Proceedings Article | 11 February 2011
KEYWORDS: Luminescence, Carbon, Fluorescence spectroscopy, Statistical analysis, Light, Confocal microscopy, Photosynthesis, Optical testing, Temperature metrology, Energy transfer
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.
