It has been largely shown that hydroxycinnamates and flavonoids, which are relevant classes of the general phenylpropanoid metabolism, serve a crucial role in the response mechanisms to several stressful agents, particularly excess light stress. Fluorescence signatures of plant tissue, namely the shape and the peak of maximum emission in the 400-600 nm waveband, depend on the relative concentration of various phenylpropanoids. As a consequence, the spectrofluorometric analysis may estimate the tissue specific distribution of secondary metabolites, a pre-requisite to assess their functional roles. Furthermore, the contribution of individual phenylpropanoids to the overall fluorescence signal may be estimated through the analysis of fluorescence images recorded at various wavelengths, i.e. multispectral fluorescence microimaging. Therefore, the analysis of i) the fluorescence signatures of cross sections treated with common fluorescent probes and ii) fluorescence imaging may both quantify and visualize the tissue-specific distribution of various hydroxycinnamates and flavonoids. Such techniques have been developed at IVALSA and IFAC, using a conventional fluorescence microscope coupled with both a multichannel spectral analyser and a CCD camera. Such techniques have been used to estimate the roles served by caffeic derivatives and flavonols in the adaptation mechanisms to excess light operating in Mediterranean species. One of the major advantages of the proposed techniques regards the very short integration time of the fluorescence signal (1-2 s) which preserves tissue from excitation light-induced damages (photo-bleaching). Moreover, the sequentially acquired fluorescence images of tissues excited under a single ultraviolet wavelength allow the contemporary but not overlapped estimation of several metabolite classes. At the same time, the contribution from chlorophyll fluorescence is not taken into account. This is a relevant improvement with respect to classical fluorescence microscopy, as the latter does not allow the visualization of secondary metabolites in tissues containing chlorophyll.
We have quantified for the first time (Agati et al. 2002. Photochem Photobiol 76; Tattini et al. 2004. New Phytol 163) the gradients of hydroxycinnamate and flavonoid accumulation throughout the whole leaf depth in Oleaceae exposed to excess light. We have also offered some conclusive data of a coordinated control system between caffeic and flavonoid metabolism operating in tissues suffering from acute doses of ultraviolet radiation. Finally, through a detailed analysis of the mesophyll distribution of mono and dihydroxy-flavonoids, a key role of flavonoids as antioxidants rather than merely UV-screeners has been shown in plants exposed to excess UV-radiation.
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