Photochemistry and Photobiology, 2008, 84: 1048–1060
Multicolor Fluorescence Imaging of Leaves—A Useful Tool for Visualizing
Systemic Viral Infections in Plants†
Mónica Pineda1, László Gáspár2, Fermı́n Morales3, Zoltán Szigeti2 and Matilde Barón*1
1
Department of Biochemistry, Cell and Molecular Biology of Plants, Estación Experimental del Zaidı́n,
Spanish Council for Scientific Research (CSIC), Granada, Spain
2
Department of Plant Physiology and Molecular Plant Biology, Eötvös Loránd University, Budapest, Hungary
3
Department of Plant Nutrition, Aula Dei Experimental Station, Spanish Council for Scientific Research (CSIC),
Zaragoza, Spain
Received 17 October 2007, accepted 13 February 2008, DOI: 10.1111/j.1751-1097.2008.00357.x
ABSTRACT
Multicolor fluorescence induced by UV light is a sensitive and
specific tool that may be used to provide information about the
primary and secondary metabolism of plants by monitoring
signals of the chlorophyll fluorescence (Chl-F) and blue-green
fluorescence (BGF), respectively. We have followed the systemic
infection of Nicotiana benthamiana plants with the Pepper mild
mottle virus (PMMoV) by means of a multicolor fluorescenceimaging system, to detect differences between two strains of
PMMoV during the infection process and to establish a
correlation between the virulence and changes induced in the
host plant. Changes in both BGF and Chl-F were monitored.
BGF increased mainly in the abaxial side of the leaf during
pathogenesis and the corresponding images showed a clear veinassociated pattern in leaves of infected plants. HPLC analysis of
leaf extracts was carried out to identify compounds emitting
BGF, and determined that chlorogenic acid was one of the main
contributors. BGF imaging was able to detect viral-induced
changes in asymptomatic (AS) leaves before detection of the
virus itself. Chl-F images confirmed our previous results of
alterations in the photosynthetic apparatus of AS leaves from
infected plants that were detected with other imaging techniques.
Fluorescence ratios F440 ⁄ F690 and F440 ⁄ F740, which increase
during pathogenesis, were excellent indicators of biotic stress.
INTRODUCTION
In the last decade, UV-excited fluorescence signals have
emerged as a sensitive and specific tool for evaluating the
physiologic state of plants before the symptoms induced by
different environmental stress factors become evident (1–3).
The excitation of leaves with long-wavelength UV radiation
(320–400 nm) results in a total of four characteristic fluorescence bands with peaks near to 440 nm (blue; F440), 520 nm
(green; F520), 690 nm (red; F690) and 740 nm (far-red; F740)
(1,2,4). Taken together, F690 and F740 are also called
chlorophyll fluorescence (Chl-F), as these fluorescence signals
are emitted by chlorophyll a (Chl). Often, F440 and F520 are
†This invited paper is part of the Series: Applications of Imaging to Biological
and Chemical Systems.
*Corresponding author email: [email protected] (Matilde Barón)
2008 The Authors. Journal Compilation. The American Society of Photobiology 0031-8655/08
1048
treated as blue-green fluorescence (BGF), as F520 is rarely
present as a distinct peak (3).
During the monitoring of multicolor fluorescence (MCF)
signals by point measurements on randomly selected leaf areas,
the information about fluorescence gradients over the whole
leaf surface is missed unless one makes time-consuming,
repeated point measurements. In contrast, the UV-excited
fluorescence imaging systems offer rapid analysis and high
spatial resolution from the leaf to the remote sensing scale
(1,2,5–7).
Blue-green fluorescence is primarily emitted from the
epidermis and from both the mesophyll cell walls and the leaf
veins by several phenolic compounds that are covalently
bound to cell wall carbohydrates. In contrast, Chl-F nearly
exclusively emanates from mesophyll cells (except for the
guard cells of the epidermis which contain some chloroplasts).
