Calcium-mediated signal transduction in transgenic Nicotiana plumbaginifolia

Over recent years evidence has rapidly accumulated to suggest that Ca 2 plays a central role in the transduction of a wide range of stimuli in plant cells. Stomata, the microscopic pores at the leaf surface which regulate gas exchange between the plant and the atmosphere, constitute an attractive model system for the study of signal transduction in plants since they respond to a variety of environmental stimuli in a readily quantifiable manner. This study describes two approaches for the study of Ca 2 -mediated signal transduction in stomatal guard cells. A novel assay for monitoring stimulus-induced changes in guard cell free cytosolic Ca 2 ([Ca2]t) was developed, using epidermal strips from a transgenic line of Nicotiana plumbaginfolia harbouring apoaequorin, the precursor of the Ca2 -sensitive photoprotein, aequorin. Results indicated that mechanical, low temperature and abscisic acid (ABA) signals directly affect stomatal behaviour, promoting rapid closure, and that elevations of guard cell [Ca 2+] Cyt play a key role in the transduction of these signals. Studies with Ca 2 channel blockers and the Ca 2 chelator EGTA, further suggested that mechanical and ABA signals primarily mobilise Ca2 from intracellular store(s), whereas influx of extracellular Ca 2 was a key component in the transduction of low temperature signals. The stomatal response to low temperature was found to be significantly influenced by the previous plant growth temperature. The second approach involved the production of transgenic lines of N. plumbaginfolia expressing apoaequorin under the control of two guard cell 'specific' promoters, the ABA-responsive cDeT6-19 promoter from Craterostigina plantagineum (Michel et al., 1994; Taylor et al., 1995) and the constitutive lipid transfer protein (LTP 1) promoter from Arabidopsis thaliana (Thoma et at., 1994), enabling stimulus-induced changes in guard cell 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 2 responses to those observed in epidermal strips, with similar sensitivities to Ca 2 antagonists. Phosphoinositide turnover, possibly stimulated by a coupling G-protein, was also implicated in the low temperature signaling pathway. The hypothesis that Ca2 plays a central role in the molecular clock mechanism in plants has also been investigated. Transgenic N. plumbaginifolia, expressing apoaequorin under the control of the CaMV 35S promoter were found to exhibit circadian oscillations in [Ca 2 ]t in free-running conditions, confirming previous findings of Johnson et al. (1995). Results obtained by chemical manipulation of the rhythm indicated a requirement for both intracellular Ca 2 -release and influx from extracellular sources for the maintenance of rhythmicity. Also implicated in the regulation of oscillatory [Ca2+]Cyt were G-proteins, calmodulin and phosphorylation / dephosphorylation reactions. Results did not support a central role for Ca 2 in the molecular clock mechanism, but did not preclude, the possibility that oscillations, entrained by daily changes in light intensity, may play a role in the photic entrainment pathway. This study also provided preliminary evidence that Ca 2 may play a role in the diurnal control of stomatal movements, since guard cell 'targeted' transgenic seedlings exhibited circadian oscillations in [Ca2 ]-dependent aequorin luminescence in freerunning conditions.