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 –


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.

A single‐pore residue renders the root anion channel SLAH2 highly nitrate selective

A single‐pore residue renders the Arabidopsis root anion channel SLAH2 highly nitrate selective

by Maierhofer T., Lind C., Huttl S., Scherzer S., Papenfuss M., Simon J., Al‐Rasheid K. A. S., Ache P., Rennenberg H., Hedrich R.,  Müller T. D., Geiger D. (2014)

Tobias Maierhofer, Christof Lind, Stefanie Hüttl, Sönke Scherzer, Melanie Papenfuß, Judy Simon, Khaled A.S. Al-Rasheid, Peter Ache, Heinz Rennenberg, Rainer Hedrich, Thomas D. Müller, Dietmar Geiger,


In Plant Cell 26: 2554– 2567 –


In contrast to animal cells, plants use nitrate as a major source of nitrogen. Following the uptake of nitrate, this major macronutrient is fed into the vasculature for long-distance transport. The Arabidopsis thaliana shoot expresses the anion channel SLOW ANION CHANNEL1 (SLAC1) and its homolog SLAC1 HOMOLOGOUS3 (SLAH3), which prefer nitrate as substrate but cannot exclude chloride ions. By contrast, we identified SLAH2 as a nitrate-specific channel that is impermeable for chloride. To understand the molecular basis for nitrate selection in the SLAH2 channel, SLAC1 and SLAH2 were modeled to the structure of HiTehA, a distantly related bacterial member. Structure-guided site-directed mutations converted SLAC1 into a SLAH2-like nitrate-specific anion channel and vice versa. Our findings indicate that two pore-occluding phenylalanines constrict the pore. The selectivity filter of SLAC/SLAH anion channels is determined by the polarity of pore-lining residues located on alpha helix 3. Changing the polar character of a single amino acid side chain (Ser-228) to a nonpolar residue turned the nitrate-selective SLAH2 into a chloride/nitrate-permeable anion channel. Thus, the molecular basis of the anion specificity of SLAC/SLAH anion channels seems to be determined by the presence and constellation of polar side chains that act in concert with the two pore-occluding phenylalanines.

The guard cell osmotic motor driving stomatal closure uses nitrate as the signal to open the major anion channel SLAC1

Date palm (Phoenix dactylifera) stomata characteristics. (a) Two‐year‐old date palms grow three to five pinnate leaves. (b) Electron micrograph of the upper leaf surface. Stomata are arranged in rows, as in grasses. The epidermis is covered with epicuticular waxes. (c, d) Laser scanning images of the date palm leaf surface stained with propidium iodide (excitation at 458 nm, fluorescence recorded at wavelengths of 490–550 nm). (d) Stomata are surrounded by lateral and polar subsidiary cells (highlighted in green in the right image, guard cells are coloured yellow). These two pairs of cells very probably produce the waxy stomatal chimney.

The desert plant Phoenix dactylifera closes stomata via nitrate‐regulated SLAC1 anion channel

by Müller H. M., Schäfer N., Bauer H., Geiger D., Lautner S., Fromm J., Riederer M., Bueno A., Nussbaumer T., Mayer K., Alquraishi S. A., Alfarhan A. H., Neher E., Al‐Rasheid K. A. S., Ache P., Hedrich R. (2017)

Heike M. Müller, Nadine Schäfer, Hubert Bauer, Dietmar Geiger, Silke Lautner, Jörg Fromm, Markus Riederer, Amauri Bueno, Thomas Nussbaumer, Klaus Mayer, Saleh A. Alquraishi, Ahmed H. Alfarhan, Erwin Neher, Khaled A. S. Al‐Rasheid, Peter Ache, Rainer Hedrich,


In New Phytol. 216(1): 150-162 –

Date palm (Phoenix dactylifera) stomata morphology. (a, b) Raster electron micrograph from a stoma on the lower leaf surface. Stomata are surrounded by a huge epicuticular wax chimney, thereby increasing the thickness of the boundary layer above the stomatal pore and, consequently, the total resistance to stomatal transpiration. The wax seems to emerge from the cells neighbouring the guard cells. (b, lower image) Magnification of a stomatal wax chimney.


