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+-dependent and Ca2+-independent signal transduction pathways. Electrophysiological studies suggest that protein phosphorylation is involved in ABA action in guard cells. Using biochemical approaches, we identified an ABA-activated and Ca2+-independent protein kinase (AAPK) from guard cell protoplasts of fava bean. Autophosphorylation of AAPK was rapidly (4 min) activated by ABA in a Ca2+- independent manner. ABA-activated autophosphorylation of 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+-independent ABA signaling pathways of guard cells.

INTRODUCTION

The plant hormone abscisic acid (ABA) regulates many im- portant 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 redistributed in response to sev- era1 stresses, including water stress (Hartung and Davies, 1991). When water stress occurs, ABA levels in leaves rise (Harris et al., 1988). lncreased ABA levels reduce stomatal aper- tures 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- up- take, and production of organic solutes (Assmann, 1993). Stomatal closure results when anion efflux, Ca2+ influx, and/or H+-ATPase inhibition depolarize the membrane potential, 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 in- hibits 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 incompletely understood. Although there are some links between ABA and cytosolic Ca2+ elevation in guard cells (McAinsh et al., 1990, 1992), vari- able 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+-dependent pathway or a Ca*+-independent pathway (Gilroy et al., 1991; MacRobbie, 1993; Allan et al., 1994).

In guard cells, electrophysiological studies indicate that in- ward K+ channels, outward K+ channels, and anion channels may be modulated by protein phosphorylation and dephos- phorylation (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+ chan- nels 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 inhibi- tors abolished both slow anion channel activity of guard cells and ABA-induced stomatal closure (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 pro- toplasts 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+-independent ABA signal- ing pathways in guard cells. To our knowledge, a plant serinelthreonine kinase whose autophosphorylation and ki- nase activity both are ABA regulated has not been described previously.

2360 The Plant Cell

RESULTS

ABA Activates a 48-kD Serine/Threonine ProteinKinase from Guard Cell Protoplasts in aCa2+-lndependent Manner

The routine yield of guard cell protoplasts (GCPs) from 30leaflets of fava bean was ~5 x 106 with 99.9% purity (calcu-lated on a cell basis by counting a sample of ~6000 cells).The purified GCPs could respond to white light by swellingand to ABA by shrinking (data not shown), as reported previ-ously (Fitzsimons and Weyers, 1986,1987), demonstrating thatthey are competent to respond to these signals.

Proteins from GCPs were assayed for autophosphorylationactivity by an in-gel assay (see Methods). When the gel wasincubated with y-32P-ATP in the presence of 100 \iM free Ca2+,two major bands at 57 and 48 kD were seen in soluble pro-teins from ABA-treated GCPs. However, only the 57-kD bandcould be detected in protein samples from GCPs treated withethanol as a control for the ABA solvent (Figure 1A), indicat-ing that autophosphorylation of the 48-kD protein was inducedby ABA. When autophosphorylation was assayed in the pres-ence 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 dis-solved in W-tris(hydroxymethyl)methyl-3-aminopropanesulfonicacid buffer (data not shown), indicating that the solvent hasno effect on the autophosphorylation reaction. These data in-dicate that autophosphorylation of the 57-kD protein is Ca2+

dependent and that ABA-induced autophosphorylation of the48-kD protein is Ca2+ independent. The ABA-induced andCa2+-independent autophosphorylation of the 48-kD proteinwas also detected, albeit weakly, in the membrane proteinsof ABA-treated GCPs (Figures 1A and 1B). When a-32P-ATPwas substituted for y-32P-ATP in the assay buffer, no radioac-tive bands were observed (Figure 1C), indicating that theradioactive bands were due to phosphorylation of these pro-teins rather than to binding of ATP

The ABA-stimulated autophosphorylation could in theory bedue to an increased amount of the 48-kD protein resulting fromde novo protein synthesis stimulated by ABA. A cytosolic pro-tein synthesis inhibitor, cycloheximide, was used to test thispossibility. When GCPs were pretreated with or without 100iaM cycloheximide for 3 min and then further treated with 10nM ABA for 15 min in the presence or absence of cyclohexi-mide, ABA-activated autophosphorylation of the 48-kD proteinwas not affected (Figure 2). These results indicate that ABA-activated autophosphorylation of the 48-kD protein is not dueto an increase in the amount of this protein.

General phosphatase inhibitors increased the amount ofABA-dependent autophosphorylation (J. Li and S.M. Assmann,unpublished data), indicating true ABA stimulation of autophos-phorylation activity. If phosphatase inhibitors had decreasedautophosphorylation, this would have suggested that ABA func-tioned by stimulating a phosphatase that then increased the

