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Guard Cell Inward K+ Channel Activity inArabidopsis Involves Expression of the Twin Channel Subunits KAT1 and KAT2

2001; Elsevier BV; Volume: 276; Issue: 5 Linguagem: Inglês

10.1074/jbc.m007303200

1083-351X

Guillaume Pilot, Benoı̂t Lacombe, Frédéric Gaymard, Isabelle Chérel, Jossia Boucherez, Jean‐Baptiste Thibaud, Hervé Sentenac,

Plant Reproductive Biology

Stomatal opening, which controls gas exchanges between plants and the atmosphere, results from an increase in turgor of the two guard cells that surround the pore of the stoma. KAT1 was the only inward K+ channel shown to be expressed in Arabidopsis guard cells, where it was proposed to mediate a K+ influx that enables stomatal opening. We report that another Arabidopsis K+channel, KAT2, is expressed in guard cells. More than KAT1, KAT2 displays functional features resembling those of native inward K+ channels in guard cells. Coexpression inXenopus oocytes and two-hybrid experiments indicated that KAT1 and KAT2 can form heteromultimeric channels. The data indicate that KAT2 plays a crucial role in the stomatal opening machinery. Stomatal opening, which controls gas exchanges between plants and the atmosphere, results from an increase in turgor of the two guard cells that surround the pore of the stoma. KAT1 was the only inward K+ channel shown to be expressed in Arabidopsis guard cells, where it was proposed to mediate a K+ influx that enables stomatal opening. We report that another Arabidopsis K+channel, KAT2, is expressed in guard cells. More than KAT1, KAT2 displays functional features resembling those of native inward K+ channels in guard cells. Coexpression inXenopus oocytes and two-hybrid experiments indicated that KAT1 and KAT2 can form heteromultimeric channels. The data indicate that KAT2 plays a crucial role in the stomatal opening machinery. inwardly rectifying K+ channel rapid amplification of cDNA ends polymerase chain reaction E. coli β-glucuronidase gene pore-forming domain domain rich in hydrophobic and acidic residues 4-morpholineethanesulfonic acid The epidermis of the aerial organs of plants presents a waxy cuticle that prevents water loss, but impedes direct access of the photosynthesizing tissues to atmospheric CO2. Pores in the epidermis, called stomata, allow atmospheric CO2 to enter the plant for photosynthesis. By providing an access to the outer atmosphere, they also allow transpiration, i.e. controlled diffusion of H2O vapor from the plant into the atmosphere, which is a driving force for the ascent of crude sap from the roots to the shoots. Regulation of the stomatal aperture allows the plant to tune and optimize uptake of CO2 and transpiration under diverse environmental conditions. Stomatal movements result from changes in turgor of the two guard cells surrounding the pore, an increase in turgor resulting in increased opening of the stomatal pore. The changes in turgor involve ion transport from and into the guard cells through K+ and anion channels. These channels are the targets of complex transduction pathways that allow the plant to regulate stomatal opening (1MacRobbie E.A.C. Philos. Trans. R. Soc. Lond. B Biol. Sci. 1998; 353: 1475-1488Crossref PubMed Scopus (230) Google Scholar). In this context, the molecular identification of guard cell ion channels was an early challenge when plant channels began to be cloned. By chance, one of the first cloned plant K+channels, KAT1 (2Anderson J.A. Huprikar S.S. Kochian L.V. Lucas W.J. Gaber R.F. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3736-3740Crossref PubMed Scopus (557) Google Scholar), was rapidly demonstrated to be endowed with functional properties compatible with a role in mediating K+ influx (3Schachtman D.P. Schroeder J.I. Lucas W.J. Anderson J.A. Gaber R.F. Science. 1992; 258: 1654-1658Crossref PubMed Scopus (366) Google Scholar) and to be expressed in guard cells (4Nakamura R.L. McKendree Jr., W.L. Hirsch R.E. Sedbrook J.C. Gaber R.F. Sussman M.R. Plant Physiol. 