Leaf chlorophyll fluorescence corrected for re-absorption by means of absorption and reflectance measurements

The in vivo emission spectra of chlorophyll (Chl) fluorescence of green leaves, taken at room temperature, show two maxima near 685 nm and 735 nm. The shape of the spectra is modified by re-absorption processes and depends on the Chl content of leaves. By performing reflectance, transmission of fluorescence measurements, we investigated the reason for the repeatedly reported increase of fluorescence ratio F685/F735 during stress or damage-induced breakdown of chlorophyll in plants. The in vivo spectra of Chl fluorescence (F), transmittance (T) and reflectance (R) were taken at room temperature at the same leaf spot. Leaves with a wide range of green colour (Chl content varied from 70 to 670 mg m(2)) were chosen from a beech (Fagus sylvatica L.) and an elm tree (Ulmus minor Miller) as well as from a wild shrub (Parthenocissus tricuspidata L.). Strict linear correlations (with a determination coefficient of r(2) > 0.92) were found between the ratio F685/F735 on the one hand, and (i) the ratio of the non-absorbed radiation (R685 + T685)/(R735 + T735), (ii) the reflectance ratio R685/R735, and (iii) the reflectance at 685 nm, on the other hand. The results demonstrate that 92% or more of the ratio F685/F735 variation in leaves during development or at damage and stress events is determined by the variation in Chl content and corresponding changes of the optical properties of leaves. The Chl fluorescence emission spectrum has been corrected at each wavelength for re-absorption by means of non-absorbed radiation (R + T) and reflectance yielding the actually emitted retrieved Chl fluorescence. The retrieved Chl fluoroescence at 685 and 735 nm is found to linearily increase with the Chl content. The shape of the retrieved Chl fluorescence spectrum was very similar to that of strongly diluted Chl alpha in solution, only the position of the emission maxima is different (673 nm and Chl alpha in ethanol solution, and 685 nm in leaves). Studies on the actually emitted <<true>> Chl fluorescence, as retrieved from room temperature and outdoor fluorescence measurements, may show new ways of plant stress detection.