Mutations in the SLAC1 anion channel slow stomatal opening and severely reduce K + uptake channel activity via enhanced cytosolic [Ca 2+ ] and increased Ca 2+ sensitivity of K + uptake channels (original) (raw)
Guard cell anion channel SLAC1 is regulated by CDPK protein kinases with distinct Ca2+ affinities
Proceedings of the National Academy of Sciences, 2010
In response to drought stress, the phytohormone abscisic acid (ABA) induces stomatal closure. Thereby the stress hormone activates guard cell anion channels in a calcium-dependent, as well as -independent, manner. Open stomata 1 protein kinase (OST1) and ABI1 protein phosphatase (ABA insensitive 1) represent key components of calcium-independent ABA signaling. Recently, the guard cell anion channel SLAC1 was identified. When expressed heterologously SLAC1 remained electrically silent. Upon coexpression with Ca 2+ -independent OST1, however, SLAC1 anion channels appear activated in an ABI1-dependent manner. Mutants lacking distinct calcium-dependent protein kinases (CPKs) appeared impaired in ABA stimulation of guard cell ion channels, too. To study SLAC1 activation via the calcium-dependent ABA pathway, we studied the SLAC1 response to CPKs in the Xenopus laevis oocyte system. Split YFP-based protein-protein interaction assays, using SLAC1 as the bait, identified guard cell expressed CPK21 and 23 as major interacting partners. Upon coexpression of SLAC1 with CPK21 and 23, anion currents document SLAC1 stimulation by these guard cell protein kinases. Ca 2+ -sensitive activation of SLAC1, however, could be assigned to the CPK21 pathway only because CPK23 turned out to be rather Ca 2+ -insensitive. In line with activation by OST1, CPK activation of the guard cell anion channel was suppressed by ABI1. Thus the CPK and OST1 branch of ABA signal transduction in guard cells seem to converge on the level of SLAC1 under the control of the ABI1/ABA-receptor complex.
F1000 - Post-publication peer review of the biomedical literature, 2000
The plant SLAC1 anion channel controls turgor pressure in the aperture-defining guard cells of plant stomata, thereby regulating exchange of water vapor and photosynthetic gases in response to environmental signals such as drought or high levels of carbon dioxide. We determined the crystal structure of a bacterial homolog of SLAC1 at 1.20Å resolution, and we have used structureinspired mutagenesis to analyze the conductance properties of SLAC1 channels. SLAC1 is a symmetric trimer composed from quasi-symmetric subunits, each having ten transmembrane helices arranged from helical hairpin pairs to form a central five-helix transmembrane pore that is gated by an extremely conserved phenylalanine residue. Conformational features suggest a mechanism for control of gating by kinase activation, and electrostatic features of the pore coupled with electrophysiological characteristics suggest that selectivity among different anions is largely a function of the energetic cost of ion dehydration. Users may view, print, copy, download and text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:
Roles of AtTPC1, vacuolar two pore channel 1, in Arabidopsis stomatal closure
Plant & cell physiology, 2010
Abscisic acid (ABA) induces production of reactive oxygen species (ROS) and nitric oxide (NO), elevation of the cytosolic free calcium ion concentration ([Ca 2 + ] cyt ) and cytosolic pH (pH cyt ), and activation of S-type anion channels in guard cells, causing stomatal closure. To investigate whether Arabidopsis Two pore channel 1 ( AtTPC1 ) that encodes the slow vacuolar (SV) channel is involved in stomatal closure, we examined stomatal movements and mobilization of second messengers in the attpc1-2 loss-of-function mutant in response to ABA, methyl jasmonate (MeJA) and Ca 2 + . Both ABA and MeJA elicited production of ROS and NO, [Ca 2 + ] cyt oscillations, cytosolic alkalization and activation of S-type anion channel currents to lead to stomatal closure in the attpc1-2 mutant as well as the wild type. Unlike the wild type, in the attpc1-2 mutant exogenous Ca 2 + neither induced stomatal closure nor activated plasma membrane S-type anion channel currents despite [Ca 2 + ] cyt elevation. These results indicate that AtTPC1 functions in response to external Ca 2 + but not to ABA and MeJA in Arabidopsis guard cells and suggest that AtTPC1 could be involved in priming of plasma membrane S-type anion channels by external Ca 2 + in Arabidopsis guard cells.
