lnvolvement of de Novo Protein Synthesis, Protein Kinase ... - NCBI

Plant Physiol. (1994) 104: 1237-1244

lnvolvement of de Novo Protein Synthesis, Protein Kinase, Extracellular Ca2+, and Lipoxygenase in Arachidonic Acid lnduction of 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Genes and lsoprenoid Accumulation in Potato (So/anum tuberosum 1.)’ Doi1 Choi* and Richard M. Bostock*

Department of Plant Pathology, University of California, Davis, California 9561 6

1990; Grosskopf et al., 1990; Conrath et al., 1991; Felix et al., 1991; Vogeli et al., 1992). Rapid responses to elicitors include perturbation of ion channels resulting primarily in K+ efflux and Ca2+and H+influx (Atkinson et al., 1990; Felix et al., 1991), generation of active oxygen species (Doke et al., 1991; Legendre et al., 1992), and ethylene production (Felix et al., 1991). Shortly following or, in some cases, concomitantly with these rapid responses is the induction of secondary metabolic pathways that lead to the biosynthesis of phytoalexins and cell wall polymers such as lignin (Bostock and Stermer, 1989; Dixon and Lamb, 1990) and pathogenesis-related proteins (Raz and Fluhr, 1992). These latter responses include the transcriptional activation of genes encoding the necessary biosynthetic enzymes (Dixon and Lamb, 1990) but also may include nontranscriptional events such as the recruitment of phytoalexin precursors from existing pools (Graham et al., 1990). The linkage of plant-generated signaling systems such as CaZ+mobilization and protein phosphorylation to the changes observed in secondary metabolism following elicitor treatment is poorly understood. Changes in protein phosphorylation pattems have been reported following elicitor treatment of plant tissues, and these changes have been proposed as early events in the activation of plant defense responses (Dietrich et al., 1990; Felix et al., 1991). Phosphorylation of plant plasma membrane proteins was observed within minutes following treatment of the membranes with inducers of proteinase inhibitors (Farmer et al., 1989, 1991). A protein kinase inhibitor, K252a, inhibited the induction of Phe ammonia-lyase activity and ethylene biosynthesis in tomato cells following treatment with a yeast extract elicitor and also inhibited microsomal protein kinase activities (Grosskopf et al., 1990). This inhibitor also suppressed elicitor-inducedCaZ+uptake and K+ efflux in parsley cells (Conrath et al., 1991). Treatment of tomato cell cultures with K-252a and another kinase inhibitor, staurosporin, blocked elicitor-induced ethylene production and extemal alkaliization and prevented elicitor-induced

A series of inhibitors were tested to determine the participation of de novo protein synthesis, protein kinase activity, extracellular Ca’+, and lipoxygenaseadivity in arachidonic acid elicitation of 3hydroxy-3-methylglutaryl coenzyme A reductase (HMCR) gene expression and sesquiterpene phytoalexin biosynthesis in potato (Solanum tuberosum 1. cv Kennebec). Cene-specific probes were used to discriminate effects on the expression of two HMCR genes (hmgl and hmg2) that respond differentially in tuber tissue following wounding or elicitor treatment. lnhibition of protein synthesis with cycloheximide completely blocked arachidonate-induced hypersensitive necrosis and browning, including HMCR gene induction and phytoalexin accumulation. This suggests that proteins necessary for coupling arachidonic acid reception to HMGR mRNA accumulation are either rapidly turned over or not present constitutively and are induced following elicitor treatment. Staurosporin, a potent inhibitor of protein kinases, and ethyleneglycol-bis(& aminoethyl ether)-N,N’-tetraacetic acid, a Ca2+chelator, inhibited arachidonate-indudion of hmg2 gene expression and phytoalexin accumulation but did not inhibit the wound-induced expression of hmgl. However, staurosporin inhibited arachidonate’s suppression of hmgl gene expression. Eicosatetraynoic acid, a lipoxygenase inhibitor that suppresses elicitor-induced phytoalexin accumulation, also inhibited arachidonate’s suppression of hmgl and induction of hmg2. The results indicate that arachidonate’s suppression of hmgl and adivation of hmg2 depend on a common intermediate or set of intermediates whose generation i s sensitive to the inhibitors tested.

