Antibodies to the CFTR modulate the turgor pressure of guard cell protoplasts via slow anion channels (original) (raw)
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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...
New Phytologist
Guard cells control the opening of stomatal pores in the leaf surface, with the use of a network of protein kinases and phosphatases. Loss-of-function of the CBLinteracting protein kinase 23 (CIPK23) was previously shown to decrease the stomatal conductance, but the molecular mechanisms underlying this response still need to be clarified. CIPK23 was specifically expressed in Arabidopsis guard cells, using an estrogeninducible system. Stomatal movements were linked to changes in ion channel activity, determined with double-barreled intracellular electrodes in guard cells and with the two-electrode voltage clamp technique in Xenopus oocytes. Expression of the phosphomimetic variant CIPK23 T190D enhanced stomatal opening, while the natural CIPK23 and a kinase inactive CIPK23 K60N variant did not affect stomatal movements. Overexpression of CIPK23 T190D repressed the activity of S-type anion channels, while their steady state activity was unchanged by CIPK23 and CIPK23 K60N. We suggest that CIPK23 enhances the stomatal conductance at favorable growth conditions, via the regulation of several ion transport proteins in guard cells. The inhibition of SLAC1-type anion channels is an important facet of this response.
SLAC1 is required for plant guard cell S-type anion channel function in stomatal signalling
Nature, 2008
Stomatal pores, formed by two surrounding guard cells in the epidermis of plant leaves, allow influx of atmospheric carbon dioxide in exchange for transpirational water loss. Stomata also restrict the entry of ozone -an important air pollutant that has an increasingly negative impact on crop yields, and thus global carbon fixation 1 and climate change 2 . The aperture of stomatal pores is regulated by the transport of osmotically active ions and metabolites across guard cell membranes 3,4 . Despite the vital role of guard cells in controlling plant water loss 3,4 , ozone sensitivity 1,2 and CO 2 supply 2,5-7 , the genes encoding some of the main regulators of stomatal movements remain unknown. It has been proposed that guard cell anion channels function as important regulators of stomatal closure and are essential in mediating stomatal responses to physiological and stress stimuli 3,4,8 . However, the genes encoding membrane proteins that mediate guard cell anion efflux have not yet been identified. Here we report the mapping and characterization of an ozone-sensitive Arabidopsis thaliana mutant, slac1. We show that SLAC1 (SLOW ANION CHANNEL-ASSOCIATED 1) is preferentially expressed in guard cells and encodes a distant homologue of fungal and bacterial dicarboxylate/malic acid transport proteins. The plasma membrane protein SLAC1 is essential for stomatal closure in response to CO 2 , abscisic acid, ozone, light/dark transitions, humidity change, calcium ions, hydrogen peroxide and nitric oxide. Mutations in SLAC1 impair slow (S-type) anion channel currents that are activated by cytosolic Ca 21 and abscisic acid, but do not affect rapid (R-type) anion channel currents or Ca 21 channel function. A low homology of SLAC1 to bacterial and fungal organic acid transport proteins, and the permeability of S-type anion channels to malate 9 suggest a vital role for SLAC1 in the function of S-type anion channels.