Ca2' B EGTAS M

ABA - + - +kD

200-

188=80-60-50-

40-

30-

20-

ABA - + - + ABAkD kD

200-120-100-80-60-

50-

40-

30-

20-

_

200-120-100 —80-60-

-« 50-

40-

30-

20-

Ca2*

Figure 1. Detection of Autophosphorylation Activity of Proteins in ABA-Treated 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 mem-brane proteins (M) from GCPs were separated on 12%SDS-polyacrylamide gels. Autophosphorylation of proteins was de-tected by autoradiography after in situ renaturation, as described inMethods. Molecular mass markers (Gibco BRL) are shown at left inkilodaltons. The stars and the arrowheads at right indicate the posi-tions of the 57- and the 48-kD autophosphorylating proteins, respectively.(A) Autophosphorylation assay was performed by incubating the gelwith y-32P-ATP in the presence of 100 |iM free Ca24.(B) Autophosphorylation assay was performed by incubating the gelwith y-32P-ATP in the presence of 450 nM EGTA(C) Autophosphorylation assay was performed by incubating the gelwith a-32P-ATP in the presence of 100 jiM free Ca2+.

number of unoccupied sites available on the kinase for auto-phosphorylation by Y-32P-ATP.

Autophosphorylation, in some cases, can affect protein ki-nase activity (Hardie, 1995). To determine whether ABAtreatment affected the kinase activity of the 48-kD protein, his-tone type III-S was used as an exogenous substrate (Harmonet al., 1987). The renatured 48-kD protein band was excisedfrom the gel and incubated in kinase assay buffer containinghistone type III-S. The 48-kD protein kinase from untreatedGCPs showed low activity toward histone type III-S (Figure 3,lanes 3 and 5). However, the phosphorylation of histone typeIII-S was markedly enhanced by the 48-kD kinase from ABA-treated GCPs. The enhanced activity of the 48-kD kinase to-ward histone type III-S was independent of the presence ofcalcium (Figure 3, lanes 4 and 6). When extracts from blankgel slices were incubated with kinase assay buffer containinghistone type III-S in the absence or presence of calcium, nophosphorylation of histone type III-S was detected (Figure 3,lanes 1 and 2). Densitometric scans performed on the autoradi-ograph shown in Figure 3 indicate that ABA stimulated kinaseactivity two- to threefold (data not shown). Therefore, theseresults show that the 48-kD kinase activity toward histone typeIII-S was indeed markedly increased by ABA treatment. Thesedata also suggest that the autophosphorylation of the 48-kD

Abscisic Acid-Activated Protein Kinase 2361

kinase activated by ABA is correlated with the enhancementof its kinase activity by ABA. Given this information, we havechosen to refer to this protein kinase as ABA-activated proteinkinase (AAPK).

To classify the 48-kD protein kinase, we analyzed phos-phoamino acids of the autophosphorylated 48-kD kinase andof histone type III-S phosphorylated by AAPK, using two-dimensional thin-layer electrophoresis. The 48-kD protein ki-nase autophosphorylated almost exclusively on serine,although a trace of phosphothreonine was also detected (Fig-ure 4A). Histone type III-S was phosphorylated on both serineand threonine residues by the 48-kD kinase (Figure 4B). Nophosphorylation of tyrosine was observed in either case. Theseresults indicate that the 48-kD protein kinase is a serine/threo-nine protein kinase.

ABA-Activated Autophosphorylation of the 48-kDKinase Is Rapid and Guard Cell Specific

To characterize further the effects of ABA on the autophos-phorylation of AAPK, we treated GCPs with variousconcentrations of ABA for different time periods. After 10 minof treatment, autophosphorylation of AAPK activated by ABAoccurred at a concentration as low as 1 u-M, with maximumeffects reached at a concentration of 10 u.M. ABA concentra-tions of 10, 25, and 50 nM all resulted in approximately equallevels of autophosphorylation (Figure 5). At 10 u,M ABA, auto-phosphorylation of AAPK activated by ABA was detected asearly as 1 min, with maximal activation occurring by 5 to 10min (Figure 6). These results show that autophosphorylationof AAPK activated by ABA is rapid and can be achieved byphysiological concentrations of ABA.

To determine whether the effect of ABA on protein autophos-phorylation was restricted to guard cells, we examined the two

ABA - - -AAPK - - +

Ca2* - + -

Histone -*•

1 2 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 (+) wereelectrophoresed and renatured. Extracts from gel slices containingthe 48-kD AAPK ( + , lanes 3 to 6) or from blank gel slices (-, lanes1 and 2) were mixed with kinase assay buffer containing Ca?1 ( + ; 100nM free Ca2', lanes 2, 5, and 6) or no Ca?1 (-; 450 »iM EGTA. lanes1. 3, and 4) and assayed for kinase activity by using histone type III-Sas an exogenous substrate. Histone type III-S is indicated with an ar-row at left.

other major cell types of leaves: mesophyll cells and epider-mal cells. The purities of mesophyll cell protoplast andepidermal 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 57-kD kinase was detected in all three of the cell types examined(Figures 7A and 7B). In contrast, ABA-activated autophosphor-ylation was detected in guard cells but not in mesophyll cellsor epidermal cells when the autophosphorylation assay wasperformed in the presence of 100 u,M free Ca2+ (Figure 7A).The same was true when the autophosphorylation assay wasperformed in the presence of 450 uM EGTA (Figure 7B). There-fore, ABA-activated autophosphorylation appears to be guardcell specific.

