MICROSPECTROFLUOROMETRY FOR LOCALIZATION OF COMPOUNDS IN LEAF TISSUES

G. Agati (G.Agati@ifac.cnr.it), P. MatteiniIstituto di Fisica Applicata “Nello Carrara” – CNR, Sesto (FI)

M. Tattini, L. TraversiIstituto per la Valorizzazione del Legno e delle Specie Arboree - CNR FI

Z. Cerovic, A. CartelatGroupe photosynthese et teledetection LURE/CNRS, Orsay France

F. Bussotti, E. Gravano, C. TaniDipartimento 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)

exc 365 nm, leaf cross section

0

2000

4000

6000

8000

10000

12000

14000

380 480 580 680 780

wavelength (nm)

fluo

resc

ence

(a.

u.)

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

punctualfluorometer

APPARATUS AND METHODS

Interferencefilter

Exc.lamp

Dichroicmirror

Epifluorescencemicroscope

CooledCCD

camera Filterwheel Optical fiber

Multichannelspectralanalyzer

Mobile mirror

800700600500400

Sample

Bandpassfilter

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 operationsbackground removingflat-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

adaxialepidermis

trichomes

470 nm ( =10 nm)

680 nm (=10 nm)

Red

Blu

ere

com

bin

ati

on

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)

0 100 200 300 400

0

20

40

60

80

100

120

Inte

nsit

y

4000

3000

2000

1000

0

Flu

ore

sce

nce

(a

.u.)

640600560520480440400

Wavelength (nm)

damaged exc=436nm

damaged exc=365nm

normal exc=365nm

20x103

15

10

5

0

Flu

ore

sce

nce

(a

.u.)

800700600500400

Wavelength (nm)

normal damaged

mesophyll exc = 365 nm

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 regimesun = 480 W/m2

shade = 70 W/m2

F580

0 32 64 96 128 160

F580 - k ·F470

trichome

b)

c)

a)

SUN LEAF SHADE LEAF

F580/F470

adaxial epidermissun shade

475

575

0

0.2

0.4

0.6

0.8

1

1.2

380 480 580 680 780wavelength (nm)

fluor

esce

nce

(a.u

.)

shade leaf

sun leaf

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 hydroxicinnamatesco-localization

Different contributions to leaf surface fluorescence

Compound accumulation (yellow fluorescence) in ozone-damaged tissues