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First published online June 12, 2003; 10.1104/pp.102.018564 Plant Physiology 132:1186-1195 (2003) © 2003 American Society of Plant Biologists Calmodulin Is Involved in Heat Shock Signal Transduction in Wheat1Institute of Molecular Cell Biology, Hebei Normal University, Shijiazhuang 050016, People's Republic of China (H.-T.L., B.L., Z.-L.S., R.-L.M., D.-Y.S., R.-G.Z.); and Institute of Agro-physics, Physiology, and Biochemistry, Hebei Academy of Agricultural Sciences, Shijiazhuang 050051, People's Republic of China (H.-T.L., B.L., X.-Z.L., R.-L.M., R.-G.Z.)
The involvement of calcium and calcium-activated calmodulin (Ca2+-CaM) in heat shock (HS) signal transduction in wheat (Triticum aestivum) was investigated. Using Fluo-3/acetoxymethyl esters and laser scanning confocal microscopy, it was found that the increase of intracellular free calcium ion concentration started within 1 min after a 37°C HS. The levels of CaM mRNA and protein increased during HS at 37°C in the presence of Ca2+. The expression of hsp26 and hsp70 genes was up-regulated by the addition of CaCl2 and down-regulated by the calcium ion chelator EGTA, the calcium ion channel blockers LaCl3 and verapamil, or the CaM antagonists N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide and chlorpromazine. Treatment with Ca2+ also increased, and with EGTA, verapamil, chlorpromazine, or trifluoperazine decreased, synthesis of HS proteins. The temporal expression of the CaM1-2 gene and the hsp26 and hsp70 genes demonstrated that up-regulation of the CaM1-2 gene occurred at 10 min after HS at 37°C, whereas that of hsp26 and hsp70 appeared at 20 min after HS. A 5-min HS induced expression of hsp26 after a period of recovery at 22°C after HS at 37°C. Taken together, these results indicate that Ca2+-CaM is directly involved in the HS signal transduction pathway. A working hypothesis about the relationship between upstream and downstream of HS signal transduction is presented.
Organisms have developed a diverse array of mechanisms for adapting to environmental changes. One of the best characterized responses is the induction of heat shock proteins (HSPs). The heat shock (HS) response has been found in almost every organism studied to date. The HSPs are synthesized by cells in response to elevated temperature but are also induced by other environmental stresses (Noven et al., 1992  ; Kilstrup et al.,
1997) and play an important role in the thermotolerance of plants
(Queitsch et al., 2000;
Burke, 2001). A connection
between HS response and oxidative stress has been observed
(Gong et al., 1997a;
Lee et al., 2000;
Larkindale and Knight, 2002;
Panchuk et al., 2002). The
HSPs are divided into several families based on their molecular mass, and most
have molecular chaperones functions (for review, see
Boston et al., 1996;
Miernyk, 1999). Angiosperms
synthesize more small HSPs (smHSPs) than other organisms. These smHSPs are
likely critical for survival of heat stress and for specific developmental
processes in plants (Waters et al.,
1996).
The changes in cytoplasmic calcium levels act as a ubiquitous signal in
eukaryotic cells. HS induced a large increase in intracellular free calcium
ion concentration ([Ca2+]i) in Chinese hamster
(Cricetulus barabensis) HA-1 fibroblasts
(Calderwood et al., 1988
The downstream events in HS signal transduction have been investigated (for
review, see Morimoto, 1998 Although some studies about upstream (primary Ca2+-CaM response) and downstream (expression of HSP genes) of HS signal transduction have been reported, a role of Ca2+-CaM in regulation of HSP gene expression and HSP synthesis has not been documented. Herein, we provide evidence for the involvement of the Ca2+-CaM signaling system in HSP gene expression or HSP synthesis and the order of signal transduction steps during HS. A possible regulatory model of Ca2+-CaM in the signal transduction pathway for heat stress is proposed.
