adjustments are crucial for plant survival; they regulate uptake of CO2 for photosynthesis, loss of water, and entrance of air pollutants such as ozone. We mapped ozone hypersensitivity, more open stomata, and stomatal CO2-insensitivity phenotypes of the Arabidopsis thaliana accession Cvi-0 to a single amino acid substitution in MITOGEN-ACTIVATED PROTEIN (MAP) KINASE 12 (MPK12). In parallel, we showed that stomatal CO2-insensitivity phenotypes of a mutant cis (CO2-insensitive) were caused by a deletion of MPK12. Lack of MPK12 impaired bicarbonate-induced activation of S-type anion channels. We demonstrated that MPK12 interacted with the protein kinase HIGH LEAF TEMPERATURE 1 (HT1)—a central node in guard cell CO2 signaling—and that MPK12 functions as an inhibitor of HT1. These data provide a new function for plant MPKs as protein kinase inhibitors and suggest a mechanism through which guard cell CO2 signaling controls plant water management.
• Roelfsema, M. and Hedrich, R. (2016) ‘Do stomata of evolutionary distant species differ in sensitivity to environmental signals?’, New phytologist, 211(3), 767–770, available: https://doi.org/10.1111/nph.14074.
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  The emergence of stomatal pores represents a major step in the evolution of land plants that enabled them to survive periodical limitations of water supply in terrestrial environments. By the closure of these pores, plants can limit transpiration and thus prevent excessive loss of water to the atmosphere. By contrast, the opening of stomata is required to enable uptake of CO2 for photosynthesis. Plants have evolved at least two regulatory mechanisms that couple stomatal opening to the demand for water and CO2. However, it is debated whether the ability to sense CO2 and humidity developed early in evolution, or if stomata gained these properties progressively. A study conducted by Franks & Britton-Harper, published in this issue of the New Phytologist (pp. 819–827), shows that CO2-sensing occurs in stomata of three evolutionary-distant fern species, thus favoring the hypothesis that stomatal responses to CO2 developed before seed plants separated from ferns c. 300 million years ago.

  @article{roelfsema2016stomata, abstract = {The emergence of stomatal pores represents a major step in the evolution of land plants that enabled them to survive periodical limitations of water supply in terrestrial environments. By the closure of these pores, plants can limit transpiration and thus prevent excessive loss of water to the atmosphere. By contrast, the opening of stomata is required to enable uptake of CO2 for photosynthesis. Plants have evolved at least two regulatory mechanisms that couple stomatal opening to the demand for water and CO2. However, it is debated whether the ability to sense CO2 and humidity developed early in evolution, or if stomata gained these properties progressively. A study conducted by Franks & Britton-Harper, published in this issue of the New Phytologist (pp. 819–827), shows that CO2-sensing occurs in stomata of three evolutionary-distant fern species, thus favoring the hypothesis that stomatal responses to CO2 developed before seed plants separated from ferns c. 300 million years ago.}, author = {Roelfsema, M.R.G. and Hedrich, R.}, journal = {New phytologist}, keywords = {ferns}, number = 3, pages = {767-770}, title = {Do stomata of evolutionary distant species differ in sensitivity to environmental signals?}, volume = 211, year = 2016 }

  %0 Journal Article %1 roelfsema2016stomata %A Roelfsema, M.R.G. %A Hedrich, R. %D 2016 %J New phytologist %N 3 %P 767-770 %R 10.1111/nph.14074 %T Do stomata of evolutionary distant species differ in sensitivity to environmental signals? %U http://onlinelibrary.wiley.com/doi/10.1111/nph.14074/full %V 211 %X The emergence of stomatal pores represents a major step in the evolution of land plants that enabled them to survive periodical limitations of water supply in terrestrial environments. By the closure of these pores, plants can limit transpiration and thus prevent excessive loss of water to the atmosphere. By contrast, the opening of stomata is required to enable uptake of CO2 for photosynthesis. Plants have evolved at least two regulatory mechanisms that couple stomatal opening to the demand for water and CO2. However, it is debated whether the ability to sense CO2 and humidity developed early in evolution, or if stomata gained these properties progressively. A study conducted by Franks & Britton-Harper, published in this issue of the New Phytologist (pp. 819–827), shows that CO2-sensing occurs in stomata of three evolutionary-distant fern species, thus favoring the hypothesis that stomatal responses to CO2 developed before seed plants separated from ferns c. 300 million years ago.
• Voss, L.J., Hedrich, R., and Roelfsema, M.R.G. (2016) ‘Current injection provokes rapid expansion of the guard cell cytosolic volume and triggers Ca2+ signals’, Molecular plant, 9(3), 471–80, available: https://doi.org/10.1016/j.molp.2016.02.004.
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  High-resolution microscopy opens the door for detailed single-cell studies with fluorescent reporter dyes and proteins. We used a confocal spinning disc microscope to monitor fluorescent dyes and the fluorescent protein Venus in tobacco and Arabidopsis guard cells. Multi-barreled microelectrodes were used to inject dyes and apply voltage pulses, which provoke transient rises in the cytosolic Ca2+ level. Voltage pulses also caused changes in the distribution of Lucifer Yellow and Venus, which pointed to a reversible increase of guard cell cytosolic volume. The dynamic cytosolic volume changes turned out to be provoked by current injection of ions. A reduction of the clamp current, by blocking K+ uptake channels with Cs+, strongly suppressed the cytosolic volume changes. Cs+ not only inhibited the expansion of the cytosol, but also inhibited hyperpolarization-induced elevations of the cytosolic Ca2+ concentration. A complete loss of voltage-induced Ca2+ signals occurred when Ca2+-permeable plasma membrane channels were simultaneously blocked with La3+. This shows that two mechanisms cause hyperpolarizationinduced elevation of the cytosolic Ca2+-concentration: (i) activation of voltage-dependent Ca2+-permeable channels, (ii) osmotically induced expansion of the cytosol, which leads to a release of Ca2+ from intracellular stores.

  @article{voss2016current, abstract = {High-resolution microscopy opens the door for detailed single-cell studies with fluorescent reporter dyes and proteins. We used a confocal spinning disc microscope to monitor fluorescent dyes and the fluorescent protein Venus in tobacco and Arabidopsis guard cells. Multi-barreled microelectrodes were used to inject dyes and apply voltage pulses, which provoke transient rises in the cytosolic Ca2+ level. Voltage pulses also caused changes in the distribution of Lucifer Yellow and Venus, which pointed to a reversible increase of guard cell cytosolic volume. The dynamic cytosolic volume changes turned out to be provoked by current injection of ions. A reduction of the clamp current, by blocking K+ uptake channels with Cs+, strongly suppressed the cytosolic volume changes. Cs+ not only inhibited the expansion of the cytosol, but also inhibited hyperpolarization-induced elevations of the cytosolic Ca2+ concentration. A complete loss of voltage-induced Ca2+ signals occurred when Ca2+-permeable plasma membrane channels were simultaneously blocked with La3+. This shows that two mechanisms cause hyperpolarizationinduced elevation of the cytosolic Ca2+-concentration: (i) activation of voltage-dependent Ca2+-permeable channels, (ii) osmotically induced expansion of the cytosol, which leads to a release of Ca2+ from intracellular stores.}, author = {Voss, L. J. and Hedrich, R. and Roelfsema, M. R. G.}, journal = {Molecular plant}, keywords = {expansion}, number = 3, pages = {471-80}, title = {Current injection provokes rapid expansion of the guard cell cytosolic volume and triggers Ca2+ signals}, volume = 9, year = 2016 }

  %0 Journal Article %1 voss2016current %A Voss, L. J. %A Hedrich, R. %A Roelfsema, M. R. G. %D 2016 %J Molecular plant %N 3 %P 471-80 %R 10.1016/j.molp.2016.02.004 %T Current injection provokes rapid expansion of the guard cell cytosolic volume and triggers Ca2+ signals %U http://www.ncbi.nlm.nih.gov/pubmed/26902185http://ac.els-cdn.com/S167420521600037X/1-s2.0-S167420521600037X-main.pdf?_tid=6f69c7e8-57eb-11e6-9221-00000aab0f6c&acdnat=1470058118_4d91eb2782ae5a415e5d1797c8feffbc %V 9 %X High-resolution microscopy opens the door for detailed single-cell studies with fluorescent reporter dyes and proteins. We used a confocal spinning disc microscope to monitor fluorescent dyes and the fluorescent protein Venus in tobacco and Arabidopsis guard cells. Multi-barreled microelectrodes were used to inject dyes and apply voltage pulses, which provoke transient rises in the cytosolic Ca2+ level. Voltage pulses also caused changes in the distribution of Lucifer Yellow and Venus, which pointed to a reversible increase of guard cell cytosolic volume. The dynamic cytosolic volume changes turned out to be provoked by current injection of ions. A reduction of the clamp current, by blocking K+ uptake channels with Cs+, strongly suppressed the cytosolic volume changes. Cs+ not only inhibited the expansion of the cytosol, but also inhibited hyperpolarization-induced elevations of the cytosolic Ca2+ concentration. A complete loss of voltage-induced Ca2+ signals occurred when Ca2+-permeable plasma membrane channels were simultaneously blocked with La3+. This shows that two mechanisms cause hyperpolarizationinduced elevation of the cytosolic Ca2+-concentration: (i) activation of voltage-dependent Ca2+-permeable channels, (ii) osmotically induced expansion of the cytosol, which leads to a release of Ca2+ from intracellular stores.

• Guzel Deger, A., Scherzer, S., Nuhkat, M., Kedzierska, J., Kollist, H., Brosche, M., Unyayar, S., Boudsocq, M., Hedrich, R., and Roelfsema, M.R.G. (2015) ‘Guard cell SLAC1-type anion channels mediate flagellin-induced stomatal closure’, The New phytologist, 208(1), 162–73, available: https://doi.org/10.1111/nph.13435.
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  During infection plants recognize microbe-associated molecular patterns (MAMPs), and this leads to stomatal closure. This study analyzes the molecular mechanisms underlying this MAMP response and its interrelation with ABA signaling. Stomata in intact Arabidopsis thaliana plants were stimulated with the bacterial MAMP flg22, or the stress hormone ABA, by using the noninvasive nanoinfusion technique. Intracellular double-barreled microelectrodes were applied to measure the activity of plasma membrane ion channels. Flg22 induced rapid stomatal closure and stimulated the SLAC1 and SLAH3 anion channels in guard cells. Loss of both channels resulted in cells that lacked flg22-induced anion channel activity and stomata that did not close in response to flg22 or ABA. Rapid flg22-dependent stomatal closure was impaired in plants that were flagellin receptor (FLS2)-deficient, as well as in the ost1-2 (Open Stomata 1) mutant, which lacks a key ABA-signaling protein kinase. By contrast, stomata of the ABA protein phosphatase mutant abi1-1 (ABscisic acid Insensitive 1) remained flg22-responsive. These data suggest that the initial steps in flg22 and ABA signaling are different, but that the pathways merge at the level of OST1 and lead to activation of SLAC1 and SLAH3 anion channels.

  @article{guzeldeger2015guard, abstract = {During infection plants recognize microbe-associated molecular patterns (MAMPs), and this leads to stomatal closure. This study analyzes the molecular mechanisms underlying this MAMP response and its interrelation with ABA signaling. Stomata in intact Arabidopsis thaliana plants were stimulated with the bacterial MAMP flg22, or the stress hormone ABA, by using the noninvasive nanoinfusion technique. Intracellular double-barreled microelectrodes were applied to measure the activity of plasma membrane ion channels. Flg22 induced rapid stomatal closure and stimulated the SLAC1 and SLAH3 anion channels in guard cells. Loss of both channels resulted in cells that lacked flg22-induced anion channel activity and stomata that did not close in response to flg22 or ABA. Rapid flg22-dependent stomatal closure was impaired in plants that were flagellin receptor (FLS2)-deficient, as well as in the ost1-2 (Open Stomata 1) mutant, which lacks a key ABA-signaling protein kinase. By contrast, stomata of the ABA protein phosphatase mutant abi1-1 (ABscisic acid Insensitive 1) remained flg22-responsive. These data suggest that the initial steps in flg22 and ABA signaling are different, but that the pathways merge at the level of OST1 and lead to activation of SLAC1 and SLAH3 anion channels.}, address = {Molecular Plant Physiology and Biophysics, Julius-von-Sachs Institute for Biosciences, Biocenter, University of Wurzburg, Julius-von-Sachs-Platz 2, D-97082, Wurzburg, Germany.}, author = {Guzel Deger, Aysin and Scherzer, Sonke and Nuhkat, Maris and Kedzierska, Justyna and Kollist, Hannes and Brosche, Mikael and Unyayar, Serpil and Boudsocq, Marie and Hedrich, Rainer and Roelfsema, M. Rob G.}, journal = {The New phytologist}, keywords = {flg22}, number = 1, pages = {162-73}, title = {Guard cell SLAC1-type anion channels mediate flagellin-induced stomatal closure}, volume = 208, year = 2015 }

  %0 Journal Article %1 guzeldeger2015guard %A Guzel Deger, Aysin %A Scherzer, Sonke %A Nuhkat, Maris %A Kedzierska, Justyna %A Kollist, Hannes %A Brosche, Mikael %A Unyayar, Serpil %A Boudsocq, Marie %A Hedrich, Rainer %A Roelfsema, M. Rob G. %C Molecular Plant Physiology and Biophysics, Julius-von-Sachs Institute for Biosciences, Biocenter, University of Wurzburg, Julius-von-Sachs-Platz 2, D-97082, Wurzburg, Germany. %D 2015 %J The New phytologist %N 1 %P 162-73 %R 10.1111/nph.13435 %T Guard cell SLAC1-type anion channels mediate flagellin-induced stomatal closure %U http://onlinelibrary.wiley.com/doi/10.1111/nph.13435/full %V 208 %X During infection plants recognize microbe-associated molecular patterns (MAMPs), and this leads to stomatal closure. This study analyzes the molecular mechanisms underlying this MAMP response and its interrelation with ABA signaling. Stomata in intact Arabidopsis thaliana plants were stimulated with the bacterial MAMP flg22, or the stress hormone ABA, by using the noninvasive nanoinfusion technique. Intracellular double-barreled microelectrodes were applied to measure the activity of plasma membrane ion channels. Flg22 induced rapid stomatal closure and stimulated the SLAC1 and SLAH3 anion channels in guard cells. Loss of both channels resulted in cells that lacked flg22-induced anion channel activity and stomata that did not close in response to flg22 or ABA. Rapid flg22-dependent stomatal closure was impaired in plants that were flagellin receptor (FLS2)-deficient, as well as in the ost1-2 (Open Stomata 1) mutant, which lacks a key ABA-signaling protein kinase. By contrast, stomata of the ABA protein phosphatase mutant abi1-1 (ABscisic acid Insensitive 1) remained flg22-responsive. These data suggest that the initial steps in flg22 and ABA signaling are different, but that the pathways merge at the level of OST1 and lead to activation of SLAC1 and SLAH3 anion channels.
• Wang, Y., Dindas, J., Rienmuller, F., Krebs, M., Waadt, R., Schumacher, K., Wu, W.-H., Hedrich, R., and Roelfsema, M.R.G. (2015) ‘Cytosolic Ca2+ signals enhance the vacuolar ion conductivity of bulging Arabidopsis root hair cells’, Molecular plant, 8(11), 1665–74, available: https://doi.org/10.1016/j.molp.2015.07.009.
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  Plant cell expansion depends on the uptake of solutes across the plasma membrane and their storage within the vacuole. In contrast to the well-studied plasma membrane, little is known about the regulation of ion transport at the vacuolar membrane. We therefore established an experimental approach to study vacuolar ion transport in intact Arabidopsis root cells, with multi-barreled microelectrodes. The subcellular position of electrodes was detected by imaging current-injected fluorescent dyes. Comparison of measurements with electrodes in the cytosol and vacuole revealed an average vacuolar membrane potential of �31 mV. Voltage clamp recordings of single vacuoles resolved the activity of voltage-independent and slowly deactivating channels. In bulging root hairs that express the Ca2+ sensor R-GECO1, rapid elevation of the cytosolic Ca2+ concentration was observed, after impalement with microelectrodes, or injection of the Ca2+ chelator BAPTA. Elevation of the cytosolic Ca2+ level stimulated the activity of voltage-independent channels in the vacuolar membrane. Likewise, the vacuolar ion conductance was enhanced during a sudden increase of the cytosolic Ca2+ level in cells injected with fluorescent Ca2+ indicator FURA-2. These data thus show that cytosolic Ca2+ signals can rapidly activate vacuolar ion channels, which may prevent rupture of the vacuolar membrane, when facing mechanical forces.

  @article{wang2015cytosolic, abstract = {Plant cell expansion depends on the uptake of solutes across the plasma membrane and their storage within the vacuole. In contrast to the well-studied plasma membrane, little is known about the regulation of ion transport at the vacuolar membrane. We therefore established an experimental approach to study vacuolar ion transport in intact Arabidopsis root cells, with multi-barreled microelectrodes. The subcellular position of electrodes was detected by imaging current-injected fluorescent dyes. Comparison of measurements with electrodes in the cytosol and vacuole revealed an average vacuolar membrane potential of �31 mV. Voltage clamp recordings of single vacuoles resolved the activity of voltage-independent and slowly deactivating channels. In bulging root hairs that express the Ca2+ sensor R-GECO1, rapid elevation of the cytosolic Ca2+ concentration was observed, after impalement with microelectrodes, or injection of the Ca2+ chelator BAPTA. Elevation of the cytosolic Ca2+ level stimulated the activity of voltage-independent channels in the vacuolar membrane. Likewise, the vacuolar ion conductance was enhanced during a sudden increase of the cytosolic Ca2+ level in cells injected with fluorescent Ca2+ indicator FURA-2. These data thus show that cytosolic Ca2+ signals can rapidly activate vacuolar ion channels, which may prevent rupture of the vacuolar membrane, when facing mechanical forces.}, author = {Wang, Yi and Dindas, Julian and Rienmuller, Florian and Krebs, Melanie and Waadt, Rainer and Schumacher, Karin and Wu, Wei-Hua and Hedrich, Rainer and Roelfsema, M. Rob G.}, journal = {Molecular plant}, keywords = {Ca2+-signals}, number = 11, pages = {1665-74}, title = {Cytosolic Ca2+ signals enhance the vacuolar ion conductivity of bulging Arabidopsis root hair cells}, volume = 8, year = 2015 }

  %0 Journal Article %1 wang2015cytosolic %A Wang, Yi %A Dindas, Julian %A Rienmuller, Florian %A Krebs, Melanie %A Waadt, Rainer %A Schumacher, Karin %A Wu, Wei-Hua %A Hedrich, Rainer %A Roelfsema, M. Rob G. %D 2015 %J Molecular plant %N 11 %P 1665-74 %R 10.1016/j.molp.2015.07.009 %T Cytosolic Ca2+ signals enhance the vacuolar ion conductivity of bulging Arabidopsis root hair cells %U http://www.ncbi.nlm.nih.gov/pubmed/26232520http://ac.els-cdn.com/S1674205215003032/1-s2.0-S1674205215003032-main.pdf?_tid=3ec57daa-8869-11e5-a16b-00000aacb35f&acdnat=1447242311_51bcc2c85b3dcfc9eabcc16868743563 %V 8 %X Plant cell expansion depends on the uptake of solutes across the plasma membrane and their storage within the vacuole. In contrast to the well-studied plasma membrane, little is known about the regulation of ion transport at the vacuolar membrane. We therefore established an experimental approach to study vacuolar ion transport in intact Arabidopsis root cells, with multi-barreled microelectrodes. The subcellular position of electrodes was detected by imaging current-injected fluorescent dyes. Comparison of measurements with electrodes in the cytosol and vacuole revealed an average vacuolar membrane potential of �31 mV. Voltage clamp recordings of single vacuoles resolved the activity of voltage-independent and slowly deactivating channels. In bulging root hairs that express the Ca2+ sensor R-GECO1, rapid elevation of the cytosolic Ca2+ concentration was observed, after impalement with microelectrodes, or injection of the Ca2+ chelator BAPTA. Elevation of the cytosolic Ca2+ level stimulated the activity of voltage-independent channels in the vacuolar membrane. Likewise, the vacuolar ion conductance was enhanced during a sudden increase of the cytosolic Ca2+ level in cells injected with fluorescent Ca2+ indicator FURA-2. These data thus show that cytosolic Ca2+ signals can rapidly activate vacuolar ion channels, which may prevent rupture of the vacuolar membrane, when facing mechanical forces.

• Kollist, H., Nuhkat, M., and Roelfsema, M.R.G. (2014) ‘Closing gaps: linking elements that control stomatal movement’, New Phytologist, 203(1), 44–62, available: https://doi.org/10.1111/nph.12832.
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  Stomata are an attractive experimental system in plant biology, because the responses of guard cells to environmental signals can be directly linked to changes in the aperture of stomatal pores. In this review, the mechanics of stomatal movement are discussed in relation to ion transport in guard cells. Emphasis is placed on the ion pumps, transporters, and channels in the plasma membrane, as well as in the vacuolar membrane. The biophysical properties of transport proteins for H+, K+, Ca2+, and anions are discussed and related to their function in guard cells during stomatal movements. Guard cell signaling pathways for ABA, CO2, ozone, microbe-associated molecular patterns (MAMPs) and blue light are presented. Special attention is given to the regulation of the slow anion channel (SLAC) and SLAC homolog (SLAH)-type anion channels by the ABA signalosome. Over the last decade, several knowledge gaps in the regulation of ion transport in guard cells have been closed. The current state of knowledge is an excellent starting point for tackling important open questions concerning stress tolerance in plants.

  @article{kollist2014closing, abstract = {Stomata are an attractive experimental system in plant biology, because the responses of guard cells to environmental signals can be directly linked to changes in the aperture of stomatal pores. In this review, the mechanics of stomatal movement are discussed in relation to ion transport in guard cells. Emphasis is placed on the ion pumps, transporters, and channels in the plasma membrane, as well as in the vacuolar membrane. The biophysical properties of transport proteins for H+, K+, Ca2+, and anions are discussed and related to their function in guard cells during stomatal movements. Guard cell signaling pathways for ABA, CO2, ozone, microbe-associated molecular patterns (MAMPs) and blue light are presented. Special attention is given to the regulation of the slow anion channel (SLAC) and SLAC homolog (SLAH)-type anion channels by the ABA signalosome. Over the last decade, several knowledge gaps in the regulation of ion transport in guard cells have been closed. The current state of knowledge is an excellent starting point for tackling important open questions concerning stress tolerance in plants.}, author = {Kollist, H. and Nuhkat, M. and Roelfsema, M. R. G.}, journal = {New Phytologist}, keywords = {review}, number = 1, pages = {44-62}, title = {Closing gaps: linking elements that control stomatal movement}, volume = 203, year = 2014 }

  %0 Journal Article %1 kollist2014closing %A Kollist, H. %A Nuhkat, M. %A Roelfsema, M. R. G. %D 2014 %J New Phytologist %N 1 %P 44-62 %R 10.1111/nph.12832 %T Closing gaps: linking elements that control stomatal movement %U http://onlinelibrary.wiley.com/doi/10.1111/nph.12832/full %V 203 %X Stomata are an attractive experimental system in plant biology, because the responses of guard cells to environmental signals can be directly linked to changes in the aperture of stomatal pores. In this review, the mechanics of stomatal movement are discussed in relation to ion transport in guard cells. Emphasis is placed on the ion pumps, transporters, and channels in the plasma membrane, as well as in the vacuolar membrane. The biophysical properties of transport proteins for H+, K+, Ca2+, and anions are discussed and related to their function in guard cells during stomatal movements. Guard cell signaling pathways for ABA, CO2, ozone, microbe-associated molecular patterns (MAMPs) and blue light are presented. Special attention is given to the regulation of the slow anion channel (SLAC) and SLAC homolog (SLAH)-type anion channels by the ABA signalosome. Over the last decade, several knowledge gaps in the regulation of ion transport in guard cells have been closed. The current state of knowledge is an excellent starting point for tackling important open questions concerning stress tolerance in plants.

