Time-resolved blue-green fluorescence of sugar beet (Beta vulgaris L.) leaves. Spectroscopic evidence for the presence of ferulic acid as the main fluorophore of the epidermis

Abstract

Sugar beet (Beta vulgaris L.) leaves emit blue-green fluorescence when excited with ultraviolet light. Comparative spectral analysis showed that the blue-green fluorescence of leaves, on both the adaxial and the abaxial side, was dominated by the blue-green fluorescence of the epidermis. A detailed investigation of the adaxial epidermis and of possible candidates for its blue-green fluorescence was carried out. The possible candidates were two hydroxycinnamic acid derivatives (ferulic acid and p-coumaric acid) and two flavonoids (kaempferol and quercetin). Each of these absorbed ultraviolet light, but ferulic acid was the only fluorophore that fluoresced similarly to epidermis. Decay-associated excitation and emission spectra were used to investigate further the blue-green fluorescence of the epidermis. In sugar beet epidermis, four kinetic components were resolved: the very fast (20-40 ps), the medium (0.8-0.9 ns), the slow (2.6-2.8 ns) and the very slow (7.1-7.6 ns) kinetic components. Several lines of evidence indicate the presence of ferulic acid as the main fluorophore of the epidermis. First, the very fast kinetic component, which contributes 70% to the overall blue-green fluorescence emitted from the epidermis, showed the spectral characteristics of ferulic acid. Second, the fluorescence lifetime of this kinetic component matched that of ferulic acid, which was 20-40 ps. Finally, the very fast kinetic component was also found in sugar beet leaves and in their phenolic extracts, and its thermal sensitivity (measured as the percentage of the blue-green fluorescence change per C°) matched that of ferulic add. The results of this work lead us to conclude that ferulic acid is the fluorophore responsible for most of the blue-green fluorescence emitted from sugar beet epidermis.