Optogenetic control of the guard cell membrane potential and stomatal movement by the light-gated anion channel

Huang S., Ding M., Roelfsema M. R. G., Dreyer I., Scherzer S., Al-Rasheid K. A. S., Gao S., Nagel G., Hedrich R., Konrad K. R. (2021)

Shouguang Huang, Meiqi Ding, M. Rob G. Roelfsema, Ingo Dreyer, Sönke Scherzer, Khaled A. S. Al-Rasheid, Shiqiang Gao, Georg Nagel, Rainer Hedrich, Kai R. Konrad,

University of Würzburg

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Science Advances 7 (28): eabg4619 – DOI: 10.1126/sciadv.abg4619

https://www.sciencedaily.com/releases/2021/07/210709193604.htm

Plants have microscopically small pores on the surface of their leaves, the stomata. With their help, they regulate the influx of carbon dioxide for photosynthesis. They also use the stomata to prevent the loss of too much water and withering away during drought.

The stomatal pores are surrounded by two guard cells. If the internal pressure of these cells drops, they slacken and close the pore. If the pressure rises, the cells move apart and the pore widens.

The stomatal movements are thus regulated by the guard cells. Signalling pathways in these cells are so complex that it is difficult for humans to intervene with them directly. However, researchers of the Julius-Maximilians-Universität (JMU) Würzburg in Bavaria, Germany, nevertheless found a way to control the movements of stomata remotely — using light pulses.

Light-sensitive protein from algae used

The researchers succeeded in doing this by introducing a light-sensitive switch into the guard cells of tobacco plants. This technology was adopted from optogenetics. It has been successfully exploited in animal cells, but the application in plant cells it is still in its infancy.

The team led by JMU biophysicist and guard cell expert Professor Rainer Hedrich describes their approach in the scientific journal Science Advances. JMU researchers Shouguang Huang (first author), Kai Konrad and Rob Roelfsema were significantly involved.

The group used a light-sensitive protein from the alga Guillardia theta as a light switch, namely the anion channel ACR1 from the group of channelrhodopsins. In response to light pulses, the switch ensures that chloride flows out of the guard cells and potassium follows. The guard cells lose internal pressure, slacken and the pore closes within 15 minutes. “The light pulse is like a remote control for the movement of the stomata,” says Hedrich.

Anion channel hypothesis confirmed

“By exposing ACR1 to light, we have bridged the cell’s own signalling chain, thus proving the hypothesis that the opening of anion channels is essential and sufficient for stomatal closure,” Hedrich summarises the results of the study. The exposure to light had almost completely prevented the transpiration of the plants.

With this knowledge, it is now possible to cultivate plants with an increased number of anion channels in the guard cells. Plants equipped in this way should close their stomata more quickly in response to approaching heat waves and thus be better able to cope with periods of drought.

“Plant anion channels are activated during stress; this process is dependent on calcium. In a follow up optogenetics project, we want to use calcium-conducting channelrhodopsins to specifically allow calcium to flow into the guard cells cell through exposure to light and to understand the mechanism of anion channel activation in detail,” Hedrich outlines the upcoming goals of his research.

Basic scientific research can also benefit from the results from Würzburg: “Our new optogenetic tool has enormous potential for research,” says the JMU professor. “With it, we can gain new insights into how plants regulate their water consumption and how carbon dioxide fixation and stomatal movements are coupled.”

GABA signalling modulates stomatal opening

GABA signalling modulates stomatal opening to enhance plant water use efficiency and drought resilience

Xu B., Long Y., Feng X., Zhu X., Sai N., Chirkova L., Betts A., Herrmann J., Edwards E. J., Okamoto M., Hedrich R., Gilliham M. (2021)

Bo XuYu LongXueying FengXujun ZhuNa SaiLarissa ChirkovaAnnette BettsJohannes HerrmannEverard J. EdwardsMamoru OkamotoRainer HedrichMatthew Gilliham

Nat Commun 12: 1952 – https://doi.org/10.1038/s41467-021-21694-3

https://www.nature.com/articles/s41467-021-21694-3

Abstract

The non-protein amino acid γ-aminobutyric acid (GABA) has been proposed to be an ancient messenger for cellular communication conserved across biological kingdoms. GABA has well-defined signalling roles in animals; however, whilst GABA accumulates in plants under stress it has not been determined if, how, where and when GABA acts as an endogenous plant signalling molecule. Here, we establish endogenous GABA as a bona fide plant signal, acting via a mechanism not found in animals. Using Arabidopsis thaliana, we show guard cell GABA production is necessary and sufficient to reduce stomatal opening and transpirational water loss, which improves water use efficiency and drought tolerance, via negative regulation of a stomatal guard cell tonoplast-localised anion transporter. We find GABA modulation of stomata occurs in multiple plants, including dicot and monocot crops. This study highlights a role for GABA metabolism in fine tuning physiology and opens alternative avenues for improving plant stress resilience.

