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2-MATERIAL AND METHODS
2.1.Plant material and chemicals
Sugar beet (Beta vulgaris cv. Monohill) was grown in a growth chamber in half-Hoagland nutrient solution.
Plants were grown at 20 °C and a PPFD of 350 |jE m" 2 s' 1 (light) and 15 °C (dark), 80% relative humidity and a
photo-period of 16 h light/8 h dark. Nucleotides, NADPH, FAD, FMN, and riboflavin were purchased from
Sigma, and used without further purifications, immediately after dissolving in pure water. The final concentration
of standards was 25 pM. Intact isolated chloroplasts were prepared as in [6].
2.2.S ingle-photon-counting measurements
Time-correlated single-photon-counting measurements of fluorescence decays were performed on the SA4 line of
the SUPERACO synchrotron in Orsay (France) as described previously [2], with some modifications. Discs of
leaves, or leaves from which the lower epidermis was striped away, were cut and enclosed in a home-made
thermostated chamber, the illuminated face pressed against a quartz window. A closed cell (0.2 to 0.5 mm optical
path) was used for chloroplast suspensions, at the place of the quartz window. The excitation part of the apparatus
consisted of a monochromator (8 nm band pass, H10, Jobin-Yvon), a UV glass filter (glass No 5840 Kopp) with
340 nm band pass interference filter (Melles Griot) or only a 420 nm band pass interference filter (Ditric Optics).
These filters were replaced by a neutral density filter when measuring scattered light. The fluorescence emission
was collected with a biconvex lens (F = 60 mm, f = 80 mm, Melles Griot) and defined by a blue glass filter
(glass No 9782 Kopp), a monochromator (9 nm band pass, HL 300 Jobin-Yvon) and an anti-UV filter (KV399,
Shott).
The counting line comprised a photomultiplier (2254QB, Philips) cooled to -40° C, a constant fraction
discriminator (7174, Enertec), a time-to-amplitude converter (TAC) (566 ORTEC), a multichannel pulse hight
analyzer (NS575, Tracor) interfaced to a microcomputer. The synchronization signal was provided by a fraction of
the excitation beam deflected to a fast photodiode followed by a delay line, a second constant fraction
discriminator, itself feeding the TAC. The excitation profile (instrumental function) was obtained by tuning the
excitation monochromator to the emission wavelength and the beam attenuated approximately by one thousand
times. The scattered excitation beam was then recorded as if it were fluorescence. Decay-histograms were acquired
until 30,000 counts were accumulated at the maximum. Iterative convolution of fluorescence decays was
performed as previously described [2] using a home-made program based on Marquardt search algorithm for non
linear parameters [17].
2.3.Analisis of decay-histograms
Decay-histograms were analyzed by applying three types of convolution models: 1) simple convolution,
assuming that there is no background nor presence of scattered light in the fluorescence decay; 2) convolution
including subtraction of background (typically between 1 and 3 counts); 3) convolution assuming the presence of
scattered light in the fluorescence decay, usually between 0 and 2%. The correctness of the convolution was
judged from the examination of the Chi 2 value, distribution of the weighted residuals between the calculated and
the experimental function, and the autocorrelation function of the residue [18]. The 3-component and 4component
4component models do not describe the recorded fluorescence decays in a satisfactory manner (data not shown).
The 5-component model or 4-component with presence of scattered light, gave satisfactory results.
The 4-component model with scattered light gave reproducible results in a variety of experimental
conditions and was independent of the starting conditions for regression (checked for 200 recorded decays), and
therefore was favoured for the presentation of results. This model is equivalent to a 5-component model in which
the very short component would be subtracted and the relative contribution to the fluorescence yield of the four
others component re-calculated. In any case, the average lifetimes and the distribution of the relative yields of the
short, medium, long and very long components was similar, if not identical, when treated with either of the two
satisfactory models. The resolution limits of our measuring system brought us to treat the very short component
as a presence of scattered light in the fluorescence, but this has also a plausible physical origin because of the
highly scattering properties of the samples.
2.4.Spectra
Decay-associated spectra (DAS) were obtained by varying the emission wavelength at which the fluorescence
decays were recorded with fixed excitation or emission wavelength [2]. Individual analysis was performed for each
decay and then a global fit was performed to all decays in a spectrum simultaneously.
Steady-state fluorescence emission spectra from 400 to 600 nm were recorded on the same apparatus by
"scrolling" the emission monochromator and integrating the number of counts during one second at each new
wavelength spaced by 2 nm. For most cases, two forward and two backward scans were averaged. Emission
spectra were corrected for instrumental response using quinine sulfate as a standard.