Subcellular components of date palm (Phoenix dactylifera) stomata, transmission electron microscopy (TEM) cross‐sections. (a) Overview of a stomatal complex with guard cells (gc), subsidiary cells (sc) and neighbouring epidermal cell. The pair of guard cells is clearly separated from epidermal cells by subsidiary cells. Subsidiary cells possess large central vacuoles (v) and mitochondria (m). Guard cells contain a large nucleus (n). (b) Detailed view of the guard cell pair. The guard cells do not share plasmodesmatal connections with the subsidiary cells, and so are symplastically isolated. Guard cells exhibit large vacuoles (v), numerous mitochondria (m) and few chloroplasts (chl). (c) Detailed view of a guard cell chloroplast containing massive starch grains (black spots). (d) Mesophyll cell showing large chloroplasts (chl) with stroma and grana thylakoids in close contact with numerous mitochondria (m), indicating high photosynthetic activity.
  • Date palm Phoenix dactylifera is a desert crop well adapted to survive and produce fruits under extreme drought and heat. How are palms under such harsh environmental conditions able to limit transpirational water loss?
  • Here, we analysed the cuticular waxes, stomata structure and function, and molecular biology of guard cells from P. dactylifera.
  • To understand the stomatal response to the water stress phytohormone of the desert plant, we cloned the major elements necessary for guard cell fast abscisic acid (ABA) signalling and reconstituted this ABA signalosome in Xenopus oocytes. The PhoenixSLAC1‐type anion channel is regulated by ABA kinase PdOST1. Energy‐dispersive X‐ray analysis (EDXA) demonstrated that date palm guard cells release chloride during stomatal closure. However, in Cl medium, PdOST1 did not activate the desert plant anion channel PdSLAC1 per se. Only when nitrate was present at the extracellular face of the anion channel did the OST1‐gated PdSLAC1 open, thus enabling chloride release. In the presence of nitrate, ABA enhanced and accelerated stomatal closure.
  • Our findings indicate that, in date palm, the guard cell osmotic motor driving stomatal closure uses nitrate as the signal to open the major anion channel SLAC1. This initiates guard cell depolarization and the release of anions together with potassium.

ABA as a central regulator and integrator of long-term changes in stomatal behaviour

The role of Arabidopsis ABA receptors from the PYR/PYL/RCAR family in stomatal acclimation and closure signal integration

by Dittrich M., Mueller H. M., Bauer H., Peirats-Llobet M., Rodriguez P. L., Geilfus C.-M., Carpentier S. C., Al Rasheid K. A. S., Kollist H., Merilo E., Herrmann J., Müller T., Ache P., Hetherington A. M., Hedrich R. (2019)

Marcus DittrichHeike M. MuellerHubert BauerMarta Peirats-LlobetPedro L. RodriguezChristoph-Martin GeilfusSebastien Christian CarpentierKhaled A. S. Al RasheidHannes KollistEbe MeriloJohannes HerrmannTobias MüllerPeter AcheAlistair M. HetheringtonRainer Hedrich,

In Nature Plants (2019) –


Stomata are microscopic pores found on the surfaces of leaves that act to control CO2 uptake and water loss. By integrating information derived from endogenous signals with cues from the surrounding environment, the guard cells, which surround the pore, ‘set’ the stomatal aperture to suit the prevailing conditions. Much research has concentrated on understanding the rapid intracellular changes that result in immediate changes to the stomatal aperture. In this study, we look instead at how stomata acclimate to longer timescale variations in their environment. We show that the closure-inducing signals abscisic acid (ABA), increased CO2, decreased relative air humidity and darkness each access a unique gene network made up of clusters (or modules) of common cellular processes. However, within these networks some gene clusters are shared amongst all four stimuli. All stimuli modulate the expression of members of the PYR/PYL/RCAR family of ABA receptors. However, they are modulated differentially in a stimulus-specific manner. Of the six members of the PYR/PYL/RCAR family expressed in guard cells, PYL2 is sufficient for guard cell ABA-induced responses, whereas in the responses to CO2, PYL4 and PYL5 are essential. Overall, our work shows the importance of ABA as a central regulator and integrator of long-term changes in stomatal behaviour, including sensitivity, elicited by external signals. Understanding this architecture may aid in breeding crops with improved water and nutrient efficiency.