Anion channels in stomatal guard cells and other plant cells are key targets within often complex signaling networks

Ion channels in plants

Hedrich R. (2012)

Rainer Hedrich,

niversity of Wuerzburg, Institute for Molecular Plant Physiology and Biophysics, Wuerzburg, Germany.

Physiol Rev 92(4): 1777-1811 – doi: 10.1152/physrev.00038.2011

https://pubmed.ncbi.nlm.nih.gov/23073631/

Abstract

Since the first recordings of single potassium channel activities in the plasma membrane of guard cells more than 25 years ago, patch-clamp studies discovered a variety of ion channels in all cell types and plant species under inspection. Their properties differed in a cell type- and cell membrane-dependent manner. Guard cells, for which the existence of plant potassium channels was initially documented, advanced to a versatile model system for studying plant ion channel structure, function, and physiology. Interestingly, one of the first identified potassium-channel genes encoding the Shaker-type channel KAT1 was shown to be highly expressed in guard cells. KAT1-type channels from Arabidopsis thaliana and its homologs from other species were found to encode the K(+)-selective inward rectifiers that had already been recorded in early patch-clamp studies with guard cells. Within the genome era, additional Arabidopsis Shaker-type channels appeared. All nine members of the Arabidopsis Shaker family are localized at the plasma membrane, where they either operate as inward rectifiers, outward rectifiers, weak voltage-dependent channels, or electrically silent, but modulatory subunits. The vacuole membrane, in contrast, harbors a set of two-pore K(+) channels. Just very recently, two plant anion channel families of the SLAC/SLAH and ALMT/QUAC type were identified. SLAC1/SLAH3 and QUAC1 are expressed in guard cells and mediate Slow- and Rapid-type anion currents, respectively, that are involved in volume and turgor regulation. Anion channels in guard cells and other plant cells are key targets within often complex signaling networks. Here, the present knowledge is reviewed for the plant ion channel biology. Special emphasis is drawn to the molecular mechanisms of channel regulation, in the context of model systems and in the light of evolution.

The nucleotide and Mg(2+) dependencies of time-dependent K(in) and K(out) channels from maize subsidiary cells were examined, showing that MgATP as well as MgADP function as channel activators

Nucleotides and Mg2+ ions differentially regulate K+ channels and non-selective cation channels present in cells forming the stomatal complex

Wolf T., Guinot D. R., Hedrich R., Dietrich P., Marten I. (2005)

Thomas WolfDavid Roger GuinotRainer HedrichPetra DietrichIrene Marten,


Molecular Plant Physiology and Biophysics, Julius-von-Sachs Institute for Bioscience, University of Wuerzburg, Germany.

===

Plant Cell Physiol. 46(10): 1682-1689 – doi: 10.1093/pcp/pci184 – Epub 2005 Aug 4 –

https://pubmed.ncbi.nlm.nih.gov/16081526/

Abstract

Voltage-dependent inward-rectifying (K(in)) and outward-rectifying (K(out)) K(+) channels are capable of mediating K(+) fluxes across the plasma membrane. Previous studies on guard cells or heterologously expressed K(+) channels provided evidence for the requirement of ATP to maintain K(+) channel activity. Here, the nucleotide and Mg(2+) dependencies of time-dependent K(in) and K(out) channels from maize subsidiary cells were examined, showing that MgATP as well as MgADP function as channel activators. In addition to K(out) channels, these studies revealed the presence of another outward-rectifying channel type (MgC) in the plasma membrane that however gates in a nucleotide-independent manner. MgC represents a new channel type distinguished from K(out) channels by fast activation kinetics, inhibition by elevated intracellular Mg(2+) concentration, permeability for K(+) as well as for Na(+) and insensitivity towards TEA(+). Similar observations made for guard cells from Zea mays and Vicia faba suggest a conserved regulation of channel-mediated K(+) and Na(+) transport in both cell types and species.

