DNA constructs of Agrobacterium-mediated transformation into <I>Nicotiana plumbaginifolia</I> were prepared. The Ca<SUP>2+</SUP>-sensitive luminescent protein, aequorin was placed in frame with the nuclear localisation signal (NLS) peptide of the simian virus SV40 Large T-antigen, <I>Xenopus leavis'</I> nucleoplasmin, and high mobility group protein, HMG1 from <I>Pisum sativum</I>. After analysing individual transformants, nucleoplasmin was shown to successfully target aequorin to nuclei. These plants, together with plants transgenic for cytosolic aequorin, were used to study Ca<SUP>2+</SUP> dynamics when exposed to the environmental stimuli, wind and cold shock. Wind induced immediate Ca<SUP>2+</SUP> transients in the cytoplasm and nucleus, while cold shock induced a Ca<SUP>2+</SUP> transient in the cytoplasm that was followed by a delayed Ca<SUP>2+</SUP> transient in the cytoplasm that was followed by a delayed transient in the nucleus. Wind and cold shock induced calmodulin gene expression that followed distinct kinetics. Rapid Amplification of cDNA Ends (3' RACE) identified the presence of two calmodulin transcripts in tobacco seedlings; <I>NpCaM-1</I> and <I>NpCaM-2</I> of which only <I>NpCaM-1</I> was induced by both stimuli. The transcripts comprise two different nucleotide sequences but encode identical polypeptides. The expression kinetics of <I>NpCaM-1</I> was related to wind and cold shock-induced nuclear and cytosolic Ca<SUP>2+</SUP> changes by using Ca<SUP>2+</SUP> agonists/antagonists. Wind-induced nuclear Ca<SUP>2+</SUP> changes correlated closely to the subsequent expression of and accumulation of <I>NpCaM-1</I> while changes in cytoplasmic Ca<SUP>2+</SUP> levels did not. Cold shock, on the other hand, did not reveal this correlation, and a role for intracellular Ca<SUP>2+</SUP> levels during subsequent expression of <I>NpCaM-1</I> remains therefore speculative. Heat shock induced a prolonged increase in nuclear Ca<SUP>2+</SUP> level. Seedlings pretreated with Ca<SUP>2+</SUP> or EGTA showed respectively enhanced or diminished subsequent thermotolerance, therefore it was suggested that these increases in Ca<SUP>2+</SUP> levels were required for the acquisition of heat-induce thermotolerance.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:654123 |
| Date | January 1998 |
| Creators | Van der Luit, Arnold H. |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/13155 |
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