The involvement of calcium and calcium-activated calmodulin (Ca 2ϩ -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 Ca 2ϩ . The expression of hsp26 and hsp70 genes was up-regulated by the addition of CaCl 2 and down-regulated by the calcium ion chelator EGTA, the calcium ion channel blockers LaCl 3 and verapamil, or the CaM antagonists N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide and chlorpromazine. Treatment with Ca 2ϩ 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 Ca 2ϩ -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 ([Ca 2ϩ ] i ) in Chinese hamster (Cricetulus barabensis) HA-1 fibroblasts (Calderwood et al., 1988). In plants, Gong et al. (1998) observed that HS caused a transient increase in [Ca 2ϩ ] i . The change in [Ca 2ϩ ] i is also involved in regulating the binding activity of the HS transcription factor (HSF) to the HS element (Mosser et al., 1990), the synthesis of HSPs (Kiang et al., 1994;Kuznetsov et al., 1998), and...

Heat shock (HS) is a common form of stress suffered by plants. It has been proposed that calmodulin (CaM) is involved in HS signal transduction, but direct evidence has been lacking. To investigate the potential regulatory function of CaM in the HS signal transduction pathway, T-DNA knockout mutants for AtCaM2, AtCaM3, and AtCaM4 were obtained and their thermotolerance tested. Of the three knockout mutant plants, there were no differences compared with wild-type plants under normal conditions. However, the AtCaM3 knockout mutant showed a clear reduction in thermotolerance after heat treatment at 45°C for 50 min. Overexpression of AtCaM3 in either the AtCaM3 knockout or wild-type background significantly rescued or increased the thermotolerance, respectively. Results from electrophoretic mobility-shift assays, real-time quantitative reverse transcription-polymerase chain reaction, and western-blot analyses revealed that, after HS, the DNA-binding activity of HS transcription factors, mRNA transcription of HS protein genes, and accumulation of HS protein were downregulated in the AtCaM3 knockout mutant and up-regulated in the AtCaM3-overexpressing transgenic lines. Taken together, these results suggest that endogenous AtCaM3 is a key component in the Ca 2+ -CaM HS signal transduction pathway.

SUMMARYAn increased concentration of cytosolic calcium ions (Ca 2+ ) is an early response by plant cells to heat shock.However, the molecular mechanism underlying the heat-induced initial Ca 2+ response in plants is unclear. In this study, we identified and characterized a heat-activated Ca 2+-permeable channel in the plasma membrane of Arabidopsis thaliana root protoplasts using reverse genetic analysis and the whole-cell patch-clamp technique. The results indicated that A. thaliana cyclic nucleotide-gated ion channel 6 (CNGC6) mediates heatinduced Ca 2+ influx and facilitates expression of heat shock protein (HSP) genes and the acquisition of thermotolerance. GUS and GFP reporter assays showed that CNGC6 expression is ubiquitous in A. thaliana, and the protein is localized to the plasma membrane of cells. Furthermore, it was found that the level of cytosolic cAMP was increased by a mild heat shock, that CNGC6 was activated by cytosolic cAMP, and that exogenous cAMP promoted the expression of HSP genes. The results reveal the role of cAMP in transduction of heat shock signals in plants. The correlation of an increased level of cytosolic cAMP in a heat-shocked plant with activation of the Ca 2+ channels and downstream expression of HSP genes sheds some light on how plants transduce a heat stimulus into a signal cascade that leads to a heat shock response.