22 
: very fast component 
is in accordance with 
;st contribution of the 
alysis was performed 
xmential factors from 
ta, common lifetimes 
ntributions of the four 
velength combination 
: (cf. Table 1.). From 
ractional contribution 
ion 375 nm, emission 
inamide fluorescence 
ides do not absorb at 
he fast and very slow 
nt was decreased and 
contributing to these 
ent and nicotinamide 
as phase (nitrogen or 
Scant change in their 
Fig. 2. By contrast, 
mosphere of nitrogen 
oresce when they are 
; nitrogen atmosphere 
:ed in the green (520 
ncreased contribution 
nee maximum, and at 
solved and defined in 
ponents were already 
; and contributions of 
vork of Goulas et al. 
lecays. In the present 
mulation of counted 
lecially important for 
ie excitation and was 
ed scattered light and 
to define well the fast 
attribution of all four 
oth sides). They have 
24] or phenolic acids 
î fluorophores of the 
iphyllic fluorophores 
e-green fluorescence 
e blue florescence in 
bution of mesophyll 
usion of Lang et al. 
region. The presence 
layer and cuticle and 
6, 24]. This seems to 
t kinetic components 
thenolics are missing 
ferulic acid is well 
t should await time- 
ed component are far 
side [23, 24] and that 
21 
20 
19 
18 
17 
16 
15 
14 
ent 
Fig. 2. Effect of gas phase on the slow and very slow component of mesophyll fluorescence. 
Fluorescence was excited with 360 nm light and decays recorded at the emission wavelength indicated on 
graph. The decays were convoluted using the "4-component with scattered light" model, and the 
fractional intensity calculated as described in Table 1. Humidified pure nitrogen (N 2 ) or air (AIR) were 
flown over the sample at a rate of 45 1/h. The temperature was 20°C. 
Because of the blue fluorescence of the epidermis, the blue/green ratio proposed as a stress signature [1, 
9] would reflect mainly changes in epidermal phenolics. For the same reasons, the well-established practice in 
biomedical studies to estimate the redox state of tissue by measuring the blue/green ratio of nucleotide 
fluorescence [14, 27] is precluded in leaves. For the study of nucleotides involved in bioenergetics of leaves, it is 
therefore preferable to use the green emission, even though nicotinamide nucleotides have a maximum emission 
in the blue. At 520 nm, the emission of NAD(P)H is still 50 %. 
Several lines of evidence indicate the presence of flavin nucleotides in the slow kinetic component at any 
given level of organization of the leaf: matched lifetime, emission maximum in the green, preferential excitation 
at 420 nm and increased fractional contribution under air. On the other hand, the very slow component shows all 
the characteristics reflecting the presence of nicotinamide nucleotides: maximal contribution to fluorescence in 
mesophyll, minimal in darkened chloroplasts (oxidized), preferential excitation at 340 nm and an increase of 
fractional contribution upon reduction under nitrogen. The apparent discrepancy concerning the lifetime is 
elevated when taking into account that in the cell the pools of nucleotides are almost totally bound to enzymes 
[13]. When bound to proteins, the lifetime of nicotinamide nucleotide increases by one order of magnitude [19, 
28] and can attain 9.5 ns for ternary complexes with substrates [29]. 
An advantage could be gained from this presence of nicotinamide and flavin nucleotides in two different 
kinetic components of fluorescence. The ratio of the fractional contribution of the slow and very slow component 
could be used as a measure of cellular redox sate. Under reducing conditions in the cell, nicotinamide nucleotides 
are reduced, the fractional contribution of the very slow component is increased, as opposed to a reduction of 
flavin nucleotides which brings a decrease in contribution of the slow component (recall that flavins are 
fluorescent when in an oxidized state). This ratio of the slow and very slow fluorescence component can be 
measured at a single wavelength, which eliminates the problem of screening and reabsorption, especially the 
reabsorption in the blue by the omnipresent chlorophyll. The most promising way to extract information on the 
redox state of nucleotides in intact leaves would be to combine the approach of actinic light induced variable 
fluorescence [6] and time-resolved measurements. 
Acknowledgements 
Part of the work described here was supported by the EUREKA project No. 380 (LASFLEUR). The visit of 
Z.G.C. and F.M. to LURE was financed by CNRS (Program Interdisciplinaire pour la Recherche sur 
lEnvironement) and the French Ministry of Foreign Affairs, respectively.