Plant Physiol. (1998) 116: 765-776

Calcium-Dependent Protein Phosphorylation May Mediate the Gibberellic Acid Response in Barley Aleurone¹

Sian Ritchie and Simon Gilroy^*

Biology Department, The Pennsylvania State University, 208 Mueller Laboratory, University Park, Pennsylvania 16802

	ABSTRACT

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Peptide substrates of well-defined protein kinases were microinjected into aleurone protoplasts of barley (Hordeum vulgare L. cv Himalaya) to inhibit, and therefore identify, protein kinase-regulated events in the transduction of the gibberellin (GA) and abscisic acid signals. Syntide-2, a substrate designed for Ca²⁺- and calmodulin (CaM)-dependent kinases, selectively inhibited the GA response, leaving constitutive and abscisic acid-regulated events unaffected. Microinjection of syntide did not affect the GA-induced increase in cytosolic [Ca²⁺], suggesting that it inhibited GA action downstream of the Ca²⁺ signal. When photoaffinity-labeled syntide-2 was electroporated into protoplasts and cross-linked to interacting proteins in situ, it selectively labeled proteins of approximately 30 and 55 kD. A 54-kD, soluble syntide-2 phosphorylating protein kinase was detected in aleurone cells. This kinase was activated by Ca²⁺ and was CaM independent, but was inhibited by the CaM antagonist N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide (250 µm), suggesting that it was a CaM-domain protein kinase-like activity. These results suggest that syntide-2 inhibits the GA response of the aleurone via an interaction with this kinase, implicating the 54-kD kinase as a Ca²⁺-dependent regulator of the GA response in these cells.

	INTRODUCTION

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The aleurone of the barley (Hordeum vulgare L.) grain secretes hydrolases that mobilize endosperm reserves during germination (for review, see Fincher, 1989; Jones and Jacobsen, 1991). The synthesis and secretion of these hydrolases (principally -amylases) is under hormonal regulation. GA stimulates the synthesis and secretion of -amylase and ABA reverses this GA effect (Fincher, 1989; Jones and Jacobsen, 1991). In addition, ABA induces a series of proteins such as RaB (van der Veen et al., 1992) and the amylase subtilisin inhibitor (Mundy and Rogers, 1986). Hence, the barley aleurone has been used extensively as a model system for the study of signal transduction in response to GA and ABA. However, the molecular basis of GA and ABA signal transduction remains poorly understood.

Changes in the levels of cytoplasmic Ca²⁺, often mediated through the regulatory protein CaM, are recognized as ubiquitous signal transduction elements in animal and plant cells (for review, see Poovaiah and Reddy, 1993; Bush, 1995). The cytoplasmic [Ca²⁺] of barley and wheat aleurone cells is elevated from 100 to around 500 nm by GA, whereas ABA reduces Ca²⁺ levels (Gilroy and Jones, 1992; Bush, 1996; Gilroy, 1996). CaM expression is also increased in response to GA (Gilroy and Jones, 1993; Schuurink et al., 1996), implicating Ca²⁺ and CaM in the GA response of these cells. Alternative regulatory events, such as changes in membrane potential and cytoplasmic pH, also seem to be critical to aleurone function (van der Veen et al., 1992; Heimovaara-Dijkstra et al., 1994a, 1994b), and thus it has become clear that distinct Ca²⁺-dependent and -independent regulatory pathways exist in this cell (Bush, 1996; Gilroy, 1996). However, the Ca²⁺- and CaM-dependent systems have proved to be central elements in the events that maintain secretory activity in response to GA and ABA in aleurone cells in vivo (Gilroy, 1996). The molecular identity of the activities regulated by such changes in Ca²⁺ and CaM has remained elusive.

