Rainer Hedrich - Academia.edu (original) (raw)

Papers by Rainer Hedrich

The Plant Cell, 2011

Cytosolic calcium homeostasis is pivotal for intracellular signaling and requires sensing of calc... more Cytosolic calcium homeostasis is pivotal for intracellular signaling and requires sensing of calcium concentrations in the cytosol and accessible stores. Numerous Ca 2+ binding sites have been characterized in cytosolic proteins. However, little is known about Ca 2+ binding inside organelles, like the vacuole. The slow vacuolar (SV) channel, encoded by Arabidopsis thaliana TPC1, is regulated by luminal Ca 2+. However, the D454/fou2 mutation in TPC1 eliminates vacuolar calcium sensitivity and increases store calcium content. In a search for the luminal calcium binding site, structure modeling indicated a possible coordination site formed by residues Glu-450, Asp-454, Glu-456, and Glu-457 on the luminal side of TPC1. Each Glu residue was replaced by Gln, the modified genes were transiently expressed in loss-of-TPC1-function protoplasts, and SV channel responses to luminal calcium were recorded by patch clamp. SV channels lacking any of the four negatively charged residues appeared altered in calcium sensitivity of channel gating. Our results indicate that Glu-450 and Asp-454 are directly involved in Ca 2+ binding, whereas Glu-456 and Glu-457 are probably involved in connecting the luminal Ca 2+ binding site to the channel gate. This novel vacuolar calcium binding site represents a potential tool to address calcium storage in plants.

Current Biology, 2018

Schäfer et al. 1 A tandem amino acid residue motif in guard cell SLAC1 anion channel of grasses a... more Schäfer et al. 1 A tandem amino acid residue motif in guard cell SLAC1 anion channel of grasses allows for the control of stomatal aperture by nitrate

Current Biology, Oct 1, 2022

iScience, Apr 1, 2022

Nutrient sensing of plants was analyzed in computational cell biology simulations Changes in exte... more Nutrient sensing of plants was analyzed in computational cell biology simulations Changes in external nutrient concentrations caused cytosolic H + and Ca 2+ signals Nutrient transporter networks exhibit transceptor-like characteristics Wet-lab experiments confirmed the conceptual predictions

Plant Biology, Jul 12, 2016

• The two-pore cation channel TPC1 operates as a dimeric channel in animal and plant endomembrane... more • The two-pore cation channel TPC1 operates as a dimeric channel in animal and plant endomembranes. Each subunit consists of two homologous Shaker-like halves, with 12 transmembrane domains in total (S1-S6, S7-S12). In plants, TPC1 channels reside in the vacuolar membrane, and upon voltage stimulation, give rise to the well-known slow-activating SV currents. • Here, we combined bioinformatics, structure modeling, site-directed mutagenesis, and in planta patch clamp studies to elucidate the molecular mechanisms of voltage-dependent channel gating in TPC1 in its native plant background. • Structure-function analysis of the Arabidopsis TPC1 channel in planta confirmed that helix S10 operates as the major voltage-sensing site, with Glu450 and Glu478 identified as possible ionpair partners for voltage-sensing Arg537. The contribution of helix S4 to voltage sensing was found to be negligible. Several conserved negative residues on the luminal site contribute to calcium binding, stabilizing the closed channel. • During evolution of plant TPC1s from two separate Shaker-like domains, the voltage-sensing function in the N-terminal Shaker-unit (S1-S4) vanished.

Proceedings of the National Academy of Sciences of the United States of America, Aug 11, 2020

This open access article is distributed under Creative Commons Attribution-NonCommercial-NoDeriva... more This open access article is distributed under Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND).

Science Advances, Jul 9, 2021

Guard cells control the aperture of plant stomata, which are crucial for global fluxes of CO 2 an... more Guard cells control the aperture of plant stomata, which are crucial for global fluxes of CO 2 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 NO 3 − 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.

Trends in Plant Science, 2021

Plant Journal, Sep 1, 2001

SummaryIn search of a K+ channel involved in phloem transport we screened a Vicia faba cotyledon ... more SummaryIn search of a K+ channel involved in phloem transport we screened a Vicia faba cotyledon cDNA library taking advantage of a set of degenerated primers, flanking regions conserved among K+ uptake channels. We cloned VFK1 (for Vicia faba K+ channel 1) characterised by a structure known from the Shaker family of plant K+ channels. When co‐expressed with a KAT1 mutant in Xenopus oocytes, heteromers revealed the biophysical properties of a K+ selective, proton‐blocked channel. Northern blot analyses showed high levels of expression in cotyledons, flowers, stem and leaves. Using in situ PCR techniques we could localise the K+ channel mRNA in the phloem. In the stem VFK1 expression levels were higher in the lower internodes. There channel transcripts increased in the light and thus under conditions of increased photosynthate allocation. VFK1 transcripts are elevated in sink leaves, and rise in source leaves during the experimental transition into sinks. Fructose‐ rather than sucrose‐ or glucose‐feeding via the petiole induced VFK1 gene activity. We therefore monitored the fructose sensitivity of the sieve tube potential through cut aphid stylets. In response to an 1 h fructose treatment the sieve tube potential shift increased from 19 mV to 53 mV per 10‐fold change in K+ concentration. Under these conditions K+ channels dominated the electrical properties of the plasma membrane. Based on the phloem localisation and expression patterns of VFK1 we conclude that this K+ channel is involved in sugar unloading and K+ retrieval.

