Plant Physiol. (1998) 116: 429-437

Heat-Shock-Induced Changes in Intracellular Ca²⁺ Level in Tobacco Seedlings in Relation to Thermotolerance¹

Ming Gong^*, Arnold H. van der Luit, Marc R. Knight, and Anthony J. Trewavas

Department of Life Sciences, Yunnan Normal University, Kunming 650092, People's Republic of China (M.G.); Institute of Cell and Molecular Biology, University of Edinburgh, Edinburgh EH9 3JH, United Kingdom (A.H.v.d.L., A.J.T.); and Department of Plant Sciences, University of Oxford, Oxford OX1 3RB, United Kingdom (M.R.K.)

	ABSTRACT

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Exposure of plants to elevated temperatures results in a complex set of changes in gene expression that induce thermotolerance and improve cellular survival to subsequent stress. Pretreatment of young tobacco (Nicotiana plumbaginifolia) seedlings with Ca²⁺ or ethylene glycol-bis(-aminoethylether)-N,N,N,N-tetraacetic acid enhanced or diminished subsequent thermotolerance, respectively, compared with untreated seedlings, suggesting a possible involvement of cytosolic Ca²⁺ in heat-shock (HS) signal transduction. Using tobacco seedlings transformed with the Ca²⁺-sensitive, luminescent protein aequorin, we observed that HS temperatures induced prolonged but transient increases in cytoplasmic but not chloroplastic Ca²⁺. A single HS initiated a refractory period in which additional HS signals failed to increase cytosolic Ca²⁺. However, throughout this refractory period, seedlings responded to mechanical stimulation or cold shock with cytosolic Ca²⁺ increases similar to untreated controls. These observations suggest that there may be specific pools of cytosolic Ca²⁺ mobilized by heat treatments or that the refractory period results from a temporary block in HS perception or transduction. Use of inhibitors suggests that HS mobilizes cytosolic Ca²⁺ from both intracellular and extracellular sources.

	INTRODUCTION

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The responses of plants to HS have received increasing attention in recent years. Elevated temperatures initiate changes in transcription and selective translation of HS mRNA encoding HSPs, thereby enhancing thermotolerance of treated plants (Nover et al., 1989; Nover, 1991; Vierling, 1991; Howarth and Ougham, 1993; Waters et al., 1996). However, the pathways by which HS signals are perceived and transduced to activate gene expression of HSPs and to induced thermotolerance are not understood.

In recent years a second-messenger Ca²⁺ was found to be involved in the perception and regulation of many responses of plants to environmental signals (Gilroy et al., 1993; Poovaiah and Reddy, 1993; Gilroy and Trewavas, 1994; Bush, 1995; Braam et al., 1996; Webb et al., 1996). [Ca²⁺]_cyt often shows significant changes in plant cells under the influence of various stress signals such as touch, wind stimulation, cold shock, wounding, and mechanical stimulation (Knight et al., 1991, 1992, 1993; 1996; Haley et al., 1995; Campbell et al., 1996; Polisensky and Braam, 1996), oxidative stress (Price et al., 1994), salinity (Lynch et al., 1989; Bush, 1996; Okazaki et al., 1996), anoxia (Subbaiah et al., 1994a; Bush, 1996; Sedbrook et al., 1996), and hypo-osmotic shock (Takahashi et al., 1997). It has been suggested that a stress-induced change in [Ca²⁺]_cyt might be one of the primary transduction mechanisms whereby gene expression and biochemical events are altered to adapt plant cells to environmental stresses (Monroy et al., 1993; Subbaiah et al., 1994a, 1994b; Monroy and Dhindsa, 1995; Braam et al., 1996).

Several authors have suggested that Ca²⁺-mediated second-messenger systems might be involved in the HS responses of animal cells (Lamarche et al., 1985; Calderwood et al., 1988; Landry et al., 1988; Mosser et al., 1990), although other results indicated that Ca²⁺ was not strictly required for some HSP synthesis (Drummond et al., 1986, 1988). In plant cells Klein and Ferguson (1987) observed that the uptake of Ca²⁺ by suspension-cultured pear cells or protoplasts was significantly enhanced during heat stress. Braam (1992) demonstrated that HS induced a strongly up-regulated expression of calmodulin-related TCH genes in cultured Arabidopsis cells, and external Ca²⁺ was required for maximal HS induction of these TCH genes. Wu et al. (1992) also indicated that pretreatment of hypocotyl segments and etiolated seedlings of Brassica napus with the Ca²⁺ ionophore A23187 or the Ca²⁺ chelator EGTA to modify Ca²⁺ homeostasis resulted in changes in the synthesis of HSPs. Using the fluorescent dye Indo-1, Biyaseheva et al. (1993) reported that HS induced a 4-fold increase in [Ca²⁺]_cyt in pea mesophyll protoplasts, but the further dynamic changes in [Ca²⁺]_cyt during HS could not be detected because of limitations in the technique.