Cell wall-bound ferulic acid has been reported to be the main
emitter of BGF in several plant species (8–10), showing that
soluble plant phenolic compounds present in the vacuole of
plant cells contribute little to the overall BGF emission in most
of the species that have been investigated so far. More recently,
chlorogenic acid (CGA) has been revealed to be one of the
main BGF fluorophores in artichoke leaves (11). This hydroxycinnamic acid (HCA) is also predominant in tobacco
plants, and its levels increase with leaf age (12,13).
The use of the fluorescence ratios F440 ⁄ F520, F440 ⁄ F690,
F440 ⁄ F740 and F690 ⁄ F740 is widespread as BGF and Chl-F
have distinct origins and can change independently in response
to stress factors. F440 ⁄ F690 and F440 ⁄ F740 are very early
stress indicators, and F440 ⁄ F520 can change after a long
exposure to stress (2,4,10). The F690 ⁄ F740 ratio has an inverse
relationship with the Chl content in the leaf (3,4,6,14,15).
Several limiting agricultural factors have been monitored
through MCF, in most cases using imaging systems—mineral
deficiencies (2,16–20), water stress (2,6,21,22) and high-temperature stress (23). Imaging is especially valuable for the
visualization of plant–pathogen interactions where, according
to the symptomatology, no uniform alteration in the foliar
metabolism is expected (24,25). Chl-F imaging (Chl-FI) has
revealed spatiotemporal changes in the photosynthetic efficiency pattern during pathogenesis in plants infected with
fungi (26–29), bacteria (30,31) and viruses (32–38). However,
there is a lack of MCF studies of the analysis of BGF signals in
Photochemistry and Photobiology, 2008, 84
pathogen-infected plants. Fungal infection has been reported
to increase F440 either by the fungus autofluorescence (39) or
by the production of plant phytoalexins (40). Chinese cabbage
infected with Turnip yellow mosaic virus showed changes in the
fluorescence ratios (41). Using MCF imaging, early visualization of small punctures made by tobacco flies on leaves as well
as a mite attack on beans was achieved (2). More recently, the
hypersensitive response (HR) to Tobacco mosaic virus (TMV)
infection was revealed by a BGF increase linked to scopoletin
accumulation (42).
In the present work, using an MCF imaging system, we
monitor a systemic viral infection in Nicotiana benthamiana
plants infected with either the Spanish or the Italian strain of
Pepper mild mottle virus (PMMoV-S, PMMoV-I). The origin
of these strains has been previously reported (43,44), respectively. The PMMoV-S strain is more virulent than PMMoV-I,
and the magnitude of the symptoms in the infected plants
correlates with the degree of virulence. In earlier studies, we
have shown that the oxygen-evolving complex of photosystem
II is the main target of this tobamovirus in the chloroplast (45–
49). Kinetic Chl-FI studies reveal a correlation between
nonphotochemical quenching and the spread of PMMoV in
asymptomatic (AS) leaves (38). Imaging thermography can
detect this infection earlier than the former technique through
the increase in leaf temperature caused by stomatal closure
(37). In order to obtain a more complete picture of the host
plant response to PMMoV infection, information about
secondary metabolites involved in signaling processes and
the response of N. benthamiana to pathogens is needed.
Hence, we have monitored changes in both BGF and Chl-F in
N. benthamiana leaves infected with either PMMoV-S or
PMMoV-I using a compact flash-lamp MCF system. Fluorescence values (F440, F520, F690 and F740) and fluorescence
ratios (F440 ⁄ F520, F440 ⁄ F690, F440 ⁄ F740 and F690 ⁄ F740),
as well as their corresponding images, of whole leaves from
control and infected plants were tracked over the course of the
infection. In an attempt to identify the compounds involved in
BGF emission, imaging was complemented by HPLC analysis
of leaf extracts.