bioRxiv (Cold Spring Harbor Laboratory), Dec 23, 2021

To fire action-potential-like electrical signals, the vacuole membrane requires the depolarizatio... more To fire action-potential-like electrical signals, the vacuole membrane requires the depolarization-activated two-pore channel TPC1, also called Slowly activating Vacuolar SV channel. The TPC1/SV channel, encoded by the TPC1 gene, functions as a voltage-dependent and Ca 2+-regulated potassium channel. TPC1 currents are activated by a rise in cytoplasmic Ca 2+ but inhibited by luminal Ca 2+. In search for species-dependent functional TPC1 channel variants, we studied polymorphic amino acids contributing to luminal Ca 2+ sensitivity. We found that the acidic residues E457, E605 and D606 of the Ca 2+-sensitive Arabidopsis AtTPC1 channel were neutralized by either asparagine or alanine in Vicia faba and many other Fabaceae as well. When expressed in the Arabidopsis loss-of-AtTPC1 function background, the wild type VfTPC1 was hypersensitive to vacuole depolarization and insensitive to blocking luminal Ca 2+. When AtTPC1 was mutated for the three VfTPC1-homologous polymorphic site residues, the Arabidopsis At-VfTPC1 channel mutant gained VfTPC1like voltage and luminal Ca 2+ insensitivity that together made vacuoles hyperexcitable. These findings indicate that natural TPC1 channel variants in plant families exist which differ in vacuole excitability and very likely respond to changes in environmental settings of their ecological niche. Significance statement Vacuolar electrical excitability and stress-related Ca 2+ signaling depends on the activity of the vacuolar cation channel TPC1. Until now, the regulatory features of AtTPC1 from the model plant Arabidopsis thaliana was believed to apply to the TPC1 channels of other species. However, here we now show that, surprisingly, the VfTPC1 channel of the economic broad bean, in contrast to AtTPC1, proves to be hyperactive and confers hyperexcitability to the vacuole. The different gating behavior is most likely related to an impaired Ca 2+ sensor site in the vacuolar pore vestibule, rising the probability to open at more negative membrane voltages. These natural variants of the TPC1 channel could help the plant adapt and respond to environmental challenges. .

Proceedings of the National Academy of Sciences of the United States of America, Mar 16, 1999

New Phytologist, 2021

Summary Soil salinity is an increasingly global problem which hampers plant growth and crop yield... more Summary Soil salinity is an increasingly global problem which hampers plant growth and crop yield. Plant productivity depends on optimal water‐use efficiency and photosynthetic capacity balanced by stomatal conductance. Whether and how stomatal behavior contributes to salt sensitivity or tolerance is currently unknown. This work identifies guard cell‐specific signaling networks exerted by a salt‐sensitive and salt‐tolerant plant under ionic and osmotic stress conditions accompanied by increasing NaCl loads. We challenged soil‐grown Arabidopsis thaliana and Thellungiella salsuginea plants with short‐ and long‐term salinity stress and monitored genome‐wide gene expression and signals of guard cells that determine their function. Arabidopsis plants suffered from both salt regimes and showed reduced stomatal conductance while Thellungiella displayed no obvious stress symptoms. The salt‐dependent gene expression changes of guard cells supported the ability of the halophyte to maintain hi...

Current Biology, 2021

Plants, as sessile organisms, gained the ability to sense and respond to biotic and abiotic stres... more Plants, as sessile organisms, gained the ability to sense and respond to biotic and abiotic stressors to survive severe changes in their environments. The change in our climate comes with extreme dry periods but also episodes of flooding. The latter stress condition causes anaerobiosis-triggered cytosolic acidosis and impairs plant function. The molecular mechanism that enables plant cells to sense acidity and convey this signal via membrane depolarization was previously unknown. Here, we show that acidosis-induced anion efflux from Arabidopsis (Arabidopsis thaliana) roots is dependent on the S-type anion channel AtSLAH3. Heterologous expression of SLAH3 in Xenopus oocytes revealed that the anion channel is directly activated by a small, physiological drop in cytosolic pH. Acidosis-triggered activation of SLAH3 is mediated by protonation of histidine 330 and 454. Super-resolution microscopy analysis showed that the increase in cellular proton concentration switches SLAH3 from an electrically silent channel dimer into its active monomeric form. Our results show that, upon acidification, protons directly switch SLAH3 to its open configuration, bypassing kinase-dependent activation. Moreover, under flooding conditions, the stress response of Arabidopsis wild-type (WT) plants was significantly higher compared to SLAH3 loss-of-function mutants. Our genetic evidence of SLAH3 pH sensor function may guide the development of crop varieties with improved stress tolerance.