• Willige, B.C., Ahlers, S., Zourelidou, M., Barbosa, I.C.R., Demarsy, E., Trevisan, M., Davis, P.A., Roelfsema, M.R.G., Hangarter, R., Fankhauser, C., and Schwechheimer, C. (2013) ‘D6PK AGCVIII kinases are required for auxin transport and phototropic hypocotyl bending in Arabidopsis’, Plant Cell, 25(5), 1674–1688, available: https://doi.org/10.1105/tpc.113.111484.
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  Phototropic hypocotyl bending in response to blue light excitation is an important adaptive process that helps plants to optimize their exposure to light. In Arabidopsis thaliana, phototropic hypocotyl bending is initiated by the blue light receptors and protein kinases phototropin1 (phot1) and phot2. Phototropic responses also require auxin transport and were shown to be partially compromised in mutants of the PIN-FORMED (PIN) auxin efflux facilitators. We previously described the D6 PROTEIN KINASE (D6PK) subfamily of AGCVIII kinases, which we proposed to directly regulate PIN-mediated auxin transport. Here, we show that phototropic hypocotyl bending is strongly dependent on the activity of D6PKs and the PIN proteins PIN3, PIN4, and PIN7. While early blue light and phot-dependent signaling events are not affected by the loss of D6PKs, we detect a gradual loss of PIN3 phosphorylation in d6pk mutants of increasing complexity that is most severe in the d6pk d6pkl1 d6pkl2 d6pkl3 quadruple mutant. This is accompanied by a reduction of basipetal auxin transport in the hypocotyls of d6pk as well as in pin mutants. Based on our data, we propose that D6PK-dependent PIN regulation promotes auxin transport and that auxin transport in the hypocotyl is a prerequisite for phot1-dependent hypocotyl bending.

  @article{willige2013agcviii, abstract = {Phototropic hypocotyl bending in response to blue light excitation is an important adaptive process that helps plants to optimize their exposure to light. In Arabidopsis thaliana, phototropic hypocotyl bending is initiated by the blue light receptors and protein kinases phototropin1 (phot1) and phot2. Phototropic responses also require auxin transport and were shown to be partially compromised in mutants of the PIN-FORMED (PIN) auxin efflux facilitators. We previously described the D6 PROTEIN KINASE (D6PK) subfamily of AGCVIII kinases, which we proposed to directly regulate PIN-mediated auxin transport. Here, we show that phototropic hypocotyl bending is strongly dependent on the activity of D6PKs and the PIN proteins PIN3, PIN4, and PIN7. While early blue light and phot-dependent signaling events are not affected by the loss of D6PKs, we detect a gradual loss of PIN3 phosphorylation in d6pk mutants of increasing complexity that is most severe in the d6pk d6pkl1 d6pkl2 d6pkl3 quadruple mutant. This is accompanied by a reduction of basipetal auxin transport in the hypocotyls of d6pk as well as in pin mutants. Based on our data, we propose that D6PK-dependent PIN regulation promotes auxin transport and that auxin transport in the hypocotyl is a prerequisite for phot1-dependent hypocotyl bending.}, address = {Department of Plant Systems Biology, Technische Universität München, 85354 Freising-Weihenstephan, Germany}, author = {Willige, B. C. and Ahlers, S. and Zourelidou, M. and Barbosa, I. C. R. and Demarsy, E. and Trevisan, M. and Davis, P. A. and Roelfsema, M. R. G. and Hangarter, R. and Fankhauser, C. and Schwechheimer, C.}, booktitle = {Plant Cell}, keywords = {blue-light-receptors}, number = 5, pages = {1674-1688}, title = {D6PK AGCVIII kinases are required for auxin transport and phototropic hypocotyl bending in Arabidopsis}, volume = 25, year = 2013 }

  %0 Journal Article %1 willige2013agcviii %A Willige, B. C. %A Ahlers, S. %A Zourelidou, M. %A Barbosa, I. C. R. %A Demarsy, E. %A Trevisan, M. %A Davis, P. A. %A Roelfsema, M. R. G. %A Hangarter, R. %A Fankhauser, C. %A Schwechheimer, C. %B Plant Cell %C Department of Plant Systems Biology, Technische Universität München, 85354 Freising-Weihenstephan, Germany %D 2013 %N 5 %P 1674-1688 %R 10.1105/tpc.113.111484 %T D6PK AGCVIII kinases are required for auxin transport and phototropic hypocotyl bending in Arabidopsis %U /brokenurl#<Go to ISI>://WOS:000321035800017 %V 25 %X Phototropic hypocotyl bending in response to blue light excitation is an important adaptive process that helps plants to optimize their exposure to light. In Arabidopsis thaliana, phototropic hypocotyl bending is initiated by the blue light receptors and protein kinases phototropin1 (phot1) and phot2. Phototropic responses also require auxin transport and were shown to be partially compromised in mutants of the PIN-FORMED (PIN) auxin efflux facilitators. We previously described the D6 PROTEIN KINASE (D6PK) subfamily of AGCVIII kinases, which we proposed to directly regulate PIN-mediated auxin transport. Here, we show that phototropic hypocotyl bending is strongly dependent on the activity of D6PKs and the PIN proteins PIN3, PIN4, and PIN7. While early blue light and phot-dependent signaling events are not affected by the loss of D6PKs, we detect a gradual loss of PIN3 phosphorylation in d6pk mutants of increasing complexity that is most severe in the d6pk d6pkl1 d6pkl2 d6pkl3 quadruple mutant. This is accompanied by a reduction of basipetal auxin transport in the hypocotyls of d6pk as well as in pin mutants. Based on our data, we propose that D6PK-dependent PIN regulation promotes auxin transport and that auxin transport in the hypocotyl is a prerequisite for phot1-dependent hypocotyl bending.
• Roelfsema, M.R.G. and Kollist, H. (2013) ‘Tiny pores with a global impact’, New Phytologist, 197(1), 11–15, available: https://doi.org/10.1111/nph.12050.
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  29th New Phytologist Symposium: Stomata 2012, in Manchester, UK, July 2012 The transition of plants from sea to terrestrial environments is undoubtedly one of the key steps during evolution. It required formation of a gas-impermeable cuticle and guard cells, which form stomatal pores that act as valves for gas-exchange between plants and surrounding environment. Closed stomata shield above-ground plant tissues from harmful factors, such as drought, air pollution and pathogenic microorganisms. Opening of these microscopic pores allows plants to assimilate vast amounts of the greenhouse gas CO2 and return more than half of the terrestrial rainfall to the atmosphere. Because of these large gas fluxes, stomata are not only of crucial importance for plant growth and agriculture, but also have a major impact on our climate (Fig. 1). No wonder that the function, development and evolution of stomata have attracted the attention of numerous scientists worldwide. The New Phytologist Symposium ‘Stomata 2012’ provided a platform for them to discuss recent progress as well as unsolved issues.

  @article{roelfsema2013pores, abstract = {29th New Phytologist Symposium: Stomata 2012, in Manchester, UK, July 2012 The transition of plants from sea to terrestrial environments is undoubtedly one of the key steps during evolution. It required formation of a gas-impermeable cuticle and guard cells, which form stomatal pores that act as valves for gas-exchange between plants and surrounding environment. Closed stomata shield above-ground plant tissues from harmful factors, such as drought, air pollution and pathogenic microorganisms. Opening of these microscopic pores allows plants to assimilate vast amounts of the greenhouse gas CO2 and return more than half of the terrestrial rainfall to the atmosphere. Because of these large gas fluxes, stomata are not only of crucial importance for plant growth and agriculture, but also have a major impact on our climate (Fig. 1). No wonder that the function, development and evolution of stomata have attracted the attention of numerous scientists worldwide. The New Phytologist Symposium ‘Stomata 2012’ provided a platform for them to discuss recent progress as well as unsolved issues.}, author = {Roelfsema, M. R. G. and Kollist, H.}, journal = {New Phytologist}, keywords = {water-stress}, number = 1, pages = {11-15}, title = {Tiny pores with a global impact}, volume = 197, year = 2013 }

  %0 Journal Article %1 roelfsema2013pores %A Roelfsema, M. R. G. %A Kollist, H. %D 2013 %J New Phytologist %N 1 %P 11-15 %R 10.1111/nph.12050 %T Tiny pores with a global impact %U http://onlinelibrary.wiley.com/doi/10.1111/nph.12050/full %V 197 %X 29th New Phytologist Symposium: Stomata 2012, in Manchester, UK, July 2012 The transition of plants from sea to terrestrial environments is undoubtedly one of the key steps during evolution. It required formation of a gas-impermeable cuticle and guard cells, which form stomatal pores that act as valves for gas-exchange between plants and surrounding environment. Closed stomata shield above-ground plant tissues from harmful factors, such as drought, air pollution and pathogenic microorganisms. Opening of these microscopic pores allows plants to assimilate vast amounts of the greenhouse gas CO2 and return more than half of the terrestrial rainfall to the atmosphere. Because of these large gas fluxes, stomata are not only of crucial importance for plant growth and agriculture, but also have a major impact on our climate (Fig. 1). No wonder that the function, development and evolution of stomata have attracted the attention of numerous scientists worldwide. The New Phytologist Symposium ‘Stomata 2012’ provided a platform for them to discuss recent progress as well as unsolved issues.

• Roelfsema, M.R.G., Hedrich, R., and Geiger, D. (2012) ‘Anion channels: master switches of stress responses’, Trends in plant science, 17(4), 221–229, available: https://doi.org/10.1016/j.tplants.2012.01.009.
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  During stress, plant cells activate anion channels and trigger the release of anions across the plasma membrane. Recently, two new gene families have been identified that encode major groups of anion channels. The SLAC/SLAH channels are characterized by slow voltage-dependent activation (S-type), whereas ALMT genes encode rapid-activating channels (R-type). Both S- and R-type channels are stimulated in guard cells by the stress hormone ABA, which leads to stomatal closure. Besides their role in ABA-dependent stomatal movement, anion channels are also activated by biotic stress factors such as microbe-associated molecular patterns (MAMPs). Given that anion channels occur throughout the plant kingdom, they are likely to serve a general function as master switches of stress responses.

  @article{roelfsema2012anion, abstract = {During stress, plant cells activate anion channels and trigger the release of anions across the plasma membrane. Recently, two new gene families have been identified that encode major groups of anion channels. The SLAC/SLAH channels are characterized by slow voltage-dependent activation (S-type), whereas ALMT genes encode rapid-activating channels (R-type). Both S- and R-type channels are stimulated in guard cells by the stress hormone ABA, which leads to stomatal closure. Besides their role in ABA-dependent stomatal movement, anion channels are also activated by biotic stress factors such as microbe-associated molecular patterns (MAMPs). Given that anion channels occur throughout the plant kingdom, they are likely to serve a general function as master switches of stress responses.}, author = {Roelfsema, M. R. G. and Hedrich, R. and Geiger, D.}, journal = {Trends in plant science}, keywords = {ABA}, number = 4, pages = {221-229}, title = {Anion channels: master switches of stress responses}, volume = 17, year = 2012 }

  %0 Journal Article %1 roelfsema2012anion %A Roelfsema, M. R. G. %A Hedrich, R. %A Geiger, D. %D 2012 %J Trends in plant science %N 4 %P 221-229 %R 10.1016/j.tplants.2012.01.009 %T Anion channels: master switches of stress responses %U http://onlinelibrary.wiley.com/doi/10.1002/9780470015902.a0002075.pub2 %V 17 %X During stress, plant cells activate anion channels and trigger the release of anions across the plasma membrane. Recently, two new gene families have been identified that encode major groups of anion channels. The SLAC/SLAH channels are characterized by slow voltage-dependent activation (S-type), whereas ALMT genes encode rapid-activating channels (R-type). Both S- and R-type channels are stimulated in guard cells by the stress hormone ABA, which leads to stomatal closure. Besides their role in ABA-dependent stomatal movement, anion channels are also activated by biotic stress factors such as microbe-associated molecular patterns (MAMPs). Given that anion channels occur throughout the plant kingdom, they are likely to serve a general function as master switches of stress responses.
• Hedrich, R., Becker, D., Geiger, D., Marten, I., and Roelfsema, M. (2012) ‘Role of ion channels in plants’, in Y., O., ed., Tokyo: Springer, 295–322, available: http://www.springer.com/de/book/9784431539926.
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  @inbook{r2012channels, address = {Tokyo}, author = {Hedrich, R. and Becker, D. and Geiger, D. and Marten, I. and Roelfsema, M.R.G.}, editor = {Y., Okada}, keywords = {plant-cell}, pages = {295-322}, publisher = {Springer}, title = {Role of ion channels in plants}, volume = {Patch clamp techniques}, year = 2012 }

  %0 Book Section %1 r2012channels %A Hedrich, R. %A Becker, D. %A Geiger, D. %A Marten, I. %A Roelfsema, M.R.G. %C Tokyo %D 2012 %E Y., Okada %I Springer %P 295-322 %T Role of ion channels in plants %U http://www.springer.com/de/book/9784431539926 %V Patch clamp techniques

• Mumm, P., Wolf, T., Fromm, J., Roelfsema, M.R.G., and Marten, I. (2011) ‘Cell Type-Specific Regulation of Ion Channels Within the Maize Stomatal Complex’, Plant and Cell Physiology, 52(8), 1365–1375, available: https://doi.org/10.1093/pcp/pcr082.
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  @article{Mumm_2011, author = {Mumm, P. and Wolf, T. and Fromm, J. and Roelfsema, M. R. G. and Marten, I.}, journal = {Plant and Cell Physiology}, keywords = {Cell}, month = {06}, number = 8, pages = {1365–1375}, publisher = {Oxford University Press ({OUP})}, title = {Cell Type-Specific Regulation of Ion Channels Within the Maize Stomatal Complex}, volume = 52, year = 2011 }

  %0 Journal Article %1 Mumm_2011 %A Mumm, P. %A Wolf, T. %A Fromm, J. %A Roelfsema, M. R. G. %A Marten, I. %D 2011 %I Oxford University Press ({OUP}) %J Plant and Cell Physiology %N 8 %P 1365--1375 %R 10.1093/pcp/pcr082 %T Cell Type-Specific Regulation of Ion Channels Within the Maize Stomatal Complex %U https://doi.org/10.1093%2Fpcp%2Fpcr082 %V 52
• Koers, S., Guzel-Deger, A., Marten, I., and Roelfsema, M.R.G. (2011) ‘Barley mildew and its elicitor chitosan promote closed stomata by stimulating guard-cell S-type anion channels’, Plant Journal, 68(4), 670–680, available: https://doi.org/10.1111/j.1365-313X.2011.04719.x.
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  Stomatal closure is known to be associated with early defence responses of plant cells triggered by microbe-associated molecular patterns (MAMPs). However, the molecular mechanisms underlying these guard-cell responses have not yet been elucidated. We therefore studied pathogen-induced changes in ion channel activity in Hordeum vulgare guard cells. Barley mildew (Blumeria graminis) hyphae growing on leaves inhibited light-induced stomatal opening, starting at 9 h after inoculation, when appressoria had developed. Alternatively, stomatal closure was induced by nano-infusion of chitosan via open stomata into the sub-stomatal cavity. Experiments using intracellular double-barreled micro-electrodes revealed that mildew stimulated S-type (slow) anion channels in guard cells. These channels enable the efflux of anions from guard cells and also promote K+ extrusion by altering the plasma membrane potential. Stimulation of S-type anion channels was also provoked by nano-infusion of chitosan. These data suggest that MAMPs of mildew hyphae penetrating the cuticle provoke activation of S-type anion channels in guard cells. In response, guard cells extrude K+ salts, resulting in stomatal closure. Plasma membrane anion channels probably represent general targets of MAMP signaling in plants, as these elicitors depolarize the plasma membrane of various cell types.

  @article{koers2011barley, abstract = {Stomatal closure is known to be associated with early defence responses of plant cells triggered by microbe-associated molecular patterns (MAMPs). However, the molecular mechanisms underlying these guard-cell responses have not yet been elucidated. We therefore studied pathogen-induced changes in ion channel activity in Hordeum vulgare guard cells. Barley mildew (Blumeria graminis) hyphae growing on leaves inhibited light-induced stomatal opening, starting at 9 h after inoculation, when appressoria had developed. Alternatively, stomatal closure was induced by nano-infusion of chitosan via open stomata into the sub-stomatal cavity. Experiments using intracellular double-barreled micro-electrodes revealed that mildew stimulated S-type (slow) anion channels in guard cells. These channels enable the efflux of anions from guard cells and also promote K+ extrusion by altering the plasma membrane potential. Stimulation of S-type anion channels was also provoked by nano-infusion of chitosan. These data suggest that MAMPs of mildew hyphae penetrating the cuticle provoke activation of S-type anion channels in guard cells. In response, guard cells extrude K+ salts, resulting in stomatal closure. Plasma membrane anion channels probably represent general targets of MAMP signaling in plants, as these elicitors depolarize the plasma membrane of various cell types.}, author = {Koers, S. and Guzel-Deger, A. and Marten, I. and Roelfsema, M. R. G.}, journal = {Plant Journal}, keywords = {mildew}, number = 4, pages = {670-680}, title = {Barley mildew and its elicitor chitosan promote closed stomata by stimulating guard-cell S-type anion channels}, volume = 68, year = 2011 }

  %0 Journal Article %1 koers2011barley %A Koers, S. %A Guzel-Deger, A. %A Marten, I. %A Roelfsema, M. R. G. %D 2011 %J Plant Journal %N 4 %P 670-680 %R 10.1111/j.1365-313X.2011.04719.x %T Barley mildew and its elicitor chitosan promote closed stomata by stimulating guard-cell S-type anion channels %U http://onlinelibrary.wiley.com/doi/10.1111/j.1365-313X.2011.04719.x/full %V 68 %X Stomatal closure is known to be associated with early defence responses of plant cells triggered by microbe-associated molecular patterns (MAMPs). However, the molecular mechanisms underlying these guard-cell responses have not yet been elucidated. We therefore studied pathogen-induced changes in ion channel activity in Hordeum vulgare guard cells. Barley mildew (Blumeria graminis) hyphae growing on leaves inhibited light-induced stomatal opening, starting at 9 h after inoculation, when appressoria had developed. Alternatively, stomatal closure was induced by nano-infusion of chitosan via open stomata into the sub-stomatal cavity. Experiments using intracellular double-barreled micro-electrodes revealed that mildew stimulated S-type (slow) anion channels in guard cells. These channels enable the efflux of anions from guard cells and also promote K+ extrusion by altering the plasma membrane potential. Stimulation of S-type anion channels was also provoked by nano-infusion of chitosan. These data suggest that MAMPs of mildew hyphae penetrating the cuticle provoke activation of S-type anion channels in guard cells. In response, guard cells extrude K+ salts, resulting in stomatal closure. Plasma membrane anion channels probably represent general targets of MAMP signaling in plants, as these elicitors depolarize the plasma membrane of various cell types.

• Jeworutzki, E., Roelfsema, M.R.G., Anschutz, U., Krol, E., Elzenga, J.T.M., Felix, G., Boller, T., Hedrich, R., and Becker, D. (2010) ‘Early signaling through the Arabidopsis pattern recognition receptors FLS2 and EFR involves Ca2+-associated opening of plasma membrane anion channels’, Plant Journal, 62(3), 367–378, available: https://doi.org/10.1111/j.1365-313X.2010.04155.x.
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  The perception of microbes by plants involves highly conserved molecular signatures that are absent from the host and that are collectively referred to as microbe-associated molecular patterns (MAMPs). The Arabidopsis pattern recognition receptors FLAGELLIN-SENSING 2 (FLS2) and EF-Tu receptor (EFR) represent genetically well studied paradigms that mediate defense against bacterial pathogens. Stimulation of these receptors through their cognate ligands, bacterial flagellin or bacterial elongation factor Tu, leads to a defense response and ultimately to increased resistance. However, little is known about the early signaling pathway of these receptors. Here, we characterize this early response in situ, using an electrophysiological approach. In line with a release of negatively charged molecules, voltage recordings of microelectrode-impaled mesophyll cells and root hairs of Col-0 Arabidopsis plants revealed rapid, dose-dependent membrane potential depolarizations in response to either flg22 or elf18. Using ion-selective microelectrodes, pronounced anion currents were recorded upon application of flg22 and elf18, indicating that the signaling cascades initiated by each of the two receptors converge on the same plasma membrane ion channels. Combined calcium imaging and electrophysiological measurements revealed that the depolarization was superimposed by an increase in cytosolic calcium that was indispensable for depolarization. NADPH oxidase mutants were still depolarized upon elicitor stimulation, suggesting a reactive oxygen species-independent membrane potential response. Furthermore, electrical signaling in response to either flg22 or elf 18 critically depends on the activity of the FLS2-associated receptor-like kinase BAK1, suggesting that activation of FLS2 and EFR lead to BAK1-dependent, calcium-associated plasma membrane anion channel opening as an initial step in the pathogen defense pathway.

