Nitrate-sensitive ATPase activity and proton pumping in stomatal protoplasts

 

 

Nitrate-sensitive ATPase activity and proton pumping in guard cell protoplasts of Commelina

by Fricker M. D., Willmer C. M. (1990)

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in J. Exp. Bot. 41(2): 193-198 – https://doi.org/10.1093/jxb/41.2.193 –

https://academic.oup.com/jxb/article-abstract/41/2/193/498816?redirectedFrom=fulltext

Abstract

ATPase activity was measured in crude homogenates of guard cell protoplasts of Commelina communis L. using a linked enzyme assay.

A low level of azide-sensitive ATPase activity was detected with a pH optimum of 6.8. This activity was stimulated by 0.01% (v/v) Triton X-100, and the pH optimum shifted to pH 7.4.

Nitrate-sensitive ATPase activity was measured in the presence of azide and showed a pH optimum around pH 8.0. Proton pumping activity in a mixed population of vesicles from GCP was monitored using fluorescence quenching of quinacrine. Mg-ATP dependent proton pumping was observed at pH 8.0, but not at pH 6.6.

The activity at pH 8.0 was inhibited by nitrate and DCCD but not vanadate.

These data indicate that activity of the tonoplast proton pump was being measured. There was, however, no evidence for a tonoplast cation (K+)/proton antiporter under these assay conditions as potassium did not reduce the initial rate of pH gradient formation or increase the rate of collapse of a pre-formed gradient after inhibition of the pump.

ATPase and phosphatase activity in stomatal protoplasts

 

 

Vanadate sensitive ATPase and phosphatase activity in guard cell protoplasts of Commelina

by Fricker M. D., Willmer C. M. (1987)

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in J. Exp. Bot. 38: 642–648 – https://doi.org/10.1093/jxb/38.4.642 –

https://academic.oup.com/jxb/article-abstract/38/4/642/457629?redirectedFrom=fulltext

Phosphatase activity was measured in extracts of guard cell protoplasts of Commelina communis L. using the artificial substrate p-nitrophenylphosphate. A pH optimum of 5.8 to 6.3 was determined.

Ammonium molybdate (Ol mol m−3) and sodium vanadate (1–0 mol m−3) gave almost complete inhibition of phosphatase activity at pH 60. ATPase assays were, therefore, conducted in the presence of 0–2 mol m −3 molybdate and vanadate was used as a specific inhibitor of plasmamembrane ATPase activity.

Vanadate sensitive ATPase activity showed a pH optimum of 6.6 and activity was stimulated by KC1. These properties are characteristic of plasmamembrane proton pumping ATPases in other systems and suggest that proton extrusion in guard cells could be mediated by a similar enzyme.

The maximum ATPase activity is sufficient to account for all the proton flux observed during the stomatal opening response.

The calcium message in stomata

 

 

Visualisation and measurement of the calcium message in guard cells

by Fricker M. D., Gilroy S., Read N. D., Trewavas A. J. (1991)

Department of Plant Sciences, University of Oxford, UK.

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In: Molecular Biology of Plant Development. W. Schuch and G. Jenkins, editor. Cambridge University Press, Cambridge –

Symp Soc Exp Biol. 45: 177-190 –

https://www.ncbi.nlm.nih.gov/pubmed/1843407

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.

Stomata

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Stomata

by Willmer C.M., Fricker M. (1996)

In : Topics in Plant Functional Biology: 2 – Eds. M. Black and B. Charlwood – Springer-Science+ Business Media B. V. –

https://books.google.be/books?id=9sjoCAAAQBAJ&pg=PA12&lpg=PA12&dq=stomata&source=bl&ots=tIpu1rPm39&sig=YGbF_YtCyNa-YR3F8_QWpf3UugI&hl=en&sa=X&ved=0ahUKEwiS3e-4ocbYAhVMmbQKHcY3DWw4HhDoAQgoMAA#v=onepage&q=stomata&f=false

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Ca and transduction pathways linking ABA perception to stomatal closure

 

Role of calcium in signal transduction of Commelina guard cells.

