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Proceedings Paper

Determination of the PSI/PSII ratio in living plant cells at room temperature by spectrally resolved fluorescence spectroscopy
Author(s): Kirstin Elgass; Martina Zell; Veronica G. Maurino; Frank Schleifenbaum
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Paper Abstract

Leaf cells of living plants exhibit strong fluorescence from chloroplasts, the reaction centers of photosynthesis. Mutations in the photosystems change their structure and can, thus, be monitored by recording the fluorescence spectra of the emitted chlorophyll light. These measurements have, up to now, mostly been carried out at low temperatures (77 K), as these conditions enable the differentiation between the fluorescence of Photosystem I (PSI) and Photosystem II (PSII). In contrast, at room temperature, energy transfer processes between the various photosynthetic complexes result in very similar fluorescence emissions, which mainly consist of fluorescence photons emitted by PSII hindering a discrimination based on spectral ROIs (regions of interest). However, by statistical analysis of high resolution fluorescence spectra recorded at room temperature, it is possible to draw conclusions about the relative PSI/PSII ratio. Here, the possibility of determining the relative PSI/PSII ratio by fluorescence spectroscopy is demonstrated in living maize plants. Bundle-sheath chloroplasts of mature maize plants have a special morphologic characteristic; they are agranal, or exhibit only rudimentary grana, respectively. These chloroplasts are depleted in PSII activity and it could be shown that PSII is progressively reduced during leaf differentiation. A direct comparison of PSII activity in isolated chloroplasts is nearly impossible, since the activity of PSII in both mesophyll- and bundle-sheath chloroplasts decays with time after isolation and it takes significantly longer to isolate bundle-sheath chloroplasts. Considering this fact the measurement of PSI/PSII ratios with the 77K method, which includes taking fluorescence spectra from a diluted suspension of isolated chloroplasts at 77K, is questionable. These spectra are then used to analyze the distribution of energy between PSI and PSII. After rapid cooling to 77K secondary biochemical influences, which attenuate the fluorescence emanated from PSI, are frozen out. Due to their characteristic morphology, maize chloroplasts of mesophyll and bundle-sheath cells are an appropriate system for demonstrating the applicability of our in vivo method which, unlike the common 77K method, does not require the isolation of chloroplasts. In mesophyll chloroplasts of higher land plants, the thylakoids have a heterogenic morphology of appressed and non-appressed membrane domains, called the grana and the stroma lamellae. PSII is enriched in the grana, whereas PSI is enriched in the stroma lamellae. Changes in chloroplast membrane structure and composition, according to changes in the PSI/ PSII ratio, can be triggered by light quality and carbon source deficiency. Here, we demonstrate the applicability of statistical analysis of fluorescence spectra to detect changes in the PSI/PSII ratio resulting from structure changes in the thylakoid membrane.

Paper Details

Date Published: 11 February 2011
PDF: 6 pages
Proc. SPIE 7902, Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues IX, 79021R (11 February 2011); doi: 10.1117/12.873752
Show Author Affiliations
Kirstin Elgass, Univ. of Tuebingen (Germany)
Martina Zell, Univ. of Cologne (Germany)
Veronica G. Maurino, Univ. of Cologne (Germany)
Frank Schleifenbaum, Univ. of Tuebingen (Germany)

Published in SPIE Proceedings Vol. 7902:
Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues IX
Daniel L. Farkas; Dan V. Nicolau; Robert C. Leif, Editor(s)

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