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The Plant Cell, Vol. 8, 2359-2368, December 1996 O 1996 American Society of Plant Physiologists An Abscisic Acid-Activated and Calcium-lndependent Protein Kinase from Guard Cells of Fava Bean Jiaxu Li and Sarah M. Assmann' Department of Biology and Plant Physiology Program, Pennsylvania State University, University Park, Pennsylvania 16802 Abscisic acid (ABA) regulation of stomatal aperture is known to involve both Ca2+-dependentand Ca2+-independentsignal transduction pathways. Electrophysiologicalstudies suggest that protein phosphorylation is involved in ABA action in guard cells. Using biochemical approaches, we identified an ABA-activated and Ca2+-independentprotein kinase (AAPK) from guard cell protoplastsof fava bean. Autophosphorylationof AAPK was rapidly (4min) activated by ABA in a Ca2+independent manner. ABA-activated autophosphorylationof AAPK occurred on serine but not on tyrosine residues and appeared to be guard cell specific. AAPK phosphorylated histone type Ill-S on serine and threonine residues, and its activity toward histone type 111-S was markedly stimulated in ABA-treated guard cell protoplasts. Our results suggest that AAPK may play an important role in the Ca2+-independentABA signaling pathways of guard cells. INTRODUCTION The plant hormone abscisic acid (ABA) regulates many important aspects of plant growth and development, such as embryogenesis, seed dormancy and germination, senescence, water status, and protein synthesis (Zeevaart and Creelman, 1988). ABA is synthesized and redistributedin response to severa1 stresses, including water stress (Hartung and Davies, 1991). When water stress occurs, ABA levels in leaves rise (Harris et al., 1988). lncreasedABA levels reduce stomatalapertures and thus inhibit transpirational water loss (Mittelheuser and van Steveninck, 1969: Weyers and Hillman, 1979). Stomatal aperture is defined by a pair of guard cells and is regulated by osmotic swelling and shrinking of these cells. Stomatal opening occurs after osmotic swelling of guard cells caused by K+ uptake (through inward K+ channels), CI- uptake, and production of organic solutes (Assmann, 1993). Stomatal closure results when anion efflux, Ca2+influx, and/or H+-ATPaseinhibition depolarize the membranepotential, thus providing the driving force for K+ efflux through outward K+ channels (Assmann, 1993). ABA inhibits inward K+ channels and activates outward K+ channels (Blatt, 1990). ABA also inhibits the plasma membrane proton pump, which provides a proton motive force for K+ and CI- uptake (Goh et al., 1996). However, the mechanisms by which ion transport proteins of guard cells are regulated by ABA are incompletelyunderstood. Although there are some links between ABA and cytosolic Ca2+elevation in guard cells (McAinsh et al., 1990, 1992), variable effects of ABA on cytosolic Ca2+ levels have been I To whom correspondence should be addressed observed, leading to the conclusion that ABA signaling can be transduced through either a Ca2+-dependentpathway or a Ca*+-independent pathway (Gilroy et al., 1991; MacRobbie, 1993; Allan et al., 1994). In guard cells, electrophysiological studies indicate that inward K+ channels, outward K+ channels, and anion channels may be modulated by protein phosphorylation and dephosphorylation (Luan et al., 1993; Li et al., 1994; Armstrong et al., 1995; Schmidt et al., 1995). In tobacco plants transformed with abi7-7, a dominant mutant gene of Arabidopsis encoding a putative protein phosphatase (Leung et al., 1994; Meyer et al., 1994), the sensitivity of both inward and outward K+ channels to ABA was reduced, and the sensitivity ot K+ channels to ABA could be restored by protein kinase inhibitors (Armstrong et al., 1995). In fava bean, protein kinase inhibitors abolished both slow anion channel activity of guard cells and ABA-inducedstomatalclosure (Schmidt et al., 1995). These studies suggest that protein kinases play an important role in the regulation of ion channels by ABA; however, to date, no protein kinases have been detected or identified in guard cells. This study was undertaken to identify protein kinases that are involved in ABA signaling pathways in guard cells. Using biochemical approaches, we have identified, in guard cell protoplasts of fava bean, a serinelthreonine protein kinase that is ABA activated and Ca2+independent. Our results imply that this kinase may be involved in Ca2+-independentABA signaling pathways in guard cells. To our knowledge, a plant serinelthreonine kinase whose autophosphorylation and kinase activity both are ABA regulated has not been described previously. 