The regulation of protein phosphorylation by kinases and phosphatases is accepted as a universal mechanism of cellular control (Cohen, 1992), and Ca²⁺ and CaM signals are frequently transduced via Ca²⁺- and CaM-dependent kinases and phosphatases (Roberts and Harmon, 1992; Roberts, 1993). Okadaic acid, a protein phosphatase inhibitor, has been found to affect both GA and ABA pathways, but not hypoxic responses in the wheat aleurone (Kuo et al., 1996). It inhibited the expression of high-pI amylase, the increase in cytoplasmic [Ca²⁺], and the cell death normally induced by GA, and antagonized the induction of the PHVA1 gene by ABA (Kuo et al., 1996). A variety of Tyr and Ser/Thr phosphatase inhibitors have also been shown to antagonize the regulation of RaB gene expression by ABA in barley aleurone (Heimovaara-Dijkstra et al., 1996). The unknown targets of these inhibitors in situ in the aleurone have made interpretation of these data difficult. However, further support for the involvement of kinases in the regulation of the hormone responses of the aleurone arises from reports that indicate that the slow vacuolar channel is regulated by phosphatase action (Bethke and Jones, 1997) and that ABA activates an aleurone mitogen-activated protein kinase-like activity (Knetsch et al., 1996), whereas GA down-regulates expression of ASPK9. ASPK9 has homology to the regulatory mitogen-activated protein kinase ERK1 of mammalian cells (Huttly and Phillips, 1995). In addition, cDNAs corresponding to putative Ca²⁺-dependent CDPKs (Roberts and Harmon, 1992; Roberts, 1993) have been detected in oat aleurone (Huttly and Phillips, 1995).

Findings from other systems also strongly suggest that protein kinase and phosphatase activities may be critical elements in the regulation of plant responses to ABA. For example, the Abi-1 and Abi-2 genes encode protein phosphatase 2C homologs, which, when defective, confer ABA insensitivity to Arabidopsis (Leung et al., 1994, 1997; Meyer et al., 1994). These phosphatase activities may regulate guard cell responses to ABA (Armstrong et al., 1995; Pei et al., 1997). ABA has also been shown to lead to rapid induction of a Ca²⁺-independent kinase (AAPK) in guard cells (Li and Assmann, 1996; Mori and Muto, 1997). Additionally, Ca²⁺-independent mutants of Arabidopsis CDPKs can induce expression driven by the ABA-regulated HVA-1 promoter in maize (Sheen, 1996). ABA has also been found to up-regulate the PKABA1 kinase in wheat embryos (Anderberg and Walker-Simmons, 1992), which has homology to kinases of oat aleurone, Aspk4 and Aspk5 (Huttly and Phillips, 1995).

We therefore initiated a screen to determine where protein kinases are involved in the regulation of the aleurone GA or ABA response, and whether Ca²⁺ is involved in these events. A range of kinase substrate peptides was introduced into the cytoplasm of aleurone protoplasts using electroporation or microinjection. These peptides could compete with the endogenous kinase substrate(s), hence blocking downstream events. This strategy has proved highly successful in, for example, defining the timing of kinase activities during mitotic progression in Tradescantia virginiana stamen hairs (Wolniak and Larsen, 1995), or highlighting the involvement of a kinase that specifically phosphorylates myosin light-chain kinase in flagellar activity (Ashizawa et al., 1995). Data are presented showing that the peptide syntide-2, a substrate designed for selectivity toward CaM kinase II (Hashimoto and Soderling, 1987), selectively inhibits the GA response in aleurone cells while leaving constitutive and ABA-regulated events unaffected. This peptide is phosphorylated by a soluble, 54-kD Ca²⁺-activated protein kinase with which it appears to interact in vivo.

	MATERIALS AND METHODS

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Protoplast Isolation

Embedding Protoplasts for Microinjection and Monitoring -Amylase Secretion from Individual Protoplasts

Microinjection of Protoplasts

Protoplasts were allowed to recover from the microinjection for 20 min before additional imaging was performed. Protoplasts that failed to maintain a turgid appearance, that exhibited disruption of normal cytoplasmic structure (typically a rapid condensation of the cytoplasm), or that failed to exhibit more than 500 counts per second LUC activity in the transient-expression experiments were not studied further. Using these criteria, the efficiency of microinjection was approximately 20%.

Confocal Microscopy and Measurement of Cytoplasmic [Ca²⁺]

FITC-dextran was visualized with the LSM-410 confocal microscope using 488-nm excitation, 488 dichroic, and 510- to 540-nm emission, selected using Zeiss interference filters. 4,6-Diamidino-2-phenylindole staining (Schuurink et al., 1996) and N-phenyl-1-naphthylamine staining (Saunders and Hepler, 1981) were visualized using 354-nm excitation, 80/20 beam splitter, and >460-nm emission. 2,7-bis-(2-Carboxyethyl)-5-(and 6)carboxyfluorescein, acetoxymethyl ester staining was performed as described by Swanson and Jones (1996) and visualized using 488-nm excitation, 488 dichroic, and 510- to 540-nm emission.