Elicitor-induced chloride efflux and anion channels in tobacco cell suspensions
Plant Physiology and Biochemistry, 1998
Membrane depolarization associated with changes in transmembrane cation and anion effluxes constitute early events in the signalling cascades which mediate elicitor-induced defense reactions in plant cells. The aim of the present study is to investigate whether membrane depolarization can be a key step in the signalling cascade by triggering the opening of voltagedependent anion channels which would mediate anion effluxes. To address this question, tobacco (Nicotiana tabacum cv. Xanthi) cells were subjected to three elicitors (pectolyase, oligogalacturonides and cryptogein) and four other depolarizing agents (vanadate, azide, cyanide and CCCP) and their plasma membrane potential was monitored. In parallel, chloride effluxes induced by these treatments were quantified. Membrane depolarizations induced by the three elicitors were of similar extent, but the intensity of elicitor-induced chloride effluxes was markedly different, pectolyase being the most potent inducer and oligogalacturonides the less active ones. The tested depolarizing agents were all as active as elicitors in terms of membrane depolarization, but again displayed very different efficiencies in the induction of chloride effluxes. The sensitivity of chloride effluxes induced by pectolyase and vanadate to several anion channel blockers was studied. The following sequence of efficiency was found: NPPB (nitrophenyl-propylamino-benzoic-acid) > 9-AC (anthracene-9-carboxylic acid) > DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid) > ETH (ethacrinic acid). The sole anion channel described up to now in tobacco cells is the plasma membrane channel TSAC (tobacco suspension anion channel). Reinvestigations of its properties in the light of the results described above suggest that this depolarization-activated channel may be a candidate to play a role in elicitation and signalling processes accompanied by membrane depolarizations. 0 Elsevier, Paris Anion channel I anion efflux I elicitor / membrane potential I Nicotiana tabacum 9-AC, anthracene-9-carboxylic acid / CCCP, carbonyl cyanide m-chlorophenylhydrazone / DIDS, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid / ETH. ethacrinic acid / NPPB, S-nitro-2,3-phenylpropylaminobenzoic acid / OGs, oligogalacturonides / PL, pectolyase Plant Physiol. Biochem., 0981-9428/98/09/O Elsevier, Paris
Pfl�gers Archiv European Journal of Physiology, 1998
More electrophysiological studies have been carried out on guard cells than on any other cell type of vascular plants. The characterization of their ion channels has been achieved using mainly the whole-cell patch-clamp technique applied to guard-cell protoplasts. The aim of this study was to obtain recordings of ion channel currents in intact guard cells and especially of slow anion channels of Arabidopsis thaliana, a species of fundamental genetic interest. Application of the discontinuous single-electrode voltage-clamp technique enabled the first characterization of K + currents in Commelina communis and of slow anion currents in C. communis and A. thaliana in intact guard cells to be made. Inward K + channels from A. thaliana were inhibited by external application of tetraethylammonium (TEA) or Ca 2+ . In the presence of K + channel blockers, slow anion channel currents were elicited in almost all guard cells tested and were confirmed by the application of anion channel blockers. In A. thaliana, only anthracene-9 carboxylic acid was able to inhibit slow anion currents, to promote stomatal opening in the dark and to reverse the effect of 25 µM abscisic acid under light. Use of a single microelectrode and preservation of cell integrity make this technique well suited for the study of ion channel regulation in species that have guard cell protoplasts with which it is difficult to form good seals.
Co-ordination of signalling elements in guard cell ion channel control
Journal of Experimental Botany, 1998
ably important for providing a plasticity of cellular response to external and environmental stimuli. Fine regulation of solutes transport across the guard Understanding the interdependence and hierarchy of cell plasma membrane for osmotic modulation is signalling elements now presents a major challenge essential for the maintenance of the proper stomatal for research in plant biology. aperture in response to environmental stimuli. The major osmotica, K+, Cl− and malate are transported Key words: Ion channels, stomatal aperture, signalling through selective ion channels in the plasma mempathway, second messengers, guard cells. brane and tonoplast of guard cells. To date, a number of ion channels have been shown to operate in the
The Plant Journal, 1994
Plasma membrane ion channels of protoplasts from tobacco cell suspensions were characterized by patchclamp experiments. In the whole-cell configuration, a voltage-dependent current with a current maximum around -90 mV was observed that displayed a reversal potential close to the Nernst potential for chloride. This whole-cell current was identified as an anion current by replacing the internal Cl-with glutamate. The tobacco _suspension anion channel (TSAC) was characterized by fast activation/deactivation and slow inactivation kinetics with voltage-dependent time constants in the range of milliseconds and seconds, respectively. Among the plant channels, TSAC exhibits original properties in terms of phosphorylation-dependent voltage regulation while sharing similarities with the fast anion channel from atomatal guard cells (GCACl). The voltage dependence of the whole-cell current reflecting the fast deactivation of the current at potentials of less than -100 mV was observed only in the presence of internal ATP or when ATP was replaced by the protein phosphatase inhibitor okadaic acid, and was suppressed by staurosporine. This suggests that protein phosphorylation may be involved in regulating the activity of the anion channel. As observed on GCACl the active auxin 1-NAA caused a time-and concentration-dependent shift of the activation potential of TSAC. In addition, TSAC reacted to the auxin agonist antibody D16 providing evidence for the recognition of the auxin signal at the outer face of the plasma membrane.