  @article{jeworutzki2010early, abstract = {The perception of microbes by plants involves highly conserved molecular signatures that are absent from the host and that are collectively referred to as microbe-associated molecular patterns (MAMPs). The Arabidopsis pattern recognition receptors FLAGELLIN-SENSING 2 (FLS2) and EF-Tu receptor (EFR) represent genetically well studied paradigms that mediate defense against bacterial pathogens. Stimulation of these receptors through their cognate ligands, bacterial flagellin or bacterial elongation factor Tu, leads to a defense response and ultimately to increased resistance. However, little is known about the early signaling pathway of these receptors. Here, we characterize this early response in situ, using an electrophysiological approach. In line with a release of negatively charged molecules, voltage recordings of microelectrode-impaled mesophyll cells and root hairs of Col-0 Arabidopsis plants revealed rapid, dose-dependent membrane potential depolarizations in response to either flg22 or elf18. Using ion-selective microelectrodes, pronounced anion currents were recorded upon application of flg22 and elf18, indicating that the signaling cascades initiated by each of the two receptors converge on the same plasma membrane ion channels. Combined calcium imaging and electrophysiological measurements revealed that the depolarization was superimposed by an increase in cytosolic calcium that was indispensable for depolarization. NADPH oxidase mutants were still depolarized upon elicitor stimulation, suggesting a reactive oxygen species-independent membrane potential response. Furthermore, electrical signaling in response to either flg22 or elf 18 critically depends on the activity of the FLS2-associated receptor-like kinase BAK1, suggesting that activation of FLS2 and EFR lead to BAK1-dependent, calcium-associated plasma membrane anion channel opening as an initial step in the pathogen defense pathway.}, author = {Jeworutzki, E. and Roelfsema, M. R. G. and Anschutz, U. and Krol, E. and Elzenga, J. T. M. and Felix, G. and Boller, T. and Hedrich, R. and Becker, D.}, journal = {Plant Journal}, keywords = {flg22}, number = 3, pages = {367-378}, title = {Early signaling through the Arabidopsis pattern recognition receptors FLS2 and EFR involves Ca2+-associated opening of plasma membrane anion channels}, volume = 62, year = 2010 }

  %0 Journal Article %1 jeworutzki2010early %A Jeworutzki, E. %A Roelfsema, M. R. G. %A Anschutz, U. %A Krol, E. %A Elzenga, J. T. M. %A Felix, G. %A Boller, T. %A Hedrich, R. %A Becker, D. %D 2010 %J Plant Journal %N 3 %P 367-378 %R 10.1111/j.1365-313X.2010.04155.x %T Early signaling through the Arabidopsis pattern recognition receptors FLS2 and EFR involves Ca2+-associated opening of plasma membrane anion channels %U http://onlinelibrary.wiley.com/doi/10.1111/j.1365-313X.2010.04155.x/full %V 62 %X The perception of microbes by plants involves highly conserved molecular signatures that are absent from the host and that are collectively referred to as microbe-associated molecular patterns (MAMPs). The Arabidopsis pattern recognition receptors FLAGELLIN-SENSING 2 (FLS2) and EF-Tu receptor (EFR) represent genetically well studied paradigms that mediate defense against bacterial pathogens. Stimulation of these receptors through their cognate ligands, bacterial flagellin or bacterial elongation factor Tu, leads to a defense response and ultimately to increased resistance. However, little is known about the early signaling pathway of these receptors. Here, we characterize this early response in situ, using an electrophysiological approach. In line with a release of negatively charged molecules, voltage recordings of microelectrode-impaled mesophyll cells and root hairs of Col-0 Arabidopsis plants revealed rapid, dose-dependent membrane potential depolarizations in response to either flg22 or elf18. Using ion-selective microelectrodes, pronounced anion currents were recorded upon application of flg22 and elf18, indicating that the signaling cascades initiated by each of the two receptors converge on the same plasma membrane ion channels. Combined calcium imaging and electrophysiological measurements revealed that the depolarization was superimposed by an increase in cytosolic calcium that was indispensable for depolarization. NADPH oxidase mutants were still depolarized upon elicitor stimulation, suggesting a reactive oxygen species-independent membrane potential response. Furthermore, electrical signaling in response to either flg22 or elf 18 critically depends on the activity of the FLS2-associated receptor-like kinase BAK1, suggesting that activation of FLS2 and EFR lead to BAK1-dependent, calcium-associated plasma membrane anion channel opening as an initial step in the pathogen defense pathway.
• Marten, I., Deeken, R., Hedrich, R., and Roelfsema, M.R.G. (2010) ‘Light-induced modification of plant plasma membrane ion transport’, Plant Biology, 12, 64–79, available: https://doi.org/10.1111/j.1438-8677.2010.00384.x.
  • [ Abstract ]
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  Light is not only the driving force for electron and ion transport in the thylakoid membrane, but also regulates ion transport in various other membranes of plant cells. Light-dependent changes in ion transport at the plasma membrane and associated membrane potential changes have been studied intensively over the last century. These studies, with various species and cell types, revealed that apart from regulation by chloroplasts, plasma membrane transport can be controlled by phytochromes, phototropins or channel rhodopsins. In this review, we compare light-dependent plasma membrane responses of unicellular algae (Eremosphaera and Chlamydomonas), with those of a multicellular alga (Chara), liverworts (Conocephalum), mosses (Physcomitrella) and several angiosperm cell types. Light-dependent plasma membrane responses of Eremosphaera and Chara are characterised by the dominant role of K+ channels during membrane potential changes. In most other species, the Ca2+-dependent activation of plasma membrane anion channels represents a general light-triggered event. Cell type-specific responses are likely to have evolved by modification of this general response or through the development of additional light-dependent signalling pathways. Future research to elucidate these light-activated signalling chains is likely to benefit from the recent identification of S-type anion channel genes and proteins capable of regulating these channels.

  @article{marten2010lightinduced, abstract = {Light is not only the driving force for electron and ion transport in the thylakoid membrane, but also regulates ion transport in various other membranes of plant cells. Light-dependent changes in ion transport at the plasma membrane and associated membrane potential changes have been studied intensively over the last century. These studies, with various species and cell types, revealed that apart from regulation by chloroplasts, plasma membrane transport can be controlled by phytochromes, phototropins or channel rhodopsins. In this review, we compare light-dependent plasma membrane responses of unicellular algae (Eremosphaera and Chlamydomonas), with those of a multicellular alga (Chara), liverworts (Conocephalum), mosses (Physcomitrella) and several angiosperm cell types. Light-dependent plasma membrane responses of Eremosphaera and Chara are characterised by the dominant role of K+ channels during membrane potential changes. In most other species, the Ca2+-dependent activation of plasma membrane anion channels represents a general light-triggered event. Cell type-specific responses are likely to have evolved by modification of this general response or through the development of additional light-dependent signalling pathways. Future research to elucidate these light-activated signalling chains is likely to benefit from the recent identification of S-type anion channel genes and proteins capable of regulating these channels.}, author = {Marten, I. and Deeken, R. and Hedrich, R. and Roelfsema, M. R. G.}, journal = {Plant Biology}, keywords = {phytochromes}, pages = {64-79}, title = {Light-induced modification of plant plasma membrane ion transport}, volume = 12, year = 2010 }

  %0 Journal Article %1 marten2010lightinduced %A Marten, I. %A Deeken, R. %A Hedrich, R. %A Roelfsema, M. R. G. %D 2010 %J Plant Biology %P 64-79 %R 10.1111/j.1438-8677.2010.00384.x %T Light-induced modification of plant plasma membrane ion transport %U http://onlinelibrary.wiley.com/doi/10.1111/j.1438-8677.2010.00384.x/epdf %V 12 %X Light is not only the driving force for electron and ion transport in the thylakoid membrane, but also regulates ion transport in various other membranes of plant cells. Light-dependent changes in ion transport at the plasma membrane and associated membrane potential changes have been studied intensively over the last century. These studies, with various species and cell types, revealed that apart from regulation by chloroplasts, plasma membrane transport can be controlled by phytochromes, phototropins or channel rhodopsins. In this review, we compare light-dependent plasma membrane responses of unicellular algae (Eremosphaera and Chlamydomonas), with those of a multicellular alga (Chara), liverworts (Conocephalum), mosses (Physcomitrella) and several angiosperm cell types. Light-dependent plasma membrane responses of Eremosphaera and Chara are characterised by the dominant role of K+ channels during membrane potential changes. In most other species, the Ca2+-dependent activation of plasma membrane anion channels represents a general light-triggered event. Cell type-specific responses are likely to have evolved by modification of this general response or through the development of additional light-dependent signalling pathways. Future research to elucidate these light-activated signalling chains is likely to benefit from the recent identification of S-type anion channel genes and proteins capable of regulating these channels.
• Roelfsema, M. and Hedrich, R. (2010) ‘Making sense out of Ca2+signals: their role in regulating stomatal movements’, Plant, cell & environment, 33(3), 305–321, available: https://doi.org/10.1111/j.1365-3040.2009.02075.x.
  • [ Abstract ]
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  Plant cells maintain high Ca2+ concentration gradients between the cytosol and the extracellular matrix, as well as intracellular compartments. During evolution, the regulatory mechanisms, maintaining low cytosolic free Ca2+ concentrations, most likely provided the backbone for the development of Ca2+-dependent signalling pathways. In this review, the current understanding of molecular mechanisms involved in Ca2+ homeostasis of plants cells is evaluated. The question is addressed to which extent the mechanisms, controlling the cytosolic Ca2+ concentration, are linked to Ca2+-based signalling. A large number of environmental stimuli can evoke Ca2+ signals, but the Ca2+-induced responses are likely to differ depending on the stimulus applied. Two mechanisms are put forward to explain signal specificity of Ca2+-dependent responses. A signal may evoke a specific Ca2+ signature that is recognized by downstream signalling components. Alternatively, Ca2+ signals are accompanied by Ca2+-independent signalling events that determine the specificity of the response. The existence of such parallel-acting pathways explains why guard cell responses to abscisic acid (ABA) can occur in the absence, as well as in the presence, of Ca2+ signals. Future research may shed new light on the relation between parallel acting Ca2+-dependent and -independent events, and may provide insights in their evolutionary origin.

  @article{roelfsema2010making, abstract = {Plant cells maintain high Ca2+ concentration gradients between the cytosol and the extracellular matrix, as well as intracellular compartments. During evolution, the regulatory mechanisms, maintaining low cytosolic free Ca2+ concentrations, most likely provided the backbone for the development of Ca2+-dependent signalling pathways. In this review, the current understanding of molecular mechanisms involved in Ca2+ homeostasis of plants cells is evaluated. The question is addressed to which extent the mechanisms, controlling the cytosolic Ca2+ concentration, are linked to Ca2+-based signalling. A large number of environmental stimuli can evoke Ca2+ signals, but the Ca2+-induced responses are likely to differ depending on the stimulus applied. Two mechanisms are put forward to explain signal specificity of Ca2+-dependent responses. A signal may evoke a specific Ca2+ signature that is recognized by downstream signalling components. Alternatively, Ca2+ signals are accompanied by Ca2+-independent signalling events that determine the specificity of the response. The existence of such parallel-acting pathways explains why guard cell responses to abscisic acid (ABA) can occur in the absence, as well as in the presence, of Ca2+ signals. Future research may shed new light on the relation between parallel acting Ca2+-dependent and -independent events, and may provide insights in their evolutionary origin.}, author = {Roelfsema, M.R.G. and Hedrich, R.}, journal = {Plant, cell & environment}, keywords = {ABA}, number = 3, pages = {305-321}, title = {Making sense out of Ca2+signals: their role in regulating stomatal movements}, volume = 33, year = 2010 }

  %0 Journal Article %1 roelfsema2010making %A Roelfsema, M.R.G. %A Hedrich, R. %D 2010 %J Plant, cell & environment %N 3 %P 305-321 %R 10.1111/j.1365-3040.2009.02075.x %T Making sense out of Ca2+signals: their role in regulating stomatal movements %U http://onlinelibrary.wiley.com/doi/10.1111/j.1365-3040.2009.02075.x/full %V 33 %X Plant cells maintain high Ca2+ concentration gradients between the cytosol and the extracellular matrix, as well as intracellular compartments. During evolution, the regulatory mechanisms, maintaining low cytosolic free Ca2+ concentrations, most likely provided the backbone for the development of Ca2+-dependent signalling pathways. In this review, the current understanding of molecular mechanisms involved in Ca2+ homeostasis of plants cells is evaluated. The question is addressed to which extent the mechanisms, controlling the cytosolic Ca2+ concentration, are linked to Ca2+-based signalling. A large number of environmental stimuli can evoke Ca2+ signals, but the Ca2+-induced responses are likely to differ depending on the stimulus applied. Two mechanisms are put forward to explain signal specificity of Ca2+-dependent responses. A signal may evoke a specific Ca2+ signature that is recognized by downstream signalling components. Alternatively, Ca2+ signals are accompanied by Ca2+-independent signalling events that determine the specificity of the response. The existence of such parallel-acting pathways explains why guard cell responses to abscisic acid (ABA) can occur in the absence, as well as in the presence, of Ca2+ signals. Future research may shed new light on the relation between parallel acting Ca2+-dependent and -independent events, and may provide insights in their evolutionary origin.
• Stange, A., Hedrich, R., and Roelfsema, M.R.G. (2010) ‘Ca2+-dependent activation of guard cell anion channels, triggered by hyperpolarization, is promoted by prolonged depolarization’, Plant Journal, 62(2), 265–276, available: https://doi.org/10.1111/j.1365-313X.2010.04141.x.
  • [ Abstract ]
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  Rapid stomatal closure is driven by the activation of S-type anion channels in the plasma membrane of guard cells. This response has been linked to Ca2+ signalling, but the impact of transient Ca2+ signals on S-type anion channel activity remains unknown. In this study, transient elevation of the cytosolic Ca2+ level was provoked by voltage steps in guard cells of intact Nicotiana tabacum plants. Changes in the activity of S-type anion channels were monitored using intracellular triple-barrelled micro-electrodes. In cells kept at a holding potential of )100 mV, voltage steps to )180 mV triggered elevation of the cytosolic free Ca2+ concentration. The increase in the cytosolic Ca2+ level was accompanied by activation of S-type anion channels. Guard cell anion channels were activated by Ca2+ with a half maximum concentration of 515 nM (SE = 235) and a mean saturation value of )349 pA (SE = 107) at )100 mV. Ca2+ signals could also be evoked by prolonged (100 sec) depolarization of the plasma membrane to 0 mV. Upon returning to )100 mV, a transient increase in the cytosolic Ca2+ level was observed, activating S-type channels without measurable delay. These data show that cytosolic Ca2+ elevation can activate S-type anion channels in intact guard cells through a fast signalling pathway. Furthermore, prolonged depolarization to 0 mV alters the activity of Ca2+ transport proteins, resulting in an overshoot of the cytosolic Ca2+ level after returning the membrane potential to )100 mV.

  @article{stange2010ca2dependent, abstract = {Rapid stomatal closure is driven by the activation of S-type anion channels in the plasma membrane of guard cells. This response has been linked to Ca2+ signalling, but the impact of transient Ca2+ signals on S-type anion channel activity remains unknown. In this study, transient elevation of the cytosolic Ca2+ level was provoked by voltage steps in guard cells of intact Nicotiana tabacum plants. Changes in the activity of S-type anion channels were monitored using intracellular triple-barrelled micro-electrodes. In cells kept at a holding potential of )100 mV, voltage steps to )180 mV triggered elevation of the cytosolic free Ca2+ concentration. The increase in the cytosolic Ca2+ level was accompanied by activation of S-type anion channels. Guard cell anion channels were activated by Ca2+ with a half maximum concentration of 515 nM (SE = 235) and a mean saturation value of )349 pA (SE = 107) at )100 mV. Ca2+ signals could also be evoked by prolonged (100 sec) depolarization of the plasma membrane to 0 mV. Upon returning to )100 mV, a transient increase in the cytosolic Ca2+ level was observed, activating S-type channels without measurable delay. These data show that cytosolic Ca2+ elevation can activate S-type anion channels in intact guard cells through a fast signalling pathway. Furthermore, prolonged depolarization to 0 mV alters the activity of Ca2+ transport proteins, resulting in an overshoot of the cytosolic Ca2+ level after returning the membrane potential to )100 mV.}, author = {Stange, A. and Hedrich, R. and Roelfsema, M. R. G.}, journal = {Plant Journal}, keywords = {Ca2+-signals}, number = 2, pages = {265-276}, title = {Ca2+-dependent activation of guard cell anion channels, triggered by hyperpolarization, is promoted by prolonged depolarization}, volume = 62, year = 2010 }

  %0 Journal Article %1 stange2010ca2dependent %A Stange, A. %A Hedrich, R. %A Roelfsema, M. R. G. %D 2010 %J Plant Journal %N 2 %P 265-276 %R 10.1111/j.1365-313X.2010.04141.x %T Ca2+-dependent activation of guard cell anion channels, triggered by hyperpolarization, is promoted by prolonged depolarization %U /brokenurl#<Go to ISI>://WOS:000276496300008 %V 62 %X Rapid stomatal closure is driven by the activation of S-type anion channels in the plasma membrane of guard cells. This response has been linked to Ca2+ signalling, but the impact of transient Ca2+ signals on S-type anion channel activity remains unknown. In this study, transient elevation of the cytosolic Ca2+ level was provoked by voltage steps in guard cells of intact Nicotiana tabacum plants. Changes in the activity of S-type anion channels were monitored using intracellular triple-barrelled micro-electrodes. In cells kept at a holding potential of )100 mV, voltage steps to )180 mV triggered elevation of the cytosolic free Ca2+ concentration. The increase in the cytosolic Ca2+ level was accompanied by activation of S-type anion channels. Guard cell anion channels were activated by Ca2+ with a half maximum concentration of 515 nM (SE = 235) and a mean saturation value of )349 pA (SE = 107) at )100 mV. Ca2+ signals could also be evoked by prolonged (100 sec) depolarization of the plasma membrane to 0 mV. Upon returning to )100 mV, a transient increase in the cytosolic Ca2+ level was observed, activating S-type channels without measurable delay. These data show that cytosolic Ca2+ elevation can activate S-type anion channels in intact guard cells through a fast signalling pathway. Furthermore, prolonged depolarization to 0 mV alters the activity of Ca2+ transport proteins, resulting in an overshoot of the cytosolic Ca2+ level after returning the membrane potential to )100 mV.
• de Carbonnel, M., Davis, P., Roelfsema, M.R.G., Inoue, S., Schepens, I., Lariguet, P., Geisler, M., Shimazaki, K., Hangarter, R., and Fankhauser, C. (2010) ‘The Arabidopsis PHYTOCHROME KINASE SUBSTRATE2 Protein Is a Phototropin Signaling Element That Regulates Leaf Flattening and Leaf Positioning’, Plant Physiology, 152(3), 1391–1405, available: https://doi.org/10.1104/pp.109.150441.
  • [ Abstract ]
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  In Arabidopsis (Arabidopsis thaliana), the blue light photoreceptor phototropins (phot1 and phot2) fine-tune the photosynthetic status of the plant by controlling several important adaptive processes in response to environmental light variations. These processes include stem and petiole phototropism (leaf positioning), leaf flattening, stomatal opening, and chloroplast movements. The PHYTOCHROME KINASE SUBSTRATE (PKS) protein family comprises four members in Arabidopsis (PKS1–PKS4). PKS1 is a novel phot1 signaling element during phototropism, as it interacts with phot1 and the important signaling element NONPHOTOTROPIC HYPOCOTYL3 (NPH3) and is required for normal phot1-mediated phototropism. In this study, we have analyzed more globally the role of three PKS members (PKS1, PKS2, and PKS4). Systematic analysis of mutants reveals that PKS2 (and to a lesser extent PKS1) act in the same subset of phototropin-controlled responses as NPH3, namely leaf flattening and positioning. PKS1, PKS2, and NPH3 coimmunoprecipitate with both phot1-green fluorescent protein and phot2-green fluorescent protein in leaf extracts. Genetic experiments position PKS2 within phot1 and phot2 pathways controlling leaf positioning and leaf flattening, respectively. NPH3 can act in both phot1 and phot2 pathways, and synergistic interactions observed between pks2 and nph3 mutants suggest complementary roles of PKS2 and NPH3 during phototropin signaling. Finally, several observations further suggest that PKS2 may regulate leaf flattening and positioning by controlling auxin homeostasis. Together with previous findings, our results indicate that the PKS proteins represent an important family of phototropin signaling proteins.

  @article{decarbonnel2010arabidopsis, abstract = {In Arabidopsis (Arabidopsis thaliana), the blue light photoreceptor phototropins (phot1 and phot2) fine-tune the photosynthetic status of the plant by controlling several important adaptive processes in response to environmental light variations. These processes include stem and petiole phototropism (leaf positioning), leaf flattening, stomatal opening, and chloroplast movements. The PHYTOCHROME KINASE SUBSTRATE (PKS) protein family comprises four members in Arabidopsis (PKS1–PKS4). PKS1 is a novel phot1 signaling element during phototropism, as it interacts with phot1 and the important signaling element NONPHOTOTROPIC HYPOCOTYL3 (NPH3) and is required for normal phot1-mediated phototropism. In this study, we have analyzed more globally the role of three PKS members (PKS1, PKS2, and PKS4). Systematic analysis of mutants reveals that PKS2 (and to a lesser extent PKS1) act in the same subset of phototropin-controlled responses as NPH3, namely leaf flattening and positioning. PKS1, PKS2, and NPH3 coimmunoprecipitate with both phot1-green fluorescent protein and phot2-green fluorescent protein in leaf extracts. Genetic experiments position PKS2 within phot1 and phot2 pathways controlling leaf positioning and leaf flattening, respectively. NPH3 can act in both phot1 and phot2 pathways, and synergistic interactions observed between pks2 and nph3 mutants suggest complementary roles of PKS2 and NPH3 during phototropin signaling. Finally, several observations further suggest that PKS2 may regulate leaf flattening and positioning by controlling auxin homeostasis. Together with previous findings, our results indicate that the PKS proteins represent an important family of phototropin signaling proteins.}, author = {de Carbonnel, M. and Davis, P. and Roelfsema, M. R. G. and Inoue, S. and Schepens, I. and Lariguet, P. and Geisler, M. and Shimazaki, K. and Hangarter, R. and Fankhauser, C.}, journal = {Plant Physiology}, keywords = {phototropin}, number = 3, pages = {1391-1405}, title = {The Arabidopsis PHYTOCHROME KINASE SUBSTRATE2 Protein Is a Phototropin Signaling Element That Regulates Leaf Flattening and Leaf Positioning}, volume = 152, year = 2010 }

  %0 Journal Article %1 decarbonnel2010arabidopsis %A de Carbonnel, M. %A Davis, P. %A Roelfsema, M. R. G. %A Inoue, S. %A Schepens, I. %A Lariguet, P. %A Geisler, M. %A Shimazaki, K. %A Hangarter, R. %A Fankhauser, C. %D 2010 %J Plant Physiology %N 3 %P 1391-1405 %R 10.1104/pp.109.150441 %T The Arabidopsis PHYTOCHROME KINASE SUBSTRATE2 Protein Is a Phototropin Signaling Element That Regulates Leaf Flattening and Leaf Positioning %U http://www.plantphysiol.org/search?author1=carbonnel&fulltext=&pubdate_year=&volume=&firstpage=&submit=yes %V 152 %X In Arabidopsis (Arabidopsis thaliana), the blue light photoreceptor phototropins (phot1 and phot2) fine-tune the photosynthetic status of the plant by controlling several important adaptive processes in response to environmental light variations. These processes include stem and petiole phototropism (leaf positioning), leaf flattening, stomatal opening, and chloroplast movements. The PHYTOCHROME KINASE SUBSTRATE (PKS) protein family comprises four members in Arabidopsis (PKS1–PKS4). PKS1 is a novel phot1 signaling element during phototropism, as it interacts with phot1 and the important signaling element NONPHOTOTROPIC HYPOCOTYL3 (NPH3) and is required for normal phot1-mediated phototropism. In this study, we have analyzed more globally the role of three PKS members (PKS1, PKS2, and PKS4). Systematic analysis of mutants reveals that PKS2 (and to a lesser extent PKS1) act in the same subset of phototropin-controlled responses as NPH3, namely leaf flattening and positioning. PKS1, PKS2, and NPH3 coimmunoprecipitate with both phot1-green fluorescent protein and phot2-green fluorescent protein in leaf extracts. Genetic experiments position PKS2 within phot1 and phot2 pathways controlling leaf positioning and leaf flattening, respectively. NPH3 can act in both phot1 and phot2 pathways, and synergistic interactions observed between pks2 and nph3 mutants suggest complementary roles of PKS2 and NPH3 during phototropin signaling. Finally, several observations further suggest that PKS2 may regulate leaf flattening and positioning by controlling auxin homeostasis. Together with previous findings, our results indicate that the PKS proteins represent an important family of phototropin signaling proteins.