New Phytologist, 2021

Summary Cytosolic calcium signals are evoked by a large variety of biotic and abiotic stimuli and... more Summary Cytosolic calcium signals are evoked by a large variety of biotic and abiotic stimuli and play an important role in cellular and long distance signalling in plants. While the function of the plasma membrane in cytosolic Ca2+ signalling has been intensively studied, the role of the vacuolar membrane remains elusive. A newly developed vacuolar voltage clamp technique was used in combination with live‐cell imaging, to study the role of the vacuolar membrane in Ca2+ and pH homeostasis of bulging root hair cells of Arabidopsis. Depolarisation of the vacuolar membrane caused a rapid increase in the Ca2+ concentration and alkalised the cytosol, while hyperpolarisation led to the opposite responses. The relationship between the vacuolar membrane potential, the cytosolic pH and Ca2+ concentration suggests that a vacuolar H+/Ca2+ exchange mechanism plays a central role in cytosolic Ca2+ homeostasis. Mathematical modelling further suggests that the voltage‐dependent vacuolar Ca2+ homeo...

PLOS Biology, 2020

The carnivorous plant Dionaea muscipula harbors multicellular trigger hairs designed to sense mec... more The carnivorous plant Dionaea muscipula harbors multicellular trigger hairs designed to sense mechanical stimuli upon contact with animal prey. At the base of the trigger hair, mechanosensation is transduced into an all-or-nothing action potential (AP) that spreads all over the trap, ultimately leading to trap closure and prey capture. To reveal the molecular basis for the unique functional repertoire of this mechanoresponsive plant structure, we determined the transcriptome of D. muscipula’s trigger hair. Among the genes that were found to be highly specific to the trigger hair, the Shaker-type channel KDM1 was electrophysiologically characterized as a hyperpolarization- and acid-activated K+-selective channel, thus allowing the reuptake of K+ ions into the trigger hair’s sensory cells during the hyperpolarization phase of the AP. During trap development, the increased electrical excitability of the trigger hair is associated with the transcriptional induction of KDM1. Conversely, ...

Nature Communications, 2019

In contrast to the plasma membrane, the vacuole membrane has not yet been associated with electri... more In contrast to the plasma membrane, the vacuole membrane has not yet been associated with electrical excitation of plants. Here, we show that mesophyll vacuoles from Arabidopsis sense and control the membrane potential essentially via the K+-permeable TPC1 and TPK channels. Electrical stimuli elicit transient depolarization of the vacuole membrane that can last for seconds. Electrical excitability is suppressed by increased vacuolar Ca2+ levels. In comparison to wild type, vacuoles from the fou2 mutant, harboring TPC1 channels insensitive to luminal Ca2+, can be excited fully by even weak electrical stimuli. The TPC1-loss-of-function mutant tpc1-2 does not respond to electrical stimulation at all, and the loss of TPK1/TPK3-mediated K+ transport affects the duration of TPC1-dependent membrane depolarization. In combination with mathematical modeling, these results show that the vacuolar K+-conducting TPC1 and TPK1/TPK3 channels act in concert to provide for Ca2+- and voltage-induced ...

Plant physiology, Jan 26, 2017

Water limitation of plants causes stomatal closure to prevent water loss by transpiration. For th... more Water limitation of plants causes stomatal closure to prevent water loss by transpiration. For this purpose, progressing soil water deficit is communicated from roots to shoots. Abscisic acid (ABA) is the key signal in stress-induced stomatal closure, however, ABA as early xylem-delivered signal is still a matter of debate. In the present study, poplar plants were exposed to water stress to investigate xylem sap sulfate and ABA, stomatal conductance and sulfate transporter (SULTR) expression. In addition, stomatal behavior and expression of ABA receptors, drought responsive genes, transcription factors and NCED3 were studied after feeding sulfate and ABA to detached poplar leaves and epidermal peels of Arabidopsis. The results show that increased xylem sap sulfate is achieved upon drought by reduced xylem unloading by PtaSULTR3;3a and PtaSULTR1;1, and by enhanced loading from parenchyma cells into the xylem via PtaALMT3b. Sulfate application caused stomatal closure in excised leaves...