We recently described a novel technology to measure [Ca²⁺]_cyt using the genetic transformation of tobacco (Nicotiana plumbaginifolia) to express apoaequorin (Knight et al., 1991). After incubation in the luminophore coelenterazine, the Ca²⁺-sensitive luminescent protein aequorin is reconstituted. Luminous plants are thus generated, the luminescence of which directly reports [Ca²⁺]_cyt (Knight et al., 1991, 1992). When Ca²⁺ binds to aequorin, the luminophore is oxidized and emits a finite amount of blue light. These transgenic tobacco plants have been used successfully to detect very rapid (transient) increases in [Ca²⁺]_cyt in response to many signals (Knight et al., 1991, 1992, 1993, 1996; Price et al., 1994; Haley et al., 1995; Campbell et al., 1996). Aequorin has also been targeted to chloroplasts, nuclei, and the vacuole membrane (Johnson et al., 1995; Knight et al., 1996; A.H. van der Luit, C. Olivari, A. Haley, M.R. Knight, and A.J. Trewavas, unpublished data), enabling intraorganellar Ca²⁺ to be measured. In earlier studies we did not detect transient increases in [Ca²⁺]_cyt by brief irrigation with hot water to mimic HS (Knight et al., 1991). On the other hand, our recent results in maize seedlings suggested that Ca²⁺ and calmodulin are involved in the regulation of intrinsic and HS-induced thermotolerance (Gong et al., 1997a, 1997b).

In view of the evidence described above, we decided to further investigate the possible role of [Ca²⁺]_cyt in HS using longer periods of stimulation on tobacco seedlings expressing transgenic aequorin.

	MATERIALS AND METHODS

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All chemicals used were obtained from Sigma, except for coelenterazine, which was purchased from Molecular Probes (Eugene, OR). Genetically transformed tobacco (Nicotiana plumbaginifolia L.), line MAQ 2.4 seedlings expressing cytosolic apoaequorin (Knight et al., 1991) and MAQ 6.3 expressing chloroplast apoaequorin (Johnson et al., 1995) were used. Sterilized tobacco seeds were germinated in plastic cuvettes containing 0.6 mL of one-half-strength Murashige-Skoog medium, 0.8% (w/v) agar, and 200 µg mL¹ kanamycin at 25°C, with a 16-h photoperiod for 2 weeks, after which time the cotyledons were fully expanded.

In Vivo Reconstitution of Aequorin and Ca²⁺-Dependent Luminescence Measurements

Stability of Aequorin to Heat Treatment

1

HS Treatments of Transgenic Tobacco Seedlings

Extracellular Ca²⁺, Ca²⁺ Chelator, and Inhibitor Treatments

Effect of External Ca²⁺ and EGTA on Thermotolerance of Tobacco Seedlings

	RESULTS

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Effect of Exogenous Ca²⁺ or EGTA on the Development of Thermotolerance of Tobacco Seedlings

View larger version (25K): [in this window] [in a new window]   Figure 1. Effects of external Ca2+ and the Ca2+ chelator EGTA on intrinsic and HS-induced thermotolerance in tobacco seedlings. Overnight pretreatment of seedlings with 10 mm Ca2+ (+Ca, open bar), 10 mm EGTA (+EGTA, checkered bar), or sterile water (control, +H2O, striped bar), subsequent heat treatment, and investigation of survival percentage were carried out as described in ``Materials and Methods''. In each case the schedule of treatments is also indicated below the figure. Each bar represents the mean ± se of three replicates, and 180 to 240 seedlings were investigated for each replicate.

In a similarly constructed experiment, seedlings pretreated with Ca²⁺, EGTA, or water were first incubated at 40°C to acquire HS-induced thermotolerance and subsequently treated at 50°C for 2 h and 20 min to induce heat injury. Viabilities were estimated again 8 d later. These data are shown in Figure 1B.