2360 The Plant Cell RESULTS Ca2' ABA Activates a 48-kD Serine/Threonine Protein Kinase from Guard Cell Protoplasts in a Ca2+-lndependent Manner The routine yield of guard cell protoplasts (GCPs) from 30 leaflets of fava bean was ~5 x 106 with 99.9% purity (calculated on a cell basis by counting a sample of ~6000 cells). The purified GCPs could respond to white light by swelling and to ABA by shrinking (data not shown), as reported previously (Fitzsimons and Weyers, 1986,1987), demonstrating that they are competent to respond to these signals. Proteins from GCPs were assayed for autophosphorylation activity by an in-gel assay (see Methods). When the gel was incubated with y-32P-ATP in the presence of 100 \iM free Ca2+, two major bands at 57 and 48 kD were seen in soluble proteins from ABA-treated GCPs. However, only the 57-kD band could be detected in protein samples from GCPs treated with ethanol as a control for the ABA solvent (Figure 1A), indicating that autophosphorylation of the 48-kD protein was induced by ABA. When autophosphorylation was assayed in the presence of 450 nM EGTA, the 57-kD band was no longer detected, but the 48-kD band could still be detected in ABA-treated GCPs (Figure 1B). Similar results were obtained when ABA was dissolved in W-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid buffer (data not shown), indicating that the solvent has no effect on the autophosphorylation reaction. These data indicate that autophosphorylation of the 57-kD protein is Ca2+ dependent and that ABA-induced autophosphorylation of the 48-kD protein is Ca2+ independent. The ABA-induced and Ca2+-independent autophosphorylation of the 48-kD protein was also detected, albeit weakly, in the membrane proteins of ABA-treated GCPs (Figures 1A and 1B). When a-32P-ATP was substituted for y-32P-ATP in the assay buffer, no radioactive bands were observed (Figure 1C), indicating that the radioactive bands were due to phosphorylation of these proteins rather than to binding of ATP The ABA-stimulated autophosphorylation could in theory be due to an increased amount of the 48-kD protein resulting from de novo protein synthesis stimulated by ABA. A cytosolic protein synthesis inhibitor, cycloheximide, was used to test this possibility. When GCPs were pretreated with or without 100 iaM cycloheximide for 3 min and then further treated with 10 nM ABA for 15 min in the presence or absence of cycloheximide, ABA-activated autophosphorylation of the 48-kD protein was not affected (Figure 2). These results indicate that ABAactivated autophosphorylation of the 48-kD protein is not due to an increase in the amount of this protein. General phosphatase inhibitors increased the amount of ABA-dependent autophosphorylation (J. Li and S.M. Assmann, unpublished data), indicating true ABA stimulation of autophosphorylation activity. If phosphatase inhibitors had decreased autophosphorylation, this would have suggested that ABA functioned by stimulating a phosphatase that then increased the ABA kD 200- M - + - + B EGTA Ca2* S ABA - + - + ABA kD kD 200- 200120100 — 80- 188= 80- 12010080- 60- 60- 50- 50- 40- 40- 40- 30- 30- 30- 20- 20- 20- _ -« 6050- Figure 1. Detection of Autophosphorylation Activity of Proteins in ABATreated GCPs. GCPs were treated with ethanol (0.02% final concentration) or 10 |iM (±)-c/s,frans-ABAfor 15 min in darkness. Soluble proteins (S) or membrane proteins (M) from GCPs were separated on 12% SDS-polyacrylamide gels. Autophosphorylation of proteins was detected by autoradiography after in situ renaturation, as described in Methods. Molecular mass markers (Gibco BRL) are shown at left in kilodaltons. The stars and the arrowheads at right indicate the positions of the 57- and the 48-kD autophosphorylating proteins, respectively. (A) Autophosphorylation assay was performed by incubating the gel with y-32P-ATP in the presence of 100 |iM free Ca24. (B) Autophosphorylation assay was performed by incubating the gel with y-32P-ATP in the presence of 450 nM EGTA (C) Autophosphorylation assay was performed by incubating the gel with a-32P-ATP in the presence of 100 jiM free Ca2+. number of unoccupied sites available on the kinase for autophosphorylation by Y-32P-ATP. Autophosphorylation, in some cases, can affect protein kinase activity (Hardie, 1995). To determine whether ABA treatment affected the kinase activity of the 48-kD protein, histone type III-S was used as an exogenous substrate (Harmon et al., 1987). The renatured 48-kD protein band was excised from the gel and incubated in kinase assay buffer containing histone type III-S. The 48-kD protein kinase from untreated GCPs showed low activity toward histone type III-S (Figure 3, lanes 3 and 5). However, the phosphorylation of histone type III-S was markedly enhanced by the 48-kD kinase from ABAtreated GCPs. The enhanced activity of the 48-kD kinase toward histone type III-S was independent of the presence of calcium (Figure 3, lanes 4 and 6). When extracts from blank gel slices were incubated with kinase assay buffer containing histone type III-S in the absence or presence of calcium, no phosphorylation of histone type III-S was detected (Figure 3, lanes 1 and 2). Densitometric scans performed on the autoradiograph shown in Figure 3 indicate that ABA stimulated kinase activity two- to threefold (data not shown). Therefore, these results show that the 48-kD kinase activity toward histone type III-S was indeed markedly increased by ABA treatment. These data also suggest that the autophosphorylation of the 48-kD Abscisic Acid-Activated Protein Kinase kinase activated by ABA is correlated with the enhancement of its kinase activity by ABA. Given this information, we have chosen to refer to this protein kinase as ABA-activated protein kinase (AAPK). To classify the 48-kD protein kinase, we analyzed phosphoamino acids of the autophosphorylated 48-kD kinase and of histone type III-S phosphorylated by AAPK, using twodimensional thin-layer electrophoresis. The 48-kD protein kinase autophosphorylated almost exclusively on serine, although a trace of phosphothreonine was also detected (Figure 4A). Histone type III-S was phosphorylated on both serine and threonine residues by the 48-kD kinase (Figure 4B). No phosphorylation of tyrosine was observed in either case. These results indicate that the 48-kD protein kinase is a serine/threonine protein kinase. ABA-Activated Autophosphorylation of the 48-kD Kinase Is Rapid and Guard Cell Specific To characterize further the effects of ABA on the autophosphorylation of AAPK, we treated GCPs with various concentrations of ABA for different time periods. After 10 min of treatment, autophosphorylation of AAPK activated by ABA occurred at a concentration as low as 1 u-M, with maximum effects reached at a concentration of 10 u.M. ABA concentrations of 10, 25, and 50 nM all resulted in approximately equal levels of autophosphorylation (Figure 5). At 10 u,M ABA, autophosphorylation of AAPK activated by ABA was detected as early as 1 min, with maximal activation occurring by 5 to 10 min (Figure 6). These results show that autophosphorylation of AAPK activated by ABA is rapid and can be achieved by physiological concentrations of ABA. To determine whether the effect of ABA on protein autophosphorylation was restricted to guard cells, we examined the two CHX ABA Ca2+ EGTA Figure 2. ABA-Activated Autophosphorylation of the 48-kD Protein Is Unaffected by Cycloheximide. After preincubation with (+) or without (-) 100 uM cycloheximide (CHX) for 3 min, the CHX samples were kept in CHX-containing solution, and both samples were then treated with ethanol (-) or ABA ( + ) as described in Figure 1. Soluble proteins from GCPs were subjected to the autophosphorylation assay in the presence of 100 uM free Ca2' or 450 ^M EGTA. The stars and arrowheads indicate the positions of the 57- and the 48-kD proteins, respectively. ABA AAPK - - Ca2* - + - 1 2 2361 + Histone -*• 3 4 5 6 Figure 3. ABA Stimulates the Kinase Activity of AAPK in a Ca2'Independent Manner. Proteins from ethanol-treated GCPs (-) or ABA-treated GCPs (+) were electrophoresed and renatured. Extracts from gel slices containing the 48-kD AAPK ( + , lanes 3 to 6) or from blank gel slices (-, lanes 1 and 2) were mixed with kinase assay buffer containing Ca?1 ( + ; 100 nM free Ca 2 ', lanes 2, 5, and 6) or no Ca?1 (-; 450 »iM EGTA. lanes 1. 