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Plant and Cell Physiology Advance Access published online on December 23, 2008
Plant and Cell Physiology, 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 e-mail: 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: Dr. Akiko Harada, Department of Biology, Osaka Medical College, 2-7, Daigaku-machi, Takatsuki 569-8686, Japan Tel: +81-72-683-1221 ex. 2963 Fax: +81-72-683-1221 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 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, 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

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

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