• Roelfsema, M. (2009) Stomata [online], Encyclopedia of Life Sciences, Wiley Online Library, available: https://doi.org/10.1002/9780470015902.a0002075.pub2.
  • [ Abstract ]
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  Stomata are small adjustable pores on the surface of aerial plant tissues. The pores open to facilitate uptake of carbon dioxide and close to limit the loss of water. Consistent with this function, stomata open in the light to enable photosynthesis and close during drought. Stomatal movements are forced by changes in the volume of two guard cells that are positioned on either side of the pore. During stomatal opening, guard cells accumulate potassium salts, causing an osmotically driven uptake of water. Owing to the influx of water the guard cells swell and bend, thereby pushing each other apart and creating an open pore in the middle. Various environmental signals, such as light, carbon dioxide and humidity, influence the ion-transport machinery within the guard cell and thus alter stomatal movement. Because of their role in regulating carbon dioxide uptake and transpiration of water, stomata are likely to play a key role in the adaption of plants to the anticipated global warming.

  @book{roelfsema2009stomata, abstract = {Stomata are small adjustable pores on the surface of aerial plant tissues. The pores open to facilitate uptake of carbon dioxide and close to limit the loss of water. Consistent with this function, stomata open in the light to enable photosynthesis and close during drought. Stomatal movements are forced by changes in the volume of two guard cells that are positioned on either side of the pore. During stomatal opening, guard cells accumulate potassium salts, causing an osmotically driven uptake of water. Owing to the influx of water the guard cells swell and bend, thereby pushing each other apart and creating an open pore in the middle. Various environmental signals, such as light, carbon dioxide and humidity, influence the ion-transport machinery within the guard cell and thus alter stomatal movement. Because of their role in regulating carbon dioxide uptake and transpiration of water, stomata are likely to play a key role in the adaption of plants to the anticipated global warming.}, author = {Roelfsema, M.R.G.}, journal = {Encyclopedia of Life Sciences}, keywords = {ion-transport}, publisher = {Wiley Online Library}, title = {Stomata}, year = 2009 }

  %0 Book %1 roelfsema2009stomata %A Roelfsema, M.R.G. %D 2009 %I Wiley Online Library %J Encyclopedia of Life Sciences %R 10.1002/9780470015902.a0002075.pub2 %T Stomata %U http://onlinelibrary.wiley.com/doi/10.1002/9780470015902.a0002075.pub2/abstract? %X Stomata are small adjustable pores on the surface of aerial plant tissues. The pores open to facilitate uptake of carbon dioxide and close to limit the loss of water. Consistent with this function, stomata open in the light to enable photosynthesis and close during drought. Stomatal movements are forced by changes in the volume of two guard cells that are positioned on either side of the pore. During stomatal opening, guard cells accumulate potassium salts, causing an osmotically driven uptake of water. Owing to the influx of water the guard cells swell and bend, thereby pushing each other apart and creating an open pore in the middle. Various environmental signals, such as light, carbon dioxide and humidity, influence the ion-transport machinery within the guard cell and thus alter stomatal movement. Because of their role in regulating carbon dioxide uptake and transpiration of water, stomata are likely to play a key role in the adaption of plants to the anticipated global warming.

• Engelmann, J.C., Deeken, R., Mueller, T., Nimtz, G., Roelfsema, M.R.G., and Hedrich, R. (2008) ‘Is gene activity in plant cells affected by UMTS-irradiation? A whole genome approach’, Advances and Applications in Bioinformatics and Chemistry, 1(1), 71–83, available: https://www.dovepress.com/is-gene-activity-in-plant-cells-affected-by-umts-irradiation-a-whole-g-peer-reviewed-article-AABC.
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  @article{engelmann2008activity, author = {Engelmann, J. C. and Deeken, R. and Mueller, T. and Nimtz, G. and Roelfsema, M. R. G. and Hedrich, R.}, journal = {Advances and Applications in Bioinformatics and Chemistry}, keywords = {gene-expression}, number = 1, pages = {71-83}, title = {Is gene activity in plant cells affected by UMTS-irradiation? A whole genome approach}, volume = 1, year = 2008 }

  %0 Journal Article %1 engelmann2008activity %A Engelmann, J. C. %A Deeken, R. %A Mueller, T. %A Nimtz, G. %A Roelfsema, M. R. G. %A Hedrich, R. %D 2008 %J Advances and Applications in Bioinformatics and Chemistry %N 1 %P 71-83 %T Is gene activity in plant cells affected by UMTS-irradiation? A whole genome approach %U https://www.dovepress.com/is-gene-activity-in-plant-cells-affected-by-umts-irradiation-a-whole-g-peer-reviewed-article-AABC %V 1
• Marten, H., Hyun, T., Gomi, K., Seo, S., Hedrich, R., and Roelfsema, M.R.G. (2008) ‘Silencing of NtMPK4 impairs CO2-induced stomatal closure, activation of anion channels and cytosolic Ca(2+)signals in Nicotiana tabacum guard cells’, Plant Journal, 55(4), 698–708, available: https://doi.org/10.1111/j.1365-313X.2008.03542.x.
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  Light-induced stomatal opening in C3 and C4 plants is mediated by two signalling pathways. One pathway is specific for blue light and involves phototropins, while the second pathway depends on photosyntheticaly active radiation (PAR). Here, the role of NtMPK4 in light-induced stomatal opening was studied, as silencing of this MAP kinase stimulates stomatal opening. Stomata of NtMPK4-silenced plants do not close in elevated atmospheric CO2, and show a reduced response to PAR. However, stomatal closure can still be induced by abscisic acid. Measurements using multi-barrelled intracellular micro-electrodes showed that CO2 activates plasma membrane anion channels in wild-type Nicotiana tabacum guard cells, but not in NtMPK4-silenced cells. Anion channels were also activated in wild-type guard cells after switching off PAR. In approximately half of these cells, activation of anion channels was accompanied by an increase in the cytosolic free Ca2+ concentration. The activity of anion channels was higher in cells showing a parallel increase in cytosolic Ca2+ than in those with steady Ca2+ levels. Both the darkness-induced anion channel activation and Ca2+ signals were repressed in NtMPK4-silenced guard cells. These data show that CO2 and darkness can activate anion channels in a Ca2+-independent manner, but the anion channel activity is enhanced by parallel increases in the cytosolic Ca2+ concentration. NtMPK4 plays an essential role in CO2- and darkness-induced activation of guard cell anion channels, through Ca2+-independent as well as Ca2+-dependent signalling pathways.

  @article{marten2008silencing, abstract = {Light-induced stomatal opening in C3 and C4 plants is mediated by two signalling pathways. One pathway is specific for blue light and involves phototropins, while the second pathway depends on photosyntheticaly active radiation (PAR). Here, the role of NtMPK4 in light-induced stomatal opening was studied, as silencing of this MAP kinase stimulates stomatal opening. Stomata of NtMPK4-silenced plants do not close in elevated atmospheric CO2, and show a reduced response to PAR. However, stomatal closure can still be induced by abscisic acid. Measurements using multi-barrelled intracellular micro-electrodes showed that CO2 activates plasma membrane anion channels in wild-type Nicotiana tabacum guard cells, but not in NtMPK4-silenced cells. Anion channels were also activated in wild-type guard cells after switching off PAR. In approximately half of these cells, activation of anion channels was accompanied by an increase in the cytosolic free Ca2+ concentration. The activity of anion channels was higher in cells showing a parallel increase in cytosolic Ca2+ than in those with steady Ca2+ levels. Both the darkness-induced anion channel activation and Ca2+ signals were repressed in NtMPK4-silenced guard cells. These data show that CO2 and darkness can activate anion channels in a Ca2+-independent manner, but the anion channel activity is enhanced by parallel increases in the cytosolic Ca2+ concentration. NtMPK4 plays an essential role in CO2- and darkness-induced activation of guard cell anion channels, through Ca2+-independent as well as Ca2+-dependent signalling pathways.}, author = {Marten, H. and Hyun, T. and Gomi, K. and Seo, S. and Hedrich, R. and Roelfsema, M. R. G.}, journal = {Plant Journal}, keywords = {MPK4}, number = 4, pages = {698-708}, title = {Silencing of NtMPK4 impairs CO2-induced stomatal closure, activation of anion channels and cytosolic Ca(2+)signals in Nicotiana tabacum guard cells}, volume = 55, year = 2008 }

  %0 Journal Article %1 marten2008silencing %A Marten, H. %A Hyun, T. %A Gomi, K. %A Seo, S. %A Hedrich, R. %A Roelfsema, M. R. G. %D 2008 %J Plant Journal %N 4 %P 698-708 %R 10.1111/j.1365-313X.2008.03542.x %T Silencing of NtMPK4 impairs CO2-induced stomatal closure, activation of anion channels and cytosolic Ca(2+)signals in Nicotiana tabacum guard cells %U http://onlinelibrary.wiley.com/doi/10.1111/j.1365-313X.2008.03542.x/full %V 55 %X Light-induced stomatal opening in C3 and C4 plants is mediated by two signalling pathways. One pathway is specific for blue light and involves phototropins, while the second pathway depends on photosyntheticaly active radiation (PAR). Here, the role of NtMPK4 in light-induced stomatal opening was studied, as silencing of this MAP kinase stimulates stomatal opening. Stomata of NtMPK4-silenced plants do not close in elevated atmospheric CO2, and show a reduced response to PAR. However, stomatal closure can still be induced by abscisic acid. Measurements using multi-barrelled intracellular micro-electrodes showed that CO2 activates plasma membrane anion channels in wild-type Nicotiana tabacum guard cells, but not in NtMPK4-silenced cells. Anion channels were also activated in wild-type guard cells after switching off PAR. In approximately half of these cells, activation of anion channels was accompanied by an increase in the cytosolic free Ca2+ concentration. The activity of anion channels was higher in cells showing a parallel increase in cytosolic Ca2+ than in those with steady Ca2+ levels. Both the darkness-induced anion channel activation and Ca2+ signals were repressed in NtMPK4-silenced guard cells. These data show that CO2 and darkness can activate anion channels in a Ca2+-independent manner, but the anion channel activity is enhanced by parallel increases in the cytosolic Ca2+ concentration. NtMPK4 plays an essential role in CO2- and darkness-induced activation of guard cell anion channels, through Ca2+-independent as well as Ca2+-dependent signalling pathways.
• Levchenko, V., Guinot, D.R., Klein, M., Roelfsema, M.R.G., Hedrich, R., and Dietrich, P. (2008) ‘Stringent control of cytoplasmic Ca2+ in guard cells of intact plants compared to their counterparts in epidermal strips or guard cell protoplasts’, Protoplasma, 233(1-2), 61–72, available: https://doi.org/10.1007/s00709-008-0307-x.
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  Cytoplasmic calcium elevations, transients, and oscillations are thought to encode information that triggers a variety of physiological responses in plant cells. Yet Ca2+ signals induced by a single stimulus vary, depending on the physiological state of the cell and experimental conditions. We compared Ca2+ homeostasis and stimulus-induced Ca2+ signals in guard cells of intact plants, epidermal strips, and isolated protoplasts. Single-cell ratiometric imaging with the Ca2+-sensitive dye Fura 2 was applied in combination with electrophysiological recordings. Guard cell protoplasts were loaded with Fura 2 via a patch pipette, revealing a cytoplasmic free Ca2+ concentration of around 80 nM at -47 mV. Upon hyperpolarization of the plasma membrane to -107 mV, the Ca2+ concentration increased to levels exceeding 400 nM. Intact guard cells were able to maintain much lower cytoplasmic free Ca2+ concentrations at hyperpolarized potentials, the average concentration at -100 mV was 183 and 90 nM in epidermal strips and intact plants, respectively. Further hyperpolarization of the plasma membrane to -160 mV induced a sustained rise of the guard cell cytoplasmic Ca2+ concentration, which slowly returned to the prestimulus level in intact plants but not in epidermal strips. Our results show that cytoplasmic Ca2+ concentrations are stringently controlled in guard cells of intact plants but become increasingly more sensitive to changes in the plasma membrane potential in epidermal strips and isolated protoplasts.

  @article{levchenko2008stringent, abstract = {Cytoplasmic calcium elevations, transients, and oscillations are thought to encode information that triggers a variety of physiological responses in plant cells. Yet Ca2+ signals induced by a single stimulus vary, depending on the physiological state of the cell and experimental conditions. We compared Ca2+ homeostasis and stimulus-induced Ca2+ signals in guard cells of intact plants, epidermal strips, and isolated protoplasts. Single-cell ratiometric imaging with the Ca2+-sensitive dye Fura 2 was applied in combination with electrophysiological recordings. Guard cell protoplasts were loaded with Fura 2 via a patch pipette, revealing a cytoplasmic free Ca2+ concentration of around 80 nM at -47 mV. Upon hyperpolarization of the plasma membrane to -107 mV, the Ca2+ concentration increased to levels exceeding 400 nM. Intact guard cells were able to maintain much lower cytoplasmic free Ca2+ concentrations at hyperpolarized potentials, the average concentration at -100 mV was 183 and 90 nM in epidermal strips and intact plants, respectively. Further hyperpolarization of the plasma membrane to -160 mV induced a sustained rise of the guard cell cytoplasmic Ca2+ concentration, which slowly returned to the prestimulus level in intact plants but not in epidermal strips. Our results show that cytoplasmic Ca2+ concentrations are stringently controlled in guard cells of intact plants but become increasingly more sensitive to changes in the plasma membrane potential in epidermal strips and isolated protoplasts.}, author = {Levchenko, V. and Guinot, D. R. and Klein, M. and Roelfsema, M. R. G. and Hedrich, R. and Dietrich, P.}, booktitle = {Protoplasma}, keywords = {ca2+-signals}, number = {1-2}, pages = {61-72}, title = {Stringent control of cytoplasmic Ca2+ in guard cells of intact plants compared to their counterparts in epidermal strips or guard cell protoplasts}, volume = 233, year = 2008 }

  %0 Journal Article %1 levchenko2008stringent %A Levchenko, V. %A Guinot, D. R. %A Klein, M. %A Roelfsema, M. R. G. %A Hedrich, R. %A Dietrich, P. %B Protoplasma %D 2008 %N 1-2 %P 61-72 %R 10.1007/s00709-008-0307-x %T Stringent control of cytoplasmic Ca2+ in guard cells of intact plants compared to their counterparts in epidermal strips or guard cell protoplasts %U /brokenurl#<Go to ISI>://WOS:000259188100007 %V 233 %X Cytoplasmic calcium elevations, transients, and oscillations are thought to encode information that triggers a variety of physiological responses in plant cells. Yet Ca2+ signals induced by a single stimulus vary, depending on the physiological state of the cell and experimental conditions. We compared Ca2+ homeostasis and stimulus-induced Ca2+ signals in guard cells of intact plants, epidermal strips, and isolated protoplasts. Single-cell ratiometric imaging with the Ca2+-sensitive dye Fura 2 was applied in combination with electrophysiological recordings. Guard cell protoplasts were loaded with Fura 2 via a patch pipette, revealing a cytoplasmic free Ca2+ concentration of around 80 nM at -47 mV. Upon hyperpolarization of the plasma membrane to -107 mV, the Ca2+ concentration increased to levels exceeding 400 nM. Intact guard cells were able to maintain much lower cytoplasmic free Ca2+ concentrations at hyperpolarized potentials, the average concentration at -100 mV was 183 and 90 nM in epidermal strips and intact plants, respectively. Further hyperpolarization of the plasma membrane to -160 mV induced a sustained rise of the guard cell cytoplasmic Ca2+ concentration, which slowly returned to the prestimulus level in intact plants but not in epidermal strips. Our results show that cytoplasmic Ca2+ concentrations are stringently controlled in guard cells of intact plants but become increasingly more sensitive to changes in the plasma membrane potential in epidermal strips and isolated protoplasts.
• Krupenina, N.A., Bulychev, A.A., Roelfsema, M.R.G., and Schreiber, U. (2008) ‘Action potential in Chara cells intensifies spatial patterns of photosynthetic electron flow and non-photochemical quenching in parallel with inhibition of pH banding’, Photochemical & Photobiological Sciences, 7(6), 681–688, available: https://doi.org/10.1039/b802243g.
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  Characean cells exposed to illumination arrange plasma-membraneH+ fluxes and photosynthesis in coordinated spatial patterns. The limited availability of CO2 in alkaline bands accounts for the lower effective quantum yield of photosystem II (ΔF/Fm′) in chloroplasts of these bands compared to acidic zones. The effect of electrically triggered action potential on the spatial distribution of photosynthetic parameters (ΔF/Fm′ and non-photochemical quenching, NPQ) and extracellular pH was studied with fluorescence imaging and pH microelectrodes. In the resting cell at a range of light intensities, the periodic profile of extracellular pH is parallel to the profile of NPQ and antiparallel to that of ΔF/Fm′. After triggering the action potential, the pH banding temporarily disappeared, but in contrast, the differences in effective quantum yield and NPQ patterns became more apparent. The transient changes in pH-banding, effective quantum yield and non-photochemical quenching are discussed in relation to alterations in intracellularCa2+ and H+ concentrations during and after the action potential.

  @article{krupenina2008action, abstract = {Characean cells exposed to illumination arrange plasma-membraneH+ fluxes and photosynthesis in coordinated spatial patterns. The limited availability of CO2 in alkaline bands accounts for the lower effective quantum yield of photosystem II (ΔF/Fm′) in chloroplasts of these bands compared to acidic zones. The effect of electrically triggered action potential on the spatial distribution of photosynthetic parameters (ΔF/Fm′ and non-photochemical quenching, NPQ) and extracellular pH was studied with fluorescence imaging and pH microelectrodes. In the resting cell at a range of light intensities, the periodic profile of extracellular pH is parallel to the profile of NPQ and antiparallel to that of ΔF/Fm′. After triggering the action potential, the pH banding temporarily disappeared, but in contrast, the differences in effective quantum yield and NPQ patterns became more apparent. The transient changes in pH-banding, effective quantum yield and non-photochemical quenching are discussed in relation to alterations in intracellularCa2+ and H+ concentrations during and after the action potential.}, author = {Krupenina, N. A. and Bulychev, A. A. and Roelfsema, M. R. G. and Schreiber, U.}, journal = {Photochemical & Photobiological Sciences}, keywords = {chara}, number = 6, pages = {681-688}, title = {Action potential in Chara cells intensifies spatial patterns of photosynthetic electron flow and non-photochemical quenching in parallel with inhibition of pH banding}, volume = 7, year = 2008 }

  %0 Journal Article %1 krupenina2008action %A Krupenina, N. A. %A Bulychev, A. A. %A Roelfsema, M. R. G. %A Schreiber, U. %D 2008 %J Photochemical & Photobiological Sciences %N 6 %P 681-688 %R 10.1039/b802243g %T Action potential in Chara cells intensifies spatial patterns of photosynthetic electron flow and non-photochemical quenching in parallel with inhibition of pH banding %U http://pubs.rsc.org/en/Content/ArticleLanding/2008/PP/b802243g#!divAbstract %V 7 %X Characean cells exposed to illumination arrange plasma-membraneH+ fluxes and photosynthesis in coordinated spatial patterns. The limited availability of CO2 in alkaline bands accounts for the lower effective quantum yield of photosystem II (ΔF/Fm′) in chloroplasts of these bands compared to acidic zones. The effect of electrically triggered action potential on the spatial distribution of photosynthetic parameters (ΔF/Fm′ and non-photochemical quenching, NPQ) and extracellular pH was studied with fluorescence imaging and pH microelectrodes. In the resting cell at a range of light intensities, the periodic profile of extracellular pH is parallel to the profile of NPQ and antiparallel to that of ΔF/Fm′. After triggering the action potential, the pH banding temporarily disappeared, but in contrast, the differences in effective quantum yield and NPQ patterns became more apparent. The transient changes in pH-banding, effective quantum yield and non-photochemical quenching are discussed in relation to alterations in intracellularCa2+ and H+ concentrations during and after the action potential.

• Sano, T., Becker, D., Ivashikina, N., Wegner, L.H., Zimmermann, U., Roelfsema, M.R.G., Nagata, T., and Hedrich, R. (2007) ‘Plant cells must pass a K+ threshold to re-enter the cell cycle’, The Plant Journal, 50(3), 401–413, available: https://doi.org/10.1111/j.1365-313X.2007.03071.x/full.
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  Potassium is an inevitable component of plant life, and potassium channels play a pivotal role in plant growth and development. The role of potassium and of K+ channels in plant cell division and cell-cycle progression, however, has not been determined so far. K+ channel blocker studies with synchronized tobacco BY-2 cells revealed that K+ uptake is required for proper cell-cycle progression during the transition from G1 to S phase. Electrophysiological studies (patch-clamp and voltage-clamp techniques) showed a cell-cycle dependency of K+ channel activities and reduced driving force for K+ uptake in dividing cells. Among the four Shaker-like K+ channel genes expressed in BY-2 cells, NKT1 represents an inwardly rectifying K+ channel that mediates K+ uptake. NKT1 is transcriptionally induced during G1 phase, while transcripts of the outward-rectifier NTORK1 dominate S phase. Elongating BY-2 cells appeared hyperpolarized (−101 ± 11 mV), and had elevated osmotic pressure and approximately twice the turgor pressure when compared with depolarized (−64 ± 8 mV) dividing cells. This indicates that cells have to gain a threshold K+ level to re-enter the cell cycle. Based on these findings, turgor regulation through modulation of K+ channel density in plant cell division and cell-cycle progression is discussed.