The Increase of [Ca2+]i during HS of Wheat (Triticum aestivum) Cells To investigate the role of [Ca2+]i upstream in HS signal transduction, we examined kinetics of change in [Ca2+]i at the early stage of HS. A thin tissue section was stripped from the sheath of the first leaf of a 10-d-old green wheat seedling and observed using laser scanning confocal microscopy (LSCM). The value of fluorescence intensity is an average value obtained by scanning >10 cells in three different repeats each experiment. The fluorescence intensity did not change (both were 15.3) if 10 µM Fluo-3 in 100 nM CaCl2 solution was observed from 22°C to 37°C (Fig. 1A, 1 and 2), so the effect of temperature on dye fluorescence in the 22°C to 37°C temperature range was negligible. The fluorescence intensity was from 14.3 to 14.6 if the tissue non-loaded was incubated from 22°C to 37°C (Fig. 1A, 3 and 4), showing that autofluorescence during HS was negligible. Treatment with 25 µM A23187 and 5 mM CaCl2 resulted in a fluorescence intensity of 227.2 (Fig. 1A, 6), whereas fluorescence intensity in tissue treated with 5 mM EGTA and 25 µM A23187 was 26 (Fig. 1A, 8). This result verified that Fluo-3-fluorescence increase does report [Ca2+]i increase. To observe clearly where the dye is located, we made a full LSCM image of a protoplast. The protoplasts were obtained from tissue treated by cellulase and incubated in 10 µM Fluo-3/AM solution at 22°C or 37°C, then observed under LSCM. The image (Fig. 1B) showed that Fluo-3 is located in the cytoplasm. The dye did not move to the vacuole or apoplast during HS treatment. The Fluo-3-fluorescence in the cytoplasm increased obviously during HS treatment (Fig. 1B, 1-4). This proved that HS caused an increase of [Ca2+] in the cytoplasm. In measurement of [Ca2+]i from wheat tissue during HS, the measured tissue was incubated in medium containing 10 µM Fluo-3/AM at 24°C in the dark for 2 h. Then, the fluorescence of the cells was observed by LSCM. Fluorescence intensity was measured every 0.5 min to a total of 10 min. Control cells maintained at 22°C remained constant in fluorescence during the experiment (Fig. 1, C and E). A significant increase in [Ca2+]i was observed in the cells during HS at 37°C (Fig. 1, D and E). The initiation of this [Ca2+]i increase occurred within 1 min of HS. After 4 min of HS, the [Ca2+]i reached a maximum 3-fold increase (Fig. 1E).
The levels of CaM protein in tissues treated with distilled water, 10 mM CaCl2, or 5 mM EGTA before HS were similar. The level of CaM protein in tissue with each treatment before HS was normalized to 100%. The concentration of CaM protein in wheat tissue treated with distilled water increased during HS at 37°C and reached a maximum 2-fold increase after 90 min of HS. Treatment with 10 mM CaCl2 promoted the increase during HS at 37°C. The accumulation of CaM protein reached a maximum 3-fold increase after 90 min of HS. The calcium ion chelator EGTA prevented CaM accumulation during HS, suggesting that CaM accumulation is dependent on calcium (Fig. 2).
Northern analysis using the wheat CaM cDNA CaM1-2 as the probe showed that the CaM1-2 is constitutively expressed, and its mRNA has a basal expression level at normal temperature (22°C). The CaM1-2 gene expression started to increase after HS at 37°C for 10 min, then reached its maximum 20 min after HS. The mRNA returned to the basal expression level after 1 h of HS.
The tissue cut from 3-d-old wheat seedlings was incubated in 1-mL solutions of 5, 10, or 50 mM CaCl2, respectively, at 22°C (non-HS temperature) for 30 min. Then, northern analysis using the wheat CaM1-2 and hsp26 cDNAs as probes was performed. Wheat CaM1-2 has a low, basal expression at 22°C (Fig. 3A), and wheat hsp26 mRNA was undetectable at 22°C (Fig. 3B). Treatment with 5 mM CaCl2 had little effect on the expression of CaM1-2 and hsp26. Treatment with 10 mM CaCl2 promoted expression of the two genes, and the effect of 50 mM CaCl2 was more marked (Fig. 3). Treatment with MgCl2 up to 50 mM did not affect expression of the genes (data not shown).