The study of elicitor action provides a paradigm for understanding the signaling that leads to the hypersensitive response and the activation of defense-related metabolism in plant-pathogen interaction. The early biochemical and molecular events that occur following elicitor treatment of plants are being investigated in severa1 systems (Rogers et al., 1988; Bostock and Stermer, 1989; Farmer et al., 1989, 1991; Peever and Higgins, 1989; Atkinson et al., 1990; Dixon and Lamb, ~

’ D.C. was supported in part by a Korean Govemment Overseas Scholarship. Research was supported by U.S. Department of Agriculture grants 88-37151-3656 and 91-37303-6504. Present address: U.S. Department of Agriculture Plant Gene Expression Center, 800 Buchanan St., Albany, CA 94710. * Corresponding author; fax 1-916-752-5674.

Abbreviations: AA, arachidonic acid; ETYA, eicosatetraynoic acid; HMGR, 3-hydroxy-3-methylglutaryl coenzyme A reductase; nPG, npropyl gallate; SGA, steroid glycoalkaloid, SHAM, salicyl hydroxamic acid. 1237

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changes in the pattem of protein phosphorylation (Felix et al., 1991). Recently, GTP-binding proteins were suggested to be involved in the rapid oxidative burst in cultured soybean cells treated with oligogalacturonides (Legendre et al., 1992). Calcium also has been proposed as an important intermediary signal in activation of defense-related responses in plants (Raz and Fluhr, 1992).Since cytoplasmic calcium levels are maintained at concentrations that are 104 to 105 times lower than extracellular levels (Gilroy et al., 1986), the release of free Ca2+from interna1 stores in the cytoplasm or its influx from extemal sources could have a profound effect on cellular metabolism. The increase in cytosolic Ca2+has been documented as an early and transient event in elicitor-treated plant cells, a key finding that lends credence to its proposed role as a second messenger in defense-related responses (Knight et al., 1991). Caz+ chelators and channel blockers have been used to demonstrate the involvement of Ca2+in elicitation of sesquiterpenoid phytoalexin synthesis in potato (Zook et al., 1987)and tobacco (Vogeli et al., 1992),electrolyte leakage and hypersensitivity expression caused by bacterial pathogens in tobacco cells (Atkinsonet al., 1990), and elicitorinducible, ethylene-dependent responses in tobacco (Raz and Fluhr, 1992). Lipid peroxidation also is an early event in elicitor-treated cells (Bostock et al., 1986; Rogers et al., 1988) and likely accounts for some of the changes observed in membrane structure and function. Although the peroxidation of membrane fatty acids occurs by severa1 mechanisms (Elstner, 1982), the reaction of polyunsaturated fatty acids with lipoxygenases can be a major source of these peroxides in injured plant tissue (Siedow, 1991). Peroxidized fatty acids are highly reactive and can be further metabolized to signal molecules such as the jasmonates (Hildebrand, 1989). Lipoxygenase activity is induced in elicitor-treated plant tissues (Peever and Higgins, 1989; Rickauer et al., 1990; Bostock et al., 1992), and treatment of potato tissue with lipoxygenase inhibitors suppresses AA-induced phytoalexin accumulation (Stelzig et al., 1983; Preisig and KuC, 1987). These results, the structural requirements of fatty acids for elicitor activity in potato (Bostock et al., 1981; Preisig and Kut, 1985), and the insensitivity of lipoxygenase-deficient potato cell lines to AA (Vaughn and Lulai, 1992) suggest that lipoxygenase participates in the coupling of elicitor reception and activation of defense-related isoprenoid metabolism. Interpretation of a number of the studies mentioned above is complicated by the somewhat crude nature of the elicitors (e.g. fungal cell wall preparations) used to effect the responses, and therefore, it is unclear whether the observed responses are specific to the elicitor active components in the preparations responsiblefor phytoalexin induction or to other components that may stimulate, in a somewhat nonspecific manner, wound or other responses that may not be directly linked to phytoalexin induction. In this study, cloned potato HMGR cDNAs corresponding to different HMGR genes (hmgl and hmg2) were used as probes to characterize components of the signal transduction pathways that lead to induction of HMGR gene expression and antimicrobial isoprenoid accumulationfollowing wounding or treatment of potato tissue with AA, a well-characterized fungal elicitor. In this system, hmgl is strongly induced