1995; 109: 371-374Crossref PubMed Scopus (211) Google Scholar), opening the way to molecular approaches. By expressing a KAT1 Cs+-resistant mutant channel in transgenicArabidopsis, evidence has been obtained that this channel functions as an inward channel (Kinchannel)1 in the guard cell plasma membrane and plays a role in stomatal opening (5Ichida A.M. Pei Z.M. Baizabal-Aguirre V.M. Turner K.J. Schroeder J.I. Plant Cell. 1997; 9: 1843-1857PubMed Google Scholar). Biochemical approaches have revealed that Ca2+-dependent protein kinases phosphorylate the KAT1 protein (6Li J. Lee Y.R. Assmann S.M. Plant Physiol. 1998; 116: 785-795Crossref PubMed Scopus (156) Google Scholar). A homolog of the animal K+ channel regulatory β-subunits has been identified and shown to interact with KAT1 (7Tang H. Vasconcelos A.C. Berkowitz G.A. Plant Cell. 1996; 8: 1545-1553PubMed Google Scholar). On the other hand, two reports (5Ichida A.M. Pei Z.M. Baizabal-Aguirre V.M. Turner K.J. Schroeder J.I. Plant Cell. 1997; 9: 1843-1857PubMed Google Scholar, 8Brüggemann L. Dietrich P. Dreyer I. Hedrich R. Planta. 1999; 207: 370-376Crossref PubMed Scopus (35) Google Scholar) pointed out differences in sensitivity to external pH and to the channel blocker Cs+between the current mediated by KAT1 after heterologous expression and the current in situ. Two nonexclusive hypotheses can be put forward to explain these differences. First, the differences could be due to the fact that information obtained with heterologous systems gives a distorted view of the functional properties in planta because of, for example, lack of interactions with endogenous proteins and/or artifactual interactions with host cell proteins (9Véry A.A. Bosseux C. Gaymard F. Sentenac H. Thibaud J.-B. Pfluegers Arch. Eur. J. Physiol. 1994; 428: 422-424Crossref PubMed Scopus (43) Google Scholar, 10Dreyer I. Antunes S. Hoshi T. Müller-Röber B. Palme K. Pongs O. Reintanz G. Hedrich R. Biophys. J. 1997; 72: 2143-2150Abstract Full Text PDF PubMed Scopus (145) Google Scholar, 11Dreyer I. Horeau C. Lemaillet G. Zimmermann S. Bush D.R. Rodriguez-Navarro A. Schachtman D.P. Spalding E.P. Sentenac H. Gaber R.F. J. Exp. Bot. 1999; 50: 1073-1087Google Scholar). The second hypothesis is that another channel is expressed in guard cells and contributes per se to the inward current by forming homomeric channels and/or in interaction with KAT1 by forming heteromeric channels. We have now identified a second gene expressed in guard cells,KAT2, for which a partial cDNA had been previously cloned (12Butt A.D. Blatt M.R. Ainsworth C.C. J. Plant Physiol. 1997; 150: 652-660Crossref Scopus (17) Google Scholar). Electrophysiological characterization of KAT2 current inXenopus oocytes revealed basic properties very similar to those of the inward current in guard cells. Regarding the sensitivity to external pH and Cs+, KAT2 is more reminiscent of the guard cell Kin channels than KAT1. Like their animal counterparts, plant Shaker channels are tetrameric proteins (13Daram P. Urbach S. Gaymard F. Sentenac H. Chérel I. EMBO J. 1997; 16: 3455-3463Crossref PubMed Scopus (116) Google Scholar). Here we also bring evidence that KAT1 and KAT2 polypeptides can assemble in heterotetramers. The results provide evidence that KAT2 is a major determinant of the inward K+ current through the guard cell membrane, a fact that should stimulate research on stomatal opening mechanisms and their regulation. The 5′-region of KAT2 cDNA (see Fig. 1 C; GenBankTM/EBI accession number AJ288900) was determined by RACE-PCR on poly(A)+ mRNA isolated from 14-day-old plantlet leaves. Amplified fragments corresponding to the 5′-end ofKAT2 cDNA were cloned and sequenced. The full-length KAT2 open reading frame was then isolated by reverse transcription-PCR on poly(A)+ mRNA using a primer hybridizing at the ATG codon and containing a Spe I site (5′-ACTAGTATGTTGAAGAGAAAGCACCTCAACAC-3′) and a reverse primer hybridizing at the stop codon and containing a Not I site (5′-GCGGCCGCTTAAGAGTTTTCATTGATGAGAATATACAAATG-3′). The amplified fragment (2.1 kilobases) was cloned at the Eco RV site of pBluescript and sequenced. Plants were grown in vitro in magenta boxes as described previously (14Gaymard F. Pilot G. Lacombe B. Bouchez D. Bruneau D. Boucherez J. Michaux-Ferriere N. Thibaud J.