A prior treatment of seedlings at 38 or 40°C substantially increased the percentage surviving the subsequent severe HS of 48 or 50°C (Fig. 1, P < 0.05). Thermotolerance of tobacco seedlings is therefore induced by several hours of incubation at these lower temperatures, as it is for other plants, including maize (Gong et al., 1997b). Pretreatment of the seedlings with 10 mm CaCl₂ enhanced the survival percentage under heat stress at 48 or 50°C as compared with the control (+H₂O, P < 0.1). The effect was observed when the seedlings were transferred directly from 25 to 48 or 50°C for heat treatment (as a result of intrinsic thermotolerance). Moreover, the effect was apparent after the seedlings were first prehardened at 38 or 40°C to induce the prior development of thermotolerance (as a result of HS-induced thermotolerance, Fig. 1). In contrast, pretreatment of the seedlings with the Ca²⁺ chelator EGTA (+EGTA) led to a greater loss of viability compared with the treatment with water (+H₂O, P <0.05). If these two treatments, Ca²⁺ and EGTA, have their anticipated effects on [Ca²⁺]_cyt, then these results suggest the possible involvement of the Ca²⁺ signal transduction chain in the subsequent development of thermotolerance. Pretreatment of the seedlings with 10 mm CaCl₂ or EGTA overnight had little effect on the growth or survival of the seedlings at 25°C with a 16-h photoperiod during 2 weeks (data not shown).

Changes of Intracellular Ca²⁺ Level during HS

View larger version (23K): [in this window] [in a new window]   Figure 2. Changes in [Ca2+]cyt in transgenic tobacco (MAQ 2.4) seedlings containing cytoplasmic aequorin during HS at 39°C (), 43°C (), and 47°C (). Each point represents the mean ± se of 10 measurements. When no error bar is indicated, the se was within the size of the symbol.

We recently constructed a new calibration curve for the particular isoform of aequorin, which has been used for transformation (Knight et al., 1996). The apoaequorin was overexpressed in Escherichia coli and calibration was determined using standard mixtures of Ca²⁺/EGTA after reconstitution with coelenterazine. Fortunately, the isoform used for transformation is among the most sensitive of the isoforms, and the dose-response curve commences below a pCa of 7 (about 100 nm) and is saturated at about 10 µm (pCa 5). This calibration curve has been used to estimate the putative increases in [Ca²⁺]_cyt resulting from HS treatment. From the luminescence data, pCa values in heat-treated tobacco seedlings were calculated, plotted, and are shown in Figure 2. In every case pCa increases substantially because of the heat treatment, although it takes 9 to 10 min before peaks are reached and the transient declines. The higher the temperature the bigger the increase in pCa. We also measured the resting pCa level at 25°C to be 7. At 39°C there is a 2-fold increase in pCa, at 43°C about a 3-fold increase in pCa, and at 47°C a 7-fold increase in pCa. The final resting pCa levels varied between 6.9 and 6.95.

To demonstrate that these changes were not the result of a nonspecific discharge of aequorin luminescence we tested the stability of aequorin to heat treatment. Purified aequorin was incubated in reconstitution buffer at 45 and 50°C and then discharged with an excess of Ca²⁺. As shown in Table I, the total luminescence of aequorin at 45 or 50°C for 0, 20, 40, or 60 min remained unchanged. Purified aequorin is therefore stable to high temperatures, and we believe the changes shown in Figure 2 represent genuine changes in cytosolic Ca²⁺.

Since the data in Figure 1 indicated that pretreatments with exogeous Ca²⁺ or EGTA modified thermotolerance, we also tested the effects of these two pretreatments on the subsequent changes in [Ca²⁺]_cyt induced by HS. The pretreatments were carried out for 4 h in the dark at 25°C before HS was applied by transferring the seedlings to 43°C. This temperature was chosen as the best compromise between the shorter time period for these experiments compared with the 2-h HS treatment at 38 and 40°C used in Figure 1. The HS effect, like many other plant processes, is dependent on both the experimental temperature and the time of exposure (Nover et al., 1989; Gong et al., 1997b). In addition, this temperature (43°C) also gave a higher HS-induced increase in [Ca²⁺]_cyt (Fig. 2) but did not lead to detectable injury to the tobacco seedlings during the heat treatment (data not shown). The results of these experiments are shown in Figure 3.