CHXA B A

Ca2+

EGTA

Figure 2. ABA-Activated Autophosphorylation of the 48-kD ProteinIs Unaffected by Cycloheximide.

After preincubation with (+) or without (-) 100 uM cycloheximide (CHX)for 3 min, the CHX samples were kept in CHX-containing solution, andboth samples were then treated with ethanol (-) or ABA ( + ) as de-scribed in Figure 1. Soluble proteins from GCPs were subjected tothe autophosphorylation assay in the presence of 100 uM free Ca2'or 450 ^M EGTA. The stars and arrowheads indicate the positions ofthe 57- and the 48-kD proteins, respectively.

ABA Activates Autophosphorylation of the 48-kDKinase Indirectly

To determine the mechanism by which ABA activates auto-phosphorylation of AAPK, we examined whether ABA activatedthe autophosphorylation of AAPK directly in GCP extracts.When ABA was added directly to the soluble fraction, solubleplus membrane fractions, or crude extract from GCPs, the au-tophosphorylation of AAPK was no longer detected (Figure8). These data indicate that ABA activates the autophosphor-ylation of AAPK indirectly (see Discussion).

Because the activity of AAPK from GCPs was markedly ac-tivated by ABA treatment (Figure 3), we next examined whetherABA treatment would increase phosphorylation of certain pro-teins in GCPs. In three in vitro phosphorylation experiments,there was no difference in the pattern of phosphorylation ofsoluble proteins between ABA-treated GCPs and untreatedGCPs (Figure 9A). In three other in vitro phosphorylation ex-periments, ABA consistently enhanced phosphorylation of

2362 The Plant Cell

V.r

P-Ser

P-ThrP-Tyr

B

P-Ser

,-N P-Thr

v. J P-Tyr

£aFigure 4. AAPK Is a Serine/Threonine Kinase.

Hydrolysates of the autophosphorylated AAPK or phosphorylated his-tone 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 electrophore-sis at pH 1.9 in the first dimension (1D) and at pH 3.5 in the seconddimension (2D). Positions of phosphoamino acid standards visualizedby ninhydrin staining are indicated as dashed circles.(A) Phosphoamino acid analysis of the autophosphorylated AAPK.(B) Phosphoamino acid analysis of histone type III-S phosphorylatedby AAPK

several soluble proteins, for example, the 115- and 90-kD pro-teins (stars in Figure 9B), and induced phosphorylation of the49- and 36-kD proteins (arrows in Figure 9B). In all in vitro phos-phorylation experiments, ABA had no apparent effect on thephosphorylation of membrane proteins (Figure 9C).

ABA elicits two types of responses in plants (Zeevaart andCreelman, 1988; Hetherington and Quatrano, 1991). The clo-sure of stomata is an example of a rapid response (withinminutes). The slow responses appear to involve RNA and pro-tein synthesis (Zeevaart and Creelman, 1988; Hetheringtonand Quatrano, 1991). Such differences in ABA response ki-netics suggest that ABA may have several modes of actionand even different receptors in various experimental systems(Zeevaart and Creelman, 1988). Autophosphorylation of AAPKwas activated rapidly (within 1 min) by physiological levels ofABA (Figures 5 and 6). The ABA-activated autophosphoryla-tion 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 in-volved in the rapid alterations in stomatal aperture that occurin response to ABA. In contrast, the near full-length cDNA clonePKABA1 (for protein kinase responsive to ABA) was identifiedfrom a cDNA library prepared from wheat embryos treated withABA for 12 hr (Anderberg and Walker-Simmons, 1992). PKABA1mRNA was detected in wheat embryos treated with ABA for48 hr or water stressed for 12 hr. Thus, ABA appears to inducethe expression of PKABA1 mRNA. Therefore, AAPK andPKABA1 are likely to be distinct protein kinases.

Recently, it has been shown that ABA induces a rapid andtransient activation of a 40- to 43-kD mitogen-activated pro-tein (MAP) kinase in barley aleurone protoplasts (Knetsch etal., 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 autophosphory-late on tyrosine and threonine residues, which are involvedin the activation of MAP kinases in response to agonist stimu-lation (Boulton et al., 1991;6egeretal., 1991; Wuetal., 1991).By contrast, our AAPK strongly phosphorylates histone (Fig-ure 3), and this phosphorylation occurs only on serine and

DISCUSSION

The in situ renaturation technique is a very powerful methodfor the detection and analysis of protein kinases from crudeprotein preparations. After SDS-PAGE and in situ renaturation,protein kinases can be detected and assayed either by auto-phosphorylation or by phosphorylation of exogenoussubstrates (Hutchcroft et al., 1991). By using this technique,we detected a 48-kD AAPK from guard cell protoplasts of favabean. Both autophosphorylation of AAPK and its kinase ac-tivity toward histone type III-S were Ca2+ independent(Figures 1 and 3). In addition, a polyclonal antibody raisedagainst the soybean Ca2+-dependent protein kinase (CDPK)failed to recognize AAPK (data not shown). Therefore, AAPKdoes not appear to be a CDPK-like protein kinase.