3, and 4) and assayed for kinase activity by using histone type III-S as an exogenous substrate. Histone type III-S is indicated with an arrow at left. other major cell types of leaves: mesophyll cells and epidermal cells. The purities of mesophyll cell protoplast and epidermal cell protoplast preparations were 99.8 and 97.1% (calculated on a cell basis by counting samples of ~6000 cells), respectively. Ca2+-dependent autophosphorylation of the 57kD kinase was detected in all three of the cell types examined (Figures 7A and 7B). In contrast, ABA-activated autophosphorylation was detected in guard cells but not in mesophyll cells or epidermal cells when the autophosphorylation assay was performed in the presence of 100 u,M free Ca2+ (Figure 7A). The same was true when the autophosphorylation assay was performed in the presence of 450 uM EGTA (Figure 7B). Therefore, ABA-activated autophosphorylation appears to be guard cell specific. ABA Activates Autophosphorylation of the 48-kD Kinase Indirectly To determine the mechanism by which ABA activates autophosphorylation of AAPK, we examined whether ABA activated the autophosphorylation of AAPK directly in GCP extracts. When ABA was added directly to the soluble fraction, soluble plus membrane fractions, or crude extract from GCPs, the autophosphorylation of AAPK was no longer detected (Figure 8). These data indicate that ABA activates the autophosphorylation of AAPK indirectly (see Discussion). Because the activity of AAPK from GCPs was markedly activated by ABA treatment (Figure 3), we next examined whether ABA treatment would increase phosphorylation of certain proteins in GCPs. In three in vitro phosphorylation experiments, there was no difference in the pattern of phosphorylation of soluble proteins between ABA-treated GCPs and untreated GCPs (Figure 9A). In three other in vitro phosphorylation experiments, ABA consistently enhanced phosphorylation of 2362 The Plant Cell P-Ser V. r P-Thr P-Tyr B P-Ser ,-N P-Thr v. J P-Tyr £a Figure 4. AAPK Is a Serine/Threonine Kinase. Hydrolysates of the autophosphorylated AAPK or phosphorylated histone type III-S were mixed with the phosphoamino acid standards (P-Ser, phosphoserine; P-Thr, phosphothreonine; P-Tyr, phosphotyrosine; Sigma) and separated on a cellulose thin-layer plate by electrophoresis at pH 1.9 in the first dimension (1D) and at pH 3.5 in the second dimension (2D). Positions of phosphoamino acid standards visualized by ninhydrin staining are indicated as dashed circles. (A) Phosphoamino acid analysis of the autophosphorylated AAPK. (B) Phosphoamino acid analysis of histone type III-S phosphorylated by AAPK several soluble proteins, for example, the 115- and 90-kD proteins (stars in Figure 9B), and induced phosphorylation of the 49- and 36-kD proteins (arrows in Figure 9B). In all in vitro phosphorylation experiments, ABA had no apparent effect on the phosphorylation of membrane proteins (Figure 9C). DISCUSSION The in situ renaturation technique is a very powerful method for the detection and analysis of protein kinases from crude protein preparations. After SDS-PAGE and in situ renaturation, protein kinases can be detected and assayed either by autophosphorylation or by phosphorylation of exogenous substrates (Hutchcroft et al., 1991). By using this technique, we detected a 48-kD AAPK from guard cell protoplasts of fava bean. Both autophosphorylation of AAPK and its kinase activity toward histone type III-S were Ca2+ independent (Figures 1 and 3). In addition, a polyclonal antibody raised against the soybean Ca2+-dependent protein kinase (CDPK) failed to recognize AAPK (data not shown). Therefore, AAPK does not appear to be a CDPK-like protein kinase. ABA elicits two types of responses in plants (Zeevaart and Creelman, 1988; Hetherington and Quatrano, 1991). The closure of stomata is an example of a rapid response (within minutes). The slow responses appear to involve RNA and protein synthesis (Zeevaart and Creelman, 1988; Hetherington and Quatrano, 1991). Such differences in ABA response kinetics suggest that ABA may have several modes of action and even different receptors in various experimental systems (Zeevaart and Creelman, 1988). Autophosphorylation of AAPK was activated rapidly (within 1 min) by physiological levels of ABA (Figures 5 and 6). The ABA-activated autophosphorylation does not seem to be due to de novo protein synthesis (Figure 2) and appears to be guard cell specific (Figure 7). These characteristics strongly suggest that the AAPK is involved in the rapid alterations in stomatal aperture that occur in response to ABA. In contrast, the near full-length cDNA clone PKABA1 (for protein kinase responsive to ABA) was identified from a cDNA library prepared from wheat embryos treated with ABA for 12 hr (Anderberg and Walker-Simmons, 1992). PKABA1 mRNA was detected in wheat embryos treated with ABA for 48 hr or water stressed for 12 hr. Thus, ABA appears to induce the expression of PKABA1 mRNA. Therefore, AAPK and PKABA1 are likely to be distinct protein kinases. Recently, it has been shown that ABA induces a rapid and transient activation of a 40- to 43-kD mitogen-activated