PLANT PHYSIOLOGY, 2001
Inward-rectifying potassium (K ϩ in) channels in guard cells have been suggested to provide a pathway for K ϩ uptake into guard cells during stomatal opening. To test the proposed role of guard cell K ϩ in channels in light-induced stomatal opening, transgenic Arabidopsis plants were generated that expressed dominant negative point mutations in the K ϩ in channel subunit KAT1. Patch-clamp analyses with transgenic guard cells from independent lines showed that K ϩ in current magnitudes were reduced by approximately 75% compared with vector-transformed controls at Ϫ180 mV, which resulted in reduction in light-induced stomatal opening by 38% to 45% compared with vector-transformed controls. Analyses of intracellular K ϩ content using both sodium hexanitrocobaltate (III) and elemental x-ray microanalyses showed that light-induced K ϩ uptake was also significantly reduced in guard cells of K ϩ in channel depressor lines. These findings support the model that K ϩ in channels contribute to K ϩ uptake during light-induced stomatal opening. Furthermore, transpirational water loss from leaves was reduced in the K ϩ in channel depressor lines. Comparisons of guard cell K ϩ in current magnitudes among four different transgenic lines with different K ϩ in current magnitudes show the range of activities of K ϩ in channels required for guard cell K ϩ uptake during light-induced stomatal opening.
Plant Physiology
Initiation of stomatal closure by various stimuli requires activation of guard cell plasma membrane anion channels, which are defined as rapid (R)- and slow (S)-type. The single-gene loss-of-function mutants of these proteins are well characterized. However, the impact of suppressing both the S- and R-type channels has not been studied. Here, by generating and studying double and triple Arabidopsis thaliana mutants of SLOW ANION CHANNEL1 (SLAC1), SLAC1 HOMOLOG3 (SLAH3), and ALUMINUM-ACTIVATED MALATE TRANSPORTER 12/QUICK-ACTIVATING ANION CHANNEL 1 (QUAC1), we show that impairment of R- and S-type channels gradually increased whole-plant steady-state stomatal conductance. Ozone-induced cell death also increased gradually in higher-order mutants with the highest levels observed in the quac1 slac1 slah3 triple mutant. Strikingly, while single mutants retained stomatal responsiveness to abscisic acid, darkness, reduced air humidity, and elevated CO2, the double mutant lacking SLAC1 and Q...
THE PLANT CELL ONLINE, 1997
Inward-rectifying K+ (K+in) channels in the guard cell plasma membrane have been suggested to function as a major pathway for K+ influx into guard cells during stomatal opening. When K+, channels were blocked with external Cs+ in wild-type Arabidopsis guard cells, light-induced stomatal opening was reduced. Transgenic Arabidopsis plants were generated that expressed a mutant of the guard cell K+, channel, KATl, which shows enhanced resistance to the Cs+ block. Stomata in these transgenic lines opened in the presence of external Cs+. Patch-clamp experiments with transgenic guard cells showed that inward K+i, currents were blocked less by Cs+ than were K+ currents in controls. These data provide direct evidence that KATl functions as a plasma membrane K+ channel in vivo and that K+, channels constitute an important mechanism for light-induced stomatal opening. In addition, biophysical properties of K+, channels in guard cells indicate that components in addition to KATl may contribute to the formation of K+in channels in vivo.
PLANT PHYSIOLOGY, 2012
Stomata account for much of the 70% of global water usage associated with agriculture and have a profound impact on the water and carbon cycles of the world. Stomata have long been modeled mathematically, but until now, no systems analysis of a plant cell has yielded detail sufficient to guide phenotypic and mutational analysis. Here, we demonstrate the predictive power of a systems dynamic model in Arabidopsis (Arabidopsis thaliana) to explain the paradoxical suppression of channels that facilitate K + uptake, slowing stomatal opening, by mutation of the SLAC1 anion channel, which mediates solute loss for closure. The model showed how anion accumulation in the mutant suppressed the H + load on the cytosol and promoted Ca 2+ influx to elevate cytosolic pH (pH i ) and free cytosolic Ca 2+ concentration ([Ca 2+ ] i ), in turn regulating the K + channels. We have confirmed these predictions, measuring pH i and [Ca 2+ ] i in vivo, and report that experimental manipulation of pH i and [Ca 2+ ] i is sufficient to recover K + channel activities and accelerate stomatal opening in the slac1 mutant. Thus, we uncover a previously unrecognized signaling network that ameliorates the effects of the slac1 mutant on transpiration by regulating the K + channels. Additionally, these findings underscore the importance of H + -coupled anion transport for pH i homeostasis.