The Plant Cell, 2001

Plant K ؉ uptake channel types differ with respect to their voltage, Ca 2 ؉ , and pH dependence. ... more Plant K ؉ uptake channel types differ with respect to their voltage, Ca 2 ؉ , and pH dependence. Here, we constructed recombinant chimeric channels between KST1, a member of the inward-rectifying, acid-activated KAT1 family, and AKT3, a member of the weakly voltage-dependent, proton-blocked AKT2/3 family. The homologous pore regions of AKT3 (amino acids 216 to 287) and KST1 (amino acids 217 to 289) have been exchanged to generate the two chimeric channels AKT3/(p)KST1 and KST1/(p)AKT3. In contrast to AKT3 wild-type channels, AKT3/(p)KST1 revealed a strong inward rectification reminiscent of that of KST1. Correspondingly, the substitution of the KST1 by the AKT3 pore led to less pronounced rectification properties of KST1/(p)AKT3 compared with wild-type KST1. Besides the voltage dependence, the interaction between the chimera and extracellular H ؉ and Ca 2 ؉ resembled the properties of the inserted rather than the respective wild-type pore. Whereas AKT3/(p)KST1 was acid activated and Ca 2 ؉ insensitive, extracellular protons and Ca 2 ؉ inhibited KST1/(p)AKT3. The regulation of the chimeric channels by cytoplasmic protons followed the respective wild-type backbone of the chimeric channels, indicating that the intracellular pH sensor is located outside the P domain. We thus conclude that essential elements for external pH and Ca 2 ؉ regulation and for the rectification of voltage-dependent K ؉ uptake channels are located within the channel pore.

The Plant Cell, 2013

Apical growth in pollen tubes (PTs) is associated with the presence of tip-focused ion gradients ... more Apical growth in pollen tubes (PTs) is associated with the presence of tip-focused ion gradients and fluxes, implying polar localization or regulation of the underlying transporters. The molecular identity and regulation of anion transporters in PTs is unknown. Here we report a negative gradient of cytosolic anion concentration focused on the tip, in negative correlation with the cytosolic Ca 2+ concentration. We hypothesized that a possible link between these two ions is based on the presence of Ca 2+-dependent protein kinases (CPKs). We characterized anion channels and CPK transcripts in PTs and analyzed their localization. Yellow fluorescent protein (YFP) tagging of a homolog of SLOW ANION CHANNEL-ASSOCIATED1 (SLAH3:YFP) was widespread along PTs, but, in accordance with the anion efflux, CPK2/CPK20/CPK17/CPK34:YFP fluorescence was strictly localized at the tip plasma membrane. Expression of SLAH3 with either CPK2 or CPK20 (but not CPK17/CPK34) in Xenopus laevis oocytes elicited S-type anion channel currents. Interaction of SLAH3 with CPK2/CPK20 (but not CPK17/CPK34) was confirmed by Förster-resonance energy transfer fluorescence lifetime microscopy in Arabidopsis thaliana mesophyll protoplasts and bimolecular fluorescence complementation in living PTs. Compared with wild-type PTs, slah3-1 and slah3-2 as well as cpk2-1 cpk20-2 PTs had reduced anion currents. Double mutant cpk2-1 cpk20-2 and slah3-1 PTs had reduced extracellular anion fluxes at the tip. Our studies provide evidence for a Ca 2+-dependent CPK2/CPK20 regulation of the anion channel SLAH3 to regulate PT growth.

The Plant Cell, 2002

The Arabidopsis phloem channel AKT3 is the founder of a subfamily of shaker-like plant potassium ... more The Arabidopsis phloem channel AKT3 is the founder of a subfamily of shaker-like plant potassium channels characterized by weak rectification, Ca 2 ؉ block, proton inhibition, and, as shown in this study, K ؉ sensitivity. In contrast to inward-rectifying, acid-activated K ؉ channels of the KAT1 family, extracellular acidification decreases AKT3 currents at the macroscopic and single-channel levels. Here, we show that two distinct sites within the outer mouth of the K ؉-conducting pore provide the molecular basis for the pH sensitivity of this phloem channel. After generation of mutant channels and functional expression in Xenopus oocytes, we identified the His residue His-228, which is proximal to the K ؉ selectivity filter (GYGD) and the distal Ser residue Ser-271, to be involved in proton susceptibility. Mutations of these sites, H228D and S271E, drastically reduced the H ؉ and K ؉ sensitivity of AKT3. Although in K ؉-free bath solutions outward K ؉ currents were abolished completely in wild-type AKT3, S271E as well as the AKT3-HDSE double mutant still mediated K ؉ efflux. We conclude that the pH-and K ؉-dependent properties of the AKT3 channel involve residues in the outer mouth of the pore. Both properties, H ؉ and K ؉ sensitivity, allow the fine-tuning of the phloem channel and thus seem to represent important elements in the control of membrane potential and sugar loading.