Fluorescent and Photoaffinity Labeling of Peptides

To generate photoaffinity forms of syntide-2 and malantide, the fluorescently labeled peptides were stirred for 4 h in the dark with benzophenone-isothiocyanate (Molecular Probes, Eugene, OR) at a 10:1 molar excess in PBS, pH 8.0 (Miller, 1991; Banks and Paquette, 1995). Labeled peptide was purified by preparative TLC as described above.

Protoplast Electroporation

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Kinase Extraction

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Kinase Assays

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Assays of autophosphorylation activities of aleurone extracts were performed as described above, except that the final volume was 50 µL, the [-³²P]ATP added was 100 nm (10,000 cpm pmol¹), and no exogenous substrate (histone or peptide) was added. Samples were incubated at room temperature for 2 min and then TCA was added to give a final concentration of 10% (w/v). After incubation on ice for 2 h the samples were centrifuged at 1,000g in a bench-top centrifuge (Tomy Kogyo Co. Ltd., Fukushima, Japan) for 10 min and the supernatants removed. The tubes were dried and 2 µL of 1 n NaOH was added. SDS-gel electrophoresis was performed using 12% polyacrylamide gels with either a Protean II or a mini-Protean II gel system (Bio-Rad) according to the manufacturer's instructions. After electrophoresis, gels were stained with Coomassie blue, sealed in polyethylene bags, and exposed to XAR-5 autoradiographic film (Kodak) overnight before being developed. In-gel kinase assays were performed as described by Kameshita and Fujisawa (1989), except that we used 12% SDS-PAGE gels, urea as the denaturing agent, and no protein was supplemented in the gel matrix.

Protein concentration was determined by the method of Bradford (1976) using BSA as a standard and the Bio-Rad protein assay kit.

	RESULTS

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Microinjection of Syntide-2 Selectively Inhibits the GA Response of Aleurone Protoplasts

Cereal aleurone cells underwent a series of well-characterized responses to GA. Expression of -amylase genes was strongly up-regulated by GA, and this can be monitored at the single-cell level using an AMY-GUS transient expression assay (Gilroy, 1996). Protoplasts were simultaneously microinjected with a plasmid containing the promoter of -amylase gene fused to a GUS gene, together with a LUC gene that has a ubiquitin promoter (and that is constitutively expressed) (Gilroy, 1996). The ratio of GUS:LUC reflects the level to which the amylase promoter was being induced by GA. The second assay we used to assess the GA response monitored the -amylase secreted by GA-responding protoplasts. -Amylase secretion can also be assayed on a single-cell basis using a starch gel assay (Hillmer et al., 1993). The third parameter monitored in the aleurone protoplasts was the development of large vacuoles characteristic of the GA response (Bush et al., 1986). Figure 1, A and B, shows that none of the microinjected peptides induced GA-like responses in protoplasts. Only syntide-2 inhibited the response of aleurone protoplasts to GA at concentrations > 25 µm (Fig. 1C). The continued GA response in protoplasts injected with peptides other than syntide, or injected with BSA, indicates that neither the microinjection procedure nor the introduction of exogenous protein into the cytoplasm inhibited the GA response. Protoplasts injected with syntide-2 remained turgid and did not exhibit the condensation of cytoplasm associated with cytotoxicity for as long as we observed them (48 h).