The osmotic motor that drives stomatal movement

Exploring biophysical and biochemical components of the osmotic motor that drives stomatal movement

by Raschke K., Hedrich R., Reckmann U., Schroeder J. I. (1988)

In Botanica Acta 101: 283-294 –

Ca2+ signals are likely to activate CPKs, which enhance the activity of S‐type anion channels and boost stomatal closure

Ca2+ signals in guard cells enhance the efficiency by which ABA triggers stomatal closure

by Huang S., Waadt R., Nuhkat M., Kollist H., Hedrich R., Roelfsema M. R. G. (2019)

Shouguang Huang, Rainer Waadt, Maris Nuhkat, Hannes Kollist,Rainer Hedrich, M. Rob G. Roelfsema,

Molecular Plant Physiology and Biophysics Julius‐von‐Sachs Institute for Biosciences Biocenter, Würzburg University, Julius‐von‐Sachs‐Platz 2, D‐97082 Würzburg, Germany


In New Phytologist


During drought, abscisic acid (ABA) induces closure of stomata via a signaling pathway that involves the Ca2+‐independent protein kinase OST1, as well as Ca2+‐dependent protein kinases (CPKs). 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 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, while 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 CPKs, which enhance the activity of S‐type anion channels and boost stomatal closure.

Optimization of photosynthesis and stomatal conductance during acclimation to heat and drought

Optimization of photosynthesis and stomatal conductance in the date palm Phoenix dactylifera during acclimation to heat and drought

by Kruse J., Adams M., Winkler B., Ghirardo A., Alfarraj S., Kreuzwieser J., Hedrich R., Schnitzler J.-P., Rennenberg H. (2019)

Jörg Kruse, Mark Adams, Barbro Winkler, Andrea Ghirardo, Saleh Alfarraj, Jürgen Kreuzwieser, Rainer Hedrich, Jörg‐Peter Schnitzler, Heinz Rennenberg,

Institute of Forest Sciences, Chair of Tree Physiology, University of Freiburg, Georges‐Köhler‐Allee 53/54, 79110 Freiburg, Germany


In New Phytologist


We studied acclimation of leaf gas exchange to differing seasonal climate and soil water availability in slow‐ growing date palm seedlings (Phoenix dactylifera). We used an extended Arrhenius‐equation to describe instantaneous temperature responses of leaf net photosynthesis (A) and stomatal conductance (G), and derived physiological parameters suitable for characterization of acclimation (Topt, Aopt and Tequ).

Optimum temperature of A (Topt) ranged between 20 ‐33°C in winter and 28 ‐45°C in summer. Growth temperature (Tgrowth) explained ~50% of the variation in Topt, which additionally depended on leaf water status at the time‐ of‐ measurement. During water‐stress, light ‐ saturated rates of A at Topt (i.e, Aopt) were reduced to 30‐80% of control levels, albeit not limited by CO2‐ supply per se.

Equilibrium temperature (Tequ), around which A/G and substomatal [CO2] are constant, remained tightly coupled with Topt. Our results suggest that acclimatory shifts in Topt and Aopt reflect a balance between maximization of photosynthesis whilst minimizing the risk of metabolic perturbations caused by imbalances in cellular [CO2].

This novel perspective on acclimation of leaf gas exchange is compatible with optimization theory, and might help elucidating other acclimation and growth strategies in species adapted to differing climates.