View larger version (19K): [in this window] [in a new window]   Figure 3. Effect of Ca2+ and EGTA pretreatments on the HS-induced changes in [Ca2+]cyt in transgenic tobacco (MAQ 2.4) seedlings containing cytoplasmic aequorin during HS at 43°C. The seedlings were pretreated with 10 mm Ca2+ (), sterile water (control, ), or 10 mm EGTA () in the dark for 4 h and subsequently heat shocked at 43°C. Each point represents the mean ± se of 8 to 10 measurements.

Pretreatment with exogenous Ca²⁺ clearly increased the rate of elevation of the pCa signal, and it peaked at a pCa of about 6.35 compared with 6.52 for the control. Treatment with Ca²⁺ also shortened the time taken to reach the peak. In contrast, the EGTA treatment severely limited the capacity of HS to increase pCa and also delayed the onset of the peak at a pCa of 6.88. These data therefore support the hypothesis deduced from the data of Figure 1 that regulation of [Ca²⁺]_cyt might represent part of the signal transduction process that leads to thermotolerance.

Although seedlings from our MAQ 2.4 line contain 95 to 99% of their aequorin in the soluble fraction of the cell, modification of the Ca²⁺ relations of organelles could contribute to the final cell response. In particular, HS is known to modify subsequent photosynthetic rates (Quinn and Williams, 1985). Since we recently reported the production of tobacco seedlings containing transgenic aequorin targeted to the chloroplast MAQ 6.3 line (Johnson et al., 1995), we decided to use these seedlings to examine any possible relationship of chloroplast Ca²⁺ to photosynthetic alterations. However, the data in Figure 4 show that there was little change in the free Ca²⁺ level of chloroplasts from these seedlings when they were heat shocked at 43 or 47°C and, therefore, the intrachloroplastic Ca²⁺ level does not seem to respond to HS, unlike [Ca²⁺]_cyt. These observations also confirm that aequorin is stable at the high temperatures used for HS.

View larger version (14K): [in this window] [in a new window]   Figure 4. Changes in [Ca2+]cyt level in transgenic tobacco (MAQ 6.3) containing chloroplast-located aequorin during HS at 43°C () or 47°C (). Each point represents the mean ± se of seven or eight measurements.

Recovery of [Ca²⁺]_cyt Responsiveness from HS

View larger version (16K): [in this window] [in a new window]   Figure 5. Changes in [Ca2+]cyt level in transgenic tobacco (MAQ 2.4) seedlings given a second HS either 5 or 8 h after the first. Three 2-week-old tobacco seedlings cultured in a cuvette were first heat shocked at 43°C for 30 min and luminescence was numerically integrated for 15 s at 5-min intervals. These seedlings were kept in the dark at 25°C for either 5 or 8 h and then heat shocked again at 43°C. Each point represents the mean ± se of eight measurements.

On the other hand, seedling refractory to HS still responded well to cold shock, wind, or touch stimulation. Transient spikes in [Ca²⁺]_cyt were observed when seedlings that had just been heat shocked at 39 or 43°C were instantly challenged with ice-cold water or stimulated by wind or touch (Table II), indicating that the heat-shocked seedlings still retained a responsiveness to these other stimuli. As before, cold shock increased pCa about 10-fold (from 100 nm to 1 µm), whereas wind increased pCa 7- to 8-fold. The seedlings that were heat shocked at 47°C for 30 min showed a much lower cold-shock-induced increase in [Ca²⁺]_cyt (Table II), most likely because HS at 47°C might lead to the eventual cellular injury of the seedlings, although this injury was not lethal (data not shown). These data do indicate that the refractory period does not directly involve modification of aequorin or an inability of the cells to regulate [Ca²⁺]_cyt.

Possible Cellular Origin for HS-Induced Increase in [Ca²⁺]_cyt

View larger version (19K): [in this window] [in a new window]   Figure 6. Effects of LaCl3, ruthenium red, and neomycin on the HS-induced changes of [Ca2+]cyt level in transgenic tobacco (MAQ 2.4) seedlings during HS at 43°C. Three microliters of 1 mm LaCl3 (), 25 µm ruthenium red (), 200 µm neomycin solution (), or sterile water (control, ) was added onto the two opened cotyledons of each seedling, and these seedlings were kept in the dark at 25°C for 4 h. After removal of the solution HS was conducted at 43°C. Each point represents the mean ± se of 8 to 10 measurements.

