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Plant and Cell Physiology Advance Access originally published online on June 30, 2007
Plant and Cell Physiology 2007 48(8):1159-1169; doi:10.1093/pcp/pcm085
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© The Author 2007. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oxfordjournals.org

Intra-Vacuolar Reserves of Membranes During Stomatal Closure: The Possible Role of Guard Cell Vacuoles Estimated by 3-D Reconstruction

Yoko Tanaka1,3, Natsumaro Kutsuna1, Yuuko Kanazawa1, Noriaki Kondo2, Seiichiro Hasezawa1 and Toshio Sano1,*

1Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwanoha, Kashiwa, Chiba 277-8562 Japan
2Division of Biosciences, Graduate School of Sciences and Engineering, Teikyo University of Science and Technology, Yatsusawa, Uenohara, Yamanashi, 409-0193 Japan

*Corresponding author: E-mail, tsano{at}k.u-tokyo.ac.jp; Fax, +81-471-36-3706.


  Abstract

Stomatal apertures are regulated by morphological changes in guard cells which have been associated with guard cell vacuolar structures. To investigate the contribution of guard cell vacuoles to stomatal movement, we examined the dynamics of vacuolar membrane structures in guard cells and evaluated the changes in vacuolar volumes and surface areas during stomatal movement. Using a transgenic Arabidopsis line expressing green fluorescent protein (GFP)–AtVAM3, we have found that the guard cell vacuolar structures became complicated during stomatal closure with the appearance of numerous intra-vacuolar membrane structures. A three-dimensional (3-D) reconstruction using our originally developed software, REANT (reconstructor and analyzer of 3-D structure), and photobleaching analysis revealed the continuity of the vacuolar structures, even when they appeared to be compartmented in confocal images of closed stomata. Furthermore, calculations of the surface area by REANT revealed an increase in vacuolar surface area during stomatal closure but a decrease in the surface area of the guard cells. Movement of a vital staining dye, FM4-64, to the vacuolar membrane was accelerated during ABA-induced stomatal closure in Vicia faba. These results suggest that the guard cell vacuoles store some portion of the excess membrane materials produced during stomatal closure as intra-vacuolar structures.

Keywords: 3-D reconstruction - Guard cell - Plasma membrane - Surface area - Vacuole - Volume change

Abbreviations: CLSM, confocal laser scanning microscopy; ER, endoplasmic reticulum; FC, fusicoccin; GFP, green fluorescent protein; PM, plasma membrane; REANT, reconstructor and analyzer of 3-D structure; VM, vacuolar membrane

3Present address: Plant Science Center, RIKEN, Suehiro-cho, Tsurumi-ku, Yokohama-City, Kanagawa, 230-0045 Japan.

(Received March 15, 2007; Accepted June 27, 2007)
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