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Plant and Cell Physiology Advance Access originally published online on November 18, 2008
Plant and Cell Physiology 2009 50(1):90-105; 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 email: journals.permissions@oxfordjournals.org

3D Confocal Laser Scanning Microscopy for the Analysis of 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: Email, aomasa{at}mail.ecc.u-tokyo.ac.jp; Fax,+81-3-5841-8175


  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 PSII) and NPQ (non-photochemical 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 mesophyll, epidermal and guard cell chloroplasts. The NPQ values decreased in the order guard cell, mesophyll and epidermal chloroplasts, except at 12 µmol m–2 s–1 actinic light, when the mesophyll value was slightly lower than that of the epidermis. 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

Abbreviations: 3D, three dimensional; a, absorption coefficient of photosynthetic pigments; A/D, analog to digital; CLSM, confocal laser scanning microscopy; iDt, dark current image integrated for t ms; EM-CCD, electron-multiplying charge-coupled device; ETR, electron transport rate; iF, chlorophyll fluorescence intensity image measured under actinic light; iFm, chlorophyll fluorescence intensity image measured during a saturation light pulse in darkness; iFm', chlorophyll fluorescence intensity image measured during the saturation light pulse under actinic light; I, irradiated light intensity; ND, neutral density; NPQ, non-photochemical quenching of chlorophyll fluorescence; PPF, photosynthetic photon flux; QA, primary quinone acceptor of PSII; QB, secondary quinone acceptor of PSII; R2, coefficient of determination; {Phi}F, chlorophyll fluorescence yield under actinic light; {Phi}Fm, chlorophyll fluorescence yield during a saturation light pulse in the dark; {Phi}Fm', chlorophyll fluorescence yield during a saturation light pulse under actinic light; {Phi}F0, minimum chlorophyll fluorescence yield after dark adaptation; {Phi}Fv, {Phi}Fm–{Phi}F0,; {Phi}Fv/{Phi}Fm, maximum photochemical yield of PSII after dark adaptation; {Phi}PSII, photochemical yield of PSII; {lambda}, wavelength of light.

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