912 
decrease in the proportion of the fast component at 460 nm. It should also be noted that the very fast component 
(or scattering, see above) disappears almost completely in chloroplasts (not shown), which is in accordance with 
the less scattering properties of organelles in solution compared to leaves. Finally, the largest contribution of the 
very slow component is seen in the mesophyll (17 %). For the results of Table 2, the analysis was performed 
individually on each decay, allowing the program to fit freely both the lifetime and pre-exponential factors from 
which fractional contributions were computed. When the global fit was applied to the data, common lifetimes 
were fitted to all decays. This accentuated even further the described changes in fractional contributions of the four 
components to fluorescence in different types of sample (data not shown). 
We further analyzed the kinetic components of the mesophyll by comparing the wavelength combination 
which favoured either nicotinamide (340/430) or flavin (375/525) nucleotide fluorescence (cf. Table 1.). From 
Table 3 it can be seen that the fast and medium components were almost un-affected. The fractional contribution 
of the slow component was increased under conditions favoring flavin fluorescence (excitation 375 nm, emission 
525 nm) and that of the very slow component doubled under conditions favouring nicotinamide fluorescence 
(excitation 340 nm, emission 430 nm). Excitation at 420 nm, where nicotinamide nucleotides do not absorb at 
all, favoured substantially the emission of the medium and slow components (Table 3). The fast and very slow 
components were not only decreased accordingly, but also the lifetime of the fast component was decreased and 
the lifetime of the very slow component increased, suggesting that the major fluorophores contributing to these 
two components are different under this type of excitation. 
The confirmation for the involvement of flavin nucleotides in the slow component and nicotinamide 
nucleotides in the very slow component came from the investigation of the effect of the gas phase (nitrogen or 
air) on mesophyll fluorescence. The fast and medium component did not show any significant change in their 
fractional contribution when the gas phase was changed and therefore are not shown in Fig. 2. By contrast, 
changes in the slow and very slow components could clearly be seen. Under the reducing atmosphere of nitrogen 
most of the nucleotides would be reduced. Pyridine nucleotides (NADH and NADPH) fluoresce when they are 
reduced, while flavin nucleotides (FMN and FAD) fluoresce when they are oxidized [21]. The nitrogen atmosphere 
brings a decrease in the fractional contribution of the slow component especially pronounced in the green (520 
nm) at the fluorescence maximum of flavins. By contrast, the very slow component has an increased contribution 
under nitrogen compared to air both at 460 nm, where pyridine nucleotides have a fluorescence maximum, and at 
520 nm, where their fluorescence is still 50 % of the maximum [19]. 
4-DISCUSSION 
In the present work four kinetic components of blue-green fluorescence could be resolved and defined in 
leaves, mesophyll and chloroplasts of sugar beet. The medium (1 ns) and slow (4 ns) components were already 
well resolved in intact spinach leaves (adaxial side) and could be discussed [2]. The lifetimes and contributions of 
the fast (0.3-0.4 ns) and very slow (8-11 ns) components differ from those found in the work of Goulas et al. 
(1990) due to several important changes in the measurements and analysis of fluorescence decays. In the present 
work the temperature and gas phase of the sample were rigorously controlled. The accumulation of counted 
photons was doubled (30.000 counts at maximum) permitting better statistics, which is especially important for 
the slow components. The excitation was made at longer wavelengths that favoured nucleotide excitation and was 
less absorbed by flavonoids [22]. Several convolution models were compared which involved scattered light and 
background. A global fit could be performed on several decays. All these changes permitted to define well the fast 
and very slow kinetic components and to analyze the spectral characteristics and relative contribution of all four 
components at different levels of leaf organization. 
The fast and medium components dominate the fluorescence of the intact leaf (on both sides). They have 
a blue maximum, and are probably composed of fluorescent flavonoids of the vacuole [23, 24] or phenolic acids 
of the cell wall [25, 2]. Total fluorescence of intact leaves is strongly influenced by these fluorophores of the 
epidermal layer (including cuticle) [8, 4] precluding the investigation on changes of mesophyllic fluorophores 
especially in the blue part of the spectrum. This is why actinic light-induced changes of blue-green fluorescence 
could not be seen in intact mature leaves [5, 6] although there were claims that most of the blue florescence in 
leaves comes from pyridine nucleotides [3]. Still, results of DAS show that the contribution of mesophyll 
fluorescence is more pronounced in the green part of the spectrum, confirming the conclusion of Lang et al. 
(1992) [4] and suggesting that light-induced changes should be searched for in this spectral region. The presence 
of "screening" compounds (plant phenolics and alkaloids) is not restricted to the epidermal layer and cuticle and 
some of them can be found in the mesophyll too, the amount depending on plant species [26, 24]. This seems to 
be the case with sugar beet, judging from the important contribution of the fast and medium kinetic components 
of mesophyll fluorescence. Unfortunately the data on fluorescence lifetimes on most plant phenolics are missing 
and, although the presence of quercetin, p-coumaric acid, kaempferol and especially ferulic acid is well 
documented in sugar beet [26], the exact assignment of the fast and medium component should await time- 
resolved studies of these compounds. All we can conclude at this point is that these short-lived component are far 
more abundant in the epidermis than in the mesophyll, and more on the adaxial than abaxial side [23,24] and that 
they are responsible for the blurred picture of total blue-green fluorescence of leaves.