Various compounds that affect the Ca2+-CaM signaling system were employed to investigate the role of Ca2+-CaM in up-regulating expression of HSP genes. Total RNA was used for northern analysis, using the hsp26 and hsp70 cDNAs as probes. In control experiments, the treatments with EGTA, LaCl3, or verapamil under non-HS condition (22°C) did not affect expression of hsp26 or hsp70. The hsp26 mRNA was undetectable, and there was low-level expression of hsp70 at 22°C (Fig. 4A). A 37°C HS increased the expression of hsp26 and hsp70. Treatment with 10 mM CaCl2 increased the expression of hsp26 and hsp70, whereas treatment with the Ca2+ chelator EGTA decreased their expression (Fig. 4B). Treatment with the calcium ion channel blockers showed that 100 µM LaCl3 only slightly lowered the expression of hsp26, whereas 300 µM LaCl3 lowered the expression level significantly (Fig. 4C). Expression of hsp26 was strongly inhibited by both 100 and 200 µM verapamil (Fig. 4D).
The expression of hsp26 and hsp70 decreased with increasing concentrations of the CaM antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W7). Treatment with 100 and 150 µM W7 had little effect on HSP gene expression, whereas treatment with higher concentrations caused a remarkable decrease in the level of hsp26 and hsp70 mRNAs (Fig. 4E). Treatment with 100 to 300 µM N-(6-aminohexyl)-1-naphthalene sulfonamide (W5), an inactive structural analog of W7, did not influence the expression of the HSP genes (Fig. 4F). Another CaM antagonist, chlorpromazine (CPZ), also down-regulated the expression of hsp26 (Fig. 4G). Control experiments showed that at 22°C, treatments with W7 or CPZ did not change the expression of hsp26 (Fig. 4H).
Proteins were isolated from wheat tissue labeled with [35S]-Met at 37°C or at 22°C (control) and were separated by SDS-PAGE (Fig. 5). At non-HS temperature, the protein patterns from tissue treated with 5 mM EGTA or 50 µM CPZ (data not shown) were similar to that from untreated tissue (Fig. 5, lane 2). In response to HS at 37°C, the synthesis of specific proteins was induced. The proteins with molecular weights corresponding to the 70- to 80-kD HSPs and 17- to 20-kD smHSPs increased during HS (Fig. 5, lane 3). Treatment with 10 mM CaCl2 at 37°C led to increased accumulation of protein bands that correlate mainly with the 70- to 90-kD HSPs and the 26- to 28-kD smHSPs (Fig. 5, lane 4). In contrast, when treated with 5 mM EGTA at 37°C, there was decreased synthesis of HS-induced proteins (Fig. 5, lane 5). The synthesis of these proteins induced by HS was also inhibited by treatment with the CaM antagonists, 50 µM CPZ or 25 µM TFP (Fig. 5, lanes 6 and 7).
CaM1-2 has a low, basal level of expression at normal temperature. However, we observed a increase in the accumulation of CaM1-2 mRNA after treatment at 37°C for only 10 min. The increased level of expression of CaM1-2 reached its maximum after 20 min of HS, then returned to basal levels after 1 h of HS (Fig. 6A). The Hsp26 expression was induced by HS treatment. At non-HS temperature, the hsp26 mRNA was undetectable and was still undetectable 10 min after HS. However, the hsp26 mRNA levels began to appear 20 min after HS treatment at 37°C. The accumulation of hsp26 mRNA increased with prolonged HS treatment (Fig. 6B). There was a low, basal level of hsp70 expression at normal temperature, and expression did not change after HS for 10 min. After HS treatment at 37°C for 20 min, the hsp70 expression levels began to increase (Fig. 6C). Similar expression patterns for hsp70 and hsp26 were observed. The activation of hsp26 or hsp70 gene expression was slower than CaM1-2 expression.
To determine the shortest HS treatment that can induce expression of hsp26, wheat seedlings were initially heat shocked at 37°C for various lengths of time and then returned to 22°C for recovery. Using this approach, we found that the induction of hsp26 expression was first detected after only 5 min of HS and peaked after 15 min of HS (Fig. 7). These results suggest that 5 min of HS is adequate to switch a key factor in the cell and initiate a signal transduction process that can carry on at a normal temperature (22°C for wheat).
Increase of [Ca2+]i and CaM Gene Expression Induced by HS
In plant cells, the list of messengers used by signaling pathways includes
Ca2+, lipids, pH, and cyclic GMP
(Sanders et al., 1999
A significant change in [Ca2+]i induced by HS has
been reported in both animal (Calderwood et
al., 1988
The HS response is ubiquitous when cells are exposed to elevated temperatures. However, little is known about how the HS signal is perceived and transduced to activate the genes encoding the HSPs. Ca2+ and CaM are proposed to be important components upstream in HS signal transduction due to the rapid response of Ca2+ and CaM to HS. More studies are needed to verify this proposal.