protein (MAP) kinase in barley aleurone protoplasts (Knetsch et al., 1996). MAP kinases phosphorylate myelin basic protein, but they do not appreciably phosphorylate histones in vitro (Hoshi et al., 1988; Sturgill and Wu, 1991; Mizoguchi et al., 1994; Suzuki and Shinshi, 1995). MAP kinases autophosphorylate on tyrosine and threonine residues, which are involved in the activation of MAP kinases in response to agonist stimulation (Boulton et al., 1991;6egeretal., 1991; Wuetal., 1991). By contrast, our AAPK strongly phosphorylates histone (Figure 3), and this phosphorylation occurs only on serine and ABA(HM) 0 1 10 25 50 Ca 2+ EGTA Figure 5. Physiological Concentrations of ABA Activate the Autophosphorylation of AAPK. GCPs were treated with ethanol (0) or various concentrations of ABA as indicated for 10 min. Soluble proteins from GCPs were subjected to the autophosphorylation assay in the presence of 100 tiM free Ca2* or 450 nM EGTA. The stars and arrowheads indicate the positions of the 57-kD protein and the 48-kD AAPK, respectively. Abscisic Acid-Activated Protein Kinase Time(min) 1 5 10 Ca,2+ 20 ABA - «_ Ca2 *>• T GCP MCP ECP ABA kD 200120- »••«* 100- EGTA 8060- ^MlMMMI ••"*" r": Wff^^^f^r 50- Figure 6. ABA Rapidly Activates the Autophosphorylation of AAPK. GCPs were treated with ethanol (-) or 10 nM ABA ( + ) for the indicated times. Soluble proteins from GCPs were subjected to the autophosphorylation assay in the presence of 100 MM free Ca2' or 450 ^iM EGTA. The stars and arrowheads indicate the positions of the 57-kD protein and the 48-kD AAPK, respectively. threonine residues (Figure 4B). AAPK autophosphorylates almost exclusively on serine, with a trace of autophosphorylation on threonine but not tyrosine (Figure 4A). These observations provide evidence that AAPK is not a MAP kinase. In an attempt to determine whether ABA can induce changes in protein phosphorylation, we examined in vivo protein phosphorylation of GCPs by incubating GCPs with 32P-orthophosphate and resolving proteins by one-dimensional SDSPAGE. Most proteins from GCPs were phosphorylated, and no significant differences in the patterns of phosphoproteins between ABA-treated GCPs and untreated GCPs were observed (data not shown). One explanation is that the phosphorylation induced or enhanced by ABA occurs in less abundant proteins, which are difficult to detect due to the relatively high background of in vivo 32P-labeling experiments and/or the limited resolving power of one-dimensional SDSPAGE (Garrison, 1993). It is also possible that it was difficult to detect changes in protein phosphorylation due to the concerted action of ABA-regulated protein phosphatases (Leung et al., 1994; Meyer et al., 1994). In some in vitro phosphorylation experiments, ABA treatments induced the phosphorylation of two soluble proteins and enhanced the phosphorylation of several other soluble proteins (Figure 9B). When the changes in phosphorylation caused by ABA treatment were observed, the phosphorylation patterns were always the same, implying specificity of the ABA effect In other replicates, ABA treatments had no effect on the phosphorylation pattern of soluble proteins (Figure 9A). It is not clear why ABA had variable effects on in vitro protein phosphorylation. However, it may be significant that variable ABA effects, for example, variable effects of ABA on cytosolic Ca2+ levels, have been reported in guard cells (Schroeder and Hagiwara, 1990; Gilroy et al., 1991) Although some studies have suggested that a rapid elevation of cytosolic Ca 24 in guard cells mediates ABA-induced stomatal closure (McAinsh et al., 1990,1992), changes in cytosolic Ca2+ levels are not always seen. Gilroy et al. (1991) found that ABA induced elevation of cytosolic Ca2' only in a minority 2363 403020- EGTA B 20Figure 7. ABA-Activated Autophosphorylation Is Guard Cell Specific. GCPs, mesophyll cell protoplasts (MCP), and epidermal cell protoplasts (ECP) were treated with ethanol (-) or 20 nM ABA (+)for 15 min. Soluble proteins (20 ng/per lane) from GCPs, mesophyll cell protoplasts, and epidermal cell protoplasts were subjected to the autophosphorylation assay in the presence of 100 nM free Ca21 or 450 nM EGTA. Molecular mass markers are shown at left in kilodaltons. The stars and arrowheads indicate the positions of the 57-kD protein and the 48-kD AAPK, respectively (A) Autophosphorylation of proteins in the presence of Ca2*. (B) Autophosphorylation of proteins in the presence of EGTA. 2364 The Plant Cell Material Treated ABA - + + + + Ca2 