MATERIALS AND METHODS
Plant growth and PMMoV infection. Nicotiana benthamiana cv. Gray
plants were cultivated in a growth chamber with 120 lmol m)2 s)1
photosynthetically active radiation, generated by white fluorescent
lamps (HPI-T 250 W; Phillips, Eindhoven, The Netherlands), with a
15 ⁄ 9 h (26 ⁄ 20C) light ⁄ dark photoperiod and relative humidity of
60%. The three lowest leaves of plants with six to seven leaves fully
expanded were carburundum-dusted and mechanically inoculated with
the Spanish or the Italian strains of PMMoV (PMMoV-S and
PMMoV-I, respectively) using 50 lL of inoculum per leaf (50 lg of
virus per mL of 20 mM sodium phosphate ⁄ biphosphate buffer pH 7.0).
Mock-inoculated plants were treated with buffer only.
At 5 (PMMoV-S-infected plants) and 7 (PMMoV-I-infected plants)
days postinoculation (dpi), visual symptoms appeared—new leaves
that developed after the inoculation showed severe curling and
wrinkling (symptomatic leaves, S). In contrast, no symptoms were
detected in leaves that were already fully expanded when the plants
were inoculated (AS leaves). Inoculated leaves showed no symptoms of
disease. In summary, we can distinguish three kinds of leaves in the
infected plants—inoculated, AS and S. Inoculated leaves were not used
in this study because they had mechanical damage. Stunting of the
plants was clearly evident at 14 dpi. PMMoV-I-infected plants were
able to recover from 21 dpi onward—plant height again increased and
plants developed new, noncurly leaves. In contrast, no new leaves
1049
appeared in PMMoV-S-infected plants after 14 dpi, and plants died at
21 dpi, except for the batch of plants in which the fluorophore content
was determined which died at 23 dpi.
Starting with the lowest leaf and numbering leaves in ascending
order, we analyzed the fifth leaf as the AS one. The second youngest
leaf at each dpi was tested as the S one.
Fluorescence imaging. Fluorescence imaging measurements were
made in control and virus-infected plants at different postinfection
times: 3, 5, 7, 10, 12, 14, 17 (for plants infected with both viral strains),
21, 24 and 28 dpi (for controls and plants infected with PMMoV-I).
The imaging system used is briefly described below, since fluorescence
excitation and detection were essentially identical to that described in
detail earlier (50).
Fluorescence-emission images of the abaxial (AB) and adaxial (AD)
surfaces of leaves were made with a compact flash-lamp fluorescence
imaging system (FL-FIS, 2, 51). Leaves were fixed in front of the flash
lamp by an air stream generated by a ventilator. Excitation light was
provided by a pulsed xenon lamp with a UV-transmission filter
(transmission maximum at 355 nm). Fluorescence images were
acquired with a CCD camera that was synchronously gated with the
flash lamp. Leaves were placed at 390 mm from the camera, to produce
images 80 mm in width. The multispectral fluorescence imaging for the
blue (F440), green (F520), red (F690) and far-red (F740) fluorescence
was acquired sequentially from an identical field of view, each with the
appropriate filter selected from a filter wheel in front of the CCD
camera. The acquisition of the fluorescence-emission images required
the accumulation of 800 images.
Images were processed by specific software (Camille 1.05 software;
Photonetics, Kehl, Germany). The raw image was corrected for
nonuniform excitation with the pixels of an image showing uniform
fluorescence (blue fluorescence image of a white paper). Black and
white images of both measured fluorescence intensity and fluorescence
ratios (F440 ⁄ F520, F440 ⁄ F690, F440 ⁄ F740 and F690 ⁄ F740, acquired
by a division of fluorescence intensity at the corresponding wavelength
of each pixel) were set at the appropriate intensity and either false color
or a black and white scale were applied by the ImageJ software
package (http://rsb.info.nih.gov/ij).