-B. Sentenac H. Cell. 1998; 94: 647-655Abstract Full Text Full Text PDF PubMed Scopus (573) Google Scholar). Total RNA extraction and Northern blotting were performed as described previously (15Lobréaux S. Massenet O. Briat J.F. Plant Mol. Biol. 1992; 19: 563-575Crossref PubMed Scopus (136) Google Scholar). Specific probes corresponding to KAT1 and KAT2 and used in Northern blot experiments were generated by PCR (cDNA fragments encoding sequences 511–587 and 524–608 of KAT1 and KAT2, respectively). Reverse transcription and PCR were performed with Superscript II (Life Technologies, Inc.) and Extra-Pol I (Eurobio), respectively, following the manufacturers' recommendations. The KAT2 promoter region was isolated from genomic DNA (ecotype Columbia) by PCR walking (16Devic M. Albert S. Delseny M. Roscoe T.J. Plant Physiol. Biochem. 1997; 35: 331-339Google Scholar) with a reverse primer introducing a unique Nco I site just upstream from the ATG codon (5′-CCATGGGGTTAGTTATAAATATAGTGATGAAACTTGTG-3′). A 1.8-kilobase fragment was isolated, cloned into pBluescript, and sequenced. The construct was digested by Bam HI andNco I and introduced into pBI320.X (from Dr. R. Derose; this plasmid bears a unique Nco I site at the initiation codon of the promoterless GUS 3′-nopaline synthase gene), leading to a translational fusion between the KAT2 promoter region and the GUS coding sequence. This construct was digested byBam HI and Sac I and introduced into the pMOG402 binary vector (from Dr. H. Hoekema). The resulting plasmid was introduced into Agrobacterium tumefaciens MP90 (17Höfgen R. Willmitzer L. Nucleic Acids Res. 1988; 20: 9877Crossref Scopus (857) Google Scholar).Arabidopsis thaliana (ecotype Columbia) was transformed with agrobacteria using the floral dip method (18Clough S.J. Bent A.F. Plant J. 1998; 16: 735-743Crossref PubMed Google Scholar). Selection of T1 seedlings was performed in vitro on the half-strength medium described by Murashige and Skoog (19Murashige T. Skoog F. Physiol. Plant. 1962; 15: 473-497Crossref Scopus (53954) Google Scholar) supplemented with 1% sucrose, 0.7% agar, and 50 μg·ml−1 kanamycin under the following conditions: 21/18 °C day/night temperature, 16-h photoperiod, and 150 microeinsteins·m−2·s−1. For GUS assay, plants were either grown in vitro on the same medium and under the same conditions as described above or grown in a greenhouse on attapulgite-peat compost (14Gaymard F. Pilot G. Lacombe B. Bouchez D. Bruneau D. Boucherez J. Michaux-Ferriere N. Thibaud J.-B. Sentenac H. Cell. 1998; 94: 647-655Abstract Full Text Full Text PDF PubMed Scopus (573) Google Scholar). GUS histochemical staining was performed as described previously (20Lagarde D. Basset M. Lepetit M. Conejero G. Gaymard F. Astruc S. Grignon C. Plant J. 1996; 9: 195-203Crossref PubMed Scopus (284) Google Scholar). Cross-sections of GUS-stained material were prepared on hydroxyethyl methacrylate (Technovit 7100, Heraus-Kulzer GmBH, Wehrheim, Germany)-embedded tissues with an Amersham Pharmacia Biotech microtome and were counterstained in purple by periodic acid-Schiff staining. KAT1 and KAT2 cDNAs were introduced into the pCi plasmid (Promega) under the control of the cytomegalovirus promoter. The resulting plasmids, pCi-KAT1 and pCi-KAT2, were injected into Xenopus oocytes (purchased from Centre de Recherches de Biochimie Macromoléculaire, CNRS, Montpellier, France) using a 10–15-μm tip diameter micropipette and a pneumatic injector (10 nl of 1 μg·μl−1plasmid solution/oocyte). Control oocytes were injected with 10 nl of empty plasmid solution. Whole-cell currents were recorded as described previously (21Lacombe B. Thibaud J.