Similarly, pretreatment of these transformed seedlings containing reconstituted aequorin with the putative intracellular Ca²⁺-channel blocker ruthenium red (25 µm) or the phospholipase C inhibitor neomycin (200 µm) greatly lowered HS-induced increases in [Ca²⁺]_cyt, as compared with the control, although the [Ca²⁺]_cyt in these seedlings was a little higher than that of seedlings pretreated with La³⁺ (Fig. 6).

	DISCUSSION

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The data presented here clearly show that HS results in a prolonged but transient increase in [Ca²⁺]_cyt in tobacco seedlings (Fig. 2). This increase was not mirrored in the chloroplasts, suggesting that HS does not modify the free Ca²⁺ level in chloroplasts (Fig. 4). Thus, presumably, the effects of HS on chloroplast activity, e.g. photosynthetic rates (Quinn and Williams, 1985), may not be mediated by chloroplast Ca²⁺, and the stability of aequorin was not affected under the HS conditions we used in these experiments (Table I).

The HS-induced increases in [Ca²⁺]_cyt gradually returned to resting levels even while HS continued (Fig. 2). Heat-shocked seedlings required recovery at 25°C for 8 h to allow a full HS-induced [Ca²⁺]_cyt response (Fig. 5). These results suggest a refractory period following HS. Price et al. (1994) proposed that the refractory period of [Ca²⁺]_cyt, which resulted from oxidative stress, was the result of a strong regulation of the pro-oxidant/antioxidant ratio. However, since refractory periods have also been observed in the responses of [Ca²⁺]_cyt to wind, touch, cold shock (Knight et al., 1991, 1992, 1996), and HS (presented here) other mechanisms are also likely to be involved in the refractory periods to these other signals. A sustained high [Ca²⁺]_cyt disturbs the intracellular phosphate-based energy metabolism and causes cytotoxicity (Hepler and Wayne, 1985). A refractory period following a stimulus-induced increase in [Ca²⁺]_cyt might prevent cells from damage that would otherwise be caused by a prolonged increase in [Ca²⁺]_cyt.

Although the heat-shocked seedlings were refractory to a second HS treatment, they retained full responsiveness (with respect to [Ca²⁺]_cyt elevation) to other stimuli such as cold shock and touch stimulation (Table II). This is very similar to the situation with oxidative stress (Price et al., 1994) and mechanical signaling (Knight et al., 1992). These observations indicate that plant cells can distinguish between different stimulus-induced increases in [Ca²⁺]_cyt and yet retain a full responsiveness of [Ca²⁺]_cyt to other stimuli, while remaining refractory to the same signal. In addition, this retention of full sensitivity to other stimuli after HS indicates that HS-induced changes in [Ca²⁺]_cyt were a positive response of tobacco seedlings to HS and not a consequence of heat injury (also indicated in Fig. 1).

Nelles (1985) reported that in corn coleoptile cells, HS led to an initial increase in membrane potential, which was followed by a steep decrease. It is known that a rapid decrease in plasma membrane potential and depolarization of the membranes will lead to the opening of plasma membrane Ca²⁺ channels and the influx of extracellular Ca²⁺ into cells (Poovaiah and Reddy, 1987, 1993). In the experiments described here external Ca²⁺ treatment enhanced the HS-induced increase in [Ca²⁺]_cyt. Conversely, the Ca²⁺ chelator EGTA and plasma membrane Ca²⁺-channel blocker La³⁺ (Tester, 1990; Monroy and Dhindsa, 1995) both significantly lowered the HS-induced increase in [Ca²⁺]_cyt (Figs. 3 and 6). These data suggest that extracellular Ca²⁺ may enter cells across plasma membranes during HS to increase [Ca²⁺]_cyt. Additionally, the putative intracellular Ca²⁺-channel inhibitor ruthenium red (Kreimer et al., 1985; Subbaiah et al., 1994a) significantly lowered the HS-induced increase in [Ca²⁺]_cyt, implying that mobilization and redistribution of intracellular Ca²⁺ are also involved in HS-induced changes in [Ca²⁺]_cyt (Fig. 6). Therefore, we suggest that the increased [Ca²⁺]_cyt observed in transformed tobacco seedlings during HS arises from both extracellular and intracellular sources. However, since ruthenium red-sensitive channels also occur in plant plasma membranes (Marshall et al., 1994) and lanthanum may enter into plant cells (Quiquampoix et al., 1990), these conclusions must be made tentatively.