Plant Physiol. Vol. 104, 1994

by wounding but is suppressed by AA, whereas hmg2 is strongly induced by AA (Choi et al., 1992). Our current model places the reductase encoded by hmgl within a channel leading to sterols and SGAs associated with wound healing and the reductase encoded by hmg2 in a channel leading to sesquiterpenoid phytoalexins associated with hypersensitive cell necrosis. Because of the clear differential response of HMGR isoforms following wounding and elicitor treatment, the gene-specific probes provided a discriminating analysis of inhibitor effects on the wound and elicitor response pathways in this system. MATERIALS A N D METHODS Chemicals, Plant Materiais, and Treatment Protocols

Certified seed grade potatoes (Solanum t u b e m u m L. cv Kennebec) were stored at 4 O C until24 h before use, and discs (22 x 5 mm) were prepared as described earlier (Bostock et al., 1981). Inhibitors were used at concentrations demonstrated in previous studies (Bostock et al., 1986; Preisig and Kut, 1987; Zook and Kut, 1987) or during the current study to strorigly inhibit arachidonate-induced phytoalexin accumulation in this system. Cycloheximide was frorn Sigma and was freshly prepared in 50 m Mes buffer (pH 5.8), containing 300 mM mannitol and 40 mM CaC12. Cycloheximide was added to a final concentration of 30 p g mL-' (approximately 100 ,UM).Potato discs were treated with the cycloheximide solution (about 20 discs/lOO mL) by immersion for 30 min in a beaker with shaking (100 rpm) at room temperature. The discs were removed and placed in Petri dishcs, and then treatments were applied to the upper surface as indicated. In some experiments, the discs were aged at 20°C for 18 h prior to treatments. In a11 experiments, the treatecl discs were incubated at 2OoC in the dark until extraction. The protein kinase inhibitor staurosporin (Tamaoki et al., 1986; Sigma) was prepared as a 1m stock solution in DMSO and applied by immersing potato discs for 30 min with shaking (1O0 rpm) in a 1 ,UMsolution (O. 1% DMSO in distilled water). Felix et al. (1991) found the 50% effective dose for staurosporin inhibition of elicitor-induced responses in tomato suspension cells to be approximately 200 nM, with nearly complete inhibition at 1 ,UM. We selected the latter concentration because our previous experience with other inhibitors indicated that responses in potato discs generally require a higher concentration of an inhibitor to exert an effect than that reported to inhibit responses in cell suspensions (Bostock et al., 1986). Control discs were immersed in an aqueous solution containing 0.1% DMSO. For EGTA treatmients, potato discs were immersed immediately after their preparation from tubers for 30 min with shaking in a 2 m EGTA solution in 25 mM Mops buffer (pH 7.4). Control discs were immersed similarly in Mops buffer. The lipoxygenase inhibitor ETYA (Cayman Chemical Co., Ann Arbor, MI) was prepared as a 59" mL-' stock solution in absolute ethand. Solutions of ETYA (1 and 5 m) were prepared immediately prior to use according to the nianufacturer's directions by diluting the stock solution with 100 rrw Tris (pH 8.0) so that the final concentration of ethanol did not exceed 5%. This solution (50 pL) was appliecl to the upper

Arachidonic Acid Signal Transduction in Potato

surface of each disc immediately after preparation. The second treatment was applied to the same surface within 30 min. Control discs were treated with 50 pL of Tris buffer. AA (Sigma) was prepared and applied to discs as described previously (Bostock et al., 1981). A11 comparisons of inhibitor effects on potato responses were made with respect to the appropriate buffer controls. Estimation of Net Protein Synthesis

Net protein synthesis in potato discs was determined by the method of Stermer and Bostock (1989). [3H]Leu (1 pCi of 1 mCi mL-'; 155 mCi pmol-'; Amersham) was mixed with nonradioactive Leu (10 m)in 10 pL and applied to potato discs that had been treated with buffer or buffer containing cycloheximide as described above. Discs were labeled for 1 h at room temperature, and the incorporation of 13H]Leuinto proteins was determined as described previously (Stermer and Bostock, 1989). Effed of Staurosporin on Phosphorylation of Potato Microsomal Membrane Proteins