Numerical data from a region of interest corresponding to the leaf
outline were processed by Camille 1.05 software. Fluorescence profiles
across the leaves (created using ImageJ) were made by drawing a line
perpendicular to the main vein, covering 266 pixels of AS leaves. The
main vein was located in the middle of the line (133 pixels to the edge
of the leaf).
Fluorophore determination. Phenolic compounds were warm (at
70C) extracted from 20 mm2 leaf disks with methanol for 30 min. The
volume of the extract solutions was diluted to 5 or 10 mL (depending
on the type of sample) with methanol, and then frozen at )80C until
they were analyzed.
Leaf extracts were analyzed by the HPLC method described
previously (11). This method revealed the presence of one (most
abundant) phenolic acid, which was identified as CGA by comparing
its retention time and UV absorption spectrum with that of CGA (pure
standard [purity ‡97%]; Fluka Chemie GmbH, Buchs, Switzerland) in
methanol. Prior to analysis, the sample extracts were thawed and
filtered through a 0.45 lm polyvinylidene fluoride filter. High-performance liquid chromatography was performed using an HPLC system
(Waters Alliance 2795 HPLC system; Waters Corp., Mildford, MA)
equipped with an online degasser, autosampler module (4C), and
column oven (40C). A diode-array detector (Waters 2996 UV–visible)
was used for analyte detection. The column used was an analytical
HPLC column (Waters Symmetry C18, 250 mm · 4.6 mm i.d.,
dp = 5 lm) with a 20-mm guard column made of the same material.
The mobile phase consisted of a gradient system of solution A, MilliQ
water-acetic acid (97.5:2.5; vol ⁄ vol) and solution B, HPLC gradient
grade methanol-acetic acid (97.5:2.5; vol ⁄ vol) at a flow rate of
1.0 mL min)1. The gradient program was as follows: linear gradient
from solution A-B (70:30) to solution A-B (51:49) in 15 min, isocratic
at solution A-B (51:49) for 10 min, followed by a linear gradient to
solution A-B (70:30) in 5 min, finally isocratic at solution A-B (70:30)
for 5 min. The injection volume was 15 lL. Automated instrumentation
was controlled by an HPLC data system (HyStar; Bruker Daltonik
GmbH, Bremen, Germany). The data were analyzed by a dataprocessing software package (HyStar PostProcessing; Bruker Daltonik
GmbH). Quantification was based on peak-area measurements
1050 Mónica Pineda et al.
at 325 nm. Standard solutions were prepared with CGA, and were
examined by HPLC in order to determine the retention time (4.1 min).
A multipoint calibration was performed from 1.25 to 40 lM. The linear
relationship was as follows: Y = 9.28X + 1.14 (R2 = 0.9999). aNaphthol (‡99% GC; Riedel-de Haën, Seelze, Germany) was used as
internal standard (retention time, tR, 23.0 min).
Statistics. The statistical analysis was carried out with specific
software (SigmaPlot 8.0 software; Systat Software, Inc., San Jose, CA).
The Student’s t-test was used to compare control with infected plants,
and PMMoV-I-infected plants with PMMoV-S-infected plants.
Graphs show mean values (obtained from at least four different
plants per leaf type) and their corresponding standard errors. Tables 1
and 2 indicate significant differences between treatments, according to
the Student’s t-test and the three significance levels tested: P < 0.05
(*), P < 0.01 (**) and P < 0.001 (***). Statistical analysis presented 6
degrees of freedom.
RESULTS
We have obtained fluorescence images (F440, F520, F690,
F740 and their corresponding ratios) from both the AB and
AD leaf surfaces of AS and S leaves in control and PMMoVinfected plants at different postinfection times. In addition,
fluorescence values averaged in the whole leaf were obtained
and compared in control and infected plants during the
infection. The statistical significance of the differences between
both kinds of plants is summarized in Tables 1 and 2 (tables
show the dpi when significant differences occur compared with
the control).
