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

Abstract

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(β-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.

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

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 mL⁻¹ 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

Reconstitution of the Ca²⁺-sensitive photoprotein aequorin was performed in vivo by adding a 3-μL droplet of 3 μm coelenterazine onto cotyledons of each tobacco seedling and keeping them in the dark at room temperature for 20 h. For luminescence measurement the remaining coelenterazine solution was removed and cuvettes containing three seedlings were placed in a sample chamber. The luminescence of the seedlings was successively integrated for 15 s using a digital chemiluminometer with a photomultiplier with a discriminator (1 kV, model 9757 AM, EMI, Ruislip, UK; Campbell, 1988).

Stability of Aequorin to Heat Treatment

To test the stability of aequorin to heat treatment, purified aequorin (Blinks, Friday Harbor, UK) was dissolved in reconstitution buffer (50 mm Tris-HCl, pH 7.4, 500 mm NaCl, 5 mm EGTA, 5 mm β-mercaptoethanol, and 0.1% [w/v] BSA) to a concentration of 60 ng mL⁻¹. Aliquots of 1 mL were heat treated at 45 or 50°C for different times. Aequorin was again discharged by adding an equal volume of 100 mm CaCl₂, and the luminescence was measured as described above.

HS Treatments of Transgenic Tobacco Seedlings

Unlike our previous method, in which transgenic tobacco seedlings were irrigated with hot water to induce HS (Knight et al., 1991), in the present experiments the cuvettes, each containing three reconstituted 2-week-old seedlings, were immersed into a water bath at the appropriate temperature. The seedlings did not, therefore, come into contact with hot water and, thus, the applied HS may mimic heat-stress situations in the field. The cuvettes were removed at specific times, and the luminescence of the seedlings was integrated numerically for 15 s at 3- or 5-min intervals in the chemiluminometer. A thermocouple was placed in the cuvette and the actual temperature in the cuvette during heat treatment was recorded. After the luminescence measurement, cuvettes were placed back into the water bath to continue HS treatment and to allow subsequent luminescence measurements. After the heat treatments, the seedlings were homogenized in reconstitution buffer and the aequorin was simply discharged with excess Ca²⁺ in the luminometer. Each seedling group produced on average about 300,000 counts ± 28,000 when loaded with coelenterazine via the cotyledons. From the luminescence data, cytosolic pCa values were calculated according to our recent method (Knight et al., 1996).

Extracellular Ca²⁺, Ca²⁺ Chelator, and Inhibitor Treatments

When CaCl₂, EGTA, LaCl₃, ruthenium red, or neomycin sulfate were used, aequorin reconstitution was performed as described above, followed by the incubation with 3 μL of the above-mentioned chemicals placed onto the cotyledons for 4 h in the dark at room temperature. After this time, the solution was drained and the seedlings were used for HS treatments and luminescence measurements.

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

Sterilized tobacco (wild-type) seeds were sown in plastic Petri dishes with three compartments, each containing 8 mL of one-half-strength Murashige-Skoog medium and 0.8% (w/v) agar, and germinated at 25°C with a 16-h photoperiod for 2 weeks. For Ca²⁺ or EGTA treatment, 5 mL of sterilized, distilled water (control), 10 mm CaCl₂, or EGTA (the pH of EGTA solution was adjusted to 6.8) was added to one of three compartments in a same Petri dish and kept overnight (15 h) at 25°C. At the end of the incubation, the solutions or distilled water were drained and the seedlings were transferred from 25 to 38 or 40°C in an incubator for 2 h for HS treatment, then returned to 25°C for a 4-h recovery period, and transferred again to 48°C for 4 h or 50°C for 2 h and 20 min for heat treatment. The control seedlings without HS treatments were transferred directly from 25 to 48 or 50°C. After the heat treatments, the seedlings were cultured again at 25°C with a 16-h photoperiod for 8 d. In preliminary experiments 8 d of recovery at 25°C for heat-treated seedlings was sufficient to easily discriminate between seedlings that were alive and those that were dead (data not shown). Dead seedlings lacking chlorophyll and turgor and with supine, often dry hypocotyls could easily be recognized, whereas viable seedlings retained turgor and green leaves and continued to grow. The data are expressed as survival percentages.

RESULTS

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

To examine whether an involvement of cytosolic Ca²⁺ can be invoked during the development of thermotolerance we pretreated tobacco seedlings with 10 mm Ca²⁺, EGTA, or water, as described in Methods. The seedlings were then divided into two batches, one of which was maintained at 25°C and the other was incubated at 38°C for 2 h to acquire HS-induced thermotolerance. After a further 4 h at 25°C both seedling batches were then incubated at 48°C for 4 h to induce heat injury. Seedling viability was then estimated 8 d later. These results are shown in Figure 1A.