Potato tuber microsomes were isolated according to the method of Stermer and Bostock (1987). Phosphorylation reactions were carried out with 1 pg of microsomal proteins in 15 pL by the method of Farmer et al. (1989). Incorporation of radioactivity into microsomal proteins was measured by slot filtration and scintillation counting according to the method of Volonte et al. (1992). Staurosporin was added to the reaction mixture to a final concentration of 1 PM. Probe Preparation, RNA Extraction, and RNA Cel Blot Analyses

DNA probes were prepared from potato HMG-COAreductase cDNAs as described earlier (Choi et al., 1992). A probe corresponding to a region that is highly conserved among HMGR genes was prepared by ScaI-NcoI digestion of the hmg3 cDNA insert (Choi et al., 1992). Gene-specific probes were prepared by PCR using oligonucleotide primers based on the determined cDNA sequences (5' region of hmgl and 3' region of hmg2). Each cDNA was amplified in 100 pL under mineral oil with 10 m Tris-HC1 (pH 8.3) at 25OC, 50 m KCl, 1.5 m MgC12, 0.01% gelatin, 200 pg mL-' BSA, 0.2 mM each deoxynucleotide triphosphate, 1p~ each specific primer, and 2.5 units of Taq polymerase (Perkin-Elmer Cetus). The PCR products were separated in 1.0% low-gelling temperature agarose (FMC, Inc., Rockland, ME) in Tris-borate EDTA buffer. Target fragments were excised from gels and purified using the Gene Clean Kit (Bio 101, Inc., Rockland, ME). Radioactive probes ([32P]CTP)were prepared from gelpurified DNA fragments by the random priming method according to the manufacturer's directions (United States Biochemical). Total RNA was isolated from the upper 1 mm of potato discs 24 h after treatment as described previously (Choi et al., 1992). Each RNA sample (20 Pg) was fractionated in 1.0% agarose gels containing formaldehyde and transferred to Nytran membranes (Amersham) after electrophoresis. A por-

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tion of the gel containing a duplicate set of samples was stained with ethidium bromide and examined under UV light. Hybridization of cDNA probes to RNA blots were camed out routinely at a stringency of 50% formamide, 1 M Na+, 42OC, and washed at the same stringency by standard procedures (Ausubel et al., 1987). The amount of labeled probe hybridized to each RNA sample was estimated with a two-dimensional radioisotope imaging system (Ambis Systems, Inc., San Diego, CA). Phytoalexin and SGA Analyses

Sesquiterpenoid phytoalexins were quantified by the semimicro method of Henfling and Ku6 (1979). Samples from the upper 1 mm of tuber discs were removed and extracted for sesquiterpenes. The concentrations of rishitin and lubimin, the major sesquiterpenoid phytoalexins in potato, were detennined by GC. SGAs were extracted from potato tuber discs according to a modification of the procedure of Allen and Ku? (1968). A 3-g sample was obtained from the upper 1 mm of tuber dscs 96 h after treatment. Samples were extracted and glycoalkaloids quantified by the spectrophotometric method described by Cadle et al. (1978). The values for SGA were calculated based on a standard curve of a-solanine (Sigma). RESULTS De Novo Protein Synthesis 1s Required for lnduction of HMCR Cenes and Phytoalexin Accumulation