The Plant Cell, 2011

Cytosolic calcium homeostasis is pivotal for intracellular signaling and requires sensing of calc... more Cytosolic calcium homeostasis is pivotal for intracellular signaling and requires sensing of calcium concentrations in the cytosol and accessible stores. Numerous Ca 2+ binding sites have been characterized in cytosolic proteins. However, little is known about Ca 2+ binding inside organelles, like the vacuole. The slow vacuolar (SV) channel, encoded by Arabidopsis thaliana TPC1, is regulated by luminal Ca 2+. However, the D454/fou2 mutation in TPC1 eliminates vacuolar calcium sensitivity and increases store calcium content. In a search for the luminal calcium binding site, structure modeling indicated a possible coordination site formed by residues Glu-450, Asp-454, Glu-456, and Glu-457 on the luminal side of TPC1. Each Glu residue was replaced by Gln, the modified genes were transiently expressed in loss-of-TPC1-function protoplasts, and SV channel responses to luminal calcium were recorded by patch clamp. SV channels lacking any of the four negatively charged residues appeared altered in calcium sensitivity of channel gating. Our results indicate that Glu-450 and Asp-454 are directly involved in Ca 2+ binding, whereas Glu-456 and Glu-457 are probably involved in connecting the luminal Ca 2+ binding site to the channel gate. This novel vacuolar calcium binding site represents a potential tool to address calcium storage in plants.

Current Biology, 2018

Schäfer et al. 1 A tandem amino acid residue motif in guard cell SLAC1 anion channel of grasses a... more Schäfer et al. 1 A tandem amino acid residue motif in guard cell SLAC1 anion channel of grasses allows for the control of stomatal aperture by nitrate

Current Biology, Oct 1, 2022

iScience, Apr 1, 2022

Nutrient sensing of plants was analyzed in computational cell biology simulations Changes in exte... more Nutrient sensing of plants was analyzed in computational cell biology simulations Changes in external nutrient concentrations caused cytosolic H + and Ca 2+ signals Nutrient transporter networks exhibit transceptor-like characteristics Wet-lab experiments confirmed the conceptual predictions

Plant Biology, Jul 12, 2016

• The two-pore cation channel TPC1 operates as a dimeric channel in animal and plant endomembrane... more • The two-pore cation channel TPC1 operates as a dimeric channel in animal and plant endomembranes. Each subunit consists of two homologous Shaker-like halves, with 12 transmembrane domains in total (S1-S6, S7-S12). In plants, TPC1 channels reside in the vacuolar membrane, and upon voltage stimulation, give rise to the well-known slow-activating SV currents. • Here, we combined bioinformatics, structure modeling, site-directed mutagenesis, and in planta patch clamp studies to elucidate the molecular mechanisms of voltage-dependent channel gating in TPC1 in its native plant background. • Structure-function analysis of the Arabidopsis TPC1 channel in planta confirmed that helix S10 operates as the major voltage-sensing site, with Glu450 and Glu478 identified as possible ionpair partners for voltage-sensing Arg537. The contribution of helix S4 to voltage sensing was found to be negligible. Several conserved negative residues on the luminal site contribute to calcium binding, stabilizing the closed channel. • During evolution of plant TPC1s from two separate Shaker-like domains, the voltage-sensing function in the N-terminal Shaker-unit (S1-S4) vanished.

Proceedings of the National Academy of Sciences of the United States of America, Aug 11, 2020

This open access article is distributed under Creative Commons Attribution-NonCommercial-NoDeriva... more This open access article is distributed under Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND).

Science Advances, Jul 9, 2021

Guard cells control the aperture of plant stomata, which are crucial for global fluxes of CO 2 an... more Guard cells control the aperture of plant stomata, which are crucial for global fluxes of CO 2 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 NO 3 − 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.

Trends in Plant Science, 2021

Plant Journal, Sep 1, 2001

SummaryIn search of a K+ channel involved in phloem transport we screened a Vicia faba cotyledon ... more SummaryIn search of a K+ channel involved in phloem transport we screened a Vicia faba cotyledon cDNA library taking advantage of a set of degenerated primers, flanking regions conserved among K+ uptake channels. We cloned VFK1 (for Vicia faba K+ channel 1) characterised by a structure known from the Shaker family of plant K+ channels. When co‐expressed with a KAT1 mutant in Xenopus oocytes, heteromers revealed the biophysical properties of a K+ selective, proton‐blocked channel. Northern blot analyses showed high levels of expression in cotyledons, flowers, stem and leaves. Using in situ PCR techniques we could localise the K+ channel mRNA in the phloem. In the stem VFK1 expression levels were higher in the lower internodes. There channel transcripts increased in the light and thus under conditions of increased photosynthate allocation. VFK1 transcripts are elevated in sink leaves, and rise in source leaves during the experimental transition into sinks. Fructose‐ rather than sucrose‐ or glucose‐feeding via the petiole induced VFK1 gene activity. We therefore monitored the fructose sensitivity of the sieve tube potential through cut aphid stylets. In response to an 1 h fructose treatment the sieve tube potential shift increased from 19 mV to 53 mV per 10‐fold change in K+ concentration. Under these conditions K+ channels dominated the electrical properties of the plasma membrane. Based on the phloem localisation and expression patterns of VFK1 we conclude that this K+ channel is involved in sugar unloading and K+ retrieval.