EGTA Figure 8. ABA Activates Autophosphorylation of AAPK Indirectly A 10 nM concentration of ABA was directly added to the soluble fraction (S), soluble plus membrane fractions (S+ M), or crude extract (CE) from untreated GCPs and incubated for 10 min. Proteins from these treatments and the soluble proteins from ethanol-treated GCPs (-) or 10 nM ABA-treated GCPs (+; 10 min) were subjected to the autophosphorylation assay in the presence of 100 \M free Ca2* or 450 ijM EGTA. The stars and arrowheads indicate the positions of the 57-kD protein and the 48-kD AAPK, respectively of Commelina communis guard cells examined and that in many guard cells, changes in cytosolic Ca2+ levels were small or undetectable, even though ABA-induced stomatal closure could still occur. ABA-induced Ca2+ elevation in patch-clamped guard cell protoplasts of fava bean was observed in only 37% of trials (Schroeder and Hagiwara, 1990). When C. communis plants were grown at 10 to 17°C, most guard cells failed to exhibit ABA-induced cytosolic Ca2+ elevation, even though ABA-induced stomatal closure could still be observed in these plants (Allan et al., 1994). Therefore, ABA signaling can be transduced through either a Ca2+-dependent pathway or a Ca2+-independent pathway (Gilroy et al., 1991; MacRobbie, 1993; Allan et al., 1994). Relatively little is known concerning the Ca2+-independent mechanisms involved in the control of stomatal closure. However, the outward K+ channels in the plasma membrane of guard cells (responsible for K+ efflux during stomatal closing) are known to be activated by ABA, and this activation is Ca2+ independent (Blatt, 1990; Lemtiri-Chlieh and MacRobbie, 1994). In guard cells of transgenic tobacco, the sensitivity of outward K+ channels to ABA was reduced by ab/7-7, and the reduced sensitivity of outward K+ channels to ABA could be reversed by protein kinase inhibitors. These results led Armstrong and co-workers to implicate a phosphatase in this ABA response (Armstrong et al., 1995). It would be premature to conclude that in vivo, AAPK activity and signaling steps upstream of this kinase are completely Ca2+ independent. However, based on the Ca2+-independent nature of both AAPK in vitro and outward K+ channel activation by ABA, we speculate that protein phosphorylation by AAPK might be involved in the modulation of outward K+ channels by ABA. The plasma membrane H+-ATPase provides the driving force for K+ and Cl~ uptake, which contribute to osmotic water uptake, guard cell swelling, and stomatal opening (Assmann, 1993). The plasma membrane H+-ATPase of oat roots can be phosphorylated in vitro by a Ca2+-dependent kinase (Schaller and Sussman, 1988). In addition, some fungal elicitors can invoke a rapid phosphatase-mediated dephosphorylation of the host plasma membrane H+-ATPase and lead to an increase in activity of this enzyme (Vera-Estrella et al., 1994). Furthermore, protein kinase C-like kinase and Ca2+/calmodulin-dependent protein kinase have been shown to be involved in the reversible phosphorylation of the plasma membrane H+-ATPase of tomato cell suspensions (Xing et al., 1996). Therefore, phosphorylation and dephosphorylation of the plasma membrane H+-ATPase may be a major means by which signals regulate the function of the H+-ATPase (Assmann and Haubrick, 1996). Blue light-dependent proton pumping in guard cells of fava bean and stomatal opening in C. benghalensis can be inhibited by calmodulin antagonists and inhibitors of Ca2+/calmodulin-dependent myosin light chain kinase (MLCK), suggesting that Ca2+/calmodulin- B M Figure 9. Effect of ABA on Protein Phosphorylation. Soluble (S) or membrane (M) proteins from ethanol-treated GCPs (-) or ABA-treated GCPs (+) were incubated with y-32P-ATP for 5 min at room temperature. Phosphorylated proteins were separated on 5 to 20% gradient SDS-polyacrylamide gels, followed by autoradiography on Kodak X-Omat AR film. Molecular mass markers are shown at left in (A) in kilodaltons. The stars indicate the positions of the proteins with ABA-enhanced phosphorylation. The arrows indicate the positions of proteins with ABA-induced phosphorylation. The dot indicates the position of a protein with no apparent change in phosphorylation, confirming equal loading of the two lanes. (A) An experiment showing no effect of ABA on phosphorylation of soluble proteins from GCPs (representative of three independent experiments). (B) An experimen; showing effects of ABA on the phosphorylation of certain soluble proteins from GCPs (representative of three independent