  @article{sano2007plant, abstract = {Potassium is an inevitable component of plant life, and potassium channels play a pivotal role in plant growth and development. The role of potassium and of K+ channels in plant cell division and cell-cycle progression, however, has not been determined so far. K+ channel blocker studies with synchronized tobacco BY-2 cells revealed that K+ uptake is required for proper cell-cycle progression during the transition from G1 to S phase. Electrophysiological studies (patch-clamp and voltage-clamp techniques) showed a cell-cycle dependency of K+ channel activities and reduced driving force for K+ uptake in dividing cells. Among the four Shaker-like K+ channel genes expressed in BY-2 cells, NKT1 represents an inwardly rectifying K+ channel that mediates K+ uptake. NKT1 is transcriptionally induced during G1 phase, while transcripts of the outward-rectifier NTORK1 dominate S phase. Elongating BY-2 cells appeared hyperpolarized (−101 ± 11 mV), and had elevated osmotic pressure and approximately twice the turgor pressure when compared with depolarized (−64 ± 8 mV) dividing cells. This indicates that cells have to gain a threshold K+ level to re-enter the cell cycle. Based on these findings, turgor regulation through modulation of K+ channel density in plant cell division and cell-cycle progression is discussed.}, author = {Sano, T. and Becker, D. and Ivashikina, N. and Wegner, L. H. and Zimmermann, U. and Roelfsema, M. R. G. and Nagata, T. and Hedrich, R.}, journal = {The Plant Journal}, keywords = {tobacco}, number = 3, pages = {401-413}, title = {Plant cells must pass a K+ threshold to re-enter the cell cycle}, volume = 50, year = 2007 }

  %0 Journal Article %1 sano2007plant %A Sano, T. %A Becker, D. %A Ivashikina, N. %A Wegner, L. H. %A Zimmermann, U. %A Roelfsema, M. R. G. %A Nagata, T. %A Hedrich, R. %D 2007 %J The Plant Journal %N 3 %P 401-413 %R 10.1111/j.1365-313X.2007.03071.x/full %T Plant cells must pass a K+ threshold to re-enter the cell cycle %U http://onlinelibrary.wiley.com/doi/10.1111/j.1365-313X.2007.03071.x/full %V 50 %X Potassium is an inevitable component of plant life, and potassium channels play a pivotal role in plant growth and development. The role of potassium and of K+ channels in plant cell division and cell-cycle progression, however, has not been determined so far. K+ channel blocker studies with synchronized tobacco BY-2 cells revealed that K+ uptake is required for proper cell-cycle progression during the transition from G1 to S phase. Electrophysiological studies (patch-clamp and voltage-clamp techniques) showed a cell-cycle dependency of K+ channel activities and reduced driving force for K+ uptake in dividing cells. Among the four Shaker-like K+ channel genes expressed in BY-2 cells, NKT1 represents an inwardly rectifying K+ channel that mediates K+ uptake. NKT1 is transcriptionally induced during G1 phase, while transcripts of the outward-rectifier NTORK1 dominate S phase. Elongating BY-2 cells appeared hyperpolarized (−101 ± 11 mV), and had elevated osmotic pressure and approximately twice the turgor pressure when compared with depolarized (−64 ± 8 mV) dividing cells. This indicates that cells have to gain a threshold K+ level to re-enter the cell cycle. Based on these findings, turgor regulation through modulation of K+ channel density in plant cell division and cell-cycle progression is discussed.
• Marten, H., Konrad, K.R., Dietrich, P., Roelfsema, M.R.G., and Hedrich, R. (2007) ‘Ca2+-dependent and -independent abscisic acid activation of plasma membrane anion channels in guard cells of Nicotiana tabacum’, Plant Physiology, 143, 28–37, available: https://doi.org/10.1104/pp.106.092643.
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  Drought induces stomatal closure, a response that is associated with the activation of plasma membrane anion channels in guard cells, by the phytohormone abscisic acid (ABA). In several species, this response is associated with changes in the cytoplasmic free Ca2+ concentration. In Vicia faba, however, guard cell anion channels activate in a Ca2+-independent manner. Because of potential differences between species, Nicotiana tabacum guard cells were studied in intact plants, with simultaneous recordings of the plasma membrane conductance and the cytoplasmic free Ca2+ concentration. ABA triggered transient rises in cytoplasmic Ca2+ in the majority of the guard cells (14 out of 19). In seven out of 14 guard cells, the change in cytoplasmic free Ca2+ closely matched the activation of anion channels, while the Ca2+ rise was delayed in seven other cells. In the remaining five cells, ABA stimulated anion channels without a change in the cytoplasmic Ca2+ level. Even though ABA could activate anion channels in N. tabacum guard cells independent of a rise in the cytoplasmic Ca2+ concentration, patch clamp experiments showed that anion channels in these cells are stimulated by elevated Ca2+ in an ATP-dependent manner. Guard cells thus seem to have evolved both Ca2+-independent and -dependent ABA signaling pathways. Guard cells of N. tabacum apparently utilize both pathways, while ABA signaling in V. faba seems to be restricted to the Ca2+-independent pathway.

  @article{marten2007ca2dependent, abstract = {Drought induces stomatal closure, a response that is associated with the activation of plasma membrane anion channels in guard cells, by the phytohormone abscisic acid (ABA). In several species, this response is associated with changes in the cytoplasmic free Ca2+ concentration. In Vicia faba, however, guard cell anion channels activate in a Ca2+-independent manner. Because of potential differences between species, Nicotiana tabacum guard cells were studied in intact plants, with simultaneous recordings of the plasma membrane conductance and the cytoplasmic free Ca2+ concentration. ABA triggered transient rises in cytoplasmic Ca2+ in the majority of the guard cells (14 out of 19). In seven out of 14 guard cells, the change in cytoplasmic free Ca2+ closely matched the activation of anion channels, while the Ca2+ rise was delayed in seven other cells. In the remaining five cells, ABA stimulated anion channels without a change in the cytoplasmic Ca2+ level. Even though ABA could activate anion channels in N. tabacum guard cells independent of a rise in the cytoplasmic Ca2+ concentration, patch clamp experiments showed that anion channels in these cells are stimulated by elevated Ca2+ in an ATP-dependent manner. Guard cells thus seem to have evolved both Ca2+-independent and -dependent ABA signaling pathways. Guard cells of N. tabacum apparently utilize both pathways, while ABA signaling in V. faba seems to be restricted to the Ca2+-independent pathway.}, author = {Marten, H. and Konrad, K. R. and Dietrich, P. and Roelfsema, M. R. G. and Hedrich, R.}, journal = {Plant Physiology}, keywords = {ABA}, pages = {28-37}, title = {Ca2+-dependent and -independent abscisic acid activation of plasma membrane anion channels in guard cells of Nicotiana tabacum}, volume = 143, year = 2007 }

  %0 Journal Article %1 marten2007ca2dependent %A Marten, H. %A Konrad, K. R. %A Dietrich, P. %A Roelfsema, M. R. G. %A Hedrich, R. %D 2007 %J Plant Physiology %P 28-37 %R 10.1104/pp.106.092643 %T Ca2+-dependent and -independent abscisic acid activation of plasma membrane anion channels in guard cells of Nicotiana tabacum %U http://www.plantphysiol.org/content/143/1/28.full.pdf+html?sid=f49d7db5-918f-49e9-9d63-48494cf28bed %V 143 %X Drought induces stomatal closure, a response that is associated with the activation of plasma membrane anion channels in guard cells, by the phytohormone abscisic acid (ABA). In several species, this response is associated with changes in the cytoplasmic free Ca2+ concentration. In Vicia faba, however, guard cell anion channels activate in a Ca2+-independent manner. Because of potential differences between species, Nicotiana tabacum guard cells were studied in intact plants, with simultaneous recordings of the plasma membrane conductance and the cytoplasmic free Ca2+ concentration. ABA triggered transient rises in cytoplasmic Ca2+ in the majority of the guard cells (14 out of 19). In seven out of 14 guard cells, the change in cytoplasmic free Ca2+ closely matched the activation of anion channels, while the Ca2+ rise was delayed in seven other cells. In the remaining five cells, ABA stimulated anion channels without a change in the cytoplasmic Ca2+ level. Even though ABA could activate anion channels in N. tabacum guard cells independent of a rise in the cytoplasmic Ca2+ concentration, patch clamp experiments showed that anion channels in these cells are stimulated by elevated Ca2+ in an ATP-dependent manner. Guard cells thus seem to have evolved both Ca2+-independent and -dependent ABA signaling pathways. Guard cells of N. tabacum apparently utilize both pathways, while ABA signaling in V. faba seems to be restricted to the Ca2+-independent pathway.
• Marten, H., Hedrich, R., and Roelfsema, M.R.G. (2007) ‘Blue light inhibits guard cell plasma membrane anion channels in a phototropin-dependent manner’, The Plant journal, 50(1), 29–39, available: https://doi.org/10.1111/j.1365-313X.2006.03026.x.
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  Guard cells respond to light through two independent signalling pathways. The first pathway is initiated by photosynthetically active radiation and has been associated with changes in the intercellular CO2 concentration, leading to inhibition of plasma membrane anion channels. The second response is blue-light-specific and so far has been restricted to the activation of plasma membrane H+-ATPases. In a search for interactions of both signalling pathways, guard cells of Vicia faba and Arabidopsis thaliana were studied in intact plants. Vicia faba guard cells recorded in CO2-free air responded to blue light with a transient outward plasma membrane current that had an average peak value of 17 pA. In line with previous reports, changes in the current–voltage relation of the plasma membrane indicate that this outward current is based on the activation of H+-ATPases. However, when V. faba guard cells were blue-light-stimulated in air with 700 ul l-1 CO2, the outward current increased to 56 pA. The increase in current was linked to inhibition of S-type anion channels. Blue light also inhibited plasma membrane anion channels in A. thaliana guard cells, but not in the phot1 phot2 double mutant. These results show that blue light inhibits plasma membrane anion channels through a pathway involving phototropins, in addition to the stimulation of guard cell plasma membrane H+-ATPases.

  @article{marten2007light, abstract = {Guard cells respond to light through two independent signalling pathways. The first pathway is initiated by photosynthetically active radiation and has been associated with changes in the intercellular CO2 concentration, leading to inhibition of plasma membrane anion channels. The second response is blue-light-specific and so far has been restricted to the activation of plasma membrane H+-ATPases. In a search for interactions of both signalling pathways, guard cells of Vicia faba and Arabidopsis thaliana were studied in intact plants. Vicia faba guard cells recorded in CO2-free air responded to blue light with a transient outward plasma membrane current that had an average peak value of 17 pA. In line with previous reports, changes in the current–voltage relation of the plasma membrane indicate that this outward current is based on the activation of H+-ATPases. However, when V. faba guard cells were blue-light-stimulated in air with 700 ul l-1 CO2, the outward current increased to 56 pA. The increase in current was linked to inhibition of S-type anion channels. Blue light also inhibited plasma membrane anion channels in A. thaliana guard cells, but not in the phot1 phot2 double mutant. These results show that blue light inhibits plasma membrane anion channels through a pathway involving phototropins, in addition to the stimulation of guard cell plasma membrane H+-ATPases.}, author = {Marten, H. and Hedrich, R. and Roelfsema, M. R. G.}, journal = {The Plant journal}, keywords = {s-type}, number = 1, pages = {29-39}, title = {Blue light inhibits guard cell plasma membrane anion channels in a phototropin-dependent manner}, volume = 50, year = 2007 }

  %0 Journal Article %1 marten2007light %A Marten, H. %A Hedrich, R. %A Roelfsema, M. R. G. %D 2007 %J The Plant journal %N 1 %P 29-39 %R 10.1111/j.1365-313X.2006.03026.x %T Blue light inhibits guard cell plasma membrane anion channels in a phototropin-dependent manner %U http://onlinelibrary.wiley.com/doi/10.1111/j.1365-313X.2006.03026.x/full %V 50 %X Guard cells respond to light through two independent signalling pathways. The first pathway is initiated by photosynthetically active radiation and has been associated with changes in the intercellular CO2 concentration, leading to inhibition of plasma membrane anion channels. The second response is blue-light-specific and so far has been restricted to the activation of plasma membrane H+-ATPases. In a search for interactions of both signalling pathways, guard cells of Vicia faba and Arabidopsis thaliana were studied in intact plants. Vicia faba guard cells recorded in CO2-free air responded to blue light with a transient outward plasma membrane current that had an average peak value of 17 pA. In line with previous reports, changes in the current–voltage relation of the plasma membrane indicate that this outward current is based on the activation of H+-ATPases. However, when V. faba guard cells were blue-light-stimulated in air with 700 ul l-1 CO2, the outward current increased to 56 pA. The increase in current was linked to inhibition of S-type anion channels. Blue light also inhibited plasma membrane anion channels in A. thaliana guard cells, but not in the phot1 phot2 double mutant. These results show that blue light inhibits plasma membrane anion channels through a pathway involving phototropins, in addition to the stimulation of guard cell plasma membrane H+-ATPases.

• Roelfsema, M.R.G., Konrad, K.R., Marten, H., Psaras, G.K., Hartung, W., and Hedrich, R. (2006) ‘Guard cells in albino leaf patches do not respond to photosynthetically active radiation, but are sensitive to blue light, CO2 and abscisic acid’, Plant, cell & environment, 29(8), 1595–1605, available: https://doi.org/10.1111/j.1365-3040.2006.01536.x.
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  Stomatal openings can be stimulated by light through two signalling pathways. The first pathway is blue light specific and involves phototropins, while the second pathway mediates a response to photosynthetically active radiation (PAR). This second pathway was studied with the use of albino Vicia faba plants and variegated leaves of Chlorophytum comosum. Treatment of V. faba with norflurazon (Nf) inhibits the synthesis of carotenoids and leads to albino leaves with guard cells that lack functional green chloroplasts. Guard cells in albino leaf patches of C. comosum, however, do contain photosynthetically active chloroplasts. Stomata in albino leaf patches of both plants did not respond to red light, although blue light could still induce stomatal opening. This shows that the response to PAR is not functioning in albino leaf patches, even though guard cells of C. comosum harbour chloroplasts. Stomata of Nf-treated plants still responded to CO2 and abscisic acid (ABA). The size of Nf-treated guard cells was increased, but impalement studies with double-barrelled microelectrodes revealed no changes in ion-transport properties at the plasma membrane of guard cells. Blue light could hyperpolarize albino guard cells by triggering outward currents with peak values of 37 pA in albino plants and 51 pA in green control cells. Because of the inhibition of carotenoid biosynthesis, Nf-treated V. faba plants contained only 4% of the ABA content found in green control plants. The ABA dose dependence of anion channel activation in guard cells was shifted in these plants, causing a reduced response to 10 µm ABA. These data show that despite the dramatic changes in physiology caused by Nf, the gross responsiveness of guard cells to blue light, CO2 and ABA remains unaltered. Stomata in albino leaf patches, however, do not respond to PAR, but require photosynthetically active mesophyll cells for this response.

  @article{roelfsema2006guard, abstract = {Stomatal openings can be stimulated by light through two signalling pathways. The first pathway is blue light specific and involves phototropins, while the second pathway mediates a response to photosynthetically active radiation (PAR). This second pathway was studied with the use of albino Vicia faba plants and variegated leaves of Chlorophytum comosum. Treatment of V. faba with norflurazon (Nf) inhibits the synthesis of carotenoids and leads to albino leaves with guard cells that lack functional green chloroplasts. Guard cells in albino leaf patches of C. comosum, however, do contain photosynthetically active chloroplasts. Stomata in albino leaf patches of both plants did not respond to red light, although blue light could still induce stomatal opening. This shows that the response to PAR is not functioning in albino leaf patches, even though guard cells of C. comosum harbour chloroplasts. Stomata of Nf-treated plants still responded to CO2 and abscisic acid (ABA). The size of Nf-treated guard cells was increased, but impalement studies with double-barrelled microelectrodes revealed no changes in ion-transport properties at the plasma membrane of guard cells. Blue light could hyperpolarize albino guard cells by triggering outward currents with peak values of 37 pA in albino plants and 51 pA in green control cells. Because of the inhibition of carotenoid biosynthesis, Nf-treated V. faba plants contained only 4% of the ABA content found in green control plants. The ABA dose dependence of anion channel activation in guard cells was shifted in these plants, causing a reduced response to 10 µm ABA. These data show that despite the dramatic changes in physiology caused by Nf, the gross responsiveness of guard cells to blue light, CO2 and ABA remains unaltered. Stomata in albino leaf patches, however, do not respond to PAR, but require photosynthetically active mesophyll cells for this response.}, author = {Roelfsema, M. R. G. and Konrad, K. R. and Marten, H. and Psaras, G. K. and Hartung, W. and Hedrich, R.}, journal = {Plant, cell & environment}, keywords = {ABA}, number = 8, pages = {1595-1605}, title = {Guard cells in albino leaf patches do not respond to photosynthetically active radiation, but are sensitive to blue light, CO2 and abscisic acid}, volume = 29, year = 2006 }

  %0 Journal Article %1 roelfsema2006guard %A Roelfsema, M. R. G. %A Konrad, K. R. %A Marten, H. %A Psaras, G. K. %A Hartung, W. %A Hedrich, R. %D 2006 %J Plant, cell & environment %N 8 %P 1595-1605 %R 10.1111/j.1365-3040.2006.01536.x %T Guard cells in albino leaf patches do not respond to photosynthetically active radiation, but are sensitive to blue light, CO2 and abscisic acid %U http://onlinelibrary.wiley.com/doi/10.1111/j.1365-3040.2006.01536.x/full %V 29 %X Stomatal openings can be stimulated by light through two signalling pathways. The first pathway is blue light specific and involves phototropins, while the second pathway mediates a response to photosynthetically active radiation (PAR). This second pathway was studied with the use of albino Vicia faba plants and variegated leaves of Chlorophytum comosum. Treatment of V. faba with norflurazon (Nf) inhibits the synthesis of carotenoids and leads to albino leaves with guard cells that lack functional green chloroplasts. Guard cells in albino leaf patches of C. comosum, however, do contain photosynthetically active chloroplasts. Stomata in albino leaf patches of both plants did not respond to red light, although blue light could still induce stomatal opening. This shows that the response to PAR is not functioning in albino leaf patches, even though guard cells of C. comosum harbour chloroplasts. Stomata of Nf-treated plants still responded to CO2 and abscisic acid (ABA). The size of Nf-treated guard cells was increased, but impalement studies with double-barrelled microelectrodes revealed no changes in ion-transport properties at the plasma membrane of guard cells. Blue light could hyperpolarize albino guard cells by triggering outward currents with peak values of 37 pA in albino plants and 51 pA in green control cells. Because of the inhibition of carotenoid biosynthesis, Nf-treated V. faba plants contained only 4% of the ABA content found in green control plants. The ABA dose dependence of anion channel activation in guard cells was shifted in these plants, causing a reduced response to 10 µm ABA. These data show that despite the dramatic changes in physiology caused by Nf, the gross responsiveness of guard cells to blue light, CO2 and ABA remains unaltered. Stomata in albino leaf patches, however, do not respond to PAR, but require photosynthetically active mesophyll cells for this response.

• Meyerhoff, O., Muller, K., Roelfsema, M.R.G., Latz, A., Lacombe, B., Hedrich, R., Dietrich, P., and Becker, D. (2005) ‘AtGLR3.4, a glutamate receptor channel-like gene is sensitive to touch and cold’, Planta, 222(3), 418–427, available: https://doi.org/10.1007/s00425-005-1551-3.
  • [ Abstract ]
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  The Arabidopsis genome encodes for 20 members of putative ligand-gated channels, termed glutamate receptors (GLR). Despite the fact that initial studies suggested a role for GLRs in various aspects of photomorphogenesis, calcium homeostasis or aluminium toxicity, their functional properties and physiological role in plants remain elusive. Here, we have focussed on AtGLR3.4, which is ubiquitously expressed in Arabidopsis including roots, vascular bundles, mesophyll cells and guard cells. AtGLR3.4 encodes a glutamate-, touch-, and cold-sensitive member of this gene family. Abiotic stress stimuli such as touch, osmotic stress or cold stimulated AtGLR3.4 expression in an abscisic acid-independent, but calcium-dependent manner. In plants expressing the Ca2+ -reporter apoaequorin, glutamate as well as cold elicited cytosolic calcium elevations. Upon glutamate treatment of mesophyll cells, the plasma membrane depolarised by about 120 mV. Both glutamate responses were transient in nature, sensitive to glutamate receptor antagonists, and were subject to desensitisation. One hour after eliciting the first calcium signal, a 50{%} recovery from desensitisation was observed, reflecting the stimulus-induced fast activation of AtGLR3.4 transcription. We thus conclude that AtGLR3.4 in particular and GLRs in general could play an important role in the Ca2+ -based, fast transmission of environmental stress.

  @article{Meyerhoff2005, abstract = {The Arabidopsis genome encodes for 20 members of putative ligand-gated channels, termed glutamate receptors (GLR). Despite the fact that initial studies suggested a role for GLRs in various aspects of photomorphogenesis, calcium homeostasis or aluminium toxicity, their functional properties and physiological role in plants remain elusive. Here, we have focussed on AtGLR3.4, which is ubiquitously expressed in Arabidopsis including roots, vascular bundles, mesophyll cells and guard cells. AtGLR3.4 encodes a glutamate-, touch-, and cold-sensitive member of this gene family. Abiotic stress stimuli such as touch, osmotic stress or cold stimulated AtGLR3.4 expression in an abscisic acid-independent, but calcium-dependent manner. In plants expressing the Ca2+ -reporter apoaequorin, glutamate as well as cold elicited cytosolic calcium elevations. Upon glutamate treatment of mesophyll cells, the plasma membrane depolarised by about 120 mV. Both glutamate responses were transient in nature, sensitive to glutamate receptor antagonists, and were subject to desensitisation. One hour after eliciting the first calcium signal, a 50{\%} recovery from desensitisation was observed, reflecting the stimulus-induced fast activation of AtGLR3.4 transcription. We thus conclude that AtGLR3.4 in particular and GLRs in general could play an important role in the Ca2+ -based, fast transmission of environmental stress.}, author = {Meyerhoff, O. and Muller, K. and Roelfsema, M. R. G. and Latz, A. and Lacombe, B. and Hedrich, R. and Dietrich, P. and Becker, D.}, journal = {Planta}, keywords = {Glutamate}, month = 10, number = 3, pages = {418–427}, title = {AtGLR3.4, a glutamate receptor channel-like gene is sensitive to touch and cold}, volume = 222, year = 2005 }

  %0 Journal Article %1 Meyerhoff2005 %A Meyerhoff, O. %A Muller, K. %A Roelfsema, M. R. G. %A Latz, A. %A Lacombe, B. %A Hedrich, R. %A Dietrich, P. %A Becker, D. %D 2005 %J Planta %N 3 %P 418--427 %R 10.1007/s00425-005-1551-3 %T AtGLR3.4, a glutamate receptor channel-like gene is sensitive to touch and cold %U https://doi.org/10.1007/s00425-005-1551-3 %V 222 %X The Arabidopsis genome encodes for 20 members of putative ligand-gated channels, termed glutamate receptors (GLR). Despite the fact that initial studies suggested a role for GLRs in various aspects of photomorphogenesis, calcium homeostasis or aluminium toxicity, their functional properties and physiological role in plants remain elusive. Here, we have focussed on AtGLR3.4, which is ubiquitously expressed in Arabidopsis including roots, vascular bundles, mesophyll cells and guard cells. AtGLR3.4 encodes a glutamate-, touch-, and cold-sensitive member of this gene family. Abiotic stress stimuli such as touch, osmotic stress or cold stimulated AtGLR3.4 expression in an abscisic acid-independent, but calcium-dependent manner. In plants expressing the Ca2+ -reporter apoaequorin, glutamate as well as cold elicited cytosolic calcium elevations. Upon glutamate treatment of mesophyll cells, the plasma membrane depolarised by about 120 mV. Both glutamate responses were transient in nature, sensitive to glutamate receptor antagonists, and were subject to desensitisation. One hour after eliciting the first calcium signal, a 50{%} recovery from desensitisation was observed, reflecting the stimulus-induced fast activation of AtGLR3.4 transcription. We thus conclude that AtGLR3.4 in particular and GLRs in general could play an important role in the Ca2+ -based, fast transmission of environmental stress.
• Roelfsema, M.R.G. and Hedrich, R. (2005) ‘In the light of stomatal opening: new insights into ’the Watergate’’, The New phytologist, 167(3), 665–691, available: https://doi.org/10.1111/j.1469-8137.2005.01460.x.
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  Stomata can be regarded as hydraulically driven valves in the leaf surface, which open to allow CO2 uptake and close to prevent excessive loss of water. Movement of these ‘Watergates’ is regulated by environmental conditions, such as light, CO2 and humidity. Guard cells can sense environmental conditions and function as motor cells within the stomatal complex. Stomatal movement results from the transport of K+ salts across the guard cell membranes. In this review, we discuss the biophysical principles and mechanisms of stomatal movement and relate these to ion transport at the plasma membrane and vacuolar membrane. Studies with isolated guard cells, combined with recordings on single guard cells in intact plants, revealed that light stimulates stomatal opening via blue light-specific and photosynthetic-active radiation-dependent pathways. In addition, guard cells sense changes in air humidity and the water status of distant tissues via the stress hormone abscisic acid (ABA). Guard cells thus provide an excellent system to study cross-talk, as multiple signaling pathways induce both short- and long-term responses in these sensory cells.