ABA(HM) 0 1 10 25 50

Ca2+

EGTA

Figure 5. Physiological Concentrations of ABA Activate the Autophos-phorylation of AAPK.

GCPs were treated with ethanol (0) or various concentrations of ABAas indicated for 10 min. Soluble proteins from GCPs were subjectedto the autophosphorylation assay in the presence of 100 tiM free Ca2*or 450 nM EGTA. The stars and arrowheads indicate the positions ofthe 57-kD protein and the 48-kD AAPK, respectively.

Abscisic Acid-Activated Protein Kinase 2363

Time(min) 1 5 10 20 Ca,2+

ABA -

Ca2

EGTA

«_*>• T »••«*

Figure 6. ABA Rapidly Activates the Autophosphorylation of AAPK.

GCPs were treated with ethanol (-) or 10 nM ABA ( + ) for the indicatedtimes. Soluble proteins from GCPs were subjected to the autophos-phorylation assay in the presence of 100 MM free Ca2' or 450 ^iMEGTA. The stars and arrowheads indicate the positions of the 57-kDprotein and the 48-kD AAPK, respectively.

threonine residues (Figure 4B). AAPK autophosphorylates al-most exclusively on serine, with a trace of autophosphorylationon threonine but not tyrosine (Figure 4A). These observationsprovide evidence that AAPK is not a MAP kinase.

In an attempt to determine whether ABA can induce changesin protein phosphorylation, we examined in vivo proteinphosphorylation of GCPs by incubating GCPs with 32P-ortho-phosphate and resolving proteins by one-dimensional SDS-PAGE. Most proteins from GCPs were phosphorylated, andno significant differences in the patterns of phosphoproteinsbetween ABA-treated GCPs and untreated GCPs were ob-served (data not shown). One explanation is that thephosphorylation induced or enhanced by ABA occurs in lessabundant proteins, which are difficult to detect due to the rel-atively high background of in vivo 32P-labeling experimentsand/or the limited resolving power of one-dimensional SDS-PAGE (Garrison, 1993). It is also possible that it was difficultto detect changes in protein phosphorylation due to the con-certed action of ABA-regulated protein phosphatases (Leunget al., 1994; Meyer et al., 1994).

In some in vitro phosphorylation experiments, ABA treat-ments induced the phosphorylation of two soluble proteins andenhanced the phosphorylation of several other soluble pro-teins (Figure 9B). When the changes in phosphorylationcaused by ABA treatment were observed, the phosphoryla-tion patterns were always the same, implying specificity of theABA effect In other replicates, ABA treatments had no effecton the phosphorylation pattern of soluble proteins (Figure 9A).It is not clear why ABA had variable effects on in vitro proteinphosphorylation. However, it may be significant that variableABA effects, for example, variable effects of ABA on cytosolicCa2+ levels, have been reported in guard cells (Schroeder andHagiwara, 1990; Gilroy et al., 1991)

Although some studies have suggested that a rapid eleva-tion of cytosolic Ca24 in guard cells mediates ABA-inducedstomatal closure (McAinsh et al., 1990,1992), changes in cyto-solic Ca2+ levels are not always seen. Gilroy et al. (1991) foundthat ABA induced elevation of cytosolic Ca2' only in a minority

B

GCP MCP ECPABA -kD

200-120-100-80-60-50-

40-

30-

20-

M̂lMMMI ••"*" r":Wff^^^f^r

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Figure 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. Solubleproteins (20 ng/per lane) from GCPs, mesophyll cell protoplasts, andepidermal cell protoplasts were subjected to the autophosphorylationassay in the presence of 100 nM free Ca21 or 450 nM EGTA. Molecu-lar mass markers are shown at left in kilodaltons. The stars andarrowheads indicate the positions of the 57-kD protein and the 48-kDAAPK, respectively(A) Autophosphorylation of proteins in the presence of Ca2*.(B) Autophosphorylation of proteins in the presence of EGTA.

2364 The Plant Cell

MaterialTreated

ABA - + + + +

Ca2

EGTA

Figure 8. ABA Activates Autophosphorylation of AAPK IndirectlyA 10 nM concentration of ABA was directly added to the soluble frac-tion (S), soluble plus membrane fractions (S+ M), or crude extract (CE)from untreated GCPs and incubated for 10 min. Proteins from thesetreatments and the soluble proteins from ethanol-treated GCPs (-) or10 nM ABA-treated GCPs (+; 10 min) were subjected to the autophos-phorylation assay in the presence of 100 \M free Ca2* or 450 ijMEGTA. The stars and arrowheads indicate the positions of the 57-kDprotein and the 48-kD AAPK, respectively