experiments). (C) An experiment showing no effect of ABA on phosphorylation of membrane proteins from GCPs (representative of six independent experiments). Abscisic Acid-Activated Protein Kinase dependent MLCK may be involved in the activation of the plasma membrane H+-ATPase (Shimazaki et al., 1992). Recently, Goh et al. (1996) showed that ABA inhibits blue light-dependent proton pumping by inactivation of the plasma membrane H+-ATPase. Considering our ABA-activated AAPK, one hypothesis is that ABA inhibits the plasma membrane H+-ATPase through phosphorylation mediated by AAPK. AAPK would then act in opposition to MLCK. Such dual regulation of ion transporters by phosphorylation at different sites or steps of a signal transduction pathway is a well-known phenomenon (Wang and Giebisch, 1991; Levitan, 1994; Li et al., 1994). It is interesting that ABA inhibition of proton pumping could only be observed in intact GCPs and not in isolated microsomes (Goh et al., 1996). Similarly, autophosphorylation of AAPK could only be detected in protein extracts from intact GCPs treated with ABA. Autophosphorylation of AAPK was not detected when ABA was directly added to the crude extract or the soluble fraction or the soluble plus membrane fractions of GCPs (Figure 8). One explanation is that ABA receptors or activators requiredfor ABA signalingwere degraded or inactivated during the extraction process. It is also possible that ABA-activated autophosphorylation of AAPK is mediated through a signaling chain that is disrupted when the cell is homogenized. Further elucidationof the guard cell signaling pathways that employ AAPK will be aided by purification of this nove1protein kinase or by cloning the gene that encodes it. METHODS Plant Material Fava bean (Vicia faba cv Long Pod) plants were grown in growth chambers with a 10-hr-light(200 pmol m-z sec-’ white light) and 14-hr-dark regime. Day and night temperatures were 21 and 18OC, respectively. The first fully expandedbifoliate leaflets from 3-week-old plants were used for experimentation. Protoplast Preparation and Purification Guard cell and epidermal cell protoplasts were isolated and purified as describedby Ling and Assmann (1992). Mesophyllcell protoplasts were isolated as described by Li and Assmann (1993). lsolated mesophyll protoplasts were further purified by flotation through a Nycodenz (Sigma)density gradient(Bush and Jones, 1988). Mesophyll protoplastswere resuspendedin 1 mL of 40% (wlw) Nycodenz solution in 0.6 M mannitol and 1 mM CaCI2.This protoplast suspension was layered in the bottom of a 15-mL centrifuge tube. Another 1 mL of 20% (w/w) Nycodenz solution in 0.6 M mannitol and 1 mM CaCI, was overlaid onto the protoplast suspension. Finally, 1 mL of 0.6 M mannitol and 1 mM CaCI2 were overlaid onto the second layer of Nycodenz. After sitting for -30 min, protoplasts accumulatingat the interface between the uppermost and middle layers were collected. These protoplasts were resuspended in 0.6 M mannitol and 1 mM CaCI, and centrifuged. The pellet was resuspendedin 0.6 M mannitol. 2365 Preparation of Soluble and Membrane Fractions Guard cell protoplasts suspended in a solution of 0.35 M mannitol, 10 mM KCI, 1 mM Mes-NaOH,pH 6.1, were treated with (?)-cis,transabscisic acid (ABA; A-1012; Sigma) from a stock ABA solution (50 mM in 95% ethanol) at room temperature in darkness. After the desired time period (15 min unless otherwise noted), protoplastswere pelleted by centrifugationand frozen in liquid nitrogen. All subsequent operations were performed at 4OC. Protoplastswere homogenizedin a buffer (50 mM Tris-HCI, pH 7.5, 1 mM MgCIZ,2 mM EDTA, 0.25 mM EGTA, and 250 mM sucrose) containing 2 mM DTT, 1 mM phenylmethylsulfonyl fluoride, 10 pglmL aprotinin, 10 pg/mL leupeptin, and 10 pglmL pepstatin.The homogenate was centrifugedat 10,OOOgfor 15 min. The resulting supernatant (crude extract) was centrifuged for 45 min at 150,OOOg. The supernatant was removed as the soluble fraction. The pellet was resuspendedin the homogenizationbuffer and recentrifuged at 150,OOOg for 45 min. The final pellet was used as the microsomal membrane fraction. 