  @article{roelfsema2005light, abstract = {Stomata can be regarded as hydraulically driven valves in the leaf surface, which open to allow CO2 uptake and close to prevent excessive loss of water. Movement of these ‘Watergates’ is regulated by environmental conditions, such as light, CO2 and humidity. Guard cells can sense environmental conditions and function as motor cells within the stomatal complex. Stomatal movement results from the transport of K+ salts across the guard cell membranes. In this review, we discuss the biophysical principles and mechanisms of stomatal movement and relate these to ion transport at the plasma membrane and vacuolar membrane. Studies with isolated guard cells, combined with recordings on single guard cells in intact plants, revealed that light stimulates stomatal opening via blue light-specific and photosynthetic-active radiation-dependent pathways. In addition, guard cells sense changes in air humidity and the water status of distant tissues via the stress hormone abscisic acid (ABA). Guard cells thus provide an excellent system to study cross-talk, as multiple signaling pathways induce both short- and long-term responses in these sensory cells.}, author = {Roelfsema, M. R. G. and Hedrich, R.}, journal = {The New phytologist}, keywords = {ABA}, number = 3, pages = {665-691}, title = {In the light of stomatal opening: new insights into 'the Watergate'}, volume = 167, year = 2005 }

  %0 Journal Article %1 roelfsema2005light %A Roelfsema, M. R. G. %A Hedrich, R. %D 2005 %J The New phytologist %N 3 %P 665-691 %R 10.1111/j.1469-8137.2005.01460.x %T In the light of stomatal opening: new insights into 'the Watergate' %U http://onlinelibrary.wiley.com/doi/10.1111/j.1469-8137.2005.01460.x/full %V 167 %X Stomata can be regarded as hydraulically driven valves in the leaf surface, which open to allow CO2 uptake and close to prevent excessive loss of water. Movement of these ‘Watergates’ is regulated by environmental conditions, such as light, CO2 and humidity. Guard cells can sense environmental conditions and function as motor cells within the stomatal complex. Stomatal movement results from the transport of K+ salts across the guard cell membranes. In this review, we discuss the biophysical principles and mechanisms of stomatal movement and relate these to ion transport at the plasma membrane and vacuolar membrane. Studies with isolated guard cells, combined with recordings on single guard cells in intact plants, revealed that light stimulates stomatal opening via blue light-specific and photosynthetic-active radiation-dependent pathways. In addition, guard cells sense changes in air humidity and the water status of distant tissues via the stress hormone abscisic acid (ABA). Guard cells thus provide an excellent system to study cross-talk, as multiple signaling pathways induce both short- and long-term responses in these sensory cells.
• Levchenko, V., Konrad, K.R., Dietrich, P., Roelfsema, M.R.G., and Hedrich, R. (2005) ‘Cytosolic abscisic acid activates guard cell anion channels without preceding Ca2+ signals’, Proceedings of the National Academy of Sciences of the United States of America, 102(11), 4203–4208, available: https://doi.org/10.1073/pnas.0500146102.
  • [ Abstract ]
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  The phytohormone abscisic acid (ABA) reports on the water status of the plant and induces stomatal closure. Guard cell anion channels play a central role in this response, because they mediate anion efflux, and in turn, cause a depolarization-induced K+ release. We recorded early steps in ABA signaling, introducing multibarreled microelectrodes in guard cells of intact plants. Upon external ABA treatment, anion channels transiently activated after a lag phase of +/-2 min. As expected for a cytosolic ABA receptor, iontophoretic ABA loading into the cytoplasm initiated a rise in anion current without delay. These ABA responses could be elicited repetitively at resting and at largely depolarized potentials (e.g., 0 mV), ruling out signal transduction by means of hyperpolarizationactivated calcium channels. Likewise, ABA stimulation did not induce a rise in the cytosolic free-calcium concentration. However, the presence of +/-100 nM background Ca2+ was required for anion channel function, because the action of ABA on anion channels was repressed after loading of the Ca2+ chelator 1,2-bis(2-aminophenoxy) ethane-N,N,N',N'-tetraacetate. The chain of events appears very direct, because none of the tested putative ABA-signaling intermediates (inositol 1,4,5 trisphosphate, inositol hexakisphosphate, nicotinic acid adenine dinucleotide phosphate, and cyclic ADP-ribose), could mimic ABA as anion channel activator. In patchclamp experiments, cytosolic ABA also evoked anion current transients carried by R- and S-type anion channels. The response was dose-dependent with half-maximum activation at 2.6 uM ABA. Our studies point to an ABA pathway initiated by ABA binding to a cytosolic receptor that within seconds activates anion channels, and in turn, leads to depolarization of the plasma membrane.

  @article{levchenko2005cytosolic, abstract = {The phytohormone abscisic acid (ABA) reports on the water status of the plant and induces stomatal closure. Guard cell anion channels play a central role in this response, because they mediate anion efflux, and in turn, cause a depolarization-induced K+ release. We recorded early steps in ABA signaling, introducing multibarreled microelectrodes in guard cells of intact plants. Upon external ABA treatment, anion channels transiently activated after a lag phase of +/-2 min. As expected for a cytosolic ABA receptor, iontophoretic ABA loading into the cytoplasm initiated a rise in anion current without delay. These ABA responses could be elicited repetitively at resting and at largely depolarized potentials (e.g., 0 mV), ruling out signal transduction by means of hyperpolarizationactivated calcium channels. Likewise, ABA stimulation did not induce a rise in the cytosolic free-calcium concentration. However, the presence of +/-100 nM background Ca2+ was required for anion channel function, because the action of ABA on anion channels was repressed after loading of the Ca2+ chelator 1,2-bis(2-aminophenoxy) ethane-N,N,N',N'-tetraacetate. The chain of events appears very direct, because none of the tested putative ABA-signaling intermediates (inositol 1,4,5 trisphosphate, inositol hexakisphosphate, nicotinic acid adenine dinucleotide phosphate, and cyclic ADP-ribose), could mimic ABA as anion channel activator. In patchclamp experiments, cytosolic ABA also evoked anion current transients carried by R- and S-type anion channels. The response was dose-dependent with half-maximum activation at 2.6 uM ABA. Our studies point to an ABA pathway initiated by ABA binding to a cytosolic receptor that within seconds activates anion channels, and in turn, leads to depolarization of the plasma membrane.}, author = {Levchenko, V. and Konrad, K. R. and Dietrich, P. and Roelfsema, M. R. G. and Hedrich, R.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, keywords = {Vicia-faba}, number = 11, pages = {4203-4208}, title = {Cytosolic abscisic acid activates guard cell anion channels without preceding Ca2+ signals}, volume = 102, year = 2005 }

  %0 Journal Article %1 levchenko2005cytosolic %A Levchenko, V. %A Konrad, K. R. %A Dietrich, P. %A Roelfsema, M. R. G. %A Hedrich, R. %D 2005 %J Proceedings of the National Academy of Sciences of the United States of America %N 11 %P 4203-4208 %R 10.1073/pnas.0500146102 %T Cytosolic abscisic acid activates guard cell anion channels without preceding Ca2+ signals %U http://www.pnas.org/content/102/11/4203.full.pdf?sid=b6ab7dcc-56ed-4da7-9028-0f3d08ab925c %V 102 %X The phytohormone abscisic acid (ABA) reports on the water status of the plant and induces stomatal closure. Guard cell anion channels play a central role in this response, because they mediate anion efflux, and in turn, cause a depolarization-induced K+ release. We recorded early steps in ABA signaling, introducing multibarreled microelectrodes in guard cells of intact plants. Upon external ABA treatment, anion channels transiently activated after a lag phase of +/-2 min. As expected for a cytosolic ABA receptor, iontophoretic ABA loading into the cytoplasm initiated a rise in anion current without delay. These ABA responses could be elicited repetitively at resting and at largely depolarized potentials (e.g., 0 mV), ruling out signal transduction by means of hyperpolarizationactivated calcium channels. Likewise, ABA stimulation did not induce a rise in the cytosolic free-calcium concentration. However, the presence of +/-100 nM background Ca2+ was required for anion channel function, because the action of ABA on anion channels was repressed after loading of the Ca2+ chelator 1,2-bis(2-aminophenoxy) ethane-N,N,N',N'-tetraacetate. The chain of events appears very direct, because none of the tested putative ABA-signaling intermediates (inositol 1,4,5 trisphosphate, inositol hexakisphosphate, nicotinic acid adenine dinucleotide phosphate, and cyclic ADP-ribose), could mimic ABA as anion channel activator. In patchclamp experiments, cytosolic ABA also evoked anion current transients carried by R- and S-type anion channels. The response was dose-dependent with half-maximum activation at 2.6 uM ABA. Our studies point to an ABA pathway initiated by ABA binding to a cytosolic receptor that within seconds activates anion channels, and in turn, leads to depolarization of the plasma membrane.

• Becker, D., Geiger, D., Dunkel, M., Roller, A., Bertl, A., Latz, A., Carpaneto, A., Dietrich, P., Roelfsema, M.R.G., Voelker, C., Schmidt, D., Mueller-Roeber, B., Czempinski, K., and Hedrich, R. (2004) ‘AtTPK4, an Arabidopsis tandem-pore K+ channel, poised to control the pollen membrane voltage in a pH- and Ca2+-dependent manner’, Proceedings of the National Academy of Sciences of the United States of America, 101(44), 15621–15626, available: https://doi.org/10.1073/pnas.0401502101.
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  @article{becker2004attpk4, author = {Becker, D. and Geiger, D. and Dunkel, M. and Roller, A. and Bertl, A. and Latz, A. and Carpaneto, A. and Dietrich, P. and Roelfsema, M. R. G. and Voelker, C. and Schmidt, D. and Mueller-Roeber, B. and Czempinski, K. and Hedrich, R.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, keywords = {pollen-tube}, number = 44, pages = {15621-15626}, title = {AtTPK4, an Arabidopsis tandem-pore K+ channel, poised to control the pollen membrane voltage in a pH- and Ca2+-dependent manner}, volume = 101, year = 2004 }

  %0 Journal Article %1 becker2004attpk4 %A Becker, D. %A Geiger, D. %A Dunkel, M. %A Roller, A. %A Bertl, A. %A Latz, A. %A Carpaneto, A. %A Dietrich, P. %A Roelfsema, M. R. G. %A Voelker, C. %A Schmidt, D. %A Mueller-Roeber, B. %A Czempinski, K. %A Hedrich, R. %D 2004 %J Proceedings of the National Academy of Sciences of the United States of America %N 44 %P 15621-15626 %R 10.1073/pnas.0401502101 %T AtTPK4, an Arabidopsis tandem-pore K+ channel, poised to control the pollen membrane voltage in a pH- and Ca2+-dependent manner %U /brokenurl#<Go to ISI>://WOS:000224922300015 %V 101
• Roelfsema, M.R.G., Levchenko, V., and Hedrich, R. (2004) ‘ABA depolarizes guard cells in intact plants, through a transient activation of R- and S-type anion channels’, The Plant journal, 37(4), 578–588, available: https://doi.org/10.1111/j.1365-313X.2003.01985.x.
  • [ Abstract ]
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  During drought, the plant hormone abscisic acid (ABA) induces rapid stomatal closure and in turn reduces transpiration. Stomatal closure is accompanied by large ion fluxes across the plasma membrane, carried by K+ and anion channels. We recorded changes in the activity of these channels induced by ABA, for guard cells of intact Vicia faba plants. Guard cells in their natural environment were impaled with double-barrelled electrodes, and ABA was applied via the leaf surface. In 45 out of 85 cells tested, ABA triggered a transient depolarization of the plasma membrane. In these cells, the membrane potential partially recovered in the presence of ABA; however, a full recovery of the membrane potentials was only observed after removal of ABA. Repetitive ABA responses could be evoked in single cells, but the magnitude of the response varied from one hormone application to the other. The transient depolarization correlated with the activation of anion channels, which peaked 5 min after introduction of the stimulus. In guard cells with a moderate increase in plasma membrane conductance (ΔG < 5 nS), ABA predominantly activated voltage-independent (slow (S)-type) anion channels. During strong responses (ΔG > 5 nS), however, ABA activated voltage-dependent (rapid (R)-type) in addition to S-type anion channels. We conclude that the combined activation of these two channel types leads to the transient depolarization of guard cells. The nature of this ABA response correlates with the transient extrusion of Cl− from guard cells and a rapid but confined reduction in stomatal aperture.

  @article{roelfsema2004depolarizes, abstract = {During drought, the plant hormone abscisic acid (ABA) induces rapid stomatal closure and in turn reduces transpiration. Stomatal closure is accompanied by large ion fluxes across the plasma membrane, carried by K+ and anion channels. We recorded changes in the activity of these channels induced by ABA, for guard cells of intact Vicia faba plants. Guard cells in their natural environment were impaled with double-barrelled electrodes, and ABA was applied via the leaf surface. In 45 out of 85 cells tested, ABA triggered a transient depolarization of the plasma membrane. In these cells, the membrane potential partially recovered in the presence of ABA; however, a full recovery of the membrane potentials was only observed after removal of ABA. Repetitive ABA responses could be evoked in single cells, but the magnitude of the response varied from one hormone application to the other. The transient depolarization correlated with the activation of anion channels, which peaked 5 min after introduction of the stimulus. In guard cells with a moderate increase in plasma membrane conductance (ΔG < 5 nS), ABA predominantly activated voltage-independent (slow (S)-type) anion channels. During strong responses (ΔG > 5 nS), however, ABA activated voltage-dependent (rapid (R)-type) in addition to S-type anion channels. We conclude that the combined activation of these two channel types leads to the transient depolarization of guard cells. The nature of this ABA response correlates with the transient extrusion of Cl− from guard cells and a rapid but confined reduction in stomatal aperture.}, author = {Roelfsema, M. R. G. and Levchenko, V. and Hedrich, R.}, journal = {The Plant journal}, keywords = {ABA}, number = 4, pages = {578-588}, title = {ABA depolarizes guard cells in intact plants, through a transient activation of R- and S-type anion channels}, volume = 37, year = 2004 }

  %0 Journal Article %1 roelfsema2004depolarizes %A Roelfsema, M. R. G. %A Levchenko, V. %A Hedrich, R. %D 2004 %J The Plant journal %N 4 %P 578-588 %R 10.1111/j.1365-313X.2003.01985.x %T ABA depolarizes guard cells in intact plants, through a transient activation of R- and S-type anion channels %U http://onlinelibrary.wiley.com/doi/10.1111/j.1365-313X.2003.01985.x/full %V 37 %X During drought, the plant hormone abscisic acid (ABA) induces rapid stomatal closure and in turn reduces transpiration. Stomatal closure is accompanied by large ion fluxes across the plasma membrane, carried by K+ and anion channels. We recorded changes in the activity of these channels induced by ABA, for guard cells of intact Vicia faba plants. Guard cells in their natural environment were impaled with double-barrelled electrodes, and ABA was applied via the leaf surface. In 45 out of 85 cells tested, ABA triggered a transient depolarization of the plasma membrane. In these cells, the membrane potential partially recovered in the presence of ABA; however, a full recovery of the membrane potentials was only observed after removal of ABA. Repetitive ABA responses could be evoked in single cells, but the magnitude of the response varied from one hormone application to the other. The transient depolarization correlated with the activation of anion channels, which peaked 5 min after introduction of the stimulus. In guard cells with a moderate increase in plasma membrane conductance (ΔG < 5 nS), ABA predominantly activated voltage-independent (slow (S)-type) anion channels. During strong responses (ΔG > 5 nS), however, ABA activated voltage-dependent (rapid (R)-type) in addition to S-type anion channels. We conclude that the combined activation of these two channel types leads to the transient depolarization of guard cells. The nature of this ABA response correlates with the transient extrusion of Cl− from guard cells and a rapid but confined reduction in stomatal aperture.

• Becker, D., Hoth, S., Ache, P., Wenkel, S., Roelfsema, M.R.G., Meyerhoff, O., Hartung, W., and Hedrich, R. (2003) ‘Regulation of the ABA-sensitive Arabidopsis potassium channel gene GORK in response to water stress’, Febs Letters, (1-2), 119–126, available: https://doi.org/10.1016/s0014-5793(03)01118-9.
  • [ Abstract ]
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  The phytohormone abscisic acid (ABA) regulates many stress-related processes in plants. In this context ABA mediates the responsiveness of plants to environmental stresses such as drought, cold or salt. In response to water stress, ABA induces stomatal closure by activating Ca2+, K+ and anion channels in guard cells. To understand the signalling pathways that regulate these turgor control elements, we studied the transcriptional control of the K+ release channel gene GORK that is expressed in guard cells, roots and vascular tissue. GORK transcription was up-regulated upon onset of drought, salt stress and cold. The wilting hormone ABA that integrates responses to these stimuli induced GORK expression in seedlings in a timeand concentration-dependent manner and this induction was dependent on extracellular Ca2+. ABA-responsive expression of GORK was impaired in the ABA-insensitive mutants abi1-1 and abi2-1, indicating that these protein phosphatases are regulators of GORK expression. Application of ABA to suspensioncultured cells for 2 min followed by a 4 h chase was su⁄cient to manifest transcriptional activation of the K+ channel gene. As predicted for a process involved in drought adaptation, only 12-24 h after the release of the stress hormone, GORK mRNA slowly decreased. In contrast to other tissues, GORK expression as well as K+ out channel activity in guard cells is ABA insensitive, allowing the plant to adjust stomatal movement and water status control separately.

  @article{becker2003, abstract = {The phytohormone abscisic acid (ABA) regulates many stress-related processes in plants. In this context ABA mediates the responsiveness of plants to environmental stresses such as drought, cold or salt. In response to water stress, ABA induces stomatal closure by activating Ca2+, K+ and anion channels in guard cells. To understand the signalling pathways that regulate these turgor control elements, we studied the transcriptional control of the K+ release channel gene GORK that is expressed in guard cells, roots and vascular tissue. GORK transcription was up-regulated upon onset of drought, salt stress and cold. The wilting hormone ABA that integrates responses to these stimuli induced GORK expression in seedlings in a timeand concentration-dependent manner and this induction was dependent on extracellular Ca2+. ABA-responsive expression of GORK was impaired in the ABA-insensitive mutants abi1-1 and abi2-1, indicating that these protein phosphatases are regulators of GORK expression. Application of ABA to suspensioncultured cells for 2 min followed by a 4 h chase was su⁄cient to manifest transcriptional activation of the K+ channel gene. As predicted for a process involved in drought adaptation, only 12-24 h after the release of the stress hormone, GORK mRNA slowly decreased. In contrast to other tissues, GORK expression as well as K+ out channel activity in guard cells is ABA insensitive, allowing the plant to adjust stomatal movement and water status control separately.}, author = {Becker, D. and Hoth, S. and Ache, P. and Wenkel, S. and Roelfsema, M. R. G. and Meyerhoff, O. and Hartung, W. and Hedrich, R.}, journal = {Febs Letters}, keywords = {K+-efflux-channel}, number = {1-2}, pages = {119-126}, title = {Regulation of the ABA-sensitive Arabidopsis potassium channel gene GORK in response to water stress}, year = 2003 }

  %0 Journal Article %1 becker2003 %A Becker, D. %A Hoth, S. %A Ache, P. %A Wenkel, S. %A Roelfsema, M. R. G. %A Meyerhoff, O. %A Hartung, W. %A Hedrich, R. %D 2003 %J Febs Letters %N 1-2 %P 119-126 %R 10.1016/s0014-5793(03)01118-9 %T Regulation of the ABA-sensitive Arabidopsis potassium channel gene GORK in response to water stress %X The phytohormone abscisic acid (ABA) regulates many stress-related processes in plants. In this context ABA mediates the responsiveness of plants to environmental stresses such as drought, cold or salt. In response to water stress, ABA induces stomatal closure by activating Ca2+, K+ and anion channels in guard cells. To understand the signalling pathways that regulate these turgor control elements, we studied the transcriptional control of the K+ release channel gene GORK that is expressed in guard cells, roots and vascular tissue. GORK transcription was up-regulated upon onset of drought, salt stress and cold. The wilting hormone ABA that integrates responses to these stimuli induced GORK expression in seedlings in a timeand concentration-dependent manner and this induction was dependent on extracellular Ca2+. ABA-responsive expression of GORK was impaired in the ABA-insensitive mutants abi1-1 and abi2-1, indicating that these protein phosphatases are regulators of GORK expression. Application of ABA to suspensioncultured cells for 2 min followed by a 4 h chase was su⁄cient to manifest transcriptional activation of the K+ channel gene. As predicted for a process involved in drought adaptation, only 12-24 h after the release of the stress hormone, GORK mRNA slowly decreased. In contrast to other tissues, GORK expression as well as K+ out channel activity in guard cells is ABA insensitive, allowing the plant to adjust stomatal movement and water status control separately.