-B. J. Membr. Biol. 1998; 166: 91-100Crossref PubMed Scopus (32) Google Scholar) using the two-electrode voltage-clamp technique, 3–7 days after injection, on oocytes continuously perfused with bath solution (see figure legends). Quantitative analyses of macroscopic current that yielded the gating parameters given in Table I were performed as described previously (21Lacombe B. Thibaud J.-B. J. Membr. Biol. 1998; 166: 91-100Crossref PubMed Scopus (32) Google Scholar).Table IComparison of functional properties of KAT1 and KAT2 channelsSelectivityP Rb/P KP Na/P KP Li/P KKAT10.351-aRef. 35.0.0021-aRef. 35.0.00131-aRef. 35.KAT20.29 ± 0.06 (4)<0.002 (4) Rb+ >≫ Na+ ≈ Li+ (Table I). Under our experimental conditions (expression level, size of patch), macroscopic KAT2 currents mimicking whole-oocyte currents could be recorded in the cell-attached patch-clamp configuration (Fig.3 C, trace c.a.). Upon patch excision, however, the KAT2 current decreased rapidly (inside-out configuration) (Fig.3 C, traces i.o.1 and i.o.2). In this configuration, unitary currents could be resolved, which are shown in Fig. 3 D. KAT2 rundown could be overcome and the initial current partly restored by cramming the patch back into the oocyte (Fig. 3 C, trace p.c.). This suggested that KAT2 required intracellular factors, available in the oocyte cytoplasm, to open. From the single-channel recordings obtained in the inside-out configuration at different potentials, we were able to determine the single-channel slope conductance of KAT2: 6.7 picosiemens in symmetrical 100 mm K+ solution (Fig.3 D), a value quite similar to that reported for KAT1 (31Hedrich R. Moran O. Conti F. Busch H. Becker D. Gambale F. Dreyer I. Küch A. Neuwinger K. Palme K. Eur. Biophys. J. 1995; 24: 107-115Crossref PubMed Scopus (84) Google Scholar,32Hoshi T. J. Gen. Physiol. 1995; 105: 309-328Crossref PubMed Scopus (195) Google Scholar). As native guard cell inwardly rectifying K+ channels are known to be stimulated upon external acidification (33Brüggemann L. Dietrich P. Becker D. Dreyer I. Palme K. Hedrich R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3298-3302Crossref PubMed Scopus (63) Google Scholar, 34Roelfsema M.R.G. Prins H.B.A. Planta. 1998; 205: 100-112Crossref PubMed Scopus (31) Google Scholar), we compared the sensitivity of KAT1 and KAT2 to external pH in parallel experiments carried out on oocytes from the same batch (Fig.4). The activation potential of KAT2 was shifted positively when the pH was decreased from 7.5 to 6.0 (Fig.4 B), leading to an increase in current amplitude at a given potential. Analyses of the correspondingG/G max versus potential curves (data not shown) showed that the G maxvalue was not changed and yielded the gating parameters (Table I). Although the apparent gating charge (z g) was not changed, the half-activation potential (E a50) was indeed shifted by approximately +15 mV when the pH was decreased from 7.5 to 6.0. By contrast, and as previously reported (35Véry A.A. Gaymard F. Bosseux C. Sentenac H. Thibaud J.-B. Plant J. 1995; 7: 321-332Crossref PubMed Scopus (149) Google Scholar), KAT1 gating parameters were left unchanged by the pH drop (Fig. 4 A), an increase in macroscopic conductance (G max) being responsible for the increase in current (Table I). Block by external Cs+ is a classical feature of plant and animal K+ channels that is believed to involve the binding of Cs+ to some site within the pore. Pore penetration by Cs+ often results in some voltage dependence of the K+ channel block (36Hille B. Ionic Channels of Excitable Membranes. Sinauer Associates, Inc., Sunderland, MA1992: 272-302Google Scholar). As previously reported (9Véry A.A. Bosseux C. Gaymard F. Sentenac H. Thibaud J.-B. Pfluegers Arch. Eur. J. Physiol. 