An important role for phosphate and the action of PHO1 in the stomatal response to ABA

PowerPoint
PHO1 is preferentially expressed in guard cells compared with mesophyll cells, and expression is induced by ABA.
(a) Localization of PHO1::GUS translational fusion protein activity in leaves. Expression was under the control of 2.1 kb of the PHO1 promoter region. Rosettes were cut at the hypocotyl and floated in water with or without 10 μm ABA for 3 h before GUS staining. In the leaf blade, GUS staining is visible in guard cells, and the intensity is increased under ABA treatment. Staining in vascular tissue of the leaf petiole can also be seen.
(b) Variation in PHO1 transcript level between wild‐type guard cell and mesophyll cell protoplasts preparations. The guard cell‐specific gene KAT1 and the mesophyll cell‐specific gene At4G26530 were also measured as controls. n =3 biological replicates; average ± SE.
(c) Expression levels of PHO1 in wild‐type guard cell protoplasts following treatment with 10 μm ABA for 40 min to 6 h, and treatment with 0–100 μm ABA for 4 h. Expression levels of the known ABA‐induced gene RAB18 are also represented. n =3 biological replicates; average ± SE.
Relative expression levels refers to transcript abundance of target genes, normalized against expression of the reference gene At1G13320 (Czechowski et al., 2005).

PHO1 Expression in Guard Cells Mediates the Stomatal Response to Abscisic Acid in Arabidopsis

Celine Z., Cecile R., Alain V., Hubert B., Rainer H., Yves P. (2012)

Céline Zimmerli, Cécile Ribot, Alain Vavasseur, Hubert Bauer, Rainer Hedrich, Yves Poirier,

Plant J. 72 (2): 199–211 – doi: 10.1111/j.1365-313X.2012.05058.x –

https://onlinelibrary.wiley.com/doi/full/10.1111/j.1365-313X.2012.05058.x

Summary

Stomatal opening and closing are driven by ion fluxes that cause changes in guard cell turgor and volume. This process is, in turn, regulated by environmental and hormonal signals, including light and the phytohormone abscisic acid (ABA). Here, we present genetic evidence that expression of PHO1 in guard cells of Arabidopsis thaliana is required for full stomatal responses to ABA. PHO1 is involved in the export of phosphate into the root xylem vessels and, as a result, the pho1 mutant is characterized by low shoot phosphate levels. In leaves, PHO1 was found expressed in guard cells and up‐regulated following treatment with ABA. The pho1 mutant was unaffected in production of reactive oxygen species following ABA treatment, and in stomatal movements in response to light cues, high extracellular calcium, auxin, and fusicoccin. However, stomatal movements in response to ABA treatment were severely impaired, both in terms of induction of closure and inhibition of opening. Micro‐grafting a pho1 shoot scion onto wild‐type rootstock resulted in plants with normal shoot growth and phosphate content, but failed to restore normal stomatal response to ABA treatment. PHO1 knockdown using RNA interference specifically in guard cells of wild‐type plants caused a reduced stomatal response to ABA. In agreement, specific expression of PHO1 in guard cells of pho1 plants complemented the mutant guard cell phenotype and re‐established ABA sensitivity, although full functional complementation was dependent on shoot phosphate sufficiency. Together, these data reveal an important role for phosphate and the action of PHO1 in the stomatal response to ABA.

OSCA1.3 regulates plant stomatal immunity

This is an unedited manuscript that has been accepted for publication. Nature Research are providing this early version of the manuscript as a service to our authors and readers. The manuscript will undergo copyediting, typesetting and a proof review before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers apply.