The Plant Journal, 2010
Stomatal pores formed by a pair of guard cells in the leaf epidermis control gas exchange and transpirational water loss. Stomatal closure is mediated by the release of potassium and anions from guard cells. Anion efflux from guard cells involves slow (S-type) and rapid (R-type) anion channels. Recently the SLAC1 gene has been shown to encode the slow, voltage-independent anion channel component in guard cells. In contrast, the R-type channel still awaits identification. Here, we show that AtALMT12, a member of the aluminum activated malate transporter family in Arabidopsis, represents a guard cell R-type anion channel. AtALMT12 is highly expressed in guard cells and is targeted to the plasma membrane. Plants lacking AtALMT12 are impaired in dark-and CO 2 -induced stomatal closure, as well as in response to the drought-stress hormone abscisic acid. Patch-clamp studies on guard cell protoplasts isolated from atalmt12 mutants revealed reduced R-type currents compared with wild-type plants when malate is present in the bath media. Following expression of AtALMT12 in Xenopus oocytes, voltage-dependent anion currents reminiscent to R-type channels could be activated. In line with the features of the R-type channel, the activity of heterologously expressed AtALMT12 depends on extracellular malate. Thereby this key metabolite and osmolite of guard cells shifts the threshold for voltage activation of AtALMT12 towards more hyperpolarized potentials. R-Type channels, like voltagedependent cation channels in nerve cells, are capable of transiently depolarizing guard cells, and thus could trigger membrane potential oscillations, action potentials and initiate long-term anion and K + efflux via SLAC1 and GORK, respectively.
Proceedings of the National Academy of Sciences, 1995
Abscisic acid (ABA) modulates the activities of three major classes of ion channels-inwardand outwardrectifying K+ channels (IK,i and IK,out, respectively) and anion channels-at the guard-cell plasma membrane to achieve a net efflux of osmotica and stomatal closure. Disruption of ABA sensitivity in wilty abil-1 mutants ofArabidopsis and evidence that this gene encodes a protein phosphatase suggest that protein (de-)phosphorylation contributes to guard-cell transport control by ABA. To pinpoint the role of ABIl, the abil-1 dominant mutant allele was stably transformed into Nicotiana benthamiana and its influence on IK,ing, IK,out, and the anion channels was monitored in guard cells under voltage clamp. Compared with guard cells from wild-type and vectortransformed control plants, expression of the abil-l gene was associated with 2to 6-fold reductions in IK,out and an insensitivity of both hc,in and IK,out to 20 ,uM ABA. In contrast, no differences between control and abil-1 transgenic plants were observed in the anion current or its response to ABA. Parallel
Cloning and Functional Expression of a Plant Voltage-Dependent Chloride Channel
THE PLANT CELL ONLINE, 1996
Plant cell membrane anion channels participate in basic physiological functions, such as cell volume regulation and signal transductlon. However, nothing is known about their molecular structum. Using a polymerase chain reaction strategy, we have cloned a tobacco cDNA (CICNtl) encoding a 780-amino acid protein with severa1 putative transmembrane domains. CIC-Ntl displays 24 to 32% amlno acid identity with members of the animal voltage-dependent chloride channel (CIC) family, whose archetype is CIC-O from the Torpedo marmorata electric organ. lnjection of ClCNtl complementary RNA into Xenopus oocytes elicited slowly activating inward currents upon membrane hyperpolarization more negative than -120 mV. These currents were carried malnly by anions, modulated by extracellular anions, and totally blocked by 10 mM extracellular calcium. The identification of CICNtl extends the CIC family to higher plants and provides a molecular probe for the study of voltage-dependent anion channels in plants.