(Assmann, 1993). The plasma membrane H+-ATPase of oatroots can be phosphorylated in vitro by a Ca2+-dependent ki-nase (Schaller and Sussman, 1988). In addition, some fungalelicitors can invoke a rapid phosphatase-mediated dephosphor-ylation of the host plasma membrane H+-ATPase and lead toan increase in activity of this enzyme (Vera-Estrella et al., 1994).Furthermore, protein kinase C-like kinase and Ca2+/calmodu-lin-dependent protein kinase have been shown to be involvedin the reversible phosphorylation of the plasma membraneH+-ATPase of tomato cell suspensions (Xing et al., 1996).Therefore, phosphorylation and dephosphorylation of theplasma membrane H+-ATPase may be a major means bywhich signals regulate the function of the H+-ATPase(Assmann and Haubrick, 1996). Blue light-dependent protonpumping in guard cells of fava bean and stomatal opening inC. benghalensis can be inhibited by calmodulin antagonistsand inhibitors of Ca2+/calmodulin-dependent myosin lightchain kinase (MLCK), suggesting that Ca2+/calmodulin-

of Commelina communis guard cells examined and that in manyguard cells, changes in cytosolic Ca2+ levels were small orundetectable, even though ABA-induced stomatal closure couldstill occur. ABA-induced Ca2+ elevation in patch-clampedguard cell protoplasts of fava bean was observed in only 37%of trials (Schroeder and Hagiwara, 1990). When C. communisplants were grown at 10 to 17°C, most guard cells failed to ex-hibit ABA-induced cytosolic Ca2+ elevation, even thoughABA-induced stomatal closure could still be observed in theseplants (Allan et al., 1994). Therefore, ABA signaling can betransduced through either a Ca2+-dependent pathway or aCa2+-independent pathway (Gilroy et al., 1991; MacRobbie,1993; Allan et al., 1994).

Relatively little is known concerning the Ca2+-independentmechanisms involved in the control of stomatal closure. How-ever, the outward K+ channels in the plasma membrane ofguard 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 ofoutward K+ channels to ABA was reduced by ab/7-7, and thereduced sensitivity of outward K+ channels to ABA could bereversed by protein kinase inhibitors. These results ledArmstrong and co-workers to implicate a phosphatase in thisABA response (Armstrong et al., 1995). It would be prematureto conclude that in vivo, AAPK activity and signaling steps up-stream of this kinase are completely Ca2+ independent.However, based on the Ca2+-independent nature of bothAAPK in vitro and outward K+ channel activation by ABA, wespeculate that protein phosphorylation by AAPK might be in-volved in the modulation of outward K+ channels by ABA.

The plasma membrane H+-ATPase provides the drivingforce for K+ and Cl~ uptake, which contribute to osmotic wa-ter uptake, guard cell swelling, and stomatal opening

BM

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 atroom temperature. Phosphorylated proteins were separated on 5 to20% gradient SDS-polyacrylamide gels, followed by autoradiographyon Kodak X-Omat AR film. Molecular mass markers are shown at leftin (A) in kilodaltons. The stars indicate the positions of the proteinswith ABA-enhanced phosphorylation. The arrows indicate the posi-tions of proteins with ABA-induced phosphorylation. The dot indicatesthe 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 ofsoluble proteins from GCPs (representative of three independentexperiments).(B) An experimen; showing effects of ABA on the phosphorylation ofcertain soluble proteins from GCPs (representative of three indepen-dent experiments).(C) An experiment showing no effect of ABA on phosphorylation ofmembrane proteins from GCPs (representative of six independentexperiments).

Abscisic Acid-Activated Protein Kinase 2365

dependent MLCK may be involved in the activation of the plasma membrane H+-ATPase (Shimazaki et al., 1992). Re- cently, 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 regu- lation 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 de- tected 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 required for ABA signaling were degraded or inacti- vated 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 elucidation of the guard cell signaling pathways that employ AAPK will be aided by purification of this nove1 protein kinase or by cloning the gene that encodes it.

METHODS

Plant Material

Fava bean (Vicia faba cv Long Pod) plants were grown in growth cham- bers 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 expanded bifoliate 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 described by Ling and Assmann (1992). Mesophyll cell 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 protoplasts were resuspended in 1 mL of 40% (wlw) Nycodenz solu- tion 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 accumulating at 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 resuspended in 0.6 M mannitol.

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,trans- abscisic 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), protoplasts were pelleted by centrifugation and frozen in liquid nitrogen. All subsequent opera- tions were performed at 4OC. Protoplasts were homogenized in 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 phenylmethylsul- fonyl fluoride, 10 pglmL aprotinin, 10 pg/mL leupeptin, and 10 pglmL pepstatin. The homogenate was centrifuged at 10,OOOg for 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 resuspended in the homogenization buffer 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 concentrations were determined using a Bio-Rad protein as- say kit, based on the method of Bradford (1976), with BSA (P7656; Sigma) as standard.