The calcium-permeable channel OSCA1.3 regulates plant stomatal immunity

Kathrin Thor, Shushu Jiang, Erwan Michard, Jeoffrey George, Sönke Scherzer, Shouguang Huang, Julian Dindas, Paul Derbyshire, Nuno Leitão, Thomas A. DeFalco, Philipp Köster, Kerri Hunter, Sachie Kimura, Julien Gronnier, Lena Stransfeld, Yasuhiro Kadota, Christoph A. Bücherl, Myriam Charpentier, Michael Wrzaczek, Daniel MacLean, Giles E. D. Oldroyd, Frank L. H. Menke, M. Rob G. Roelfsema, Rainer Hedrich, José Feijó, Cyril Zipfel, (2020)

 Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China

In Nature, 2020 – DOI: 10.1038/s41586-020-2702-1

https://www.nature.com/articles/s41586-020-2702-1#citeas

Abstract

Perception of biotic and abiotic stresses often leads to stomatal closure in plants1,2. Rapid influx of calcium ions (Ca2+) across the plasma membrane plays an important role in this response, but the identity of Ca2+ channels involved has remained elusive3,4. Here, we report that the Arabidopsis thaliana Ca2+-permeable channel OSCA1.3 controls stomatal closure during immunity. OSCA1.3 is rapidly phosphorylated upon perception of pathogen-associated molecular patterns (PAMPs). Biochemical and quantitative phospho-proteomics analyses reveal that the immune receptor-associated cytosolic kinase BIK1 interacts with and phosphorylates the N-terminal cytosolic loop of OSCA1.3 within minutes of treatment with the peptidic PAMP flg22 derived from bacterial flagellin. Genetic and electrophysiological data reveal that OSCA1.3 is permeable to Ca2+, and that BIK1-mediated phosphorylation on its N-terminus increases this channel activity. Importantly, OSCA1.3 and its phosphorylation by BIK1 are critical for stomatal closure during immunity. Notably, OSCA1.3 does not regulate stomatal closure upon perception of abscisic acid – a plant hormone associated with abiotic stresses. Our study thus identifies a long sought-after plant Ca2+ channel and its activation mechanisms underlying stomatal closure during immune signaling, and suggests specificity in Ca2+ influx mechanisms in response to different stresses.

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Corrections & amendments

Publisher Correction: The calcium-permeable channel OSCA1.3 regulates plant stomatal immunity

https://doi.org/10.1038/s41586-020-2954-9

Correction to: Nature https://doi.org/10.1038/s41586-020-2702-1

Published online 26 August 2020

Check for updates

Kathrin Thor, Shushu Jiang, Erwan Michard, Jeoffrey George, Sönke Scherzer, Shouguang Huang, Julian Dindas, Paul Derbyshire, Nuno Leitão, Thomas A. DeFalco, Philipp Köster, Kerri Hunter, Sachie Kimura, Julien Gronnier, Lena Stransfeld, Yasuhiro Kadota, Christoph A. Bücherl, Myriam Charpentier, Michael Wrzaczek, Daniel MacLean, Giles E. D. Oldroyd, Frank L. H. Menke, M. Rob G. Roelfsema, Rainer Hedrich, José Feijó & Cyril Zipfel

In this Article, owing to an error in the production process, a grant was omitted from the Acknowledgements section, which should have stated that work in the J.F. laboratory was supported by the National Institutes of Health (NIH R01 GM131043). The Article has been corrected online.
(19) (PDF) Publisher Correction: The calcium-permeable channel OSCA1.3 regulates plant stomatal immunity. Available from: https://www.researchgate.net/publication/345983684_Publisher_Correction_The_calcium-permeable_channel_OSCA13_regulates_plant_stomatal_immunity [accessed Nov 24 2020].

Ca2+‐dependent activation of guard cell anion channels

Schematic representation of Ca2+‐ and voltage‐dependent regulation of S‐type anion channels in Nicotiana tabacum guard cells.
Ion channels are represented by butterfly motifs, transporters by filled circles and the membrane potential by plus or minus symbols. The arrows through the motifs indicate the direction of ion flow under physiological conditions. The lines connecting the symbols represent regulatory interactions: stimulation and inhibition are indicated by arrowheads and perpendicular line ends, respectively. Proven interactions are given in black, and possible regulatory events are shown as grey lines. The scheme shows that hyperpolarization can have a twofold effect on the activity of S‐type anion channels: S‐type anion channels are inhibited by hyperpolarization, but stimulated by a hyperpolarization‐induced increase in the cytosolic Ca2+ level. In addition, the cytosolic free Ca2+ concentration feeds back on the Ca2+ transporters and possibly on Ca2+ channels as well.
Ca2+‐dependent activation of inward current with a depolarizing voltage step.
(a) A guard cell of Nicotiana tabacum was stimulated with a 100 sec voltage step to 0 mV [top trace in (a)]. The cytosolic free Ca2+ concentration decreased during the depolarization, followed by a transient increase after returning to the holding potential of −100 mV [middle trace in (a)]. Depolarization of the plasma membrane caused only small changes in current [lower trace in (a); note that outward K+ channels are blocked by Cs+ in the electrodes], whereas a large transient increase in current occurs after returning to the holding potential of −100 mV.
(b) False‐coloured images representing the cytosolic free Ca2+ concentration in the same guard cell as in (a). Images were obtained before (closed circle), during (closed square) and after (open circle and open square) the depolarizing voltage step [symbols correspond to (a)]. Colour codes are linked to the cytosolic free Ca2+ concentration in the scale bar on the left. Note the high cytosolic free Ca2+ concentration at the tip of the guard cell, corresponding to the site of micro‐electrode impalement.