The involvement of Ca2+ in activation of HSF
(Mosser et al., 1990
Some compounds such as EGTA, La3+, and all other inhibitors were
used to investigate the role of Ca2+ and CaM in this study. These
compounds do affect living cells. In particular, La3+ has been
shown to drastically perturb [Ca2+]i homeostasis
(Plieth 2001
Evidence is given that there is considerable interlinking between heat and
oxidative stress responses (Gong et al., 1997;
Dat et al., 1998
Our experimental results establish the kinetics of the [Ca2+]i increase induced by HS and the expression of wheat CaM1-2, hsp26, and hsp70 genes. These results define the order of the signal transduction steps during and immediately after HS. The increase of [Ca2+]i induced by HS at 37°C occurs very quickly, taking only 1 min after HS (Fig. 1). The level of CaM1-2 mRNA significantly increased 10 min after HS, but the increase in expression of hsp26 and hsp70 was detected 20 min after HS (Fig. 6). The expression of CaM1-2 appears to increase more rapidly than expression of the HSP genes. The different temporal expression between CaM and HSP genes indicates that CaM is located upstream in HS signal transduction.
Only 5 min of HS at 37°C was needed to induce hsp26 gene
expression if a recovery time of 55 min at 22°C after HS was allowed
(Fig. 7). However, a longer
time of 20 min HS at 37°C was needed for expression of hsp26 gene
if without recovery time (Fig.
6B). This result is consistent with previous work done with
soybeans (Glycine max) by Kimpel et al.
(1990
Our previous work has shown that there is a CaM-binding site within maize
cytoplasmic HSP70 and that HSP70 binds CaM in a Ca2+-dependent
manner (Sun et al., 2000
It is possible that there are several different pathways of HS signal
transduction in cells. Mosser et al.
(1990
Reagents
All enzymes were purchased from Promega (Madison, WI) or Sino-American
Biotechnology Company (Luoyang, People's Republic of China). Both the reverse
transcription-PCR system and agarose are from Promega. W7, W5, CPZ, TFP, and
verapamil were obtained from Sigma (St. Louis). Nylon membranes were the
product of Gelman Instrument Co. (Ann Arbor, MI). [35S]-Met was the
product of Amersham Biosciences UK, Ltd. (Little Chalfont, Buckinghamshire,
UK). [
Seeds of winter wheat (Triticum aestivum L. cv 90-80) were imbibed overnight (12 h) in distilled water at 22°C. The soaked seeds were sown on three layers of filter paper wetted with distilled water for germination in the dark at 22°C for 3 d. Segments (1.5 cm long) excised from 3-d-old etiolated seedlings were used in all experiments. The tissue segments were placed wound-side down in 1 mL of different solutions (distilled water as control, CaCl2, EGTA, verapamil, LaCl3, W7, W5, CPZ, and TFP, respectively; the concentrations of compounds are described in the figure legends) at 22°C for 20 min, and then the tissue was subjected to a direct HS by placing in a controlled temperature incubator at 37°C for 1 h. In the experiments on kinetics of gene expression during HS, tissue incubated in distilled water was heat shocked in an incubator at 37°C for different times. In the recovery experiment, the tissue was heat shocked at 37°C for different times, then returned to 22°C. All treated tissues were immediately frozen in liquid N2.