Proceedings of the National Academy of Sciences, 2009
In response to drought stress the phytohormone ABA (abscisic acid) induces stomatal closure and, therein, activates guard cell anion channels in a calcium-dependent as well as-independent manner. Two key components of the ABA signaling pathway are the protein kinase OST1 (open stomata 1) and the protein phosphatase ABI1 (ABA insensitive 1). The recently identified guard cell anion channel SLAC1 appeared to be the key ion channel in this signaling pathway but remained electrically silent when expressed heterologously. Using split YFP assays, we identified OST1 as an interaction partner of SLAC1 and ABI1. Upon coexpression of SLAC1 with OST1 in Xenopus oocytes, SLAC1-related anion currents appeared similar to those observed in guard cells. Integration of ABI1 into the SLAC1/OST1 complex, however, prevented SLAC1 activation. Our studies demonstrate that SLAC1 represents the slow, deactivating, weak voltage-dependent anion channel of guard cells controlled by phosphorylation/dephosphorylation. ABA signaling ͉ S-type anion channel ͉ OST1/ABI1
The Plant Cell, 2020
Upon recognition of microbes, pattern recognition receptors (PRRs) activate pattern-triggered immunity. FLAGELLIN SENSING2 (FLS2) and BRASSINOSTEROID INSENSITIVE1-ASSOCIATED KINASE1 (BAK1) form a typical PRR complex that senses bacteria. Here, we report that the kinase activity of the malectin-like receptor-like kinase STRESS INDUCED FACTOR 2 (SIF2) is critical for Arabidopsis (Arabidopsis thaliana) resistance to bacteria by regulating stomatal immunity. SIF2 physically associates with the FLS2-BAK1 PRR complex and interacts with and phosphorylates the guard cell SLOW ANION CHANNEL1 (SLAC1), which is necessary for abscisic acid (ABA)-mediated stomatal closure. SIF2 is also required for the activation of ABA-induced S-type anion currents in Arabidopsis protoplasts, and SIF2 is sufficient to activate SLAC1 anion channels in Xenopus oocytes. SIF2-mediated activation of SLAC1 depends on specific phosphorylation of Ser 65. This work reveals that SIF2 functions between the FLS2-BAK1 initial immunity receptor complex and the final actuator SLAC1 in stomatal immunity.
Plant Physiology, 2013
Cytosolic Ca 2+ in guard cells plays an important role in stomatal movement responses to environmental stimuli. These cytosolic Ca 2+ increases result from Ca 2+ influx through Ca 2+-permeable channels in the plasma membrane and Ca 2+ release from intracellular organelles in guard cells. However, the genes encoding defined plasma membrane Ca 2+-permeable channel activity remain unknown in guard cells and, with some exceptions, largely unknown in higher plant cells. Here, we report the identification of two Arabidopsis (Arabidopsis thaliana) cation channel genes, CNGC5 and CNGC6, that are highly expressed in guard cells. Cytosolic application of cyclic GMP (cGMP) and extracellularly applied membrane-permeable 8-Bromoguanosine 39,59-cyclic monophosphate-cGMP both activated hyperpolarization-induced inward-conducting currents in wild-type guard cells using Mg 2+ as the main charge carrier. The cGMP-activated currents were strongly blocked by lanthanum and gadolinium and also conducted Ba 2+ , Ca 2+ , and Na + ions. cngc5 cngc6 double mutant guard cells exhibited dramatically impaired cGMP-activated currents. In contrast, mutations in CNGC1, CNGC2, and CNGC20 did not disrupt these cGMP-activated currents. The yellow fluorescent protein-CNGC5 and yellow fluorescent protein-CNGC6 proteins localize in the cell periphery. Cyclic AMP activated modest inward currents in both wild-type and cngc5cngc6 mutant guard cells. Moreover, cngc5 cngc6 double mutant guard cells exhibited functional abscisic acid (ABA)-activated hyperpolarization-dependent Ca 2+-permeable cation channel currents, intact ABA-induced stomatal closing responses, and whole-plant stomatal conductance responses to darkness and changes in CO 2 concentration. Furthermore, cGMPactivated currents remained intact in the growth controlled by abscisic acid2 and abscisic acid insensitive1 mutants. This research demonstrates that the CNGC5 and CNGC6 genes encode unique cGMP-activated nonselective Ca 2+-permeable cation channels in the plasma membrane of Arabidopsis guard cells. Plants lose water via transpiration and take in CO 2 for photosynthesis through stomatal pores. Each stomatal pore is surrounded by two guard cells, and stomatal movements are driven by the change of turgor pressure in guard cells. The intracellular second messenger Ca 2+ functions in guard cell signal transduction (Schroeder