1994; 428: 422-424Crossref PubMed Scopus (43) Google Scholar, 31Hedrich R. Moran O. Conti F. Busch H. Becker D. Gambale F. Dreyer I. Küch A. Neuwinger K. Palme K. Eur. Biophys. J. 1995; 24: 107-115Crossref PubMed Scopus (84) Google Scholar), addition of 0.5 mm Cs+ to the external medium resulted in a voltage-dependent block of the KAT1 current (Table I). In parallel experiments on the same batch of oocytes, addition of Cs+ resulted in a voltage-independent block of KAT2 inward currents (Fig. 5,A and B; and Table I). The inhibition constant of this block, estimated from the data shown in Fig. 5 B, is 2.5 mm. It is worth noting that the sequences of the pore domains of KAT1 and KAT2 differ by a single amino acid, Phe266 in KAT1 corresponding to Leu in KAT2 (Fig.1 B). In this context, a KAT1-F266L mutant channel (a gift from R. Hedrich, University of Würzburg, Würzburg, Germany), i.e. a KAT1 mutant with the pore sequence of KAT2, was studied. The Cs+ sensitivity of the mutant channel is clearly voltage-dependent and reminiscent of that of KAT1 (Fig. 5 C). Therefore, the pore domain sequence is not the only determinant of KAT1 and KAT2 sensitivity to Cs+. Further pharmacological characterization revealed that KAT2 has a lower sensitivity to external Ba2+than KAT1 and a sensitivity to external tetraethylammonium similar to that of KAT1 (Table I). Single-point mutations in the KAT1 P domain have been recently reported to yield dominant-negative mutants (37Baizabal-Aguirre V.M. Clemens S. Uozumi N. Schroeder J.I. J. Membr. Biol. 1999; 167: 119-125Crossref PubMed Scopus (60) Google Scholar). We introduced a mutation in KAT1 (W253G) that produced electrically silent channels in Xenopus oocytes. Coexpression of this mutant with the wild-type KAT1 channel inXenopus oocytes resulted in a lower inward current than did the expression of the wild-type channel alone (Fig.6 A). Such an effect indicates that the KAT1-W253G mutant has a dominant-negative capability,i.e. that it can interact with the wild-type polypeptide, leading to formation of channels that are not functional or not targeted to the membrane. Interestingly, coexpression of the KAT1-W253G mutant with KAT2 decreased the inward current as well (Fig.6 A), providing evidence that the polypeptides encoded byKAT1 and KAT2 can interact and form heterotetrameric channels. Plant K+ channels have been shown to form tetramers through interactions involving the cytoplasmic C-terminal domain (13Daram P. Urbach S. Gaymard F. Sentenac H. Chérel I. EMBO J. 1997; 16: 3455-3463Crossref PubMed Scopus (116) Google Scholar). We therefore investigated the possibility of interactions between the C-terminal domains of KAT1 and KAT2 using the two-hybrid system in yeast (38Fields S. Song O. Nature. 1989; 340: 245-246Crossref PubMed Scopus (4863) Google Scholar) and obtained positive results (Fig. 6 B). Coexpression of the wild-type KAT1 and KAT2 polypeptides in oocytes resulted in an inward current that activated at a threshold potential between those of KAT1 and KAT2. We failed to find any typical feature of this current (i.e. a feature that would not be reminiscent of KAT1 or KAT2 properties). Interestingly, however, the current monitored in control oocytes injected with 10 ng of either KAT1 or KAT2 plasmid was significantly smaller than that monitored in oocytes coinjected with 5 ng of each plasmid, suggesting some synergy. The physiological significance of this effect cannot be assessed at the present time. However, in the animal field, similar observations due to interaction between channel subunits have been shown to play a rolein situ in the organism (39Krapivinsky G. Gordon E.A. Wickman K. Velimirovic B. Krapivinsky L. Clapham D.E. Nature. 1995; 374: 135-141Crossref PubMed Scopus (758) Google Scholar, 40Duprat F. Lesage F. Guillemare E. Fink M. Hugnot J.P. Bigay J. Lazdunski M. Romey G. Barhanin J. Biochem. Biophys. Res. Commun. 1995; 212: 657-663Crossref PubMed Scopus (133) Google Scholar). KAT1 and KAT2 belong to the Shaker-like family of K+channels (41Zimmermann S. Sentenac H. Curr. Opin. Plant Biol. 1999; 2: 477-482Crossref PubMed Scopus (59) Google Scholar), of which nine members have been identifi