Blue-green fluorescence emission of leaves
The most obvious changes in the BGF emission of both AS
and S leaves from infected plants occurred on the AB surface
(Fig. 1). BGF emitted by AS leaves from infected plants and
their equivalent controls increased with leaf age. The AB
surface of AS leaves from PMMoV-infected plants showed
higher BGF emission compared to control values from 10 and
12 dpi onward (PMMoV-S and -I, respectively; Fig. 1 left
panel, Table 1). In addition, BGF emission from plants
infected with PMMoV-S was higher compared to those
infected with PMMoV-I. BGF emission decreased to values
similar to that of the control in the last PMMoV-I-infection
steps (21–28 dpi), coinciding with the recovery phase in these
plants.
The increase in BGF fluorescence from leaf surfaces during
pathogenesis was even higher in S leaves from infected plants.
Table 1. Summary of the statistical analysis of the intensity of the fluorescence emitted in the blue, green, red and far-red region by intact leaves of
control and PMMoV-infected Nicotiana benthamiana plants.
PMMoV
Asymptomatic leaf
Symptomatic leaf
Strain
Abaxial surface
Adaxial surface
Abaxial surface
Adaxial surface
-I
-S
-I
-S
-I
-S
-I
-S
12–24**
10–14**,17***
12–14*, 17**, 21*, 24***
10***, 12–14**, 17***
.5*, 7**, 10***, 12** 14–17*
NS
.7*, 10–14**, 17*
.17*
NS
5**
NS
5–7*; 14–17*
.7*, 10–12**, 14–17*
NS
.7*, 10–12**, 14–17*
NS
10–17***, 21–24*, 28**
7*, 10–14***, 17**
10–17***, 21–28**
5*, 7–14***, 17**
.10*, 17–21*
.5**, 7–10*, 17*
.10**, 14*, 17**, 21*
.7*, 10**, 14–17**
10**, 14**, 17–21*, 24**
7**, 10*, 12–14***, 17*
14*, 21–24**
7**, 10*, 12–14***, 17*
.7**, 10*, 12–14**, 17–21*, 28**
.7**, 12–14**, 17*
.7**, 10*, 12–17**, 21*, 28*
.7**, 12***, 14**, 17***
F440
F520
F690
F740
Table displays days postinoculation where significant differences compared with controls were found at P < 0.05 (*), P < 0.01 (**) and
P < 0.001 (***). Degrees of freedom: 6. Arrows indicate increase ( ) or decrease (.) in fluorescence emission of PMMoV-infected plants
compared with controls. Asymptomatic leaves = old leaves which remain symptomless during the whole infection process; F440 = blue
fluorescence; F520 = green fluorescence; F690 = red fluorescence; F740 = far-red fluorescence; PMMoV-I and -S = Italian and Spanish strains
of the Pepper mild mottle virus; NS = no significant differences were found at any dpi assayed; symptomatic leaves = young leaves displaying
curling from the first week of infection.
Table 2. Summary of the statistical analysis of the different fluorescence ratios of control and PMMoV-infected Nicotiana benthamiana plants.