Tissue sections of 1.5 cm from 3-d-old etiolated wheat seedlings were used in all experiments except measurement of [Ca2+]i. For measurement of [Ca2+]i, a thin tissue section was needed to load the dye and make the LSCM observations. A thin segment with intact cell layers could be obtained from the leaf sheath of 10-d-old green wheat seedlings. We had to use the 10-d-old green seedlings for investigation of [Ca2+]i, although the 3-d-old wheat etiolated seedlings were used in all other experiments. The seedlings were grown in pots in growth chamber at 22°C day/18°C night under a fluorescent light (approximately 250 µmol m–1 s–1) with a 12-h photoperiod. Fluo-3/AM was used as the Ca2+-sensitive fluorescent probe. Thin tissue sections, about three cell layers thick and 0.5 cm long, were stripped from the sheath of the first leaf of a 10-d-old green seedling and rinsed with an isotonic solution three times, then incubated in medium containing 10 µM Fluo-3/AM at 24°C in the dark for 2 h. A section loaded with Fluo-3/AM was placed on outer surface of a glass tube, through which water was circulated from a bath with the aid of a pump. The temperature of the tissue on the outer surface of the glass tube was able to reach 37°C within 2 min and maintained at 37°C, whereas the warm water was circulated from water bath at 39°C into an inner glass tube. The subepidermal leaf sheath cells were observed under LSCM (MRC-1024 with a four-line argon laser box, Bio-Rad Laboratories, Hercules, CA). Excitation filter (488 ± 10 nm) and emission filter (530 ± 40 nm) were used in this experiment. The scan mode was XY-T (three dimensional). The change of fluorescence intensity in the cells with time was recorded with the Lasersharp 2000 time lapse program (Bio-Rad Laboratories). After that, the kinetics of fluorescence intensity were measured with the software Laserpix 4.0 (Bio-Rad Laboratories).
The plasmid encoding the soybean (Glycine max) hsp70 cDNA
was kindly provided by Professor Joe L. Key (Botany Department, University of
Georgia, Athens; Roberts and Key,
1991
According to the published sequence
(Joshi et al., 1997
The tissues frozen in liquid N2 were ground with a mortar and
pestle. The extraction of total RNA was performed essentially as described by
Ausubel et al. (1998
For isolation of CaM, wheat tissue treated with Ca2+ or EGTA at
37°C was ground in liquid N2, then homogenized in buffer (50
mM Tris-HCl [pH 8.0], 1 mM EGTA, 0.5 mM
phenylmethylsulfonyl fluoride, 20 mM NaHSO4, and 0.15
M NaCl) at 1:1 (w/v). The homogenates were disintegrated by
sonication for total of 2 min, treated in a water bath of 90°C to 95°C
for 3 min followed by cooling, then centrifuged at 10,000g for 30
min. The supernatants were used for measurement of protein quantity and CaM
concentration. It has been reported that the content of apoplastic CaM is only
2.7% of total CaM in cells (Ye and Sun,
1988
Wheat tissues were incubated in 1 mL of various treatment solutions (as
described in the figure legends) and heat shocked at 37°C or kept at
22°C as control with gentle shaking. Forty microcuries of
[35S]-Met was added to each sample after 2 h at 37°C. Labeling
was carried out for another 2 h at 37°C. The labeled tissues were rinsed
thoroughly with rinse buffer (1 mM K-PO4 [pH 7.5], 1%
[w/v] Suc, and 5 mM Met), then immediately placed into liquid
N2. For protein isolation, the tissues were ground in liquid
N2 and then homogenized in homogenization buffer (50 mM
Tris-HCl [pH 7.5], 2% [w/v]
We thank Professor Hillel Fromm (Weizmann Institute of Science, Rehovot, Israel) for the wheat CaM1-2 cDNA and Professor Joe L. Key (Botany Department, University of Georgia, Athens) for the soybean hsp70 cDNA. We also thank Dr. Jan A. Miernyk (Plant Genetics Research Unit, U.S. Department of Agriculture, Agricultural Research Service, Columbia, MO) for critical reading of the manuscript and comments. Received December 6, 2002; returned for revision January 14, 2003; accepted March 17, 2003.
Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.102.018564.
1 This work was supported by the National Natural Science Foundation of China
(grant no. 3977075), by the Natural Science Foundation of Hebei Province,
China (grant no. 301447), and by the National Key Basic Research Special
Funds, China (G1999011700). * Corresponding author; e-mail zhourengang{at}163.com; fax 0086-311-7042490.
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TOP
ABSTRACT
RESULTS
-32P]dCTP was from the Beijing Yahui Bio-medical
Technology Company (Beijing, People's Republic of China). The Random Primer
DNA Labeling Kit was from the Beijing Dingguo Biotechnology Development Center
(Beijing, People's Republic of China). All other reagents used were of
analytical purity. 
-mercaptoethanol, and 5 mM EDTA).
Homogenates were centrifuged at 12,000g at 4°C for 20 min. The
supernatants were separated by 12.5% (w/v) SDS-PAGE. After gels were dried
under vacuum, autoradiography was performed using Kodak x-ray film at
–80°C. 
