The use of chlorophyll fluorescence excitation spectra for the non-destructive in situ assessment of UV-absorbing compounds in leaves

In this study a method was designed to assess non-destructively the type of UV-screening compounds present in the leaf epidermis. The method is based on the recording and calculation of the ratio of UV-excitation spectra of chlorophyll fluorescence (FER) from the adaxial and abaxial sides of biracial leaves, or from older and younger segments of monocotyledonous leaves. The logarithm of this ratio (logFER) matched the absorption spectrum of the UV-absorbers present in the leaf, as confirmed by its overlap with the absorption spectrum of the methanolic extract of the leaf or of the isolated epidermis. By using the logFER approach, it was possible to demonstrate that the concentration but not the classes of compounds present in the epidermis that are responsible for UV-screening is affected by the side and the age of the leaves. In contrast, measurements from the leaves of seven dicots and one monocot indicated large difference in the classes of these compounds between species. Finally, it was shown that the logFER in the UV is independent of the emission wavelength, and that the method can be used for quantitative measurements. This method expands to the spectral domain the use of ChlF for the estimation of the leaf epidermal transmittance.