Treatment of potato discs with a 100 PM solution of cycloheximide inhibited de novo protein synthesis by 99% relative to the corresponding controls in freshly prepared discs (unaged) and discs aged for 18 h prior to treatment. Cycloheximide treatment also completely abolished the AA-induced hypersensitive response and browning of the tissue and strongly affected HMGR transcript accumulation (Fig. 1).AAinduced levels of total HMGR and hmg2 transcripts were strongly inhibited by cycloheximide in either unaged (cf. lanes 1 and 2 in Fig. 1) or aged potato discs (cf. lanes 4 and 5 in Fig. 1).Wound-induced levels of total HMGR transcripts were inhibited by 83% following cycloheximide and AA treatment of unaged potato discs (cf. lanes 1 and 3 in Fig. 1). In aged discs, the suppression of hmgl transcript levels by AA treatment was inhibited by cycloheximide, and the hmgl transcript levels in the cycloheximide plus AA treatment exceeded those for the control by a factor of 1.4 (lanes 4 and 5 in Fig. 1). The levels of total HMGR and hmg2 transcripts in the wound-only treatment were similar to those in the cycloheximideand AA treatment (Fig. 1).Inhibition of protein synthesis and HMGR gene expression in AA-treated discs resulted in no accumulation of sesquiterpene phytoalexins. lnvolvement of Protein Kinase and Ca2+ in the lnduction of HMCR Gene Expression and lsoprenoid Accumulation by AA

Staurosporin is a potent inhibitor of different groups of protein kinases (Tamaoki et al., 1986). To investigate the role of protein kinase in the induction of HMGR gene expression

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Unaged discs Aged discs 1

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Total HMGR mRNA

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Figure 1. Effect of cycloheximide on HMCR mRNA accumulation by AA and wounding. Freshly prepared (unaged) or aged potato discs were pretreated with cycloheximide (100 MM) and then treated with water or AA. RNA for gel blot analyses was extracted 24 h after treatment of fresh discs and 12 h after treatment of aged discs. RNA (20 Mg/lane) was hybridized with 32P-labeled HMCR DNA probes corresponding to a conserved region (Total HMCR mRNA), an hmg!-specific sequence, or an bmg2-specific sequence as indicated. Radioactivity was quantified by radioisotope image analysis. Lane 1, RNA from unaged potato discs treated with cycloheximide and AA. Lane 2, RNA from unaged potato discs treated with buffer and AA. Lane 3, RNA from unaged potato discs treated with buffer and water. Lane 4, RNA from aged potato discs treated with cycloheximide and AA. Lane 5, RNA from aged potato discs treated with buffer and AA. Lane 6, RNA from aged potato discs treated with buffer and water.

and isoprenoid accumulation by AA, potato discs were prepared and immediately immersed in a 1 ^M solution of staurosporin. The discs were removed, and then the upper surfaces were treated with AA or water. The staurosporin pretreatment did not inhibit the wound induction of total HMGR mRNA or of hmgl mRNA (cf. lanes 1 and 2 in Fig. 2). However, this treatment inhibited AA induction of total HMGR mRNA (31% of the AA-induced levels) and of hmg2 mRNA (46% of AA; cf. lanes 3 and 4 in Fig. 2). AA suppressed wound induction of hmgl mRNA (20% of buffer control; cf. lanes 1 and 3 in Fig. 2) as previously reported (Choi et al., 1992), but this suppression was partially overcome by pretreatment of the discs with staurosporin (74% of buffer control; lane 4 in Fig. 2). In vitro phosphorylation experiments indicated that staurosporin at the concentration used in vivo inhibited the incorporation of radioactivity into microsomal proteins by 78%. EGTA is a Ca2+ ion chelator that was previously shown to inhibit AA-induced phytoalexin accumulation in potato (Zook and Kuc, 1987). Pretreatment of potato discs in a 2 mM solution of EGTA alone resulted in a 2.7-fold increase in total HMGR transcripts and increased the wound-induced levels of hmgl and hmg2 mRNA levels above the buffer control (cf. lanes 1 and 6 in Fig. 2). However, EGTA inhibited AAinduced levels of total HMGR (48% of AA) and of hmg2 mRNA (34% of AA; cf. lanes 3 and 7 in Fig. 2). Pretreatment of the tuber discs with EGTA and staurosporin (lane 5 in Fig. 2) resulted in a stronger inhibition of the AA effect on total HMGR (21% of AA) and hmg2 (23% of AA) mRNA levels than when these inhibitors were tested individually. These