Intracellular Ca²⁺ mobilization is often mediated by another second messenger, InsP₃ (Cote and Crain, 1993; Allen et al., 1995; Bush, 1995). Our data show that the phospholipase C inhibitor neomycin reduces the magnitude of the HS-induced increase in [Ca²⁺]_cyt (Fig. 6). Neomycin is believed to inhibit the hydrolysis of phosphoinositides, thereby preventing the production of InsP₃ and InsP₃-mediated mobilization of intracellular Ca²⁺ (Phillippe, 1994). InsP₃ could therefore be involved in the HS-induced mobilization and redistribution of intracellular Ca²⁺ in plant cells. It is found that the HS responses of cultured animal cells also involve altered mobilization of InsP₃ (Calderwood et al., 1988).

Although many environmental stresses lead to the increase in [Ca²⁺]_cyt (see the introduction), these changes in [Ca²⁺]_cyt exhibit enormous variability in amplitude, kinetics, and spatial distribution of [Ca²⁺]_cyt. For example, touch, wind stimulation, and cold shock all cause sharp spikes in [Ca²⁺]_cyt in tobacco seedlings within 15 s (Knight et al., 1991, 1992, 1996), oxidative and salt stresses cause relatively lower transients of [Ca²⁺]_cyt, lasting for several minutes (Price et al., 1994; Bush, 1996; Okazaki et al., 1996), and anoxia induces increases in [Ca²⁺]_cyt, lasting for several hours (Subbaiah et al., 1994a; Sedbrook et al., 1996). Our data presented here show that in tobacco seedlings HS induces a lower but prolonged increase in [Ca²⁺]_cyt, lasting for 10 to 20 min (Figs. 2 and 4-6). These temporal, spatial, and amplitude variations in stress-induced increases in [Ca²⁺]_cyt under different environmental stresses may allow plant cells to distinguish one kind of stress from another and to induce distinct gene expression to adapt to a particular stress. This possibility, however, awaits further investigation.

We recently reported that Ca²⁺ and calmodulin may be involved in the acquisition of the HS-induced thermotolerance in maize seedlings. The acquisition of the HS-induced thermotolerance requires the entry of extracellular Ca²⁺ into cells across the plasma membrane and the mediation of intracellular calmodulin (Gong et al., 1997b). In addition, we also found that external Ca²⁺ treatments enhanced intrinsic thermotolerance in maize seedlings, which was associated with increased activities of antioxidative systems during heat stress, and EGTA treatments had the opposite effect (Gong et al., 1997a). Braam (1992) found that HS treatment strongly up-regulated expression of calmodulin-related TCH genes in cultured Arabidopsis cells and that external Ca²⁺ was required for maximal HS induction of these genes. Conversely, EGTA treatment inhibited the HS-induced expression of TCH genes. TCH genes are considered to play some important roles in the perception, response, and adaptation of plants to various environmental stresses (Xu et al., 1995, 1996; Braam et al., 1996).

In our present experiments modification of [Ca²⁺]_cyt levels in tobacco seedlings led to a change of thermotolerance. External Ca²⁺ treatments, which enhanced the HS-induced increases in [Ca²⁺]_cyt (Fig. 3), also enhanced intrinsic and HS-induced thermotolerance in tobacco seedlings (Fig. 1). In contrast, EGTA treatment, which chelates extracellular Ca²⁺ and greatly lowered the HS-induced increase in [Ca²⁺]_cyt (Fig. 3), also decreased the intrinsic and HS-induced thermotolerance compared with the controls (Fig. 1). These results imply the physiological importance of Ca²⁺ in generating thermotolerance in tobacco seedlings. As discussed above, these increases are a positive response of tobacco seedlings to heat stress and are not due to injury. HS-induced increases in [Ca²⁺]_cyt therefore seems to act as a signal to trigger some of the biochemical and physiological events that enable plants to adapt following heat stress.

	FOOTNOTES

   Received June 25, 1997; accepted October 7, 1997.

	ABBREVIATIONS

Abbreviations: [Ca²⁺]_cyt, cytosolic free Ca²⁺ level. HS, heat shock. HSP(s), heat-shock protein(s). InsP₃, inositol-1,4,5 trisphosphate.

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