• Roelfsema, M.R.G., Hanstein, S., Felle, H.H., and Hedrich, R. (2002) ‘CO2 provides an intermediate link in the red light response of guard cells’, The Plant Journal, 32(1), 65–75, available: https://doi.org/10.1046/j.1365-313X.2002.01403.x.
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  Guard cells in intact leafs display light-induced membrane potential changes, which alter the direction of K+-transport across the plasma membrane (Roelfsema et al., 2001). A beam of blue light, but not red light, directed at the impaled guard cell triggers this response, while both light qualities induce opening of stomata. To gain insight into this apparent contradiction, we explored the possible interaction between red light and CO2. Guard cells in the intact plant were impaled with double-barrelled electrodes and illuminated with red light. Cells that were hyperpolarized in CO2-free air, depolarized after a switch to air with 700 µl l−1 CO2, in a reversible manner. As a result, K+-fluxes across the plasma membrane changed direction, to favour K+ extrusion and stomatal closure in the presence of CO2. Concurrent with the depolarization, an inward current across the plasma membrane appeared, most likely due to activation of anion channels. Guard cell responses to CO2 could be recorded in darkness as well as in red light. However, in darkness some cells spontaneously depolarized, these cells hyperpolarized again in red light. Here, red light was projected on a large area of the leaf and decreased the intracellular CO2 concentration by about 250 µl l−1, as measured with a miniature CO2 sensor placed in the substomatal cavity. We conclude, that in intact leaves the red light response of guard cells is mediated through a decrease of the intercellular CO2 concentration.

  @article{TPJ:TPJ1403, abstract = {Guard cells in intact leafs display light-induced membrane potential changes, which alter the direction of K+-transport across the plasma membrane (Roelfsema et al., 2001). A beam of blue light, but not red light, directed at the impaled guard cell triggers this response, while both light qualities induce opening of stomata. To gain insight into this apparent contradiction, we explored the possible interaction between red light and CO2. Guard cells in the intact plant were impaled with double-barrelled electrodes and illuminated with red light. Cells that were hyperpolarized in CO2-free air, depolarized after a switch to air with 700 µl l−1 CO2, in a reversible manner. As a result, K+-fluxes across the plasma membrane changed direction, to favour K+ extrusion and stomatal closure in the presence of CO2. Concurrent with the depolarization, an inward current across the plasma membrane appeared, most likely due to activation of anion channels. Guard cell responses to CO2 could be recorded in darkness as well as in red light. However, in darkness some cells spontaneously depolarized, these cells hyperpolarized again in red light. Here, red light was projected on a large area of the leaf and decreased the intracellular CO2 concentration by about 250 µl l−1, as measured with a miniature CO2 sensor placed in the substomatal cavity. We conclude, that in intact leaves the red light response of guard cells is mediated through a decrease of the intercellular CO2 concentration.}, author = {Roelfsema, M. Rob G. and Hanstein, Stefan and Felle, Hubert H. and Hedrich, Rainer}, journal = {The Plant Journal}, keywords = {potential}, number = 1, pages = {65–75}, publisher = {Blackwell Science Ltd}, title = {CO2 provides an intermediate link in the red light response of guard cells}, volume = 32, year = 2002 }

  %0 Journal Article %1 TPJ:TPJ1403 %A Roelfsema, M. Rob G. %A Hanstein, Stefan %A Felle, Hubert H. %A Hedrich, Rainer %D 2002 %I Blackwell Science Ltd %J The Plant Journal %N 1 %P 65--75 %R 10.1046/j.1365-313X.2002.01403.x %T CO2 provides an intermediate link in the red light response of guard cells %U http://dx.doi.org/10.1046/j.1365-313X.2002.01403.x %V 32 %X Guard cells in intact leafs display light-induced membrane potential changes, which alter the direction of K+-transport across the plasma membrane (Roelfsema et al., 2001). A beam of blue light, but not red light, directed at the impaled guard cell triggers this response, while both light qualities induce opening of stomata. To gain insight into this apparent contradiction, we explored the possible interaction between red light and CO2. Guard cells in the intact plant were impaled with double-barrelled electrodes and illuminated with red light. Cells that were hyperpolarized in CO2-free air, depolarized after a switch to air with 700 µl l−1 CO2, in a reversible manner. As a result, K+-fluxes across the plasma membrane changed direction, to favour K+ extrusion and stomatal closure in the presence of CO2. Concurrent with the depolarization, an inward current across the plasma membrane appeared, most likely due to activation of anion channels. Guard cell responses to CO2 could be recorded in darkness as well as in red light. However, in darkness some cells spontaneously depolarized, these cells hyperpolarized again in red light. Here, red light was projected on a large area of the leaf and decreased the intracellular CO2 concentration by about 250 µl l−1, as measured with a miniature CO2 sensor placed in the substomatal cavity. We conclude, that in intact leaves the red light response of guard cells is mediated through a decrease of the intercellular CO2 concentration.
• Roelfsema, M. and Hedrich, R. (2002) ‘Studying guard cells in the intact plant: modulation of stomatal movement by apoplastic factors’, New Phytologist, 153(3), 425–431, available: https://doi.org/10.1046/j.0028-646X.2001.Documedoc.doc.x.
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  Here, we discuss why guard cells in intact plants respond to environmental signals in a different way than guard cells in epidermal strips, or protoplasts thereof. In intact leaves stomatal opening is counteracted by epidermal cells that press against the guard cells. Changes in the turgor of epidermal cells therefore can alter the stomatal aperture. In addition, stomatal opening may be modulated by the solute composition of the guard cell wall. Changes in apoplastic K+, Cl− and Ca2+ occur after light–dark transitions, but not in such a way that it would support stomatal opening. Organic anions may play a role, since they enhance the open probability of anion channels in the plasma membrane. Furthermore, studies with auxin-resistant and abscisic acid-insensitive mutants show that light-induced stomatal opening is modulated by these hormones. Using the newly developed method in which guard cells in the intact plant are impaled with double-barreled electrodes, the role of these apoplastic factors now can be studied on single guard cells that are still in their natural environment.

  @article{roelfsema2002studying, abstract = {Here, we discuss why guard cells in intact plants respond to environmental signals in a different way than guard cells in epidermal strips, or protoplasts thereof. In intact leaves stomatal opening is counteracted by epidermal cells that press against the guard cells. Changes in the turgor of epidermal cells therefore can alter the stomatal aperture. In addition, stomatal opening may be modulated by the solute composition of the guard cell wall. Changes in apoplastic K+, Cl− and Ca2+ occur after light–dark transitions, but not in such a way that it would support stomatal opening. Organic anions may play a role, since they enhance the open probability of anion channels in the plasma membrane. Furthermore, studies with auxin-resistant and abscisic acid-insensitive mutants show that light-induced stomatal opening is modulated by these hormones. Using the newly developed method in which guard cells in the intact plant are impaled with double-barreled electrodes, the role of these apoplastic factors now can be studied on single guard cells that are still in their natural environment.}, author = {Roelfsema, M.R.G. and Hedrich, R.}, journal = {New Phytologist}, keywords = {epidermal-strip}, number = 3, pages = {425-431}, title = {Studying guard cells in the intact plant: modulation of stomatal movement by apoplastic factors}, volume = 153, year = 2002 }

  %0 Journal Article %1 roelfsema2002studying %A Roelfsema, M.R.G. %A Hedrich, R. %D 2002 %J New Phytologist %N 3 %P 425-431 %R 10.1046/j.0028-646X.2001.Documedoc.doc.x %T Studying guard cells in the intact plant: modulation of stomatal movement by apoplastic factors %U http://onlinelibrary.wiley.com/doi/10.1046/j.0028-646X.2001.Documedoc.doc.x/full %V 153 %X Here, we discuss why guard cells in intact plants respond to environmental signals in a different way than guard cells in epidermal strips, or protoplasts thereof. In intact leaves stomatal opening is counteracted by epidermal cells that press against the guard cells. Changes in the turgor of epidermal cells therefore can alter the stomatal aperture. In addition, stomatal opening may be modulated by the solute composition of the guard cell wall. Changes in apoplastic K+, Cl− and Ca2+ occur after light–dark transitions, but not in such a way that it would support stomatal opening. Organic anions may play a role, since they enhance the open probability of anion channels in the plasma membrane. Furthermore, studies with auxin-resistant and abscisic acid-insensitive mutants show that light-induced stomatal opening is modulated by these hormones. Using the newly developed method in which guard cells in the intact plant are impaled with double-barreled electrodes, the role of these apoplastic factors now can be studied on single guard cells that are still in their natural environment.

• Roelfsema, M.R.G., Steinmeyer, R., Staal, M., and Hedrich, R. (2001) ‘Single guard cell recordings in intact plants: light-induced hyperpolarization of the plasma membrane’, The Plant journal, 26(1), 1–13, available: https://doi.org/10.1046/j.1365-313x.2001.01000.x/full.
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  Guard cells are electrically isolated from other plant cells and therefore offer the unique possibility to conduct current- and voltage-clamp recordings on single cells in an intact plant. Guard cells in their natural environment were impaled with double-barreled electrodes and found to exhibit three physiological states. A minority of cells were classified as far-depolarized cells. These cells exhibited positive membrane potentials and were dominated by the activity of voltage-dependent anion channels. All other cells displayed both outward and inward rectifying K+-channel activity. These cells were either depolarized or hyperpolarized, with average membrane potentials of −41 mV (SD 16) and −112 mV (SD 19), respectively. Depolarized guard cells extrude K+ through outward rectifying channels, while K+ is taken up via inward rectifying channels in hyperpolarized cells. Upon a light/dark transition, guard cells that were hyperpolarized in the light switched to the depolarized state. The depolarization was accompanied by a 35 pA decrease in pump current and an increase in the conductance of inward rectifying channels. Both an increase in pump current and a decrease in the conductance of the inward rectifier were triggered by blue light, while red light was ineffective. From these studies we conclude that light modulates plasma membrane transport through large membrane potential changes, reversing the K+-efflux via outward rectifying channels to a K+-influx via inward rectifying channels.

  @article{roelfsema2001single, abstract = {Guard cells are electrically isolated from other plant cells and therefore offer the unique possibility to conduct current- and voltage-clamp recordings on single cells in an intact plant. Guard cells in their natural environment were impaled with double-barreled electrodes and found to exhibit three physiological states. A minority of cells were classified as far-depolarized cells. These cells exhibited positive membrane potentials and were dominated by the activity of voltage-dependent anion channels. All other cells displayed both outward and inward rectifying K+-channel activity. These cells were either depolarized or hyperpolarized, with average membrane potentials of −41 mV (SD 16) and −112 mV (SD 19), respectively. Depolarized guard cells extrude K+ through outward rectifying channels, while K+ is taken up via inward rectifying channels in hyperpolarized cells. Upon a light/dark transition, guard cells that were hyperpolarized in the light switched to the depolarized state. The depolarization was accompanied by a 35 pA decrease in pump current and an increase in the conductance of inward rectifying channels. Both an increase in pump current and a decrease in the conductance of the inward rectifier were triggered by blue light, while red light was ineffective. From these studies we conclude that light modulates plasma membrane transport through large membrane potential changes, reversing the K+-efflux via outward rectifying channels to a K+-influx via inward rectifying channels.}, author = {Roelfsema, M. R. G. and Steinmeyer, R. and Staal, M. and Hedrich, R.}, journal = {The Plant journal}, keywords = {Vicia-faba}, number = 1, pages = {1-13}, title = {Single guard cell recordings in intact plants: light-induced hyperpolarization of the plasma membrane}, volume = 26, year = 2001 }

  %0 Journal Article %1 roelfsema2001single %A Roelfsema, M. R. G. %A Steinmeyer, R. %A Staal, M. %A Hedrich, R. %D 2001 %J The Plant journal %N 1 %P 1-13 %R 10.1046/j.1365-313x.2001.01000.x/full %T Single guard cell recordings in intact plants: light-induced hyperpolarization of the plasma membrane %U http://onlinelibrary.wiley.com/doi/http://onlinelibrary.wiley.com/doi/10.1046/j.1365-313x.2001.01000.x/full %V 26 %X Guard cells are electrically isolated from other plant cells and therefore offer the unique possibility to conduct current- and voltage-clamp recordings on single cells in an intact plant. Guard cells in their natural environment were impaled with double-barreled electrodes and found to exhibit three physiological states. A minority of cells were classified as far-depolarized cells. These cells exhibited positive membrane potentials and were dominated by the activity of voltage-dependent anion channels. All other cells displayed both outward and inward rectifying K+-channel activity. These cells were either depolarized or hyperpolarized, with average membrane potentials of −41 mV (SD 16) and −112 mV (SD 19), respectively. Depolarized guard cells extrude K+ through outward rectifying channels, while K+ is taken up via inward rectifying channels in hyperpolarized cells. Upon a light/dark transition, guard cells that were hyperpolarized in the light switched to the depolarized state. The depolarization was accompanied by a 35 pA decrease in pump current and an increase in the conductance of inward rectifying channels. Both an increase in pump current and a decrease in the conductance of the inward rectifier were triggered by blue light, while red light was ineffective. From these studies we conclude that light modulates plasma membrane transport through large membrane potential changes, reversing the K+-efflux via outward rectifying channels to a K+-influx via inward rectifying channels.
• Roelfsema, M.R.G., Steinmeyer, R., and Hedrich, R. (2001) ‘Discontinuous single electrode voltage-clamp measurements: assessment of clamp accuracy in Vicia faba guard cells’, Journal of experimental botany, 52(362), 1933–1939, available: https://doi.org/10.1093/jexbot/52.362.1933.
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  The use of a discontinuous single electrode voltage‐clamp (dSEVC) offers an attractive alternative to the patch‐clamp technique, since whole‐cell measurements can be performed with a single sharp electrode. Comparison of current–voltage relations, however, revealed a weaker voltage dependence of channels measured with the dSEVC compared to patch clamp. The accuracy of the dSEVC was tested on Vicia faba guard cells impaled with double‐barrelled electrodes. The actual clamp potential was measured independently of the dSEVC, at the second barrel. The weaker voltage dependence of ion channels appeared to be due to an overestimation of the clamp potential by the dSEVC. The deviation between the intended and actual clamp potential showed a linear relationship with the injected current; on average a 126 mV deviation was found for a clamp current of 1 nA. The deviation was probably caused by a slow settling capacity at the electrode, not compensated by the dSEVC amplifier. It is concluded that the dSEVC method in its current state is only suited for the study of small ion conductances in plant cells.

  @article{roelfsema2001discontinuous, abstract = {The use of a discontinuous single electrode voltage‐clamp (dSEVC) offers an attractive alternative to the patch‐clamp technique, since whole‐cell measurements can be performed with a single sharp electrode. Comparison of current–voltage relations, however, revealed a weaker voltage dependence of channels measured with the dSEVC compared to patch clamp. The accuracy of the dSEVC was tested on Vicia faba guard cells impaled with double‐barrelled electrodes. The actual clamp potential was measured independently of the dSEVC, at the second barrel. The weaker voltage dependence of ion channels appeared to be due to an overestimation of the clamp potential by the dSEVC. The deviation between the intended and actual clamp potential showed a linear relationship with the injected current; on average a 126 mV deviation was found for a clamp current of 1 nA. The deviation was probably caused by a slow settling capacity at the electrode, not compensated by the dSEVC amplifier. It is concluded that the dSEVC method in its current state is only suited for the study of small ion conductances in plant cells.}, author = {Roelfsema, M. R. G. and Steinmeyer, R. and Hedrich, R.}, journal = {Journal of experimental botany}, keywords = {single-electrode}, number = 362, pages = {1933-1939}, title = {Discontinuous single electrode voltage-clamp measurements: assessment of clamp accuracy in Vicia faba guard cells}, volume = 52, year = 2001 }

  %0 Journal Article %1 roelfsema2001discontinuous %A Roelfsema, M. R. G. %A Steinmeyer, R. %A Hedrich, R. %D 2001 %J Journal of experimental botany %N 362 %P 1933-1939 %R 10.1093/jexbot/52.362.1933 %T Discontinuous single electrode voltage-clamp measurements: assessment of clamp accuracy in Vicia faba guard cells %U https://academic.oup.com/jxb/article/52/362/1933/601748/Discontinuous-single-electrode-voltage-clamp %V 52 %X The use of a discontinuous single electrode voltage‐clamp (dSEVC) offers an attractive alternative to the patch‐clamp technique, since whole‐cell measurements can be performed with a single sharp electrode. Comparison of current–voltage relations, however, revealed a weaker voltage dependence of channels measured with the dSEVC compared to patch clamp. The accuracy of the dSEVC was tested on Vicia faba guard cells impaled with double‐barrelled electrodes. The actual clamp potential was measured independently of the dSEVC, at the second barrel. The weaker voltage dependence of ion channels appeared to be due to an overestimation of the clamp potential by the dSEVC. The deviation between the intended and actual clamp potential showed a linear relationship with the injected current; on average a 126 mV deviation was found for a clamp current of 1 nA. The deviation was probably caused by a slow settling capacity at the electrode, not compensated by the dSEVC amplifier. It is concluded that the dSEVC method in its current state is only suited for the study of small ion conductances in plant cells.
• Hedrich, R. and Roelfsema, M.R.G. (2001) ‘Plant ion transport’, in Encyclopedia of Life Sciences, Nature Publishing Group, available: https://doi.org/10.1038/npg.els.0001307.
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  Plant ion transport plays a key role in major physiological processes, such as nutrient uptake and redistribution, movement, growth and microbe interaction. Changes in the activity and density of ion-pumps, channels and carriers represent essential membrane-delimited steps in these processes.

  @inbook{hedrich2001plant, abstract = {Plant ion transport plays a key role in major physiological processes, such as nutrient uptake and redistribution, movement, growth and microbe interaction. Changes in the activity and density of ion-pumps, channels and carriers represent essential membrane-delimited steps in these processes.}, author = {Hedrich, R. and Roelfsema, M. R. G.}, booktitle = {Encyclopedia of life sciences}, keywords = {co-transport}, publisher = {Nature Publishing Group}, title = {Plant ion transport}, year = 2001 }

  %0 Book Section %1 hedrich2001plant %A Hedrich, R. %A Roelfsema, M. R. G. %B Encyclopedia of life sciences %D 2001 %I Nature Publishing Group %R 10.1038/npg.els.0001307 %T Plant ion transport %U http://onlinelibrary.wiley.com/doi/10.1038/npg.els.0001307/abstract %X Plant ion transport plays a key role in major physiological processes, such as nutrient uptake and redistribution, movement, growth and microbe interaction. Changes in the activity and density of ion-pumps, channels and carriers represent essential membrane-delimited steps in these processes.
• Szyroki, A., Ivashikina, N., Dietrich, P., Roelfsema, M.R.G., Ache, P., Reintanz, B., Deeken, R., Godde, M., Felle, H., Steinmeyer, R., Palme, K., and Hedrich, R. (2001) ‘KAT1 is not essential for stomatal opening’, Proceedings of the National Academy of Sciences of the United States of America, 98(5), 2917–21, available: http://www.ncbi.nlm.nih.gov/pubmed/11226341http://www.pnas.org/content/98/5/2917.full.pdf.
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  @article{szyroki2001essential, author = {Szyroki, A. and Ivashikina, N. and Dietrich, P. and Roelfsema, M. R. G. and Ache, P. and Reintanz, B. and Deeken, R. and Godde, M. and Felle, H. and Steinmeyer, R. and Palme, K. and Hedrich, R.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, keywords = {K+-uptake-channel}, number = 5, pages = {2917-21}, title = {KAT1 is not essential for stomatal opening}, volume = 98, year = 2001 }

  %0 Journal Article %1 szyroki2001essential %A Szyroki, A. %A Ivashikina, N. %A Dietrich, P. %A Roelfsema, M. R. G. %A Ache, P. %A Reintanz, B. %A Deeken, R. %A Godde, M. %A Felle, H. %A Steinmeyer, R. %A Palme, K. %A Hedrich, R. %D 2001 %J Proceedings of the National Academy of Sciences of the United States of America %N 5 %P 2917-21 %T KAT1 is not essential for stomatal opening %U http://www.ncbi.nlm.nih.gov/pubmed/11226341http://www.pnas.org/content/98/5/2917.full.pdf %V 98

• Ache, P., Becker, D., Ivashikina, N., Dietrich, P., Roelfsema, M.R.G., and Hedrich, R. (2000) ‘GORK, a delayed outward rectifier expressed in guard cells of Arabidopsis thaliana, is a K+-selective, K+-sensing ion channel’, FEBS letters, 486(2), 93–98, available: http://www.ncbi.nlm.nih.gov/pubmed/11113445http://www.sciencedirect.com/science?_ob=MImg&_imagekey=B6T36-41V8DVS-2-9&_cdi=4938&_user=616166&_pii=S0014579300022481&_origin=gateway&_coverDate=12%2F08%2F2000&_sk=995139997&view=c&wchp=dGLzVtz-zSkzV&md5=0fa62edfd3998d4cfcb55543cc51f722&ie=/sdarticle.pdf.
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  Here we report on the molecular identification, guard cell expression and functional characterization of AtGORK, an Arabidopsis thaliana guard cell outward rectifying K(+) channel. GORK represents a new member of the plant Shaker K(+) channel superfamily. When heterologously expressed in Xenopus oocytes the gene product of GORK mediated depolarization-activated K(+) currents. In agreement with the delayed outward rectifier in intact guard cells and protoplasts thereof, GORK is activated in a voltage- and potassium-dependent manner. Furthermore, the single channel conductance and regulation of GORK in response to pH changes resembles the biophysical properties of the guard cell delayed outward rectifier. Thus GORK very likely represents the molecular entity for depolarization-induced potassium release from guard cells.

  @article{ache2000delayed, abstract = {Here we report on the molecular identification, guard cell expression and functional characterization of AtGORK, an Arabidopsis thaliana guard cell outward rectifying K(+) channel. GORK represents a new member of the plant Shaker K(+) channel superfamily. When heterologously expressed in Xenopus oocytes the gene product of GORK mediated depolarization-activated K(+) currents. In agreement with the delayed outward rectifier in intact guard cells and protoplasts thereof, GORK is activated in a voltage- and potassium-dependent manner. Furthermore, the single channel conductance and regulation of GORK in response to pH changes resembles the biophysical properties of the guard cell delayed outward rectifier. Thus GORK very likely represents the molecular entity for depolarization-induced potassium release from guard cells.}, author = {Ache, P. and Becker, D. and Ivashikina, N. and Dietrich, P. and Roelfsema, M. R. G. and Hedrich, R.}, journal = {FEBS letters}, keywords = {K+-efflux-channel}, number = 2, pages = {93-98}, title = {GORK, a delayed outward rectifier expressed in guard cells of Arabidopsis thaliana, is a K+-selective, K+-sensing ion channel}, volume = 486, year = 2000 }

  %0 Journal Article %1 ache2000delayed %A Ache, P. %A Becker, D. %A Ivashikina, N. %A Dietrich, P. %A Roelfsema, M. R. G. %A Hedrich, R. %D 2000 %J FEBS letters %N 2 %P 93-98 %T GORK, a delayed outward rectifier expressed in guard cells of Arabidopsis thaliana, is a K+-selective, K+-sensing ion channel %U http://www.ncbi.nlm.nih.gov/pubmed/11113445http://www.sciencedirect.com/science?_ob=MImg&_imagekey=B6T36-41V8DVS-2-9&_cdi=4938&_user=616166&_pii=S0014579300022481&_origin=gateway&_coverDate=12%2F08%2F2000&_sk=995139997&view=c&wchp=dGLzVtz-zSkzV&md5=0fa62edfd3998d4cfcb55543cc51f722&ie=/sdarticle.pdf %V 486 %X Here we report on the molecular identification, guard cell expression and functional characterization of AtGORK, an Arabidopsis thaliana guard cell outward rectifying K(+) channel. GORK represents a new member of the plant Shaker K(+) channel superfamily. When heterologously expressed in Xenopus oocytes the gene product of GORK mediated depolarization-activated K(+) currents. In agreement with the delayed outward rectifier in intact guard cells and protoplasts thereof, GORK is activated in a voltage- and potassium-dependent manner. Furthermore, the single channel conductance and regulation of GORK in response to pH changes resembles the biophysical properties of the guard cell delayed outward rectifier. Thus GORK very likely represents the molecular entity for depolarization-induced potassium release from guard cells.