60th the soluble and membrane fractions were used immediately. Protein Determination Protein concentrationswere determined using a Bio-Rad protein assay kit, based on the method of Bradford (1976), with BSA (P7656; Sigma) as standard. Detection of Protein Autophosphorylation Autophosphorylationof proteinsin SDS-polyacrylamide gels was performed as described by Wang and Chollet(1993),basedon the method of Kameshita and Fujisawa (1989). Duplicate samples of proteins extractedfrom protoplastswere electrophoresedon a 12% SDS-polyacrylamide gel (Laemmli, 1970). The gel was cut into two identical halves.The gels were washed twice with 50 mM Tris-HCI, pH 8.0, containing 20% (vlv) 2-propanol for 1 hr per wash, and then with 50 mM Tris-HCI, pH 8.0, containing5 mM 2-mercaptoethanoland 0.1 mM EDTA (buffer A) for 1 hr at room temperature. Proteins in the gel were denatured by incubating the gel in buffer A containing 8 M urea for 2 incubations of 1 hr each at roomtemperature. Proteins were then renatured using buffer A containing 0.04% (v/v) Tween 40 at 4% for 18 hr. After preincubation at room temperature for 30 min with 40 mM Hepes-NaOH, pH 7.5, 10 mM MgCIz,0.45 mM EGTA. and 2 mM DTT (buffer B) in the absence or presence of 0.55 mM CaCI,, the gels were incubated with buffer B containing 10 pCilmL V-~~P-ATP (3000 Cilmmol;Amersham) for 1 hr at room temperature. The gels were then washed extensively with 5% trichloroacetic acid and 1% sodium pyrophosphateuntil radioactivity in the used wash solutionwas barely detectable.The gels were then stained with Coomassie Brilliant Blue R 250. After destaining, the gels were air dried between two sheets of cellophane and exposed to Kodak X-Omat AR film for 1 to 3 days at room temperature. Detection of Protein Kinase Activity Protein kinase activity was determined based on the methbd of Yao et al. (1995), using histone type Ill-S (Sigma) as an exogenous substrate (Harmon et al., 1987). Proteins from untreated or ABA-treated protoplasts were subjected to SDS-PAGE, denaturation. and 2366 The Plant Cell renaturation. Portions' of replicate lanes containing the autophosphorylating 48-kD bands were excised. The gel slices were put into a 1.5" microcentrifuge tube containing 200 pL of buffer B in the absence or presence of 0.55 mM CaCI, and crushed with a pestle (Fisher Scientific, Pittsburgh, PA). After centrifugation, the supernatant was removed, and histone type Ill-S was added to give a final concentration of 0.05 mg/mL. The reaction was initiated by the addition of 10 pCi y-3zP-ATF!After 5 min at room temperature, the reaction was terminated by the addition of cold acetone (Bennett,1980). Precipitated proteinswere resuspendedin electrophoresissample buffer and boiled for 3 min. The samples were then electrophoresed on a 12% SDS-polyacrylamide gel. Phosphorylated histone type Ill-S was detected. by autoradiography, as .described above. Phosphoamino Acid Anaiysis Histone type Ill-S was phosphorylated and subjected to SDS-PAGE, as described above. Autophosphorylation of proteins by the in-gel assay was performed as given above. Phosphorylated histone type Ill-S and autophosphorylatedproteins were transferred to lmmobilonP membrane (Millipore, Bedford, MA) at 80 V for 2 hr in transfer buffer (25 mM Tris, 192 mM glycine, 20% methanol, 0.1% SDS). The lmmobilon membrane was then thoroughly washed with water and exposed to x-ray film. Pieces of lmmobilon containingthe 48-kD autophosphorylating band or the phosphorylated histone type Ill-S band were cut out and hydrolyzed in 6 M HCI at llO°C for 1 hr, as described by Kamps and Sefton (1989). The hydrolysatewas lyophilized,and phosphoamino acids were analyzedon a cellulosethin-layerplate (EM Science, Gibbstown, NJ) by two-dimensional electrophoresis (Boyle et al., 1991). REFERENCES Allan, A.C., Fricker, M.D., Ward, J.L., Beale, M.H., and Trewavas, A.J. (1994).Two transduction pathwaysmediate rapid effects of abscisic acid in Commelina guard cells. Plant Cell 6, 1319-1328. Anderberg, R.J., and Walker-Simmons, M.K. (1992). lsolation of a wheat cDNA clone for an abscisic acid-inducible transcript with homology to protein kinases. Proc. Natl. Acad. Sci. USA 89, 10183-10187. Armstrong, F., Leung, J., Grabov, A., Brearley, J., Giraudat, J., and Blatt, M.R. (1995). 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Assmann Plant Cell 1996;8;2359-2368 DOI 10.1105/tpc.8.12.2359 This information is current as of August 3, 2017 Permissions https://www.copyright.com/ccc/openurl.do?sid=pd_hw1532298X&issn=1532298X&WT.mc_id=pd_hw1532298X eTOCs Sign up for eTOCs at: http://www.plantcell.org/cgi/alerts/ctmain CiteTrack Alerts Sign up for CiteTrack Alerts at: http://www.plantcell.org/cgi/alerts/ctmain Subscription Information Subscription Information for The Plant Cell and Plant Physiology is available at: http://www.aspb.org/publications/subscriptions.cfm © American Society of Plant Biologists ADVANCING THE SCIENCE OF PLANT BIOLOGY