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Studies using stably transformed tobacco plants containing very low levels of PI-PLC in their guard cells show that this enzyme plays a role in the events associated with the inhibition of stomatal opening by ABA, but not in the cellular reactions that are responsible for ABA-induced stomatal closure. However, Commelina communis guard cells microinjected with the InsP3 antagonist, heparin, fail to close on addition of ABA. There are three possible explanations for this apparent data mismatch. The differences may be indicative of species-specific signalling pathways, the presence of a PI-PLC isoform(s) that is not down-regulated in these transgenic lines and/or they may reflect differences between short-term (acute) administration of an inhibitor and long-term (chronic) effects of gene manipulation. It is possible that the guard cell is a robust signalling system that is able to adapt or compensate for the chronic loss of PI-PLC, but which is unable to adjust quickly to acute loss of this component. It would be interesting to investigate this possibility further using either transient manipulation of gene expression or through the use of an inducible promoter.

The calcium-releasing second messenger inositol 1,4,5-trisphosphate is involved in the regulation of stomatal aperture by ABA. In other signalling pathways, inositol 1,4,5-trisphosphate is generated by the action of phospholipase C. We have studied the importance of phospholipase C in guard cell ABA-signalling pathways. Immunolocalisation of a calcium-activated phospholipase C confirmed the presence of phospholipase C in tobacco guard cells. Transgenic tobacco plants with considerably reduced levels of phospholipase C in their guard cells were only partially able to regulate their stomatal apertures in response to ABA. These results suggest that phospholipase C is involved in the amplification of the calcium signal responsible for reductions in stomatal aperture in response to ABA. As full ABA-induced inhibition of stomatal opening was not observed, our results support a role for the action of other calcium-releasing second messengers in the guard cell ABA-signalling pathway. It is not known whether these different calcium-releasing second messengers act in the same or parallel ABA-signalling pathways.

The calcium-releasing second messenger inositol 1,4,5-trisphosphate is involved in the regulation of stomatal aperture by ABA. In other signalling pathways, inositol 1,4,5-trisphosphate is generated by the action of phospholipase C. We have studied the importance of phospholipase C in guard cell ABA-signalling pathways. Immunolocalisation of a calcium-activated phospholipase C confirmed the presence of phospholipase C in tobacco guard cells. Transgenic tobacco plants with considerably reduced levels of phospholipase C in their guard cells were only partially able to regulate their stomatal apertures in response to ABA. These results suggest that phospholipase C is involved in the amplification of the calcium signal responsible for reductions in stomatal aperture in response to ABA. As full ABA-induced inhibition of stomatal opening was not observed, our results support a role for the action of other calcium-releasing second messengers in the guard cell ABA-signalling pathway. It is not known whether these different calcium-releasing second messengers act in the same or parallel ABA-signalling pathways.

Inositol 1,4,5-trisphosphate (InsP3) is an important component of calcium-based signal transduction pathways in eukaryotic cells. Hydrolysis of phosphatidylinositol 4,5-bisphosphate by phosphoinositide-specific phospholipase C (PI-PLC) generates InsP3, which mobilizes calcium from endomembrane stores, and diacylglycerol. We have used a combined molecular and cell physiological approach to examined the role of the PI-PLC/InsP3-mediated calcium mobilizing pathway in the signal transduction pathway(s) by which stomatal guard cells respond to environmental stimuli such as abscisic acid (ABA) and drought. We have identified a guard cell PI-PLC that is specifically inhibited by an aminosteroid, U-73122; ABA-induced stomatal closure and ABA-induced oscillations in guard cell cytosolic free calcium are both inhibited by U-73122 (Staxen et al., 1999, Proc. Natl. Acad. Sci. USA 96 1779-1784). In addition, microinjection of the competitive InsP3 inhibitor heparin directly into the cytosol of guard cells also inhibits the ABA-induced reduction in guard cell turgor. These data, together with the results of our studies using Nicotiana rustica plants transformed with sense and antisense constructs of the PI-PLC, suggest a role for the PI-PLC/InsP3-mediated calcium-mobilizing pathway in the response of stomata to ABA and drought.

Inositol 1,4,5-trisphosphate (InsP3) is an important component of calcium-based signal transduction pathways in eukaryotic cells. Hydrolysis of phosphatidylinositol 4,5-bisphosphate by phosphoinositide-specific phospholipase C (PI-PLC) generates InsP3, which mobilizes calcium from endomembrane stores, and diacylglycerol. We have used a combined molecular and cell physiological approach to examined the role of the PI-PLC/InsP3-mediated calcium mobilizing pathway in the signal transduction pathway(s) by which stomatal guard cells respond to environmental stimuli such as abscisic acid (ABA) and drought. We have identified a guard cell PI-PLC that is specifically inhibited by an aminosteroid, U-73122; ABA-induced stomatal closure and ABA-induced oscillations in guard cell cytosolic free calcium are both inhibited by U-73122 (Staxen et al., 1999, Proc. Natl. Acad. Sci. USA 96 1779-1784). In addition, microinjection of the competitive InsP3 inhibitor heparin directly into the cytosol of guard cells also inhibits the ABA-induced reduction in guard cell turgor. These data, together with the results of our studies using Nicotiana rustica plants transformed with sense and antisense constructs of the PI-PLC, suggest a role for the PI-PLC/InsP3-mediated calcium-mobilizing pathway in the response of stomata to ABA and drought.

Image of a fire on the west side of Flin Flon Lake, Flin Flon Manitoba. Photograph courtesy of F. Aitken.

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Image of a large crowd at Flin Flon Sports Day at Lakebottom Park. Photograph courtesy of F. Aitken.

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Image of construction at the Hudson Bay Mining and Smelting Company (H.B.M.S.), Flin Flon, Manitoba. Photograph courtesy of F. Aitken.

Image of a large crowd at Flin Flon Sports Day at Lakebottom Park. Photograph courtesy of F. Aitken.

Image of the sinking for the north main shaft at the Hudson Bay Mining and Smelting Company (H.B.M.S), Flin Flon, Manitoba. Photograph courtesy of F. Aitken.

Image of the construction of buildings at Hudson Bay Mining and Smelting Company (H.B.M.S.), Flin Flon, Manitoba. Photograph courtesy of F. Aitken.

Image of the south main changehouse at the Hudson Bay Mining and Smelting Company (H.B.M.S.), Flin Flon, Manitoba. Photograph courtesy of F. Aitken.