title: Visualisation and measurement of the calcium message in guard cells.
abstract: We have applied several novel technologies to investigate the role of cytosolic free calcium [Ca2+]i in signal transduction in guard cells of Commelina communis L. Fluorescence ratio imaging and photometry together with the fluorescent Ca2+ indicator Indo-1 were used to directly visualise and measure dynamic spatial and temporal changes in [Ca2+]i in response to various exogenous stimuli. More subtle manipulation of the Ca2+ signal transduction pathway was achieved through the use of photoactivateable, caged Ca2+ and caged inositol-1,4,5-triphosphate (InsP3) released directly into the cytoplasm of the guard cell after microinjection. In these experiments, changes in [Ca2+]i were simultaneously monitored with the fluorescent Ca2+ indicator, Fluo-3. Resting levels of [Ca2+]i (100-200 nM) increased in response to elevated [Ca2+]e, lowering [K+]e, application of the ionophore A-23187 or cytosolic release of either Ca2+ or InsP3 from their caged forms. Stomatal closure was triggered if [Ca2+]i increased above a threshold of about 600 nM. Abscisic acid (ABA) had little effect on [Ca2+]i in the majority of cells studied, being elevated in only a minority of cells investigated. However, stomatal closure occurred in all cases after ABA application. This suggests that ABA acts through both Ca(2+)-independent and Ca(2+)-dependent pathways. The imaging data revealed a substantial heterogeneity in [Ca2+]i within the guard cell. Cytoplasmic regions, particularly near the nucleus, often showed marked elevations and sometimes oscillations. The origin and kinetics of the Ca2+ fluxes leading to the dynamic spatial patterns is discussed along with several new approaches directed towards identification of the source of the Ca2+. These methods include optical sectioning and 3-D reconstruction of both the endomembrane system and [Ca2+]i in living guard cells using confocal microscopy. Overall, our data is consistent with multiple sources for [Ca2+]i, including uptake across the plasma membrane and InsP3- or Ca(2+)-induced Ca2+ release from internal stores.
authors.affiliation: Department of Plant Sciences, University of Oxford, UK.
authors.forename: M D
authors.initials: MD
authors.lastname: Fricker
authors.forename: S
authors.initials: S
authors.lastname: Gilroy
authors.forename: N D
authors.initials: ND
authors.lastname: Read
authors.forename: A J
authors.initials: AJ
authors.lastname: Trewavas
mesh_terms: D002118:Calcium / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D005453:Fluorescence; D007700:Kinetics; D010783:Photometry; D059828:Plant Cells; D010944:Plants / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*
chemical_list: D002118:Calcium