View larger version (39K): [in this window] [in a new window]   Figure 1. The effect of microinjection of protein kinase peptide substrates on GA-induced amylase secretion, vacuolation, and amylase-GUS and EM-GUS expression. A and B, Freshly isolated protoplasts were microinjected with 50 µm of the indicated protein kinase substrate peptides or BSA and then treated for 24 h with (+GA) or without (GA) 5 µm GA. C, Freshly isolated protoplasts were microinjected with a range of syntide-2 concentrations and then treated for 24 h with 5 µm GA. Secretion of amylase, development of GA-induced vacuolated morphology, and amylase-GUS expression were then assessed on a single-cell basis. D, Freshly isolated protoplasts were co-microinjected with 50 µm syntide-2 or BSA and the EM-GUS construct and treated with or without 10 µm ABA. Induction of EM-GUS expression was then assessed on a single-cell basis. Protoplasts were scored as showing induction of amylase-GUS or EM-GUS expression if they exhibited a GUS:LUC of more than 4000 (Gilroy, 1996). Protoplasts were scored as secreting amylase if they showed a cleared "halo" of >25 µm in the starch thin-film assay (Hillmer et al., 1992). Vacuolated protoplasts were those showing development to stage 3 or 4 as defined by Bush et al. (1986). The responses of at least nine microinjected protoplasts per treatment are shown.

Microinjection of syntide-2 did not inhibit ABA-regulated or constitutive gene expression. Protoplasts injected with syntide-2 showed expression of the constitutive ubiquitin-LUC construct in transient-expression assays to levels similar (>500 counts per second) to what was seen in nonsyntide-2-injected controls (data not shown). ABA-regulated gene expression was monitored using a construct of the promoter of the ABA-responsive EM gene fused to a GUS gene (Jacobsen and Close, 1991; Gilroy, 1996). Figure 1D shows that in response to ABA, syntide-2-injected protoplasts exhibited levels of induction of the Em promoter to levels equivalent to those seen in noninjected or BSA-injected controls.

Syntide-2 Does Not Prevent Changes in Cytosolic Ca²⁺

View larger version (22K): [in this window] [in a new window]   Figure 2. The effect of microinjection of syntide-2 on GA-induced cytoplasmic Ca2+ elevation. Freshly isolated protoplasts were microinjected with dextran-conjugated indo-1, with or without 50 µm syntide-2. These protoplasts were then treated without hormone (control) or with 5 µm GA and their cytoplasmic [Ca2+] was monitored at the indicated times using confocal ratio imaging. The results represent the mean ± se of at least nine microinjected protoplasts per treatment.

Microinjection Loads Syntide-2 into the Cytosol of Aleurone Protoplasts

View larger version (94K): [in this window] [in a new window]   Figure 3. Cytoplasmic distribution of fluorescently labeled syntide-2 loaded into protoplasts by microinjection. A, Fluorescence from a protoplast microinjected with FITC-dextran. B, Fluorescence from a protoplast microinjected with 1 µm cytoplasmic concentration of fluorescently labeled syntide-2. C, Membranous components of protoplasts visualized by incubation with 5 µm N-phenyl-1-naphthylamine. D, Nuclear fluorescence from a protoplast stained with 4,6-diamidino-2-phenylindole. E, Fluorescence from vacuoles labeled with 2,7-bis-(2-carboxyethyl)-5-(and 6)carboxyfluorescein, acetoxymethyl ester. Freshly isolated protoplasts were microinjected with FITC-labeled 10-kD dextran fluorescein (A) or fluorescein labeled syntide-2 (B), treated for 24 h with 5 µm GA, and then fluorescence visualized using the Zeiss LSM-410 confocal microscope. C to E, Protoplasts were treated for 24 h with 5 µm GA and then stained for the appropriate compartment. Results are representative of more than 10 replicates. v, Vacuole; n, nucleus. The scale bar represents 10 µm.

Interactions between Syntide-2 and Aleurone Proteins in Vivo

View larger version (43K): [in this window] [in a new window]   Figure 4. The effect of electroporation of protein kinase substrate peptides on GA-induced amylase secretion. Freshly isolated protoplasts were loaded with 25 µm syntide-2, malantide, or BSA by electroporation and treated for 48 h with or without 5 µm GA. Secreted amylase activity was then assessed. A set of nonelectroporated control protoplasts was treated identically. Results represent the mean ± se of three separate experiments.

Having established that protoplasts loaded with syntide-2 by electroporation showed the expected inhibition of the GA response (Fig. 4), we then used this as a tool in combination with a photoaffinity technique to label the proteins targeted by syntide-2. Syntide-2 or malantide were fluorescently tagged and then covalently labeled with the UV-activated cross-linking agent benzophenone-4-isothiocyanate. When irradiated with UV, this agent cross-links the peptide and whatever molecule it is interacting with in vivo. Protoplasts were loaded with this photoaffinity, fluorescent syntide-2 or malantide by electroporation.