bioRxiv (Cold Spring Harbor Laboratory), Dec 23, 2021

To fire action-potential-like electrical signals, the vacuole membrane requires the depolarizatio... more To fire action-potential-like electrical signals, the vacuole membrane requires the depolarization-activated two-pore channel TPC1, also called Slowly activating Vacuolar SV channel. The TPC1/SV channel, encoded by the TPC1 gene, functions as a voltage-dependent and Ca 2+-regulated potassium channel. TPC1 currents are activated by a rise in cytoplasmic Ca 2+ but inhibited by luminal Ca 2+. In search for species-dependent functional TPC1 channel variants, we studied polymorphic amino acids contributing to luminal Ca 2+ sensitivity. We found that the acidic residues E457, E605 and D606 of the Ca 2+-sensitive Arabidopsis AtTPC1 channel were neutralized by either asparagine or alanine in Vicia faba and many other Fabaceae as well. When expressed in the Arabidopsis loss-of-AtTPC1 function background, the wild type VfTPC1 was hypersensitive to vacuole depolarization and insensitive to blocking luminal Ca 2+. When AtTPC1 was mutated for the three VfTPC1-homologous polymorphic site residues, the Arabidopsis At-VfTPC1 channel mutant gained VfTPC1like voltage and luminal Ca 2+ insensitivity that together made vacuoles hyperexcitable. These findings indicate that natural TPC1 channel variants in plant families exist which differ in vacuole excitability and very likely respond to changes in environmental settings of their ecological niche. Significance statement Vacuolar electrical excitability and stress-related Ca 2+ signaling depends on the activity of the vacuolar cation channel TPC1. Until now, the regulatory features of AtTPC1 from the model plant Arabidopsis thaliana was believed to apply to the TPC1 channels of other species. However, here we now show that, surprisingly, the VfTPC1 channel of the economic broad bean, in contrast to AtTPC1, proves to be hyperactive and confers hyperexcitability to the vacuole. The different gating behavior is most likely related to an impaired Ca 2+ sensor site in the vacuolar pore vestibule, rising the probability to open at more negative membrane voltages. These natural variants of the TPC1 channel could help the plant adapt and respond to environmental challenges. .

Proceedings of the National Academy of Sciences of the United States of America, Mar 16, 1999

New Phytologist, 2021

Summary Soil salinity is an increasingly global problem which hampers plant growth and crop yield... more Summary Soil salinity is an increasingly global problem which hampers plant growth and crop yield. Plant productivity depends on optimal water‐use efficiency and photosynthetic capacity balanced by stomatal conductance. Whether and how stomatal behavior contributes to salt sensitivity or tolerance is currently unknown. This work identifies guard cell‐specific signaling networks exerted by a salt‐sensitive and salt‐tolerant plant under ionic and osmotic stress conditions accompanied by increasing NaCl loads. We challenged soil‐grown Arabidopsis thaliana and Thellungiella salsuginea plants with short‐ and long‐term salinity stress and monitored genome‐wide gene expression and signals of guard cells that determine their function. Arabidopsis plants suffered from both salt regimes and showed reduced stomatal conductance while Thellungiella displayed no obvious stress symptoms. The salt‐dependent gene expression changes of guard cells supported the ability of the halophyte to maintain hi...

Current Biology, 2021

Plants, as sessile organisms, gained the ability to sense and respond to biotic and abiotic stres... more Plants, as sessile organisms, gained the ability to sense and respond to biotic and abiotic stressors to survive severe changes in their environments. The change in our climate comes with extreme dry periods but also episodes of flooding. The latter stress condition causes anaerobiosis-triggered cytosolic acidosis and impairs plant function. The molecular mechanism that enables plant cells to sense acidity and convey this signal via membrane depolarization was previously unknown. Here, we show that acidosis-induced anion efflux from Arabidopsis (Arabidopsis thaliana) roots is dependent on the S-type anion channel AtSLAH3. Heterologous expression of SLAH3 in Xenopus oocytes revealed that the anion channel is directly activated by a small, physiological drop in cytosolic pH. Acidosis-triggered activation of SLAH3 is mediated by protonation of histidine 330 and 454. Super-resolution microscopy analysis showed that the increase in cellular proton concentration switches SLAH3 from an electrically silent channel dimer into its active monomeric form. Our results show that, upon acidification, protons directly switch SLAH3 to its open configuration, bypassing kinase-dependent activation. Moreover, under flooding conditions, the stress response of Arabidopsis wild-type (WT) plants was significantly higher compared to SLAH3 loss-of-function mutants. Our genetic evidence of SLAH3 pH sensor function may guide the development of crop varieties with improved stress tolerance.