Plant Physiol. Vol. 104, 1994

results suggest that both Ca2+ and protein kinase activity are required for full induction of HMGR genes by AA treatment. Since there was no detectable effect of staurosporin on wound-induced levels of total HMGR transcripts or on hmglspecific transcripts, it would appear either that activation of protein kinases is not prerequisite for HMGR gene expression following wounding or that levels of kinase activity sufficient to sustain the wound-induced HMGR expression are not accessible to these inhibitors. Similar patterns of initiation of sesquiterpenoid phytoalexin accumulation were detected in AA-treated potato discs with or without staurosporin pretreatment during the first 33 h of the experiment (Fig. 3). By 44 h a significant difference was apparent, with staurosporin inhibiting the elicitor-induced phytoalexin levels by 70%. However, by 80 h the phytoalexin levels in the inhibitor-pretreated discs acquired the same levels as in the elicitor-only treatment. SGA levels also were analyzed from the potato discs treated with staurosporin and EGTA. Staurosporin pretreatment suppressed SGA levels, but EGTA pretreatment had no significant effect on SGA accumulation relative to the control (Fig. 4). Effect of ETYA on HMGR Gene Expression and Isoprenoid Accumulation Induced by AA

Lipoxygenase activity appears to be involved in wound induction of hmgl mRNA and SGA accumulation in potato tuber tissue, and methyl jasmonate may be an important lipoxygenase metabolite involved in regulating this response (Choi et al., 1994). To test for the involvement of lipoxygenase activity in the AA induction of hmg2 mRNA and sesquiterpene phytoalexin accumulation, potato discs were treated with ETYA and then analyzed. ETYA was used within a concentration range (1-5 ITIM; 50-250 nmol/disc surface) that previously had been shown to inhibit AA elicitor activity

1 2

3 4

5 6 7

Total HMGR mRNA hmgl

hmg2

Figure 2. Effect of staurosporin and ECTA on HMGR mRNA accumulation. RNA samples for gel blot analyses were extracted from potato discs 20 h after treatment and hybridized with 32P-labeled HMCR DNA probes as described in Figure 1 and in "Materials and Methods." Potato discs were treated immediately after preparation (unaged) with buffer or buffer and inhibitor and then treated with water or AA (0.17^mol/disc)as indicated. Representative data from two independent experiments are presented. Radioactivity was quantified by radioisotope image analysis. Lane 1, Buffer only control. Lane 2, Staurosporin (1 MM). Lane 3, AA (0.17 ^mol/disc). Lane 4, Staurosporin plus AA. Lane 5, Staurosporin plus ECTA (2 mM) plus AA. Lane 6, ECTA. Lane 7, ECTA plus AA.

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Arachidonic Acid Signal Transduction in Potato

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Figure 3. The effect of staurosporin on AA-induced sesquiterpene phytoalexin accumulation. Rishitin and lubimin were determined following treatment of potato discs with AA (O)or staurosporin and AA (O). Potato discs were treated immediately after preparation (unaged)with 0.1% DMSO or 1 p~ staurosporin in 0.1% DMSO and then treated with AA (0.17 pmolldisc). No phytoalexin accumulation occurs in the absence of AA. Results presented are from one experiment and are the means and SE of three samples for each point. The experiment was performed twice with similar results. f. wt., Fresh weight.

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(Preisig and KuC, 1987). RNA gel blot analyses of samples taken at different times after treatment with ETYA and AA revealed that the kmgl mRNA suppression (Fig. 5A) and kmg2 mRNA induction (Fig. 58) by AA were delayed. However, by 48 h the discs had overcome the ETYA effect and apparently responded to the applied AA. ETYAs effect on the wound induction of the different HMGR genes varied in experiments, ranging from no effect (50 nmol/disc surface) to partia1 suppression (250 nmolldisc surface). ETYA only partially inhibited the arachidonate-induced phytoalexin accumulation when discs were sampled 96 h after treatment (20 f 5% inhibition at 250 nmol of ETYA/disc surface).

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Figure 5. The effect of ETYA on the AA-induced expression of hmgl and hmg2. RNA was extracted from potato discs treated immediately after preparation with Tris buffer, AA (50 pgldisc), or ETYA (50 pL/disc of 5 m M ETYA in Tris buffer) plus AA. The RNA samples were hybridized on gel blots with "P-labeled hmgl- and hmg2-specific probes, and radioactivity was quantified by radioisotope image analysis. A, Accumulation of hmgl mRNA; 6, accumulation of hmg2 mRNA.