PMMoV
Strain
F440 ⁄ F520
F440 ⁄ F690
F440 ⁄ F740
F690 ⁄ F740
-I
-S
-I
-S
-I
-S
-I
-S
Asymptomatic leaf
Abaxial surface
Symptomatic leaf
Adaxial surface
Abaxial surface
Adaxial surface
14–28**
.17**, 28*
NS
NS
12–14*, 17**
.17**
NS
.17*
7*, 10**, 12–14***, 17–21**, 24* 5*, 7**, 10***, 12–17**, 21*, 28* 10–17***, 21–24* 7*, 10–14***, 17–21**, 28**
12–14*, 17**
NS
7*, 10**, 12–17*
7–10*, 12***, 14**, 17*
10–12***, 14–21**, 24*
7**, 10***, 12–14**, 17–21*
10–17***, 21–24* 7*, 10–14***, 17–21**, 28*
14*, 17**
14*
7*, 10**, 12–17*
7–10*, 12–14***, 17**
10–12*
.12*, 14**, 28*
NS
.12–14*, 28*
17*
NS
10–12*, 14–17** .7*, 17*
Table displays days postinoculation where significant differences compared with controls were found at P < 0.05 (*), P < 0.01 (**) and
P < 0.001 (***). Degrees of freedom: 6. Arrows indicate increase ( ) or decrease (.) in fluorescence emission of PMMoV-infected plants
compared with controls. Asymptomatic leaves = old leaves which remain symptomless during the whole infection process; F440 = blue
fluorescence; F520 = green fluorescence; F690 = red fluorescence; F740 = far-red fluorescence; PMMoV-I and -S = Italian and Spanish strains
of the Pepper mild mottle virus; NS = no significant differences were found at any dpi assayed; symptomatic leaves = young leaves displaying
curling from the first week of infection.
Photochemistry and Photobiology, 2008, 84
1051
Figure 1. Time-course of blue (F440), green (F520), red (F690), and far-red (F740) fluorescence emission of asymptomatic (left) and symptomatic
(right) leaves of Nicotiana benthamiana control and PMMoV-infected plants. Fluorescence signals integrated over the entire leaf at every dpi
assayed are displayed; error bars indicate standard error. Abaxial and adaxial surfaces of the leaves are shown. Data are mean ± standard error of
n = 4.
PMMoV-S caused the outstanding signals from 5 to 7 dpi
onward (F520 and F440, respectively). In the case of PMMoVI infection, the first significant BGF increase was recorded at
10 dpi. During the recovery phase of these plants, BGF values
of the S leaves decreased to values similar to that of the control
plants (Fig. 1 right panel, Table 1).
Chlorophyll fluorescence emission of leaves
Values of Chl-F integrated over the entire leaf that were
emitted both by AS and by S leaves were higher on the AB leaf
side in all cases (Fig. 1). Chl-F decreased with the leaf age of
AS leaves in infected plants as well as in their corresponding
controls from the middle stage of the infection onward (Fig. 1
left panel). In general, no significant differences in Chl-F
emission from these kinds of leaves could be established
between PMMoV-S-infected and control plants (except the
F740 signal from the AB surface at 17 dpi; Table 1). However,
PMMoV-I-infected plants showed a significant decrease in
Chl-F from the AB surface at 5–17 dpi (F690), as well as in the
AD surface at 7–17 dpi (F690 and F740). These differences
disappeared during the recovery phase in both sides of the leaf.
1052 Mónica Pineda et al.
The S leaves from infected plants also showed a decrease in
Chl-F compared to the control (Fig. 1 right panel). The AB
side of S leaves from PMMoV-S-infected plants exhibited the
first significant changes as early as 5 dpi (F690, Table 1), while
the AD surface showed significant differences only from 7 dpi
(F690 and F740). PMMoV-I-infected plants had lower Chl-F
values from 7 to 10 dpi onward (AD and AB surfaces,
respectively). During the late recovery phase (from 24 dpi on)
in PMMoV-I-infected plants, the Chl-F emission decreased to
values similar to that of the control (see Table 1 for statistical
significance).
cence signals recorded across these leaves (Fig. 3). The AB leaf
surface was chosen because its fluorescence signal was more
prominent.
Blue-green fluorescence emission from control leaves was
the lowest. BGF levels in AS leaves from PMMoV-infected
plants had higher values along the whole profile, were more
evident in the vein area and were highest in the case of
PMMoV-S-infected plants. Three peaks could be distinguished, corresponding to Type I and II veins. On the other
hand, control leaves had the highest Chl-F values. The main
veins had the lowest Chl-F values in AS leaves in both the
control and PMMoV-infected plants (Fig. 3).