Ca2+‐dependent activation of guard cell anion channels, triggered by hyperpolarization, is promoted by prolonged depolarization

Stange A., Hedrich R., Roelfsema M. R. G. (2010)

Annette Stange, Rainer Hedrich, M. Rob G. Roelfsema,

The Plant Journal 62(2) – https://doi.org/10.1111/j.1365-313X.2010.04141.x

https://onlinelibrary.wiley.com/doi/full/10.1111/j.1365-313X.2010.04141.x

Time dependence of depolarization evoked Ca2+ responses.
(a) Nicotiana tabacum guard cell stimulated with depolarizing voltage steps from −100 to 0 mV for the duration of 1, 10 and 100 sec (top trace). The cytosolic free Ca2+ concentrations (middle trace), calculated from the FURA2 fluorescent ratio, showed only minor changes during the 1 sec pulse and a decrease during the 10 sec pulse. However, the 100 sec voltage step to 0 mV caused a transient overshoot of the cytosolic Ca2+ level after returning to −100 mV. The transient increase in the cytosolic free Ca2+ concentration correlated with a transient increase of inward current (lower trace).
(b) False‐coloured images representing the cytosolic free Ca2+ concentration in the same guard cell as in (a). Images were obtained before (closed circle), during (closed square) and after (open circle and open square) the depolarizing voltage step [symbols correspond to (a)]. Colour codes are linked to the cytosolic free Ca2+ concentration in the scale bar on the left. Note that depolarization triggers changes in the cytosolic free Ca2+ concentration throughout the cell, which are most obvious in the area surrounding the nucleus.

Summary

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.

The chloroplasts of adaxial GCs of stomata have a highly expressive functional organization and that the photosynthetic apparatus of adaxial GCs is well adapted to elevated light intensities

Evidence for the functional organization of chloroplast in adaxial guard cells of Vicia faba leaves by single cell analysis

by Goh C. H, Hedrich R., Nam H. G. (2002)

Chang-Hyo Goha, Rainer Hedrichb, Hong Gil Nama

a Division of Molecular and Life Science, Pohang University of Science and Technology, San 31, Nam-Gu, Hyoja-Dong, Pohang, Kyungbuk, 790-784 South Korea

b Lehrstuhl für Molekulare Pflanzenphysiologie und Biophysik, Julius-von-Sachs Institut, Universität Würzburg, Julius-von-Sachs Platz 2, D-97082 Wurzburg, Germany

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In Plant Sci. 162: 965–972 – doi: 10.1016/S0168-9452(02)00047-X –

https://www.sciencedirect.com/science/article/pii/S016894520200047X?via%3Dihub

Abstract

The functional organization of chloroplasts in adaxial guard cells (GCs) of Vicia faba leaves was investigated by the saturation pulse method of chlorophyll fluorescence induction using single GC pairs. Quantitative imaging analysis of chlorophyll fluorescence performed on adaxial GCs upon dark–light transition revealed a large chlorophyll fluorescence transient. Adaxial GCs displayed PS II quantum yields and relative electron transport rates (ETR) comparable to the abaxial GCs. The slow chlorophyll fluorescence transients in the two cells provided further evidence for the activation of the Calvin cycle in GC chloroplasts on both sides of the leaf. The ETR upon increasing light intensity was, however, differentially expressed in the two cell types. When the rates were normalized to their maximum ETR activity, half-saturation was achieved at 76 and 45 μmol m−2 s−1 in adaxial and abaxial GCs, respectively. These results provide unequivocal evidence that the chloroplasts of adaxial GCs have a highly expressive functional organization and that the photosynthetic apparatus of adaxial GCs is well adapted to elevated light intensities.

