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Plant and Cell Physiology Advance Access originally published online on December 23, 2008
Plant and Cell Physiology 2009 50(2):360-373; doi:10.1093/pcp/pcn203
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© The Author 2008. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oxfordjournals.org

Measurement of Changes in Cytosolic Ca2+ in Arabidopsis Guard Cells and Mesophyll Cells in Response to Blue Light

Akiko Harada1,2,* and Ken-ichiro Shimazaki1

1Department of Biology, Graduate School of Sciences, Kyushu University, Fukuoka, 810-8560 Japan

*Corresponding author: E-mail, bio006{at}art.osaka-med.ac.jp.


  Abstract

Phototropins (phot1 and phot2) are blue light (BL) receptors that mediate responses including phototropism, chloroplast movement and stomatal opening, and increased cytosolic Ca2+. BL absorbed by phototropins activates plasma membrane H+-ATPase in guard cells, resulting in membrane hyperpolarization, and drives K+ uptake and stomatal opening. However, it is unclear whether the phototropin-mediated Ca2+ increase activates the H+-ATPase. Here, we determined cytosolic Ca2+ concentrations in guard cell protoplasts (GCPs) from Arabidopsis transformed with aequorin. Cytosolic Ca2+ increased rapidly in response to BL in GCPs from both the wild type and phot1 phot2 double mutants, but was mostly suppressed by an inhibitor of photosynthetic electron flow (DCMU). With depleted external K+, we observed another slower Ca2+ increase, which was phototropin- dependent. Fusicoccin, a H+-ATPase activator, mimicked the effect of BL. The slow Ca2+ increase thus appears to result from membrane hyperpolarization. The slow Ca2+ increase was suppressed by external K+ and was restored by blockers of inward-rectifying K+ channels, CsCl and tetraethylammonium, suggesting the preferential uptake of K+ over Ca2+. Such efficient K+ uptake in response to BL was not found in mesophyll cells. Both the fast and the slow Ca2+ increases were inhibited by Ca2+ channel blockers (CoCl2 and LaCl3) and a chelating agent (EGTA). These results indicate that the phototropin-mediated Ca2+ increase was not observed prior to H+-ATPase activation in guard cells and that Ca2+ entered guard cells via Ca2+ channels through photosynthesis and phototropin-mediated membrane hyperpolarization.

Keywords: Arabidopsis thaliana - Blue light - Ca2+ metabolism - Guard cells - K+ metabolism - Phototropins

Abbreviations: BL, blue light; BTP, bis(tris[hydroxymethyl]methylamino) propane; DMSO, dimethylsulfoxide; FC, fusicoccin; GCP, guard cell protoplast; K+in channel, inward-rectifying K+ channel; Ler, Landsberg erecta; MCP, mesophyll cell protoplast; RL, red light; RT–PCR, reverse transcription–PCR; TEA, tetraethylammonium; WS, Wassilewskija

2Present address: Department of Biology, Osaka Medical College, Takatsuki, 569-8686 Japan.

(Received September 22, 2008; Accepted December 18, 2008)
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