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Figure 4. Accumulation of SCA following treatment with stauros-

porin or EGTA. Potato discs were treated immediately after preparation with water, staurosporin, AA, Mops buffer, or EGTAIMops, and the accumulation of SCA was determined 96 h after treatment. Each value is the mean and SE of three samples. f. wt., Fresh weight.

In this study, inhibitors were used to identify components of the signal transduction path coupling AA reception with HMGR gene expression and sesquiterpenoid phytoalexin accumulation. The results indicate that de novo protein synthesis, protein kinase activity, extracellular Caz+,and lipoxygenase activity are required for the AA effects on specific HMGR genes and phytoalexin accumulation. Our experiments with cycloheximide are consistent with earlier work with similar inhibitors that demonstrated a requirement of de novo protein synthesis in unaged potato discs for expression of hypersensitive cell death (Furuichi et al., 1979). Earlier studies, however, did not address the issue of protein synthesis in the events linking AA reception with gene activation. Our results indicate that protein synthesis is required for both the AA and wound induction of HMGR

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gene expression, as well as the suppression of hmgl mRNA levels by AA in aged discs. This suggests that proteins necessary for coupling AA reception to HMGR mRNA accumulation are either rapidly tumed over or are not present constitutively and are induced following elicitor treatment. Furthermore, the results with aged discs indicate that the necessary proteins for gene activation following elicitor treatment are not induced by wounding alone. Nonetheless, the induction of HMGR mRNA accumulation is more rapid in aged discs (Choi et al., 1992), and perhaps some of the wound-induced proteins enhance the efficiency or rate of transcription in the responding cells. Stermer and Bostock (1989) demonstrated that AA can rapidly stimulate protein synthesis in potato discs. Many of the induced proteins are pathogenesis-related proteins and enzymes involved in the biosynthesis of secondary metabolites, but others likely are factors necessary for the transcriptional activation or suppression of the corresponding genes. The results with staurosporin suggest that protein kinase activities also are involved in AA's effect on HMGR mRNA levels. We also tested another kinase inhibitor, K-252a (Rüegg and Burgess, 1989), and found it was virtually identical with staurosporin in its effects on potato responses. The effect of the kinase inhibitors on AA elicitor activity was similar to that of cycloheximide in that the kinase inhibitors interfered with both the induction of hmg2 and the suppression of hmgl. The HMGR transcript levels in the wound-only treatments were unaffected by kinase inhibitors. Staurosporin and K-252a are potent inhibitors of cAMP- and cGMP-dependent protein kinases and protein kinase C in mammalian cells (Tamaoki et al., 1986; Rüegg and Burgess, 1989). In our study, staurosporin strongly inhibited phosphorylation of potato tuber microsomal proteins, and both staurosporin and K-252a had similar effects on HMGR gene expression. Staurosporin's effect was only transient, and phytoalexin levels were the same as in the AA-treated control discs by 80 h after treatment. Although the reason for this is uncertain, possible explanations are that (a) staurosporin is unstable and is degraded within 80 h in vivo and (b) during the aging process potato discs synthesize a new set of kinases that can respond to AA. Although staurosporin did not inhibit wound induction of hmgl mRNA levels, it strongly inhibited SGA accumulation, suggesting the involvement of protein kinases in other steps in wound-induced sterol biosynthesis. Experiments with specific protein kinase substrates (Kemp et al., 1975) or with inhibitors specific to certain types of kinases may be informative about the nature of the specific kinase(s) that mediates AA's effect on HMGR gene expression and phytoalexin accumulation. lnvolvement of Extracellular Caz+ in HMGR Gene lnduction and lsoprenoid Biosynthesis

A study by Zook et al. (1987) indicated that extracellular Ca2+is involved in AA elicitation of phytoalexin accumulation in potato tuber. The results from this study corroborate that role but indicate that extracellular Ca2+participates in elicitation of phytoalexin synthesis as early as HMGR gene expression. EGTA can specifically reduce the availability of extracellular Ca2+ (Gilroy et al., 1986). In our experiments,