New Phytologist, 2021

Summary Cytosolic calcium signals are evoked by a large variety of biotic and abiotic stimuli and... more Summary Cytosolic calcium signals are evoked by a large variety of biotic and abiotic stimuli and play an important role in cellular and long distance signalling in plants. While the function of the plasma membrane in cytosolic Ca2+ signalling has been intensively studied, the role of the vacuolar membrane remains elusive. A newly developed vacuolar voltage clamp technique was used in combination with live‐cell imaging, to study the role of the vacuolar membrane in Ca2+ and pH homeostasis of bulging root hair cells of Arabidopsis. Depolarisation of the vacuolar membrane caused a rapid increase in the Ca2+ concentration and alkalised the cytosol, while hyperpolarisation led to the opposite responses. The relationship between the vacuolar membrane potential, the cytosolic pH and Ca2+ concentration suggests that a vacuolar H+/Ca2+ exchange mechanism plays a central role in cytosolic Ca2+ homeostasis. Mathematical modelling further suggests that the voltage‐dependent vacuolar Ca2+ homeo...

PLOS Biology, 2020

The carnivorous plant Dionaea muscipula harbors multicellular trigger hairs designed to sense mec... more The carnivorous plant Dionaea muscipula harbors multicellular trigger hairs designed to sense mechanical stimuli upon contact with animal prey. At the base of the trigger hair, mechanosensation is transduced into an all-or-nothing action potential (AP) that spreads all over the trap, ultimately leading to trap closure and prey capture. To reveal the molecular basis for the unique functional repertoire of this mechanoresponsive plant structure, we determined the transcriptome of D. muscipula’s trigger hair. Among the genes that were found to be highly specific to the trigger hair, the Shaker-type channel KDM1 was electrophysiologically characterized as a hyperpolarization- and acid-activated K+-selective channel, thus allowing the reuptake of K+ ions into the trigger hair’s sensory cells during the hyperpolarization phase of the AP. During trap development, the increased electrical excitability of the trigger hair is associated with the transcriptional induction of KDM1. Conversely, ...

Nature Communications, 2019

In contrast to the plasma membrane, the vacuole membrane has not yet been associated with electri... more In contrast to the plasma membrane, the vacuole membrane has not yet been associated with electrical excitation of plants. Here, we show that mesophyll vacuoles from Arabidopsis sense and control the membrane potential essentially via the K+-permeable TPC1 and TPK channels. Electrical stimuli elicit transient depolarization of the vacuole membrane that can last for seconds. Electrical excitability is suppressed by increased vacuolar Ca2+ levels. In comparison to wild type, vacuoles from the fou2 mutant, harboring TPC1 channels insensitive to luminal Ca2+, can be excited fully by even weak electrical stimuli. The TPC1-loss-of-function mutant tpc1-2 does not respond to electrical stimulation at all, and the loss of TPK1/TPK3-mediated K+ transport affects the duration of TPC1-dependent membrane depolarization. In combination with mathematical modeling, these results show that the vacuolar K+-conducting TPC1 and TPK1/TPK3 channels act in concert to provide for Ca2+- and voltage-induced ...

Plant physiology, Jan 26, 2017

Water limitation of plants causes stomatal closure to prevent water loss by transpiration. For th... more Water limitation of plants causes stomatal closure to prevent water loss by transpiration. For this purpose, progressing soil water deficit is communicated from roots to shoots. Abscisic acid (ABA) is the key signal in stress-induced stomatal closure, however, ABA as early xylem-delivered signal is still a matter of debate. In the present study, poplar plants were exposed to water stress to investigate xylem sap sulfate and ABA, stomatal conductance and sulfate transporter (SULTR) expression. In addition, stomatal behavior and expression of ABA receptors, drought responsive genes, transcription factors and NCED3 were studied after feeding sulfate and ABA to detached poplar leaves and epidermal peels of Arabidopsis. The results show that increased xylem sap sulfate is achieved upon drought by reduced xylem unloading by PtaSULTR3;3a and PtaSULTR1;1, and by enhanced loading from parenchyma cells into the xylem via PtaALMT3b. Sulfate application caused stomatal closure in excised leaves...