Figure 5 (lanes 1 and 2) shows that when the above procedure was performed without subsequent UV illumination, no cross-linking was evident and little protein labeling could be observed after SDS-PAGE of the protoplast extracts. However, with UV activation, both photoaffinity-labeled malantide and syntide-2 became cross-linked to a range of proteins. Most of these proteins were labeled identically by both syntide-2 and malantide. Because malantide is not inhibitory to the GA response (Figs. 1A and 4), the proteins labeled in common between syntide and malantide probably reflect nonspecific interactions with aleurone cell components. However, 33- and 41-kD proteins were selectively labeled by malantide, whereas syntide-2 showed enhanced cross-linking to 30- and 55-kD proteins. The proteins showing selective interaction (labeling) with syntide-2 represent tentative in vivo candidates for the site of action of syntide-2. UV irradiation of unelectroporated protoplasts incubated in the photoaffinity syntide-2 or malantide showed no detectable labeling of protoplast proteins (data not shown). This indicates that the labeled proteins seen in Figure 5 (lanes 3 and 4) are most likely intracellular in origin.

View larger version (98K): [in this window] [in a new window]   Figure 5. Fluorograph of proteins labeled in vivo by photoaffinity syntide-2 or photoaffinity malantide. Protoplasts were electroporated with fluorescent, photoaffinity-labeled syntide-2 or malantide and allowed to recover for 1 h. The protoplasts were then frozen in liquid N2 to freeze molecular interactions, and total proteins were extracted in the dark (lanes 1 and 2). In parallel experiments the peptide-loaded protoplasts were UV irradiated to cross-link the photoaffinity peptides to closely associated proteins and then extracted (lanes 3 and 4). Proteins were then separated by SDS-PAGE. Peptide-labeled proteins were visualized using the fluorescein attached to the photoaffinity peptides. Note the 30- and 55-kD proteins selectively labeled by syntide-2 (arrows) and the 33- and 41-kD specific to malantide (stars). The positions of molecular mass markers are indicated in kilodaltons on the left.

Biochemical Identification of the Target of Syntide-2

View larger version (40K): [in this window] [in a new window]   Figure 6. The inhibition of histone and endogenous protein phosphorylation by syntide-2. Syntide-2 was added to in vitro substrate phosphorylation assays carried out without (A) or with (B) 25 µm histone. The samples were precipitated with 10% TCA and processed for SDS-PAGE and autoradiography. C, Phosphorylation levels from the 70- and 40-kD endogenous substrate bands and histone was assessed by cutting sections of the gel that corresponded to bands on the autoradiograph, and counting duplicate samples in a scintillation counter. Results are typical of two independent experiments. , Histone; , 70 kD; , 40 kD.

We next tested whether syntide-2 was a substrate of aleurone kinase(s). The same range of kinase substrate peptides that was microinjected into protoplasts (Fig. 1A), as well as histone, was assayed for phosphorylation in vitro for protein kinase assays by crude aleurone extracts. Syntide-2 and histone were phosphorylated in a Ca²⁺-activated manner, the activity being predominantly in the soluble fraction (Fig. 7A). The other peptides were phosphorylated to much lesser extents even when assayed for much longer times (up to 20 min; data not shown). None of the other peptides showed Ca²⁺-activated phosphorylation. The Michaelis-Menten kinetics of syntide-2 phosphorylation (Fig. 7B) suggest a kinase activity with an apparent K_m of 8 µm for syntide-2.

View larger version (17K): [in this window] [in a new window]   Figure 7. Peptide phosphorylation activity in barley aleurone extract. A, Microsomal (pellet) and soluble (supernatant) fractions were prepared and their kinase activity assessed using 25 µm substrate peptides or histone. Assays were carried out in the presence of 50 µm CaCl2 (+Ca) or 1 mm EGTA (Ca). B, A range of syntide-2 concentrations was used on the soluble fraction in assays containing 50 µm CaCl2, and the level of 32P incorporation into the peptide during the initial 2 min of reaction was calculated. The reaction rate was linear over this time period. The results are the mean ± se of two experiments, with two replicates per experiment.

