A new approach to visualize secondary metabolites in plants
G. Conéjéro, P.Talamond et J-L Verdeil
Plate-forme d’Histocytologie et Imagerie Cellulaire Végétale (http://phiv.cirad.fr/)
UMR Biochimie & Physiologie Moléculaire des Plantes, UMR Développement et Adaptation des Plantes INRA CIRAD Montpellier
France
The autofluorescence of some secondary metabolites in plants can be used to study their tissular and cellular localization by an original
approach of spectral imaging in multiphonic microscopy. So, the 5-caffeoyl quinic acid (chlorogenic acid, an abundant phenolic compound in coffea plant) can
be detected in the cells of leaf of Coffea canephora, without staining or disturbance of the environment, only on a base of this specific spectral analysis. Its
localization allows to make hypotheses on the cellular function.
v
ep
pp
pp
ep
With epifluorescence or confocal microscope, we can observe the
autofluorescence of plant tissues which results from several fluorophores. The
emission spectra of these endogenous fluorophores overlap widely but the spectral
analysis allows to discriminate between these various fluorescences (Zimmermann
et al., 2003, Berg 2004). The Linear Unmix method of confocal and multiphoton
microscopes can then be used to separate dyes with overlapping fluorescent
emission spectra that could not be separated by conventional confocal methods.
The multiphotonic microscope allows particularly to excite some polyphenolic
compound metabolites like UV laser. The figure 1 shows the autofluorescence of
living young leaf of Coffea canephora, specie which contains very high level of
chlorogenic acid (Mondolot et al, 2006).The epidermis and some cells of
parenchyma shows a strong blue fluorescence. In these cells, we can observed
vesicles with the same fluorescence (in red: chlorophyll).
ep
A
ep
B
Figure 1: Autofluorescence of living young leaf cross- section of Coffea canephora observed
with a epifluorescence microscope (A, filter exc: 340-380 nm, em: 425-800 nm) and
with a multiphotonic microscope (B, exc: laser IR 720 nm, em: channel 435-485 nm in blue,
Channel 670-700 nm in red). ep: epidermis, pp: palisade parenchyma, v: vesicle.
ep
B
A
ep
Residual channel
crystal
Magnification on vesicles
C
overlay
ep
A
B
Figure 2: Spectral analysis on cross-section of living leaf of Coffea
canephora by multiphoton microscopy, exc: 720 nm. A: Images
gallery obtained after Lambda acquisition between 360 and 600 nm
(spectral resolution: 10 nm), B: manual research of emission spectra
on cells, one on the adaxial epidermis (green) and one in the palisade
parenchyma (red). Magnification on vesicle observed in cells of
parenchyma. C: Spectral deconvolution (linear unmixing) from the
two emission spectra found in the section and a residual channel
(white) (left: split, right: overlay of 3 channels).
On a crystal of chlorogenic acid, we obtain a specific spectral signature with a wide peak between 450
and 500 nm (Fig.3 A). The figure 3B shows the result of linear unmixing on cross-section of C. caneph
from this spectrum (pink), The distribution of this fluorescence in epidermis cells and particularly
in vesicles (Fig.3 C spectral acquisition and D linear unmixing) is similar to that observed in figure 2.
This specific fluorescence of chlorogenic acid is not observed by linear unmixing on cross-section of
a control specie of coffea with very small content of chlorogenic acid (E).
Emission spectrum of
chlorogenic acid
ep
ep
C
The spectral imaging allows to found an emission spectrum with a peak
between 450 and 500 nm in cells of epidermis and in parenchyma (vesicles and
chloroplasts) (Fig.2A,B). The second peak was found in parenchyma. The
calculated picture obtained by advanced linear unmixing shows the
distribution of both fluorescences in cross-section of leaf (Fig. 2C) and one
residual channel generated to visualize undeterminated fluorescences.
D
E
Figure 3: Spectral analysis of purified powder of chlorogenic acid (A), linear unmixing on cross
section of young leaf of Coffea canephora (B) with specific spectrum of chlorogenic acid (pink),
chlorophyll (blue) and unknown fluorescence (yellow). Magnification in vesicles of epidermis cells:
spectral acquisition (C) and linear unmixing (D) . Control: linear unmixing on cross-section of young
leaf of Coffea pseudozanguebarie, (E).
Conclusion:Multiphoton microscopy (allowing to reproduce uv excitation), combined with spectral analysis open the way to image autofluorescent molecules in living
organs. Based on the methodology developed in our lab, we were able to localize chlorogenic acid in coffea leaves at the cellular scale, demonstrating the great potential of
multiphoton microscopy combined with spectral deconvolution. Several putative functions have been attributed to chlorogenic acid . Its localization in the vacuole of epidermis
cells, in vesicles and chloroplasts of mesophyl cells strengthen the hypothesis of its involvement in leaf protection against uv damages or pathogens.
Materials and methods: Cross-sections (50 µm) were obtained from young leaves of Coffea canephora and Coffea pseudozanguebarie using a vibratome and then dipped in phosphate buffer salin and ascorbic acid. Epi-fluorescence
microscopy was done on Leica DM6000 equipped with Q-Imaging camera. A multiphoton microscope ZEISS 510 META NLO equipped with a laser COHERENT Chameleon Ultra II Ti-Sapphire (Montpellier RIO Imaging) was used to obtain
emission spectra from fresh leaves. Spectral analysis was carried out using the autofluorescence properties of phenolic compounds on UV light, without any dyes. Reference spectrum was obtained from Lambda acquisition of a powder of
chlorogenic acid with META system of multiphoton microscope (dichroic mirror KP 650, BP 365-600 nm). The Avanced Linear Unmixing function of LSM 510 Meta (Emission Finger printing method) allows to visualize the fluorescence of
phenolic compounds in cells from reference spectra.
Ref: Zimmermann et al FEBS lett.546,87-92 (2003), Berg RH, J Microscopy, 214,174-181(2004),
Mondolot et al., Annals of Botany, 98, 33-40 (2006).