Preliminary Studies On The Molecular Mechanism For Calcium Initial Response In Heat Shock Signal Transduction

Our privous work have proved that [Ca²⁺]cyto increased during heat shock （HS） in wheat （Triticum aestivum L.） seedlings, the levels of both calmodulin （CaM） protein and CaM mRNA increased during HS, and that Ca²⁺ and CaM affected the interaction between heat shock transcription factor （HSF） and heat shock element （HSE）, the expression of HSP genes, and the thermotolerance of plants. In Arabidopsis （Arabidopsis thaliana）, as a model plant, the increase in [Ca²⁺]cyto and the expression of CaM gene induced by HS was observed, and Ca²⁺ and CaM were able to promote the expression of HSP genes. Real time quantitative PCR （real time PCR） analysis indicated that the expression of AtCaM3 and AtCaM7 genes was up-regulated during HS and the up-regulation of AtCaM3 expression occurred earlier than that of AtCaM7 or HSP18.2. It was suggested that AtCaM3 is probably involved in HS signal transduction.Our further work indicated that AtCBK3 （CaM-binding protein kinase） played an important role in transducing the HS signaling from CaM to HSF. It was found that AtCBK3 interacted with AtHSFA1 both in vitro and in vivo, and that AtHSFA1 can be phosphorylated by AtCBK3 in vitro in the presence of Ca²⁺ and CaM. The transgenic Arabidopsis lines overexpressing the AtCBK3 gene and the T-DNA insertion null mutant lines of AtCBK3 showed improvement and decline in basal thermotolerancec respectively. AtPP7, a CaM-binding protein phosphatase, might also play an important role in transducing the HS signaling from CaM to HSF. It was proved that AtPP7 was able to interact with CaM and HSF in vitro. The T-DNA insertion AtPP7 knockout lines impaired the basal thermotolerance of plant. The transgenic Arabidopsis lines overexpressing the AtPP7 gene showed improvement in basal thermotolerance. All these results suggested that the AtCBK3 and AtPP7 are important members downstream of CaM in heat shock signal transduction.Based our research results, we suggested that Ca²⁺-CaM is involve in heat shock signaling, and proposed a new pathway of HS signal transduction: Ca²⁺-CaM pathway. The HS signals were perceived by an as yet unidentified receptor; receptor activation was followed by an increase in [Ca²⁺]cyto. This elevated level of [Ca²⁺]cyto then directly activated CaM, and promoted the expression and accumulation of CaM. Activated CaM regulated the activity of AtCBK3 and AtPP7 by interacting with them. AtCBK3 and AtPP7 affected the HSF activity by phosphorylating or dephosphorylating the HSF. Activation of HSF promoted the transcription and translation of HSP genes. In the end, the thermotolerance of plants was changed. Whereas how did the initial response of HS signal transduction work? And how did HS signals cause the increase in [Ca²⁺]cyto during HS? We proposed two possible ways based the results in preliminary experiments: one way was the mobilization of Ca²⁺ from intracellular Ca²⁺ store pools promoted by IP3-PLC; the other way was the mobilization of Ca²⁺ from extracellular Ca²⁺ sources by Ca²⁺ channels in plasma-membrane.The role of inositol 1,4,5-trisphosphate （IP3） in transducing HS signals in Arabidopsis was examined. The whole-plant IP3 level increased within 3 min of HS at 37℃. Using the transgenic Arabidopsis plants which have AtHSP18.2 promoter::GUS fusion gene, it was found that the level of ?-glucuronidase （GUS） activity was up-regulated by the addition of IP3 at both non-HS and HS. The GUS activity was down-regulated by U-73122, a phospholipase C inhibitor. The level of [Ca²⁺]cyto in suspension-cultured Arabidopsis cells expressing apoaequorin increased during HS at 37℃. Treatment with U-73122 prevented the increase of [Ca²⁺]cyto to some extent. The expression of six PLC genes （PLC1-5, 7） from Arabidopsis was differentially regulated by HS at 37℃. The expression of PLCa and PLCb genes increased during HS. The location of C2 domain of PLCb in onion cell which transformed with 35S::C2（PLCb）-YFP was changed after HS. This fact was suggested that the location of C2 domain is functionally changed due to HS. These results showed possible involvement of IP3-PLC pathway in the increase of [Ca²⁺]cyto in HS signal transduction in higher plants. The role of ion channels in plasma-membrane in HS signaling was detected by using the patch-clamp technique. At first, the protoplasts were isolated from the elongation region of 7-day-old Arabidopsis root tip where had been proved the most sensitive part responding to HS of the whole seedlings. In patch-clamp analysis, a hyperpolarization-activated current was recorded, so the activity of hyperpolarization-activated-voltage-dependence Ca²⁺ channels in the plasma membrane （PM） of root cells was investigated. It was also found that the currents at -200 mV before HS were much lower than that after HS, suggesting that Ca²⁺ channels in root cell plasma membrane may be involved in the increase of [Ca²⁺]cyto in HS signaling. In order to investigate the key gene for the increase of [Ca²⁺]cyto in HS signaling, a group of gene family （AtCNGC） null mutants by T-DNA insertation were screened to study the function of ion channel genes in HS signal transduction. It was found that knock out of the AtCNGCx （cngcx-1） impaired the induced thermotolerance of the plants. Using patch-clamp technique, in cngcx-1, there was not detected the increase of currents after HS which was found in Col, suggesting that AtCNGCx is a probable Ca²⁺ channel gene in PM involved in HS signal transduction.