907 
SPECTRAL AND TIME-RESOLVED ANALYSIS OF BLUE-GREEN 
FLUORESCENCE EMITTED BY PLANTS 
Z. G. CEROVIC, F. MORALES & I. MOYA 
Laboratoire pour l'Utilisation du Rayonnement Eléctromagnétique, 
Bât 209 D, Université de Paris XI, 
91405 Orsay, (France) 
ABSTRACT 
Synchrotron radiation and the time-correlated single-photon-counting technique were used to investigate the 
spectral and time-resolved characteristics of blue-green fluorescence of leaves, mesophyll and chloroplasts. Four 
kinetic components were resolved. Decay-associated spectra and comparative analysis showed that the fluorescence 
of leaves, on both sides, was dominated by the fast (0.3 ns) and the medium (1 ns) kinetic components 
comprising fluorophores emitting principally in the blue and present in the epidermal layer. In the mesophyll, 
these two faster components have two maxima, in the blue and in the green part of the spectrum, with a shift of 
the blue maxima to longer wavelengths when compared to leaves. The slow component (3.5 ns) was green related 
with a strong indication for the presence of flavins. The very slow component (9 ns) had a maximal fractional 
intensity in mesophyll and was blue related. The excitation and emission characteristics and the effect of 
anaerobic atmosphere on the fractional intensities of the slow and very slow component showed that they contain 
fluorescence of flavin nucleotides and nicotinamide nucleotide, respectively. Time-resolved measurements could 
be a mean to extract the information on nucleotide fluorescence from the overall leaf fluorescence, and therefore to 
evaluate changes in mesophyll redox sate. 
KEY WORDS: decay-associated spectra, flavin nucleotides, photosynthesis, plant phenolics, pyridine 
nucleotides 
1-INTRODUCTION 
Blue-green fluorescence (BGF) of plants, excited by UV light, has received recently an increasing interest [1-7]. It 
was shown that its emission spectrum depends on plant type [1] and plant anatomy [8, 4]. For instance, emission 
from adaxial side of bifacial leaves differs substantially from abaxial side [8,4]. Blue-green fluorescence was also 
shown to depend on environmental factors experienced by the plant, like water stress [9] or nutrient concentration 
in soil [3]. 
Thanks to all these findings BGF became a new potential signature for remote sensing of plants [10] 
comparable to red fluorescence. But BGF is a sum of emission by several fluorophores, unlike red fluorescence. 
Red fluorescence depends only on one fluorophore, the chlorophyll, whose fluorescence yield, although 
complicated by various quenching components, can be related directly to photosynthetic efficiency [11]. In 
addition, the potential blue-green fluorophores are distributed in different compartments of the leaf and the plant 
cell, further complicating the quest for the origins and causes for BGF variability. 
The substances that are candidates for BGF can be divided in two main classes. The first class comprises 
aromatic compounds located in the vacuole and cell walls of the epidermis and that are related to the secondary 
plant metabolism and UV-protection [8, 12]. The second class is represented by the cofactors of the metabolism, 
pyridine and flavine nucleotides, which are directly related to the redox state and fluxes of the plant cell [13]. The 
fluorescence of these compounds is readily used in biomedical and microbiological studies [14-16]. The 
fluorescence of pyridine nucleotides could be complementary to red, chlorophyll, fluorescence but their 
detectability in intact leaves has received opposite conclusions [2-6]. 
In the work of Goulas et al. (1990) the problem was approached by combining spectrofluorimetric 
measurements with lifetime measurements in the sub-nanosecond domain to differentiate more precisely the 
components of BGF. In the present study, this combined kinetic spectrofluorimetric approach was further used for 
a comparative analysis of leaves, mesophyll (leaves without epidermis) and isolated chloroplasts of the 
industrially important species sugar beet (Beta vulgaris L.). Decay-associated spectra (DAS) of blue-green 
fluorescence were recorded and analyzed by both an individual and global computation approach. Possible origins 
and characteristics of the four resolved kinetic component of fluorescence are discussed, and a way to access 
changes in redox state of nucleotides in leaves is proposed.