microspectrofluorometry for localization of compounds in leaf tissues g. agati...
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MICROSPECTROFLUOROMETRY FOR LOCALIZATION OF COMPOUNDS IN LEAF TISSUES
G. Agati ([email protected]), 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