Imaging of multicolor fluorescence of leaves
To analyze the viral-induced spatiotemporal changes in
fluorescence emission at different wavelengths, we compared
the images obtained before (5 dpi) and after (17 dpi, once the
fluorescence emission changes were very evident) changes in
fluorescence (Fig. 2) in both S and AS leaves from control and
PMMoV-infected plants.
The F440 pattern from control leaves, equivalent to the AS
in infected plants, did not significantly change over the
experimental period (Fig. 2a). Areas showing the highest
fluorescence emission corresponded to the vascular tissues of
the AB leaf surface. At 5 dpi, AS leaves from infected plants
were very similar to that of the controls. However, an increase
in F440 could be detected at 17 dpi in both the veinal and
interveinal tissues of AS leaves of infected plants, and was
more apparent on the AB surface and in the case of PMMoVS-infected plants. Viral-induced changes on the F520 pattern
were very similar to those of F440.
During the first days after infection, the F690 and F740
pattern (Fig. 2a) of AS leaves from infected plants appeared to
be similar to that of control plants; however, at 17 dpi, Chl-F
emission of both leaf sides decreased in the whole leaf
compared to that of control plants. A fluorescence gradient
pattern appeared where the highest Chl-F values were found in
the leaf areas surrounding the main veins.
Control leaves corresponding to the S leaves in infected
plants displayed a very homogeneous BGF pattern on both
surfaces during the period analyzed (Fig. 2b). The S leaf of
PMMoV-infected plants did not differ from controls at 5 dpi
(increased values recorded in the AD surface were not
significant). However, a BGF increase in the whole leaf (both
surfaces) was evident at 17 dpi. The vein areas which had the
highest values were more apparent on the AB surface of the S
leaves and in PMMoV-S-infected plants, as in the case of AS
leaf patterns of infected plants. In contrast to the defined ChlF pattern from AS leaves of infected plants, no clear pattern
emerged from the F690 and F740 images of S leaves (Fig. 2b).
Profiles of asymptomatic leaves at 17 dpi
Fluorescence profiles of the AS leaf surface at 17 dpi were
plotted to visualize the distribution of the different fluores-
Fluorescence ratios of leaves
In AS leaves, the values of F440 ⁄ F520 ratio decreased with leaf
age in all cases (Fig. 4 left panel). A significant increase in this
ratio in infected plants compared to the controls was reported
in the AB side from 12 and 14 dpi onward (PMMoV-S and -Iinfected plants, respectively) (Table 2).
F440 ⁄ F690 and F440 ⁄ F740 were the fluorescence ratios
that reported the PMMoV infection the earliest, and showed
significant increases with respect to control values in the AD
surface at 5 (F440 ⁄ F690) and 7 dpi (F440 ⁄ F740). In the case
of PMMoV-I infection, these ratios were similar to that of the
control plants during the symptom-recovery phase (Fig. 4 left
panel, Table 2). In the case of PMMoV-S infection, an increase
in both ratios was detected at 12 dpi (AB surface, F440 ⁄ F690),
and remained high during the whole infection process.
F690 ⁄ F740 values were generally higher in the AB surface
than on the AD surface (Fig. 4 left panel). This ratio slightly
decreased on both leaf surfaces until it was about 10–12 dpi in
control and infected plants; from this time onward the ratio
rose. Notably, AS leaves for both kinds of infected plants did
not have a lower Chl content than the controls, except for the
AB side of the AS leaves of PMMoV-S-infected plants at the
last infection point (Table 2). Moreover, the AD surface of AS
leaves of PMMoV-I-infected plants had lower values of
F690 ⁄ F740 than controls at various times (12, 14, 28 dpi;
Table 2).
In the case of S leaves (Fig. 4 right panel), every fluorescence ratio assayed was a good indicator of the stress induced
by PMMoV infection in N. benthamiana plants, except for
F440 ⁄ F520. However, none of the ratios could detect significant differences between S leaves of infected plants and the
equivalent controls before visual symptoms appeared. Alterations in both F440 ⁄ F690 and F440 ⁄ F740 ratios due to the
infection with both viral strains could be detected as early as
7 dpi (Fig. 4 right panel, Table 2). The recovery phase of
PMMoV-I-infected plants was registered as a decrease in these
ratios.