The Plant Cell, 2001

Plant K ؉ uptake channel types differ with respect to their voltage, Ca 2 ؉ , and pH dependence. ... more Plant K ؉ uptake channel types differ with respect to their voltage, Ca 2 ؉ , and pH dependence. Here, we constructed recombinant chimeric channels between KST1, a member of the inward-rectifying, acid-activated KAT1 family, and AKT3, a member of the weakly voltage-dependent, proton-blocked AKT2/3 family. The homologous pore regions of AKT3 (amino acids 216 to 287) and KST1 (amino acids 217 to 289) have been exchanged to generate the two chimeric channels AKT3/(p)KST1 and KST1/(p)AKT3. In contrast to AKT3 wild-type channels, AKT3/(p)KST1 revealed a strong inward rectification reminiscent of that of KST1. Correspondingly, the substitution of the KST1 by the AKT3 pore led to less pronounced rectification properties of KST1/(p)AKT3 compared with wild-type KST1. Besides the voltage dependence, the interaction between the chimera and extracellular H ؉ and Ca 2 ؉ resembled the properties of the inserted rather than the respective wild-type pore. Whereas AKT3/(p)KST1 was acid activated and Ca 2 ؉ insensitive, extracellular protons and Ca 2 ؉ inhibited KST1/(p)AKT3. The regulation of the chimeric channels by cytoplasmic protons followed the respective wild-type backbone of the chimeric channels, indicating that the intracellular pH sensor is located outside the P domain. We thus conclude that essential elements for external pH and Ca 2 ؉ regulation and for the rectification of voltage-dependent K ؉ uptake channels are located within the channel pore.

The Plant Cell, 2013

Apical growth in pollen tubes (PTs) is associated with the presence of tip-focused ion gradients ... more Apical growth in pollen tubes (PTs) is associated with the presence of tip-focused ion gradients and fluxes, implying polar localization or regulation of the underlying transporters. The molecular identity and regulation of anion transporters in PTs is unknown. Here we report a negative gradient of cytosolic anion concentration focused on the tip, in negative correlation with the cytosolic Ca 2+ concentration. We hypothesized that a possible link between these two ions is based on the presence of Ca 2+-dependent protein kinases (CPKs). We characterized anion channels and CPK transcripts in PTs and analyzed their localization. Yellow fluorescent protein (YFP) tagging of a homolog of SLOW ANION CHANNEL-ASSOCIATED1 (SLAH3:YFP) was widespread along PTs, but, in accordance with the anion efflux, CPK2/CPK20/CPK17/CPK34:YFP fluorescence was strictly localized at the tip plasma membrane. Expression of SLAH3 with either CPK2 or CPK20 (but not CPK17/CPK34) in Xenopus laevis oocytes elicited S-type anion channel currents. Interaction of SLAH3 with CPK2/CPK20 (but not CPK17/CPK34) was confirmed by Förster-resonance energy transfer fluorescence lifetime microscopy in Arabidopsis thaliana mesophyll protoplasts and bimolecular fluorescence complementation in living PTs. Compared with wild-type PTs, slah3-1 and slah3-2 as well as cpk2-1 cpk20-2 PTs had reduced anion currents. Double mutant cpk2-1 cpk20-2 and slah3-1 PTs had reduced extracellular anion fluxes at the tip. Our studies provide evidence for a Ca 2+-dependent CPK2/CPK20 regulation of the anion channel SLAH3 to regulate PT growth.

The Plant Cell, 2002

The Arabidopsis phloem channel AKT3 is the founder of a subfamily of shaker-like plant potassium ... more The Arabidopsis phloem channel AKT3 is the founder of a subfamily of shaker-like plant potassium channels characterized by weak rectification, Ca 2 ؉ block, proton inhibition, and, as shown in this study, K ؉ sensitivity. In contrast to inward-rectifying, acid-activated K ؉ channels of the KAT1 family, extracellular acidification decreases AKT3 currents at the macroscopic and single-channel levels. Here, we show that two distinct sites within the outer mouth of the K ؉-conducting pore provide the molecular basis for the pH sensitivity of this phloem channel. After generation of mutant channels and functional expression in Xenopus oocytes, we identified the His residue His-228, which is proximal to the K ؉ selectivity filter (GYGD) and the distal Ser residue Ser-271, to be involved in proton susceptibility. Mutations of these sites, H228D and S271E, drastically reduced the H ؉ and K ؉ sensitivity of AKT3. Although in K ؉-free bath solutions outward K ؉ currents were abolished completely in wild-type AKT3, S271E as well as the AKT3-HDSE double mutant still mediated K ؉ efflux. We conclude that the pH-and K ؉-dependent properties of the AKT3 channel involve residues in the outer mouth of the pore. Both properties, H ؉ and K ؉ sensitivity, allow the fine-tuning of the phloem channel and thus seem to represent important elements in the control of membrane potential and sugar loading.