| It is now well established that cold-triggered calcium influx mediates cold-induced gene expression and development of freezing tolerance (cold acclimation). In this thesis, cold signaling events both upstream and downstream of calcium influx were examined.; First, it was shown that the studies on calcium mediation of cold acclimation in alfalfa cell suspension cultures could be applied to intact seedlings of Arabidopsis. Calcium chelators and channel blockers caused a strong reduction in the cold-induced accumulation of kin1 and kin2 transcripts, suggesting that calcium influx was an essential event during cold signaling and that the source of calcium for this influx was largely the calcium-rich cell wall. Evidence suggesting the involvement of calcium-dependent protein kinases (CDPKs) was also obtained.; Second, the nature of events upstream of calcium influx was explored. For this study, transgenic Brassica napus seedlings possessing both the endogenous cold-inducible BN115 gene and the coding part of β-glucuronidase (GUS) gene placed under the control of the BN115 promoter were used. Thus cold-activation of the BN115 promoter drove the expression of both BN115 at the transcriptional level and the GUS enzyme activity at the translational level. Cold-activation of BN115 was inhibited by chemicals which cause membrane fluidization, cytoskeletal stabilization and inhibition of Ca2+ influx, and mimicked at 25°C by chemicals causing membrane rigidification, cytoskeletal destabilization and Ca2+ influx. Inhibitors of protein and lipid kinases prevented cold-activation of BN115, but inhibition of protein phosphatases activated BN115 at 25°C.; Third, given the increasing importance of mitogen-activated protein kinases (MAPKs) in signal transduction, the nature of molecular mechanisms that lead to cold-activation of a previously reported MAPK, SAMK, was investigated. During this study, the first plant MAPK activated by heat shock was discovered and named HAMK (H&barbelow;eat-shock-a&barbelow;ctivated M&barbelow;APK&barbelow;). It was shown that cold-activation of SAMK is mediated by cold-induced membrane rigidification, whereas the heat shock-activation of HAMK occurs through heat shock-induced membrane fluidization. Whereas activation of both SAMK and HAMK is blocked by an actin microfilament stabilizer, it is mimicked at 25°C by chemical destabilizers of microtubules or actin microfilaments. All of these events are inhibited by blocking the influx of extracellular Ca 2+. Cold-activation of SAMK and heat-activation of HAMK was prevented by treatment of cells with inhibitors of CDPKs. Thus, cold and heat shock are sensed by structural changes in the plasma membrane, which transduces the signal via cytoskeletal rearrangements to the opening of calcium channels, leading to Ca2+ influx, activation of CDPKs and activation of distinct MAPK cascades. |