Gilroy S.,Gilroy

Fricker M. D.,Mark_Fricker_2127_1865

Read N. D.,

 Trewavas A. J.trewavas

 (1991)

in Plant Cell, 3, 333344. –

CrossRef |PubMedCAS |

http://www.ncbi.nlm.nih.gov/pubmed/12324599

Abstract

The role of cytosolic Ca2+ in signal transduction in stomatal guard cells of Commelina communis was investigated using fluorescence ratio imaging and photometry.

By changing extracellular K+, extracellular Ca2+, or treatment with Br-A23187, substantive increases in cytosolic Ca2+ to over 1 micromolar accompanied stomatal closure. The increase in Ca2+ was highest in the cytoplasm around the vacuole and the nucleus.

Similar increases were observed when the cells were pretreated with ethyleneglycol-bis-(o-aminoethyl)tetraacetic acid or the channel blocker La3+, together with the closing stimuli. This suggests that a second messenger system operates between the plasma membrane and Ca2+-sequestering organelle(s).

The endogenous growth regulator abscisic acid elevated cytosolic Ca2+ levels in a minority of cells investigated, even though stomatal closure always occurred.

Ca2+-dependent and Ca2+-independent transduction pathways linking abscisic acid perception to stomatal closure are thus indicated.

Rapid effects of abscisic acid in Commelina stomata

 

 

Two transduction pathways mediate rapid effects of abscisic acid in Commelina guard cells.

by Allan A. C., Fricker M. D., Ward J. L., Beale M. H., Trewavas A. J. (1994)

in Plant Cell, 6, 1319–1328. – doi: http:/​/​dx.​doi.​org/​10.​1105/​tpc.​6.​9.​1319

CrossRef | – http://www.plantcell.org/content/6/9/1319

Abstract

Commelina guard cells can be rapidly closed by abscisic acid (ABA), and it is thought that this signal is always transduced through increases in cytosolic calcium. However, when Commelina plants were grown at 10 to 17[deg]C, most guard cells failed to exhibit any ABA-induced increase in cytosolic calcium even though all of these cells closed.

At growth temperatures of 25[deg]C or above, ABA-induced closure was always associated with an increase in cytosolic calcium. This suggests that there may be two transduction routes for ABA in guard cells; only one involves increases in cytosolic calcium. Activation of either pathway on its own appears to be sufficient to cause closure.

Because the rates of ABA accumulation and transport in plants grown at different temperatures are likely to be different, we synthesized and microinjected caged ABA directly into guard cells. ABA was released internally by UV photolysis and subsequently caused stomatal closure.

This result suggests a possible intracellular locale for the hypothesized ABA receptor.

See: The Plant Cell

Rapid effects of ABA in Commelina stomata

 

Two transduction pathways mediate rapid effects of abscisic acid in Commelina guard cells

by Allan A. C., Fricker M. D., Ward J. L., Beale M. H., Trewavas A. J. (1994)

in Plant Cell 6, 1319–1328. doi: 10.2307/3869829 –

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text

Abstract

Commelina guard cells can be rapidly closed by abscisic acid (ABA), and it is thought that this signal is always transduced through increases in cytosolic calcium. However, when Commelina plants were grown at 10 to 17[deg]C, most guard cells failed to exhibit any ABA-induced increase in cytosolic calcium even though all of these cells closed. At growth temperatures of 25[deg]C or above, ABA-induced closure was always associated with an increase in cytosolic calcium.

This suggests that there may be two transduction routes for ABA in guard cells; only one involves increases in cytosolic calcium.

Activation of either pathway on its own appears to be sufficient to cause closure. Because the rates of ABA accumulation and transport in plants grown at different temperatures are likely to be different, we synthesized and microinjected caged ABA directly into guard cells. ABA was released internally by UV photolysis and subsequently caused stomatal closure. This result suggests a possible intracellular locale for the hypothesized ABA receptor.

See the text: The Plant Cell