Plant Physiol. Vol. 104, 1994

the effect of EGTA was different between the wound-only and elicitor treatments. Wound induction of hmgl mRNA levels WilS not inhibited and was even enhanced by Ca2+ depletion, as were the transcript levels for hmg:'. Findings from recent studies with Ca2+/calmodulinantagonists suggest that Ca2' and calmodulin-like proteins are elements of the pathway mediating elicitor-induced accumulation of sesquiterpenoid phytoalexins in tobacco cell-suspension cultures (Vogeli et al., 1992) and indicate that elicitor-induced responses in tobacco cells are different in their sensitivities to calcium/calmodulin antagonists. Elicitor-inducible sesquiterpene cyclase is strongly suppressed by calcium/calmodulin antagonists, whereas elicitor-inducible Phe ammonia-lyase is unaffected by the same inhibitors. Ate Different Lipoxygenases lnvolved in Wouncl and AA lndlaction of HMGR Gene Expression and lsoprenoid Accumulation?

ETYA inhibited both wound and AA induction of HMGR gene expression and also inhibited phytoalexin accumulation to some degree. The lipoxygenase inhibitors SHAM and nPG were previously shown to inhibit AA elicitation of phytoalexins and other responses in potato (Stelzig 12t al., 1983; Bostock et al., 1986; Preisig and KuC, 1987). Recently, we demonstrated that SHAM and nPG were very effective in inhibiting the wound induction of hmgl expression and that this inhibition could be overcome by treatment with the lipoxygcnase pathway product methyl jasmonate (Choi et al., 1994). However, in our experiments AA-induced hmg2 mRNA levels and phytoalexin accumulation were not affected significantly by SHAM and nPG (data not shown). The reason for our inability to reproduce results of earlier studies of the phytoalexin response, in spite of repeated attempts, is unclear but may be related to differences in the potatoes used in the various studies with respect to physiological age and, hence, in their responsiveness to elicitors and other stimuli. Preisig and Kuk (1987) noted that the effect of SHAM on phytoalexin induction by AA was transient, and by 96 h the levels of rishitin and lubimin in the inhibitortreated discs approached those in the discs treated only with AA. Thus, differences in the physiological responsiveness of tubers could impact on time-course experiments. Another possibility for the apparent differences in the sensitivities of the wound and elicitor response pathways is related to the notion that, since different lipoxygenase isozymes have different catalytic properties, they likely also have different physiological roles (Siedow, 1991).SHAM and nPG irihibit lipoxygenases by different mechanisms (Peterman and Siedow, 1983) and appear to be scimewhat less specific than ETYA, an acetylenic analog of AA. that inhibits lipoxygenase activity by irreversibly binding to the enzyme. ETYA may discriminate lipoxygenase isozymes and was reported to inhibit 12-lipoxygenasein human cells but not the 5-lipoxygenase in rabbit cells (Tobias and Harnilton, 1978). It is possible, then, that different potato lipoxygenases have different sensitivities to inhibitors, and some inhibitors may strongly affect a lipoxygenase that is essential for jasmonate biosynthesis but be less effective on the putative lipoxygenase involved in the AA response. ETYA only delayed the elicitor

Arachidonic Acid Signal Transduction in Potato responses, similar to the effect of kinase inhibitors, suggesting that lipoxygenase activity recovers from the inhibitor treatment during the period of observation. Cloning and further characterization of lipoxygenase genes will provide materials that will aid in understanding the contribution of specific lipoxygenases and lipoxygenase-mediated products to wound- and defense-related metabolism in potato and other species. In summary, our results are consistent with the hypothesis that AA affects HMGR gene expression, and ultimately phytoalexin synthesis, indirectly through its activation of secondary signals. Furthermore, A A s suppression of hmgl and activation of hmg2 appear to depend on a common intennediate or set of intermediates whose generation is sensitive to the inhibitors tested. These experiments further demonstrate that with respect to isoprenoid metabolism the wound and elicitor response pathways rely on different signal transduction paths. ACKNOWLEDCMENT Our appreciation is extended to B.L. Ward for his significant contributions toward the cloning and characterization of the HMGR cDNAs used in this study. Received August 9, 1993; accepted December 1, 1993. Copyright Clearance Center: 0032-0889/94/l04/1237/08.

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