Many protein kinases show a highly active autophosphorylating activity. Therefore, we used this property as an initial screen for candidate protein kinases for the syntide-2 phosphorylating activity in extracts of aleurone layers. The same autophosphorylation profile was seen when proteins were extracted from protoplasts (data not shown) or intact tissue (aleurone layers). Layers were therefore routinely used for biochemical experiments. Figure 8A shows that two major autophosphorylating bands of 54 and 68 kD were visible on autoradiographs. Fainter signals at around 60 and 50 kD were sometimes visible, but they varied in relative intensity between experiments. The degree of syntide-2 phosphorylation did not change between experiments, regardless of whether or not these intermediate bands were visible on autoradiographs. Autophosphorylation of the 54-kD band was Ca²⁺ activated, whereas that of the 68-kD band was not (Fig. 8B). The 54-kD activity was found predominantly in the soluble fraction, whereas the 68-kD activity was found in both soluble and membrane-associated fractions. When autophosphorylation was assayed "in gel," the only kinase activity detected was that of a Ca²⁺-dependent 54-kD protein (Fig. 8C). The pattern of autophosphorylation was identical in samples extracted from aleurone layers that had been treated with CaCl₂, GA, or ABA for up to 24 h.

View larger version (45K): [in this window] [in a new window]   Figure 8. Autophosphorylation characteristics of barley aleurone kinase activities. A, Autophosphorylating activities of kinases in soluble or microsomal fractions extracted from aleurone layers. Assays were carried out in the presence of 50 µm CaCl2 using 50 µg of protein per fraction. B, Autophosphorylation activities of kinases in a crude fraction were carried out in the presence of 50 µm CaCl2 (+) or 500 µm EGTA (). C, Aleurone layers were treated for 24 h with no hormones (), 5 µm GA, or 5 µm ABA. Crude extracts of proteins were then prepared, run on 12% SDS-PAGE, renatured, and assayed for autophosphorylating activity in gel. For these assays incubation with [32P]ATP at a concentration of 1 µm (50,000 dpm pmol1) was carried out in the presence of 50 µm CaCl2 (+Ca) or 1 mm EGTA (Ca). Note that only the 54-kD kinase is renatured under these conditions. The positions of molecular mass markers are indicated in kilodaltons on the left.

The CaM antagonist W7 (250 µm) inhibited autophosphorylation of the 54-kD kinase and syntide-2 phosphorylation (Fig. 9), whereas the much-less-active analog N-(6-aminohexyl)-1-naphthalene-sulfonamide (W5) had no effect on either parameter (data not shown). W7 did not affect autophosphorylation of the 68-kD kinase. Exogenous spinach or bovine CaM had no effect on autophosphorylation or syntide-2 phosphorylation.

View larger version (29K): [in this window] [in a new window]   Figure 9. The effect of protein kinase inhibitors, CaM antagonist W7, and exogenous CaM on autophosphorylation and syntide-2 phosphorylation activities in the barley aleurone. Four protein kinase inhibitors, W7, or CaM (bv, bovine; sp, spinach [Sigma]) were added to autophosphorylation and syntide-2 phosphorylation assays at the indicated concentrations. Aleurone layers were extracted and assays were carried out in the presence of 50 µm CaCl2 as described in ``Materials and Methods''. A, Autophosphorylation of the 68- and 54-kD proteins was visualized by autoradiography; B, the effect of protein kinase inhibitors on syntide-2 phosphorylation expressed as a percentage of control (no inhibitor) is shown below the appropriate inhibitor lane on the autoradiograph. The inhibitors used were obtained from Calbiochem: H89, specific for cAMP-dependent protein kinase; KN93, specific for Ca2+/CaM-dependent protein kinase II; bisindolylmaleimide (bis), specific for protein kinase C; and 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7), a broad-range Ser/Thr kinase inhibitor. The experiments were repeated twice with two replicates each. The largest se for syntide-2 phosphorylation was 12%.

A range of protein kinase inhibitors was also added to syntide-2 phosphorylation and autophosphorylation assays. Figure 9 shows that all of the inhibitors used (H89, bisindolylmaleimide, and 1-(5-isoquinolinesulfonyl)-2-methylpiperazine) (H7), except KN93, inhibited syntide-2 phosphorylation at high concentrations (100 µm). KN93 did not affect syntide-2 phosphorylation or autophosphorylation of the 54-kD band but it partially affected autophosphorylation of the 68-kD band. Conversely, H89 had no affect on autophosphorylation of the 68-kD band, whereas it did inhibit autophosphorylation of the 54-kD band. H89 also inhibited syntide-2 phosphorylation with an IC₅₀ of 25 µm.