• Roelfsema, M.R.G., Staal, M., and Prins, H.B.A. (1998) ‘Blue light-induced apoplastic acidification of Arabidopsis thaliana guard cells: Inhibition by ABA is mediated through protein phosphatases’, Physiologia Plantarum, 103(4), 466–474, available: https://doi.org/10.1034/j.1399-3054.1998.1030404.x.
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  The phytohormone abscisic acid (ABA) inhibits blue light-induced apoplastic acidification of guard cells. The signal transduction pathway of ABA, mediating this response, was studied using ABA-insensitive (abi) mutants of Arabidopsis thaliana. Apoplastic acidification was monitored with a flat tipped pH-electrode placed on epidermal strips, in which only guard cells were viable. Blue light-induced apoplastic acidification was reduced by vanadate and diethylstilbestrol (DES), indicating involvement of plasma membrane-bound H+-ATPases. In wild type epidermal strips, ABA reduced blue light-induced acidification to 63%. The inhibition did not result from an increased cytoplasmic free Ca2+ concentration in guard cells, since factors that increase the Ca2+ concentration stimulated apoplastic acidification. Apoplastic acidification was not inhibited by ABA in abi1 and abi2 mutants. In abi1 epidermal strips ABA had no effect on the acidification rate, while it stimulated apoplastic acidification in abi2. The ABA response in both mutants could be partially restored with protein kinase and phosphatase inhibitors. The abi1 guard cells became ABA responsive in the presence of okadaic acid, a protein phosphatase inhibitor. In abi2 guard cells the wild type ABA response was partially restored by K-252a, a protein kinase inhibitor. Apoplastic inhibition is thus mediated through the protein phosphatases encoded by ABI1 and ABI2. The results with protein kinase and protein phosphatase inhibitors indicate that ABI1 and ABI2 are involved in separate signal transduction pathways.

  @article{roelfsema1998lightinduced, abstract = {The phytohormone abscisic acid (ABA) inhibits blue light-induced apoplastic acidification of guard cells. The signal transduction pathway of ABA, mediating this response, was studied using ABA-insensitive (abi) mutants of Arabidopsis thaliana. Apoplastic acidification was monitored with a flat tipped pH-electrode placed on epidermal strips, in which only guard cells were viable. Blue light-induced apoplastic acidification was reduced by vanadate and diethylstilbestrol (DES), indicating involvement of plasma membrane-bound H+-ATPases. In wild type epidermal strips, ABA reduced blue light-induced acidification to 63%. The inhibition did not result from an increased cytoplasmic free Ca2+ concentration in guard cells, since factors that increase the Ca2+ concentration stimulated apoplastic acidification. Apoplastic acidification was not inhibited by ABA in abi1 and abi2 mutants. In abi1 epidermal strips ABA had no effect on the acidification rate, while it stimulated apoplastic acidification in abi2. The ABA response in both mutants could be partially restored with protein kinase and phosphatase inhibitors. The abi1 guard cells became ABA responsive in the presence of okadaic acid, a protein phosphatase inhibitor. In abi2 guard cells the wild type ABA response was partially restored by K-252a, a protein kinase inhibitor. Apoplastic inhibition is thus mediated through the protein phosphatases encoded by ABI1 and ABI2. The results with protein kinase and protein phosphatase inhibitors indicate that ABI1 and ABI2 are involved in separate signal transduction pathways.}, author = {Roelfsema, M. R. G. and Staal, M. and Prins, H. B. A.}, journal = {Physiologia Plantarum}, keywords = {blue-light}, number = 4, pages = {466-474}, title = {Blue light-induced apoplastic acidification of Arabidopsis thaliana guard cells: Inhibition by ABA is mediated through protein phosphatases}, volume = 103, year = 1998 }

  %0 Journal Article %1 roelfsema1998lightinduced %A Roelfsema, M. R. G. %A Staal, M. %A Prins, H. B. A. %D 1998 %J Physiologia Plantarum %N 4 %P 466-474 %R 10.1034/j.1399-3054.1998.1030404.x %T Blue light-induced apoplastic acidification of Arabidopsis thaliana guard cells: Inhibition by ABA is mediated through protein phosphatases %U http://onlinelibrary.wiley.com/doi/10.1034/j.1399-3054.1998.1030404.x/full %V 103 %X The phytohormone abscisic acid (ABA) inhibits blue light-induced apoplastic acidification of guard cells. The signal transduction pathway of ABA, mediating this response, was studied using ABA-insensitive (abi) mutants of Arabidopsis thaliana. Apoplastic acidification was monitored with a flat tipped pH-electrode placed on epidermal strips, in which only guard cells were viable. Blue light-induced apoplastic acidification was reduced by vanadate and diethylstilbestrol (DES), indicating involvement of plasma membrane-bound H+-ATPases. In wild type epidermal strips, ABA reduced blue light-induced acidification to 63%. The inhibition did not result from an increased cytoplasmic free Ca2+ concentration in guard cells, since factors that increase the Ca2+ concentration stimulated apoplastic acidification. Apoplastic acidification was not inhibited by ABA in abi1 and abi2 mutants. In abi1 epidermal strips ABA had no effect on the acidification rate, while it stimulated apoplastic acidification in abi2. The ABA response in both mutants could be partially restored with protein kinase and phosphatase inhibitors. The abi1 guard cells became ABA responsive in the presence of okadaic acid, a protein phosphatase inhibitor. In abi2 guard cells the wild type ABA response was partially restored by K-252a, a protein kinase inhibitor. Apoplastic inhibition is thus mediated through the protein phosphatases encoded by ABI1 and ABI2. The results with protein kinase and protein phosphatase inhibitors indicate that ABI1 and ABI2 are involved in separate signal transduction pathways.
• Roelfsema, M.R.G. and Prins, H.B.A. (1998) ‘The membrane potential of Arabidopsis thaliana guard cells; depolarizations induced by apoplastic acidification’, Planta, 205(1), 100–112, available: https://doi.org/10.1007/s004250050301.
  • [ Abstract ]
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  The apoplastic pH of guard cells probably acidifies in response to light, since light induces proton extrusion by both guard cells and epidermal leaf cells. From the data presented here, it is concluded that these apoplastic pH changes will affect K+ fluxes in guard cells of Arabidopsis thaliana (L.) Heynh. Guard cells of this species were impaled with double-barrelled microelectrodes, to measure the membrane potential (Em) and the plasma-membrane conductance. Guard cells were found to exhibit two states with respect to their Em, a depolarized and a hyperpolarized state. Apoplastic acidification depolarized Em in both states, though the origin of the depolarization differed for each state. In the depolarized state, the change in Em was the result of a combined pH effect on instantaneously activating conductances and on the slow outward rectifying K+ channel (s-ORC). At a more acidic apoplastic pH, the current through instantaneously activated conductances became more inwardly directed, while the maximum conductance of s-ORC decreased. The effect on s-ORC was accompanied by an acceleration of activation and deactivation of the channel. Experiments with acid loading of guard cells indicated that the effect on s-ORC was due to a lowered intracellular pH, caused by apoplastic acidification. In the hyperpolarized state, the pH-induced depolarization was due to a direct effect of the apoplastic pH on the inward rectifying K+ channel. Acidification shifted the threshold potential of the channel to more positive values. This effect was accompanied by a decrease in activation times and an increase of deactivation times, of the channel. From the changes in Em and membrane conductance, the expected effect of acidification on K+ fluxes was calculated. It was concluded that apoplastic acidification will increase the K+-efflux in the depolarized state and reduce the K+-influx in the hyperpolarized state.

  @article{roelfsema1998membrane, abstract = {The apoplastic pH of guard cells probably acidifies in response to light, since light induces proton extrusion by both guard cells and epidermal leaf cells. From the data presented here, it is concluded that these apoplastic pH changes will affect K+ fluxes in guard cells of Arabidopsis thaliana (L.) Heynh. Guard cells of this species were impaled with double-barrelled microelectrodes, to measure the membrane potential (Em) and the plasma-membrane conductance. Guard cells were found to exhibit two states with respect to their Em, a depolarized and a hyperpolarized state. Apoplastic acidification depolarized Em in both states, though the origin of the depolarization differed for each state. In the depolarized state, the change in Em was the result of a combined pH effect on instantaneously activating conductances and on the slow outward rectifying K+ channel (s-ORC). At a more acidic apoplastic pH, the current through instantaneously activated conductances became more inwardly directed, while the maximum conductance of s-ORC decreased. The effect on s-ORC was accompanied by an acceleration of activation and deactivation of the channel. Experiments with acid loading of guard cells indicated that the effect on s-ORC was due to a lowered intracellular pH, caused by apoplastic acidification. In the hyperpolarized state, the pH-induced depolarization was due to a direct effect of the apoplastic pH on the inward rectifying K+ channel. Acidification shifted the threshold potential of the channel to more positive values. This effect was accompanied by a decrease in activation times and an increase of deactivation times, of the channel. From the changes in Em and membrane conductance, the expected effect of acidification on K+ fluxes was calculated. It was concluded that apoplastic acidification will increase the K+-efflux in the depolarized state and reduce the K+-influx in the hyperpolarized state.}, author = {Roelfsema, M. R. G. and Prins, H. B. A.}, journal = {Planta}, keywords = {potassium-channel}, number = 1, pages = {100-112}, title = {The membrane potential of Arabidopsis thaliana guard cells; depolarizations induced by apoplastic acidification}, volume = 205, year = 1998 }

  %0 Journal Article %1 roelfsema1998membrane %A Roelfsema, M. R. G. %A Prins, H. B. A. %D 1998 %J Planta %N 1 %P 100-112 %R 10.1007/s004250050301 %T The membrane potential of Arabidopsis thaliana guard cells; depolarizations induced by apoplastic acidification %U https://link.springer.com/article/10.1007/s004250050301 %V 205 %X The apoplastic pH of guard cells probably acidifies in response to light, since light induces proton extrusion by both guard cells and epidermal leaf cells. From the data presented here, it is concluded that these apoplastic pH changes will affect K+ fluxes in guard cells of Arabidopsis thaliana (L.) Heynh. Guard cells of this species were impaled with double-barrelled microelectrodes, to measure the membrane potential (Em) and the plasma-membrane conductance. Guard cells were found to exhibit two states with respect to their Em, a depolarized and a hyperpolarized state. Apoplastic acidification depolarized Em in both states, though the origin of the depolarization differed for each state. In the depolarized state, the change in Em was the result of a combined pH effect on instantaneously activating conductances and on the slow outward rectifying K+ channel (s-ORC). At a more acidic apoplastic pH, the current through instantaneously activated conductances became more inwardly directed, while the maximum conductance of s-ORC decreased. The effect on s-ORC was accompanied by an acceleration of activation and deactivation of the channel. Experiments with acid loading of guard cells indicated that the effect on s-ORC was due to a lowered intracellular pH, caused by apoplastic acidification. In the hyperpolarized state, the pH-induced depolarization was due to a direct effect of the apoplastic pH on the inward rectifying K+ channel. Acidification shifted the threshold potential of the channel to more positive values. This effect was accompanied by a decrease in activation times and an increase of deactivation times, of the channel. From the changes in Em and membrane conductance, the expected effect of acidification on K+ fluxes was calculated. It was concluded that apoplastic acidification will increase the K+-efflux in the depolarized state and reduce the K+-influx in the hyperpolarized state.

• Roelfsema, M.R.G. and Prins, H.B.A. (1997) ‘Ion channels in guard cells of Arabidopsis thaliana (L.) Heynh.’, Planta, 202(1), 18–27, available: https://doi.org/10.1007/s004250050098.
  • [ Abstract ]
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  Despite the availability of many mutants for signal transduction, Arabidopsis thaliana guard cells have so far not been used in electrophysiological research. Problems with the isolation of epidermal strips and the small size of A. thaliana guard cells were often prohibiting. In the present study these difficulties were overcome and guard cells were impaled with double-barreled microelectrodes. Membrane-potential recordings were often stable for over half an hour and voltage-clamp measurements could be conducted. The guard cells were found to exhibit two states. The majority of the guard cells had depolarized membrane potentials, which were largely dependent on external K+ concentrations. Other cells displayed spontaneous transitions to a more hyperpolarized state, at which the free-running membrane potential (Em) was not sensitive to the external K+ concentration. Two outward-rectifying conductances were identified in cells in the depolarized state. A slow outward-rectifying channel (s-ORC) had properties resembling the K+-selective ORC of Vicia faba guard cells (Blatt, 1988, J Membr Biol 102: 235–246). The activation and inactivation times and the activation potential, all depended on the reversal potential (Erev) of the s-ORC conductance. The s-ORC was blocked by Ba2+ (K1/2 = 0.3–1.3mM) and verapamil (K1/2 = 15–20 μM). A second rapid outward-rectifying conductance (r-ORC) activated instantaneously upon stepping the voltage to positive values and was stimulated by Ba2+. Inward-rectifying channels (IRC) were only observed in cells in the hyperpolarized state. The activation time and activation potential of this channel were not sensitive to the external K+ concentration. The slow activation of the IRC (t1/2 ≈ 0.5 s) and its negative activation potential (Vthreshold = −155 mV) resemble the values found for the KAT1 channel expressed in Saccharomyces cerevisiae (Bertl et al., 1995, Proc Natl Acad Sci USA 92: 2701–2705). The results indicate that A. thaliana guard cells provide an excellent system for the study of signal transduction processes.

  @article{roelfsema1997channels, abstract = {Despite the availability of many mutants for signal transduction, Arabidopsis thaliana guard cells have so far not been used in electrophysiological research. Problems with the isolation of epidermal strips and the small size of A. thaliana guard cells were often prohibiting. In the present study these difficulties were overcome and guard cells were impaled with double-barreled microelectrodes. Membrane-potential recordings were often stable for over half an hour and voltage-clamp measurements could be conducted. The guard cells were found to exhibit two states. The majority of the guard cells had depolarized membrane potentials, which were largely dependent on external K+ concentrations. Other cells displayed spontaneous transitions to a more hyperpolarized state, at which the free-running membrane potential (Em) was not sensitive to the external K+ concentration. Two outward-rectifying conductances were identified in cells in the depolarized state. A slow outward-rectifying channel (s-ORC) had properties resembling the K+-selective ORC of Vicia faba guard cells (Blatt, 1988, J Membr Biol 102: 235–246). The activation and inactivation times and the activation potential, all depended on the reversal potential (Erev) of the s-ORC conductance. The s-ORC was blocked by Ba2+ (K1/2 = 0.3–1.3mM) and verapamil (K1/2 = 15–20 μM). A second rapid outward-rectifying conductance (r-ORC) activated instantaneously upon stepping the voltage to positive values and was stimulated by Ba2+. Inward-rectifying channels (IRC) were only observed in cells in the hyperpolarized state. The activation time and activation potential of this channel were not sensitive to the external K+ concentration. The slow activation of the IRC (t1/2 ≈ 0.5 s) and its negative activation potential (Vthreshold = −155 mV) resemble the values found for the KAT1 channel expressed in Saccharomyces cerevisiae (Bertl et al., 1995, Proc Natl Acad Sci USA 92: 2701–2705). The results indicate that A. thaliana guard cells provide an excellent system for the study of signal transduction processes.}, author = {Roelfsema, M. R. G. and Prins, H. B. A.}, journal = {Planta}, keywords = {potential}, number = 1, pages = {18-27}, title = {Ion channels in guard cells of Arabidopsis thaliana (L.) Heynh.}, volume = 202, year = 1997 }

  %0 Journal Article %1 roelfsema1997channels %A Roelfsema, M. R. G. %A Prins, H. B. A. %D 1997 %J Planta %N 1 %P 18-27 %R 10.1007/s004250050098 %T Ion channels in guard cells of Arabidopsis thaliana (L.) Heynh. %U https://link.springer.com/article/10.1007/s004250050098 %V 202 %X Despite the availability of many mutants for signal transduction, Arabidopsis thaliana guard cells have so far not been used in electrophysiological research. Problems with the isolation of epidermal strips and the small size of A. thaliana guard cells were often prohibiting. In the present study these difficulties were overcome and guard cells were impaled with double-barreled microelectrodes. Membrane-potential recordings were often stable for over half an hour and voltage-clamp measurements could be conducted. The guard cells were found to exhibit two states. The majority of the guard cells had depolarized membrane potentials, which were largely dependent on external K+ concentrations. Other cells displayed spontaneous transitions to a more hyperpolarized state, at which the free-running membrane potential (Em) was not sensitive to the external K+ concentration. Two outward-rectifying conductances were identified in cells in the depolarized state. A slow outward-rectifying channel (s-ORC) had properties resembling the K+-selective ORC of Vicia faba guard cells (Blatt, 1988, J Membr Biol 102: 235–246). The activation and inactivation times and the activation potential, all depended on the reversal potential (Erev) of the s-ORC conductance. The s-ORC was blocked by Ba2+ (K1/2 = 0.3–1.3mM) and verapamil (K1/2 = 15–20 μM). A second rapid outward-rectifying conductance (r-ORC) activated instantaneously upon stepping the voltage to positive values and was stimulated by Ba2+. Inward-rectifying channels (IRC) were only observed in cells in the hyperpolarized state. The activation time and activation potential of this channel were not sensitive to the external K+ concentration. The slow activation of the IRC (t1/2 ≈ 0.5 s) and its negative activation potential (Vthreshold = −155 mV) resemble the values found for the KAT1 channel expressed in Saccharomyces cerevisiae (Bertl et al., 1995, Proc Natl Acad Sci USA 92: 2701–2705). The results indicate that A. thaliana guard cells provide an excellent system for the study of signal transduction processes.

• Roelfsema, M.R.G. and Prins, H.B.A. (1995) ‘Effect of abscisic acid on stomatal opening in isolated epidermal strips of abi mutants of Arabidopsis thaliana’, Physiologia plantarum, 95(3), 373–378, available: https://doi.org/10.1111/j.1399-3054.1995.tb00851.x.
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  Abscisic acid-insensitive mutants of Arabidopsis thaliana L. var. Landsberg erecta were selected for their decreased sensitivity to ABA during germination. Two of these mutants, abi-1 and abi-2, display a wilty phenotype as adult plants, indicating disturbed water relations. Experiments were undertaken to find out if this results from insensitivity of mutant stomates to ABA. Growth conditions and methods to isolate epidermal strips were optimized to study stomatal movement. Wild type stomates required external ionic conditions comparable to those found for other species such as Commelina communis. The largest light-induced opening of A. thaliana stomates was found at an external KCl concentration of 50 mM. Stomatal apertures were increased by lowering external Ca2+ to 0.05 mM. The apertures of stomates incubated with 10 μM ABA were not altered by changes in Ca2+ from 0.05 to 1.0 mM. Stomates of all abi mutants showed a light-stimulated stomatal opening. The opening of wild type and abi-3 stomates was inhibited by ABA, while stomates of abi-1 and abi-2 did not respond to ABA. The insensitivity of abi-1 and abi-2 stomates to ABA may thus explain the observed disturbed water relations.

  @article{roelfsema1995effect, abstract = {Abscisic acid-insensitive mutants of Arabidopsis thaliana L. var. Landsberg erecta were selected for their decreased sensitivity to ABA during germination. Two of these mutants, abi-1 and abi-2, display a wilty phenotype as adult plants, indicating disturbed water relations. Experiments were undertaken to find out if this results from insensitivity of mutant stomates to ABA. Growth conditions and methods to isolate epidermal strips were optimized to study stomatal movement. Wild type stomates required external ionic conditions comparable to those found for other species such as Commelina communis. The largest light-induced opening of A. thaliana stomates was found at an external KCl concentration of 50 mM. Stomatal apertures were increased by lowering external Ca2+ to 0.05 mM. The apertures of stomates incubated with 10 μM ABA were not altered by changes in Ca2+ from 0.05 to 1.0 mM. Stomates of all abi mutants showed a light-stimulated stomatal opening. The opening of wild type and abi-3 stomates was inhibited by ABA, while stomates of abi-1 and abi-2 did not respond to ABA. The insensitivity of abi-1 and abi-2 stomates to ABA may thus explain the observed disturbed water relations.}, author = {Roelfsema, M. R. G. and Prins, H. B. A.}, journal = {Physiologia plantarum}, keywords = {ABA}, number = 3, pages = {373-378}, title = {Effect of abscisic acid on stomatal opening in isolated epidermal strips of abi mutants of Arabidopsis thaliana}, volume = 95, year = 1995 }

  %0 Journal Article %1 roelfsema1995effect %A Roelfsema, M. R. G. %A Prins, H. B. A. %D 1995 %J Physiologia plantarum %N 3 %P 373-378 %R 10.1111/j.1399-3054.1995.tb00851.x %T Effect of abscisic acid on stomatal opening in isolated epidermal strips of abi mutants of Arabidopsis thaliana %U http://onlinelibrary.wiley.com/doi/10.1111/j.1399-3054.1995.tb00851.x/full %V 95 %X Abscisic acid-insensitive mutants of Arabidopsis thaliana L. var. Landsberg erecta were selected for their decreased sensitivity to ABA during germination. Two of these mutants, abi-1 and abi-2, display a wilty phenotype as adult plants, indicating disturbed water relations. Experiments were undertaken to find out if this results from insensitivity of mutant stomates to ABA. Growth conditions and methods to isolate epidermal strips were optimized to study stomatal movement. Wild type stomates required external ionic conditions comparable to those found for other species such as Commelina communis. The largest light-induced opening of A. thaliana stomates was found at an external KCl concentration of 50 mM. Stomatal apertures were increased by lowering external Ca2+ to 0.05 mM. The apertures of stomates incubated with 10 μM ABA were not altered by changes in Ca2+ from 0.05 to 1.0 mM. Stomates of all abi mutants showed a light-stimulated stomatal opening. The opening of wild type and abi-3 stomates was inhibited by ABA, while stomates of abi-1 and abi-2 did not respond to ABA. The insensitivity of abi-1 and abi-2 stomates to ABA may thus explain the observed disturbed water relations.