Detection of Protein Autophosphorylation

Autophosphorylation of proteins in SDS-polyacrylamide gels was per- formed as described by Wang and Chollet (1993), based on the method of Kameshita and Fujisawa (1989). Duplicate samples of proteins extracted from protoplasts were electrophoresed on a 12% SDS-poly- acrylamide gel (Laemmli, 1970). The gel was cut into two identical halves. The gels were washed twice with 50 mM Tris-HCI, pH 8.0, con- taining 20% (vlv) 2-propanol for 1 hr per wash, and then with 50 mM Tris-HCI, pH 8.0, containing 5 mM 2-mercaptoethanol and 0.1 mM EDTA (buffer A) for 1 hr at room temperature. Proteins in the gel were dena- tured by incubating the gel in buffer A containing 8 M urea for 2 incubations of 1 hr each at room temperature. Proteins were then rena- tured 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 pyrophosphate until radioactivity in the used wash solution was 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 sub- strate (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 au- tophosphorylating 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 superna- tant 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 addi- tion of 10 pCi y-3zP-ATF! After 5 min at room temperature, the reaction was terminated by the addition of cold acetone (Bennett, 1980). Precipi- tated proteins were resuspended in electrophoresis sample buffer and boiled for 3 min. The samples were then electrophoresed on a 12% SDS-polyacrylamide gel. Phosphorylated histone type Ill-S was de- tected. 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 as- say was performed as given above. Phosphorylated histone type Ill-S and autophosphorylated proteins were transferred to lmmobilon P mem- brane (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 containing the 48-kD autophosphorylat- ing 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 hydrolysate was lyophilized, and phosphoamino acids were analyzed on a cellulose thin-layer plate (EM Science, Gibbs- town, NJ) by two-dimensional electrophoresis (Boyle et al., 1991).

In Vitro Protein Phosphorylation

Soluble proteins were diluted to a final volume of 100 pL in phosphory- lation buffer (25 mM Tris-HCI, pH 7.5, 5 mM MgC12, 0.25 mM EGTA, 0.1 mM DTT). Mewbrane proteins were resuspended in the same buffer containing 0.1% (w/v) Triton X-100. The phosphorylation reaction was initiated by the addition of 15 pCi y3*P-ATP and 10 nmol ATI? After 5 min at room temperature, the reaction was terminated by the addi- tion of cold acetone. Precipitated proteins were resuspended in electrophoresis sample buffer and boiled for 5 min (soluble fraction) or 1 min (membrane fraction). 60th soluble and membrane proteins were analyzed by gradient SDS-PAGE (5 to 20% acrylamide). The gels were dried and subjected to autoradiography as described above.

ACKNOWLEDGMENTS

We thank Dr. Richard Frisque for use of his equipment for phos- phoamino acid analysis, Dr. Jennifer Swenson for help with phosphoamino acid analysis, and Dr. Alice Harmon for providing CDPK antibodies. This research was supported by National Science Foun- dation Grant No. MCB-9316319 to S.M.A.

Received August 23, 1996; accepted.October 7, 1996.

REFERENCES

Allan, A.C., Fricker, M.D., Ward, J.L., Beale, M.H., and Trewavas, A.J. (1994). Two transduction pathways mediate rapid effects of ab- scisic 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 ho- mology to protein kinases. Proc. Natl. Acad. Sci. USA 89,

Armstrong, F., Leung, J., Grabov, A., Brearley, J., Giraudat, J., and Blatt, M.R. (1995). Sensitivity to abscisic acid of guard cell K+ chan- nels is suppressed by abil-1, a mutant Awbidopsis gene encoding a putative protein phosphatase. Proc. Natl. Acad. Sci. USA 92,

Assmann, S.M. (1993). Signal transduction in guard cells. Annu. Rev. Cell 6101. 9, 345-375.

Assmann, S.M., and Haubrick, L.L. (1996). Transport proteins of the plant plasma membrane. Curr. Opin. Cell Biol. 8, 458-467.

Bennett, J. (1980). Chloroplast phosphoproteins: Evidence for a thylakoid-bound phosphoprotein phosphatase. Eur. J. Biochem. 104,

Blatt, M.R. (1990). Potassium channel current in intact stomatal guard cells: Rapid enhancement by abscisic acid. Planta 180, 445-455.

Boulton, T.G., Nye, S., Robbins, D., Ip, N., Radziejewska, E., Morgenbesser, S., DePinho, R., Panayotatos, N., Cobb, M., and Yancopoulos, G. (1991). ERKs: A family of protein-serinenhreonine kinases that are activated and tyrosine phosphorylated in response to insulin and NGF. Cell 65, 663-675.

Boyle, W.J., van der Geer, P., and Hunter, T. (1991). Phosphopep tide mapping and phosphoamino acid analysis by two-dimensional separation on thin-layer cellulose plates. Methods Enzymol. 201,

Bradford, M.M. (1976). A rapid and sensitive method for the quantita- tion of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-254.

Bush, D.S., and Jones, R.L. (1988). Cytoplasmic calcium and a-amylase secretion from barley aleurone protoplasts. Eur. J. Cell

Fitzsimons, P.J., and Weyers, J.D.B. (1986). Volume changes of Com- melina communis guard cell protoplasts in response to K+, light and C02. Physiol. Plant. 66, 463-468.

Fitzsimons, P.J., and Weyers, J.D.B.'(1987). Aesponses of Comme- lina communis L. guard cell protoplasts in response to abscisic acid.

Garrison, J.C. (1993). Study of protein phosphorylation in intact cells. In Protein Phosphorylation: A Practical Approach, D.G. Hardie, ed (Oxford, UK: Oxford University Press), pp. 1-29.

Gilroy, S., Fricker, M.D., Read, N.D., and Trewavas, A.J. (1991). Role of calcium in signal transduction of Commelina guard cells. Plant Cell 3, 333-344.