The biophysical mechanisms that control stomatal movements differ between ferns and seed plants

Fig. 1 Ion channels in guard cells of the ferns Polypodium vulgare (upper panels) and Asplenium scolopendrium (lower panels). (a) Bright field images of
fern stomata; note the large number of chloroplasts in the guard cells of both P. vulgare and A. scolopendrium. (b) Voltage-induced activation of ion
channels in fern guard cells. Guard cells were impaled with double-barreled microelectrodes and the membrane potential was clamped from a holding
potential of 100 mV, in 2-s pulses and with 20-mV steps, to more positive or negative values. Current traces are superimposed. Note that the timedependent changes in ion currents display all hallmarks of K+ efflux and uptake channels, known from seed plant guard cells. (c) Current–voltage relation
of fern guard cells, determined at the start (open symbols) and end (closed symbols) of 2-s test pulses applied from a holding potential of 100 mV as
shown in (b). Average data are shown (n = 40, P. vulgare; n = 15, A. scolopendrium); error bars, SE.

Guard cells in fern stomata are connected by plasmodesmata, but control cytosolic Ca2+ levels autonomously

by Voss L. J., McAdam S. A. M., Knoblauch M., Rathje J. M., Brodribb T., Hedrich R., Roelfsema M. R. G. (2018)

Lena J. Voss1, Scott A. M. McAdam2,3, Michael Knoblauch4, Jan M. Rathje1, Tim Brodribb2, Rainer Hedrich1, M. Rob G. Roelfsema1,

1 Molecular Plant Physiology and Biophysics, Julius-von-Sachs Institute for Biosciences, Biocenter, W€urzburg University, Julius-von-Sachs-Platz 2, D-97082 Wurzburg, Germany;

2 School of Biological Science, University of Tasmania, Hobart, TAS 7001, Australia;

3 Botany and Plant Pathology, Purdue University, 915 West State Street, West Lafayette, IN 47907, USA;

4 School of Biological Sciences, Washington State University, PO Box 644236, Pullman, WA 99164-4236, USA

===

In New Phytologist 219: 206–215 – doi: 10.1111/nph.15153 –

https://nph.onlinelibrary.wiley.com/doi/pdf/10.1111/nph.15153

Fig. 2 Hyperpolarization-induced elevation of the cytosolic free Ca2+ concentration in fern guard cells. (a, e) Color-coded images of Polypodium vulgare
(a) and Asplenium scolopendrium (e) guard cells showing changes in Oregon Green-BAPTA (OG-BAPTA) fluorescence intensity relative to the value
measured at the start of the experiment. Images were obtained before (right panel), during (middle panels) and after clamping the membrane potential
from 100 to –200 mV. The time from the start of the experiment is shown below each panel; bars, 20 lm. (b) Color-coded images of the cytosolic free
Ca2+ concentration (based on FURA2 signals) in a P. vulgare guard cell, before (upper panel), during (middle panel) and after (lower panel) stimulation of
the cell with a 200-mV voltage pulse. The color code is linked to cytosolic Ca2+ concentrations in the calibration bar below the panels and the time from
the start of the experiment is indicated at the left bottom of each panel; bar, 20 lm. (c) Time-dependent changes in the cytosolic free Ca2+ concentration
induced by a voltage pulse to 200 mV (indicated by the black area in the bar below the graph). Average data are shown for 15 P. vulgare guard cells;
error bars, SE. (d) Frequency distribution of the magnitude of the rise in the cytosolic free Ca2+ concentration induced by voltage pulses to 180 (filled
bars), 200 (diagonally striped bars) or –220 mV (horizontal striped bars). Data were obtained with FURA2-loaded P. vulgare guard cells as in (b) and (c).

Summary

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.

Fig. 3 Cytosolic connections between guard
cells in fern stomata. (a, d) Color-coded
images of Polypodium vulgare (a) and
Polypodium glycyrrhiza (d) stomata for
which the upper guard cell was current
injected with Lucifer Yellow (LY). Note that,
in time, the fluorescent dye moved into the
lower (non-impaled) guard cell. The
calibration bars on the right link the color
code to the relative fluorescence intensity,
which was set to 100% at the region of
interest at t = 20 min. The time from the start
of the experiments is shown below each
panel. Bars, 20 lm. (b, c) Time-dependent
increase in fluorescence intensity of LY
current injected into a single guard cell
(closed symbols) of fern stomata and slowly
appearing in the neighboring guard cell
(open symbols). Data are the average of 15
experiments with P. vulgare (b) and eight
experiments with P. glycyrrhiza (c); error
bars, SE.

