1. MICROSPECTROFLUOROMETRY FOR LOCALIZATION OF COMPOUNDS IN LEAF TISSUES G. Agati (G.Agati@ifac.cnr.it), P. Matteini Istituto di Fisica Applicata Nello Carrara – CNR, Sesto (FI) M. Tattini, L. Traversi Istituto per la Valorizzazione del Legno e delle Specie Arboree - CNR FI Z. Cerovic, A. Cartelat Groupe photosynthese et teledetection LURE/CNRS, Orsay France F. Bussotti, E. Gravano, C. Tani Dipartimento di Biologia Vegetale, UNIFI supported by: CNR Target Project on Biotechnology; CNR-CNRS bilateral cooperation project n. 11409; Universita degli Studi di Firenze Localization of compounds in leaf tissues is an important tool for 2)understanding mechanisms of response to stress conditions 3)understanding the functional roles of particular classes of compounds (polyphenols) 1)optimizing fluorescence monitoring of vegetation (blue-green and red-Chl fluorescence signatures) Fluorescence from endogenous compounds in leaf tissues Red fluorescence (single fluorophore) chlorophyll-a Blue-green fluorescence (several fluorophores) Phenylpropanoids hydroxycinnamates coumarins flavonoids Cofactors pyridine nucleotides flavins Others alkaloids quinones Adapted from Magritte, Les tables de la loi, 1961 FLIDAR punctual fluorometer APPARATUS AND METHODS Interference filter Exc. lamp Dichroic mirror Epifluorescence microscope Cooled CCD camera Filter wheel Optical fiber Multichannel spectral analyzer Mobile mirror Sample Bandpass filter Acquisition and analysis of fluorescence spectra Gaussian deconvolution for band separation and quantification Visualization of the multispectral image Image elaboration by a suitable computation function Digital imaging allows suitable image elaboration: math operations background removing flat-field correction false color representation recombination Imaging by a charge- coupled device (CCD) camera with narrow-pass (10 nm) optical filters for band separation Sequential acquisition of fluorescence images at the spectral bands of interest Autofluorescence imaging in Triticum aestivum L. (wheat) ( exc = 365 nm) 20 m adaxial epidermis trichomes 470 nm ( =10 nm) 680 nm ( =10 nm) RedBlue recombination exc = 436 nm em = 580 nm cuticle guard cells sclerenchyma bands exc = 365 nm em = 470 nm cell wall hydroxicinnamates Blue merging Red Autofluorescence, exc = 436 nm, em = 10 nm) 680 nm 2-bands merging 546 nm Line Profile Distance (Pixel) 0100200 300 400 0 20 40 60 80 100 120 Intensity Localization of flavonoids in leaf tissues of Phillyrea latifolia L. to understand their functional role in the acclimation mechanisms to excess light stress comparison between sun and shade plants Flavonoid fluorescence must be induced ( e.g. by Narturstoff reagent) Light regime sun = 480 W/m 2 shade = 70 W/m 2 F580 0 32 64 96 128 160 F580 - k ·F470 trichome b) c) a) SUN LEAF SHADE LEAF F580/F470 adaxial epidermis sun shade 475 575 Suitable fluorescence image acquisition and elaboration permits to evidence the tissue specific localization of flavonoids and their large difference between sun and shade leaves. Studying the plant response to ozone stress in Acer pseudoplatanus L. 100 m Chl and hydroxicinnamates co-localization Different contributions to leaf surface fluorescence Compound accumulation (yellow fluorescence) in ozone-damaged tissues