10183-10187.

9520-9524.

85-89.

110-149.

BiOl. 46, 466-469.

J. EXP. BOt. 38, 992-1001.

Goh, C.-H., Kinoshita, T., Oku, T., and Shimazaki, K. (1996). Inhibi- tion of blue light-dependent H+ pumping by abscisic acid in Vicia guard-cell protoplasts. Plant Physiol. 111, 433-440.

Hardie, D.G. (1995). Cellular functions of protein kinases. In The Pro- tein Kinase FactsBook, G. Hardie and S. Hanks, eds (London, UK: Academic Press), pp. 48-56.

Abscisic Acid-Activated Protein Kinase 2367

Harmon, A.C., Putnam-Evans, C., and Cormier, M.J. (1987). A calcium-dependent but calmodulin-independent protein kinase from soybean. Plant Physiol. 83, 830-837.

Harris, M.J., Outlaw, W.H., Jr., Mertens, R., and Weiler, E.W. (1988). Water-stress-induced changes in the abscisic acid content of guard cells and other cells of Vicia faba L. leaves as determined by enzyme- amplified immunoassay, Proc. Natl. Acad. Sci. USA 85,2584-2588.

Hartung, W., and Davies, W.J. (1991). Drought-induced changes in physiology and ABA. In Abscisic Acid: Physiology and Biochemis- try, W.J. Davies and H.G. Jones, eds (Oxford, UK: BlOS Scientific),

Hetherington, A.M., and Quatrano, R.S. (1991). Mechanisms of ac- tion of abscisic acid at the cellular level. New Phytol. 119, 9-32.

Hoshi, M., Nishida, E., and Sakai, H. (1988). Activation of a Caz++- inhibitable protein kinase that phosphorylates microtuble-associated protein 2 in vitro by growth factors, phorbol esters, and serum in quiescent cultured human fibroblasts. J. Biol. Chem. 263,5396-5401.

Hutchcroft, J.E., Anostario, M., Jr., Harrison, M.L., and Geahlen, R.L. (1991). Renaturation and assay of protein kinases after elec- trophoresis in sodium dodecyl sulfate-polyacrylamide gels. Methods Enzymol. 200, 417-423.

Kameshita, I . , and Fujisawa, H . (1989). A sensitive method for de- tection of calmodulin-dependent protein kinase I1 activity in sodium dodecyl sulfate-polyacrylamide gel. Anal. Biochem. 183, 139-143.

Kamps, M.P., and Sefton, B.M. (1989). Acid and base hydrolysis of phosphoproteins bound to lmmobilon facilitates analysis of phos- phoamino acids in gel-fractionated proteins. Anal. Biochem. 176, 22-27.

Knetsch, M.L.W., Wang, M., Snaar-Jagalska, B.E., and Heimovaara- Dijkstra, S. (1996). Abscisic acid induces mitogen-activated pro- tein kinase activation in barley aleurone protoplasts. Plant Cell 8, 1061-1067.

Laemmli, U.K. (1970). Cleavage of structural proteins during the as- sembly of the head of bacteriophage T4. Nature 277, 680-685.

Lemtiri-Chlieh, F., and MacRobbie, E.A.C. (1994). Role of calcium in the modulation of Vicia guard cell potassium channels by abscisic acid: A patch clamp study. J. Membr. Biol. 137, 99-107.

Leung, J., Bouvier-Durand, M., Morris, P.-C., Guerrier, D., Chefdor, F., and Giraudat, J. (1994). Arabidopsis ABA-response gene ABI7: Features of a calcium-modulated protein phosphatase. Science 264,

Levitam I.B. (1994). Modulation of ion channels by protein phosphory- lation and dephosphorylation. Annu. Rev. Physiol. 56, 193-212.

Li, W., and Assmann, S.M. (1993). Characterization of a G-protein- regulated outward K ' current in mesophyll cells of Vicia faba L. Proc. Natl. Acad. Sci USA 90, 262-266.

Li, W., Luan, S., Schreiber, S.L., and Assmann, S.M. (1994). Evi- dente for protein phosphatase 1 and 2A regulation of Kt channels in two types of leaf cells. Plant Physiol. 106, 963-970.

Ling, V., and Assmann, S.M. (1992). Cellular distribution of calmodulin and calmodulin-binding proteins in Vicia faba L. Plant Physiol. 100,

Luan, S., Li, W., Rusnak, F., Assmann, S.M., and Schreiber, S.L. (1993). lmmunosuppressants implicate protein phosphatase regu- lation of Kt channels in guard cells. Proc. Natl. Acad. Sci. USA 90,

MacRobbie, E.A.C. (1993). Ca2' and cell signalling in guard cells. Se-

pp. 63-79.

1448-1452.

970-978.

2202-2206.

min. Cell Biol. 4. 113-122.

.McAinsh, M.R., Brownlee, C., and Hetherington, A.M. (1990). Ab- scisic acid-induced elevation of guard cell cytosolic Ca2+ precedes stomatal closure. Nature 343, 186-188.