	DISCUSSION

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The high selectivity of protein kinases for their substrate peptides allowed us to use these peptides as inhibitors of kinase action in vivo. We reasoned that the phosphorylation of the synthetic peptide by a kinase would competitively inhibit the phosphorylation of the "normal" in vivo target, and hence block the effect of the in vivo substrate phosphorylation. In aleurone we found that microinjection or electroporation of syntide-2, but not the other kinase substrate peptides tested, led to a reduction in the amylase secretion, the vacuolation, and the activation of amylase gene transcription normally associated with the GA response. Constitutive gene expression (ubiquitin-LUC) and the ABA response (induction of EM-GUS) were unaffected by concentrations of syntide-2 that inhibited the GA response. These results suggest that this peptide is highly selective for affecting transduction of the GA signal, possibly through competitive inhibition of an aleurone protein kinase.

GA causes an elevation of cytosolic [Ca²⁺] in aleurone (Bush and Jones, 1987, 1988; Gilroy and Jones, 1992; Bush, 1996; Gilroy, 1996), which is essential for maintaining secretory activity in these cells (Gilroy, 1996). This increase was not detectably altered by the microinjected syntide-2, suggesting that an event downstream of the GA-regulated Ca²⁺ signal was affected by the peptide. Although we cannot determine from this result whether syntide-2 is inhibiting a Ca²⁺-dependent step in the GA response, this is a strong possibility. In barley aleurone we have found that syntide-2 phosphorylation was enhanced approximately 6-fold in the presence of CaCl₂. Syntide-2 was designed as a selective substrate for mammalian CaM kinase II (Hashimoto and Soderling, 1987). Very few CaM-modulated protein phosphorylation activities have been found in plants (Roberts and Harmon, 1992), and a plant CaM kinase II homolog has not been reported. However, a family of plant Ca²⁺-regulated protein kinases (CDPKs) containing an endogenous CaM-like domain has been characterized from a range of plant sources (e.g. Harper et al., 1991; Roberts and Harmon, 1992; DasGupta, 1994; Saha and Singh, 1995; Furumoto et al., 1996) and two putative CDPKs were found in a PCR-based screen of kinases in mRNA from oat aleurone (Huttly and Phillips, 1995). Proposed sites of Ca²⁺-regulated events in the aleurone cell include fluxes into the ER to sustain amylase production (Bush et al., 1993; Gilroy and Jones, 1993), regulation of vacuolar ion channel activity (Bethke and Jones, 1994), and targeting and fusion of secretory vesicles with the plasma membrane (Zorec and Tester, 1992). Exocytosis in plant cells has been proposed to require elevated Ca²⁺ localized at the zone of vesicle fusion, where Ca²⁺-dependent lipid-binding proteins, such as annexins, may play a critical role in the exocytotic event (Battey and Blackbourn, 1993). It is possible the 54-kD kinase may act to regulate one of these activities.

The kinase inhibitors KN93 and H89 revealed that inhibition of autophosphorylation of the 54-kD band was closely correlated with inhibition of syntide-2 phosphorylation, suggesting that the 54-kD kinase may be the Ca²⁺-dependent syntide-2 phosphorylating activity in vivo. Thus, KN93 failed to inhibit both autophosphorylation of the 54-kD protein and syntide-2 phosphorylation but had a partial effect on the autophosphorylation of the 68-kD kinase. Conversely, H89 left the 68-kD band unaffected but inhibited both syntide-2 phosphorylation and autophosphorylation of the 54-kD band. The in vivo photoaffinity labeling of a 55-kD protein by syntide-2 (Fig. 5) is noteworthy in this context, because this labeled protein has a similar molecular mass to that predicted for the 54-kD syntide-2 kinase coupled to syntide-2. Peptide-based photoaffinity probes have previously been used to successfully photoaffinity label protein kinases for which they are a specific substrate (Miller, 1991). These data led us to the hypothesis that the 54-kD kinase activity was responsible for the phosphorylation of syntide-2 in vitro and, hence, was a possible target for the effect of syntide-2 on the GA response in vivo. This idea is reinforced by the observation that in vitro, syntide-2 competed with endogenous substrates for phosphorylation by the Ca²⁺-activated kinase at a concentration range (>10 µm) similar to that which affected the GA response when microinjected into protoplasts in vivo.

