This study describes two approaches for the study of Ca<SUP>2+</SUP>-mediated signal transduction in stomatal guard cells. A novel assay for monitoring stimulus-induced changes in guard cell cytosolic Ca<SUP>2+</SUP> ([Ca<SUP>2+</SUP>]<SUB>cyt</SUB>) was developed, using epidermal strips from a transgenic line of <I>Nicotiana plumbaginifolia</I> harbouring apoqequorin, the precursor of the Ca<SUP>2+</SUP>-sensitive photoprotein, aequorin. Results indicated that mechanical, low temperature and abscisic acid (AGA) signals, directly affected stomatal behaviour, promoting rapid closure, and that elevations of guard cell [Ca<SUP>2+</SUP>]<SUB>cyt</SUB> play a key role in the transduction of these signals. Studies with Ca<SUP>2+</SUP> channel blockers and the Ca<SUP>2+</SUP> chelator EGTA, further suggested that mechanical ABA signals primarily mobilise Ca<SUP>2+</SUP> from intracellular store(s), whereas influx of extracellular Ca<SUP>2+</SUP> was a key component in the transduction of low temperature signals. The stomatal response to low temperature was found to be significantly influenced by previous plant growth temperature. The second approach involved the production of transgenic lines of <I>N. plumbaginifolia</I> expressing apoaequorin under the control of two guard cell 'specific' promoters, the ABA-responsivecDeT6-19 promoter from <I>C.plantagineum</I> (Michel <I>et al</I>., 1994; Taylor <I>et al</I>., 1995) and the constitutive lipid transfer protein (LTP1) promoter from <I>A. thaliana</I> (Thoma <I>et al</I>., 1994), enabling stimulus-induced changes in guard cell [Ca<SUP>2+</SUP>]<SUB>cyt</SUB> to be monitored in intact seedlings. ABA-induction of cDET6-19 promoter activity did not affect the ability of stomata to respond to further applications of ABA. Results showed that mechanical and cold shock signals induced similar Ca<SUP>2+</SUP> responses to those observed in epidermal strips, with similar sensitivities to Ca<SUP>2+</SUP> antagonists. Studies with inhibitors of other putative components of the plant Ca<SUP>2+</SUP> signaling pathway also implicated phosphoinositide turnover, possibly stimulated by a coupling G-protein, in the low temperature signaling pathway. The hypothesis that Ca<SUP>2+</SUP> plays a central role in the molecular clock mechanism in plants has also been investigated. Transgenic <I>N. plumbaginifolia</I> seedlings expressing apoaequorin under the control of the CaMV 35S promoter were found to exhibit circadian oscillations in [Ca<SUP>2+</SUP>]<SUB>cyt</SUB> in free-running conditions, confirming previous findings of Johson <I>et al</I> (1995).
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:663995 |
| Date | January 1997 |
| Creators | Wood, Nicola T. |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/13242 |
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