McAinsh, M.R., Brownlee, C., and Hetherington, A.M. (1992). Visualizing changes in cytosolic free Ca2+ during the response of stomatal guard cells to abscisic acid. Plant Cell 4, 1113-1122.

Meyer, K., Leube, M.P., and Grill, E. (1994). A protein phosphatase 2C involved in ABA signal transduction in Arabidopsis tbaliana. Science 264, 1452-1455.

Mittelheuser, C.J., and van Steveninck, R.F.M. (1969). Stomatal clo- sure and inhibition of transpiration induced by (RS)-abscisic acid. Nature 221, 281-282.

Mizoguchi, T., Gotoh, Y., Nishida, E., Yamaguchi-Shinozaki, K., Hayashida, N., Iwasaki, T., Kamada, H., and Shinozaki, K. (1994). Characterization of two cDNAs that encode MAP kinase homologues in Arabidopsis tbaliana and analysis of the possible role of auxin in activating such kinase activities in cultured cells. Plant J. 5, 1 1 1-1 22.

Schaller, G.E., and Sussman, M.R. (1988). Phosphorylation of the plasma-membrane H+-ATPase of oat roots by a calcium-stimulated protein kinase. Planta 173, 509-518.

Schmidt, C., Schelle, I., Liao, Y., and Schroeder, J.I. (1995). Strong regulation of slow anion channels and abscisic acid signaling in guard cells by phosphorylation and dephosphorylation events. Proc. Natl. Acad. Sci. USA 92, 9535-9539.

Schroeder, J.I., and Hagiwara, S. (1990). Repetitive increases in cyto- solic Caz+ of guard cells by abscisic acid activation of nonselective Ca2+ permeable channels. Proc. Natl. Acad. Sci. USA 87,

Seger, R., Ahn, N.G., Boulton, T.G., Yancopoulos, G.D., Panayotatos, N., Radziejewska, E., Ericsson, L., Bratlien, R.L., Cobb, M.H., and Krebs, E.G. (1991). Microtubule-associated pro- tein 2 kinases, ERKI and ERK2, undergo autophosphorylation on both tyrosine and threonine residues: lmplications for their mecha- nism of activation. Proc. Natl. Acad. Sci. USA 88, 6142-6146.

Shimazaki, K., Kinoshita, T., and Nishimura, M. (1992). lnvolvement of Caz+/calmodulin-dependent myosin light chain kinase in blue light-dependent H+ pumping of guard Cell prOtOplaStS frOm vicia faba L. Plant Physiol. 99, 1416-1421.

Sturgill, T.W., and Wu, J. (1991). Recent progress in characterization of protein kinase cascades for phosphorylation of ribosomal pro- tein S6. Biochim. Biophys. Acta 1092, 350-357.

Suzuki, K., and Shinshi, H. (1995). Transient activation and tyrosine phosphorylation of a protein kinase in tobacco cells treated with a fungal elicitor. Plant Cell 7, 639-647.

9305-9309.

Vera-Estrella, R., Barkla, B.J., Higgins, V.J., and Blumwald, E. (1994). Plant defense response to fungal pathogens: Activation of host plasma membrane H+-ATPase by elicitorinduced enzyme dephos- phorylation. Plant Physiol. 104, 209-215.

Wang, W., and Giebisch, G. (1991). Dual modulation of renal ATP- sensitive K+ channel by protein kinases A and C. Proc. Natl. Acad. Sci. USA 88, 9722-9725.

Wang, Y.-H., and Chollet, R. (1993). Partia1 purification and charac- terization of phosphoenolpyruvate carboxylase protein-serine kinase from illuminated maize leaves. Arch. Biochem. Biophys. 304, 496-502.

2368 The Plant Cell .

Weyers, J.D.B., and Hillman, J.R. (1979). Sensitivity of Commelina stomata to abscisic acid. Planta 146, 623-628.

Wu, J., Rossomando, A. J., Her, J.H., Delvecchio, R., Weber, M. J., and Sturgill, T.W. (1991). Autophosphorylation in vitro of recombinant 42-kilodalton mitcgen-activated protein kinase on tyrosine. Proc. Natl. Acad. Sci. USA 88, 9506-9512.

Xing, T., Higgins, V.J., and Blumwald, E. (1996). Regulation of plant defense response to funga1 pathogens: Two types of protein kinases

in the reversible phosphorylation of the host plasma membrane H+- ATPase. Plant Cell 8, 555-564.

Yao, B., Zhang, Y., Delikat, S., Mathias, S., Basu, S., and Kolesnick, R. (1995). Phosphorylation of Raf by ceramide-activated protein ki- nase. Nature 378, 307-310.

Zeevaart, J.A.D., and Creelman, R.A. (1966). Metabolism and phys- iology of abscisic acid. Annu. Rev. Plant Physiol. Plant MOI. Biol. 39, 439-473.

DOI 10.1105/tpc.8.12.2359 1996;8;2359-2368Plant Cell

J. Li and S. M. AssmannBean.

An Abscisic Acid-Activated and Calcium-Independent Protein Kinase from Guard Cells of Fava

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