The precise identity of the 54-kD kinase and the molecular basis of its Ca²⁺ activation remain to be determined. Although the Ca²⁺ dependence of many animal kinases is mediated by CaM (Hanson and Schulman, 1992), these CaM-activated processes are generally inhibited by the CaM antagonist W7 in the range of 0.1 to 10 µm. Proteins containing a CaM-like domain, e.g. CDPK, often require 100 to 200 µm W7 for inhibition (Roberts and Harmon, 1992). W7 inhibited the 54-kD kinase activity with an IC₅₀ of 250 µm, suggesting that the enzyme contains a CaM-like domain (Roberts and Harmon, 1992) consistent with its in-gel Ca²⁺ activation (Fig. 8C). The Ca²⁺ activation of this kinase probably does not represent tightly bound co-purifying CaM because this CaM would have had to remain attached to the kinase despite extraction in 1 mm EDTA and SDS-PAGE. Spinach or bovine brain CaM had no effect on either autophosphorylation or histone phosphorylation activity of the 54-kD kinase, further suggesting that the kinase is of the CDPK class. Both the spinach and bovine brain CaM used in these experiments activated cyclic nucleotide phosphodiesterase (assayed according to the method of Cheung [1971]; data not shown). Additionally, bovine CaM enhances the activity of an aleurone Ca²⁺-activated phosphatase (data not shown) and the Ca²⁺-ATPase of the aleurone ER (Gilroy and Jones, 1993), and spinach CaM has been shown to alter the ABA response of aleurone protoplasts (Gilroy, 1996) and to regulate ion channels at the aleurone tonoplast (Bethke and Jones, 1994). Thus, it is unlikely that the failure of these CaMs to activate the 54-kD kinase can be attributed to a general inactivity toward CaM-regulated events or a lack of potential to influence aleurone enzymes.

Despite this circumstantial evidence that the 54-kD kinase is of the CDPK class, no proteins in crude, soluble, or microsomal fractions cross-reacted with antibody against soybean CDPK (A.C. Harmon and S. Gilroy, unpublished data). Furthermore, the kinase does not exhibit the Ca²⁺-dependent mobility shift on SDS-PAGE characteristic of CDPK (data not shown). Since the discovery and characterization of CDPK from soybean (Putnam-Evans et al., 1990; Harper et al., 1991), several kinases have been identified, forming a family of CDPK-like activities (Hrabak et al., 1996, and refs. therein). However, not all CDPKs share the same characteristics. For example, in Arabidopsis CDPKs with varying numbers of EF hand Ca²⁺-binding sites have been characterized (Hrabak et al., 1996). Similarly, a carrot kinase that contains the CaM-like domain of CDPK did not require Ca²⁺ for maximum activity when expressed in Escherichia coli (Furumoto et al., 1996), and CDPK-like proteins from potato tubers (MacIntosh et al., 1996) and groundnut (DasGupta, 1994) were both recognized by an anti-soybean CDPK monoclonal antibody but did not show Ca²⁺-dependent mobility shift on SDS-PAGE. Thus, the failure of the 54-kD kinase to exhibit all of the characteristics of classic CDPKs does not preclude it from being a CaM-like domain kinase. Current research is aimed at defining the precise molecular identity of this kinase.

	FOOTNOTES

   Received August 15, 1997; accepted November 3, 1997.

	ABBREVIATIONS

Abbreviations: CaM, calmodulin. CDPK, calmodulin-like domain protein kinase. FITC, fluorescein isothiocyanate. H89, N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide. IC₅₀, inhibitor concentration for 50% inhibition. KN93, N-[2-(N(4-chlorocinnamyl)-N-methylaminomethyl)phenyl]-N-[2-hydroxyethyl]-4-methoxybenzenesulfonamide) . LUC, luciferase. W7, N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide.

	ACKNOWLEDGMENT

The authors thank Elison Blancaflor for critical reading of the manuscript.

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