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 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

In our original study (Knight et al., 1991) in which MAQ 2.4 aequorin-transformed seedlings were used, we reported that irrigation of tobacco seedlings with hot water at temperatures up to 55°C only induced slight or no detectable changes in luminescence. At the commencement of this investigation we repeated and confirmed those observations (data not shown). However, when the transformed tobacco seedlings cultured in cuvettes were heat shocked at 39, 43, or 47°C, for periods up to 35 min, a significant increase in [Ca²⁺]_cyt from these seedlings was observed (Fig. 2). This increase in [Ca²⁺]_cyt lasted for 10 to 20 min, depending on the temperature used, and was followed by a gradual decrease, which approached original resting levels (Fig. 2). Continued HS treatment did not elicit further increases in [Ca²⁺]_cyt. Measurement of luminescence required removal of the cuvettes containing seedlings from the HS treatment bath for the 15-s luminescence measurements. So that we could assess the effect of transient removal on the temperature of the seedlings, thermocouples were introduced into blank cuvettes containing the requisite volume of agar and temperatures were recorded continuously. The quoted HS temperatures 39, 43, and 47°C are therefore the weighted average of the temperatures experienced by the seedlings throughout the whole 35-min measurement period. The temperature fluctuation in the cuvettes during 15 s of luminescence measurement was about 2 to 3°C and lasted 80 to 90 s (data not shown). In addition, HS treatment at 39, 43, and 47°C for 35 min did not lead to any lethal injury to the seedlings, and all of the seedlings could survive after the treatments (data not shown).

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⁺.

Table I.

Effect of HS on Ca²⁺-dependent luminescence of purified aequorin

HS	45°C	50°C
min	counts s−1
0	107,160  ± 987	108,317  ± 741
20	105,516  ± 1662	109,200  ± 1008
40	103,673  ± 1280	109,788  ± 860
60	106,831  ± 1036	109,928  ± 1788

Solutions of purified aequorin were heat treated at a given temperature for the times shown and then discharged with excess CaCl₂. The values are means ± se of five measurements.

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.

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.

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

As shown in Figure 2, the changes in [Ca²⁺]_cyt indicate that HS-induced increases in [Ca²⁺]_cyt only lasted 15 to 20 min even though the seedlings continued to be stimulated by high temperatures. This implies that a possible refractory period follows HS in which no further HS-induced change in [Ca²⁺]_cyt can be elicited. We investigated this possibility by application of HS, followed by a return to 25°C, and then HS every 1 h. Only after a further 5-h period at 25°C could we start to detect a recovery in sensitivity to HS, as shown in Figure 5, and full recovery required 8 h. This time course is similar to the loss of the refractory period induced by exogenously added hydrogen peroxide to induce oxidative stress (Price et al., 1994).

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.

Table II.

Effects of prior HS of transgenic tobacco seedlings (MAQ 2.4) on their subsequent response to cold shock and wind signaling

HS	25°C, Control	39°C, 35 min	43°C, 35 min	47°C, 30 min
	pCa
Background	7.0  ± 0.02	7.01  ± 0.01	7.0  ± 0.02	7.0  ± 0.01
Cold-shock-induced increase in [Ca2+]cyt	6.0  ± 0.1	6.1  ± 0.1	5.9  ± 0.07	6.4  ± 0.03
Wind-stimulus-induced increase in [Ca2+]cyt	6.3  ± 0.03		6.1  ± 0.03

Three tobacco seedlings in a cuvette were first heat shocked in a waterbath at a given temperature for the given time as shown in Table II, taken out, and cooled down for 5 min at 25°C. Then the cuvette was placed into the sample chamber of the chemiluminometer. For cold shock 1 mL of ice-cold water was injected gently into the cuvette and the cold shock-induced Ca²⁺-dependent luminescence of the seedlings was recorded numerically for 15 s. For wind stimulation, 10 mL of air was injected rapidly over the seedlings by a port in the sample chamber with a syringe and the wind-induced luminescence of the seedlings was measured numerically for 15 s. Remaining aequorin was estimated at the end of the treatment and cytosolic pCa was calculated. The values are means ± se of 6 to 10 replicates.

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

To investigate the sources for the increased [Ca²⁺]_cyt under HS, the transformed tobacco seedlings containing reconstituted cytosolic aequorin were pretreated with several Ca²⁺-signaling inhibitors. As shown in Figure 6, seedlings pretreated with 1 mm LaCl₃, a putative plasma membrane Ca²⁺-channel blocker, demonstrated a much lower HS-induced increase in [Ca²⁺]_cyt.

Figure 6.

Effects of LaCl_3, 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

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.

Abbreviations:

[Ca²⁺]_cyt

cytosolic free Ca²⁺ level

HS

heat shock

HSP(s)

heat-shock protein(s)

InsP₃

inositol-1,4,5 trisphosphate