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Plant and Cell Physiology Advance Access published online on November 18, 2008
Plant and Cell Physiology, doi:10.1093/pcp/pcn174
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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

3D confocal laser scanning microscopy for analyzing chlorophyll fluorescence parameters of chloroplasts in intact leaf tissues

Kenji Omasa, Atsumi Konishi, Hikaru Tamura and Fumiki Hosoi

Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo, Tokyo 113-8675, Japan

Corresponding author Prof. Kenji Omasa, Department of Biological and Environmental Engineering, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo, Tokyo 113-8657, Japan, Tel: +81-3-5841-5340, Fax: +81-3-5841-8175, e-mail: aomasa{at}mail.ecc.u-tokyo.ac.jp


  Abstract

We analyzed the chlorophyll fluorescence parameters in a 3D cellular arrangement in vivo by using a modified Nipkow disk–type confocal laser-scanning microscope (CLSM). We first defined the 3D values of {Phi}PSII (photochemical yield of photosystem II) and NPQ (nonphotochemical quenching) in mesophyll, epidermal, and guard cell chloroplasts from the leaf surface to several tens of microns in depth. We also used this CLSM method to analyze the relationships between actinic light intensity and the chlorophyll fluorescence parameters for Boston fern and broad bean leaf specimens. As the actinic light intensity increased, the mean {Phi}PSII values decreased and the NPQ values increased in all chloroplasts of Boston fern and broad bean leaf. These values differed with cell type and species. The Boston fern chloroplasts had lower {Phi}PSII values than the broad bean chloroplasts, and vice versa for the NPQ values. The {Phi}PSII values of Boston fern chloroplasts decreased in the order of mesophyll, epidermal, and guard cell chloroplasts. The NPQ values decreased in the order of guard cell, mesophyll, and epidermal chloroplasts, except at 12 µmol m–2 s–1 actinic light, when the mesophyll value was slightly lower than the epidermal one. The trend in the {Phi}PSII and NPQ values of broad bean mesophyll and guard cell chloroplasts was opposite to that of Boston fern chloroplasts. As 3D CLSM can provide the {Phi}PSII and NPQ values of each chloroplast in a 3D cellular arrangement, this method has potential for investigating differences in the functions of chloroplasts in vivo.

Keywords: 3D - Chlorophyll fluorescence - Chloroplast - CLSM - NPQ - {Phi}PSII

(Received April 2, 2008; Accepted November 12, 2008)
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