Voltage clamp experiments with fern guard cells revealed similar properties of voltagedependent 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.

Fig. 4 Electron microscopy images of a Polypodium glycyrrhiza stoma. (a)
Transverse section through a P. glycyrrhiza stoma; note that the nucleus
(1) is located in the periphery of the guard cell, close to the stomatal split
(2) and surrounded by multiple chloroplasts (3). Lipid droplets (4) are lined
up in close proximity to the guard cell wall. (b, c) Close up of the cell wall
interconnecting both guard cells; note the plasmodesma spanning the cell
wall between the guard cells.

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.

SLAH3 is a regulatory target of chitin receptor-associated kinase PBL27 in microbial stomatal closure

PBL27 interacts with and phosphorylates SLAH3.
(A) Confocal microscopy of N. benthamiana leaves transiently expressing the indicated split-YFP constructs. Representative images are shown. (B) Co-immunoprecipitation of PBL27 and SLAH3 transiently expressed in N. benthamiana leaves. These experiments were performed at least twice with similar results. Expected sizes of PBL27-T7 and BIK1-T7 fusion proteins correspond to 57 kDa and 46 kDa, respectively. SLAH3-FLAG used for immune-precipitation has an expected size of 73 kDa. (C) PBL27 trans-phosphorylates SLAH3-N and SLAH3-C. In vitro kinase assay incubating equal amounts of recombinant His-MBP-PBL27, His-MBP-PBL27 K112E (kinase dead) with recombinant His-SLAH3-N or GST-SLAH3-C (GST-SLAH3-C-His). Autoradiogram, left panel; Coomassie colloidal blue (CCB) stained membrane, right panel. These experiments were repeated three times with similar results. (D) Chitin-activated PBL27 trans-phosphorylates SLAH3-N. Transgenic pbl27-1/pPBL27::PBL27−3 × HA Arabidopsis seedlings were treated (+) or not (-) with 1 mg/ml chitin for 10 min. Total proteins were subjected to immunoprecipitation with anti-HA beads followed by immunoblot analysis with anti-HA to reveal PBL27−3 × HA (upper panel). Immuno-precipitated PBL27−3 × HA was incubated with recombinant His-SLAH3-N for in vitro kinase assay. Autoradiogram, left panel; Coomassie colloidal blue (CCB) stained membrane, right panel. Col-0 seedlings were used as a control. These experiments were repeated three times with similar results. (E) Chitin induces SLAH3 phosphorylation. Transgenic Arabidopsis slah3-1/35S::SLAH3−3 × FLAG transgenic were treated (+) or not (-) with 1 mg/ml chitin for 30 min. Total proteins were subjected to immunoprecipitation with anti-FLAG beads. The phosphorylated form of SLAH3−3 × FLAG was shifted upward in Phos-tag SDS-PAGE. After phosphatase treatment, the shifted band of SLAH3−3 × FLAG dispersed, indicating the SLAH3−3 × FLAG was phosphorylated after treatment with chitin. The white arrow indicates the phosphorylated form of SLAH3-FLAG. The bands were detected with an anti-FLAG antibody. This experiment was repeated three times with similar results.

Anion channel SLAH3 is a regulatory target of chitin receptor-associated kinase PBL27 in microbial stomatal closure

by Liu Y., Maierhofer T., Rybak K., Sklenar J., Breakspear A., Johnston M. G., Fliegmann J., Huang S., Roelfsema M. R. G., Felix G., Faulkner C., Menke F. L. H., Geiger D., Hedrich R. , Robatzek S. (2019)

Yi LiuTobias MaierhoferKatarzyna RybakJan SklenarAndy BreakspearMatthew G JohnstonJudith FliegmannShouguang HuangM Rob G RoelfsemaGeorg FelixChristine FaulknerFrank LH MenkeDietmar GeigerRainer Hedrich , Silke Robatzek,

In eLife 2019;8:e44474 – https://doi.org/10.7554/eLife.44474.001

https://elifesciences.org/articles/44474?utm_source=miragenews&utm_medium=miragenews&utm_campaign=news

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.