Increases in F690 ⁄ F740 were observed in S leaves from
PMMoV-infected plants. The AD surface of leaves from
control and infected plants displayed lower values of
F690 ⁄ F740 than the AB surface (Fig. 4 right panel).
Figure 2. Images of blue (F440), green (F520), red (F690) and far-red (F740) fluorescence emission of asymptomatic (a) and symptomatic (b) leaves
of Nicotiana benthamiana control and PMMoV-infected plants. Abaxial and adaxial surfaces of the leaves are shown. Images obtained before
(5 dpi) and after (17 dpi) fluorescence changes occurred are displayed. The color scale applied is shown for each panel. Different sizes of
asymptomatic (old) and symptomatic (young) leaves are due to leaf age.
Photochemistry and Photobiology, 2008, 84
1053
1054 Mónica Pineda et al.
Figure 3. Infection-induced changes in leaf fluorescence emission on the abaxial surface of asymptomatic leaves at 17 dpi. Fluorescence profiles are
shown along a 266-pixel line drawn in the middle of an asymptomatic leaf, and centered on the midrib (vein Type I; for Nicotiana benthamiana vein
classification, see Roberts et al. [55]). Vertical dotted lines represent the positions of vein Types I and II. Fluorescence emission was normalized to
maximum values.
Imaging of fluorescence ratios of leaves
Figure 5 displays the images corresponding to the fluorescence
ratios of both AS and S leaves before (5 dpi) and after (17 dpi,
once changes in ratios values were very evident) the biggest
changes in the fluorescence ratios.
A lower F440 ⁄ F520 due to leaf aging was evident in the
images of AS leaves from infected plants and their controls
(Fig. 5a). No significant differences were found between
control and infected plants at 5 dpi; however, images at
17 dpi displayed higher F440 ⁄ F520 values in PMMoV-infected
plants compared to the controls, especially in the vein areas of
the AB surface.
At 5 dpi, images of the F440 ⁄ F690 ratio did not show
differences between control and infected plants (Fig. 5a).
Nevertheless, a clear rise in this ratio in AS leaves of infected
plants was evident at 17 dpi, and was more prominent on the
AD surface. For this parameter, a declining gradient was
detectable from the vein (which had the highest values of the
ratio) to the rest of the leaf blade. Images of F440 ⁄ F740
displayed a very similar situation, albeit with higher values
than F440 ⁄ F690 (Fig. 5a).
During the infection, no significant decreases in Chl content
(Table 2) were evident in the F690 ⁄ F740 images from AS
leaves of infected plants compared to the control (Fig. 5a),
except for the AB surface of the AS leaves from PMMoV-Sinfected plants, where Chl loss was evident at 17 dpi. The AD
surface of the leaves generally had lower values of F690 ⁄ F740
than the AB surface.
In the case of S leaves, no clear trend emerged from the
F440 ⁄ F520 images (Fig. 5b). The only remarkable fact was
that the AD surface of the PMMoV-S-infected plants
had lower F440 ⁄ F520 values compared to the controls at
17 dpi.
For F440 ⁄ F690 and F440 ⁄ F740 an increase in these ratios
was clear in the images of S leaves from infected plants only at
17 dpi, with the vascular tissues emitting the highest fluorescence (Fig. 5b). This vein-associated pattern, very similar to
that found in AS leaves of infected plants, was more obvious
on the AB surface of the S leaves. Increases in F690 ⁄ F740
Photochemistry and Photobiology, 2008, 84
1055
Figure 4. Time course of F440 ⁄ F520, F440 ⁄ F690, F440 ⁄ F740 and F690 ⁄ F740 fluorescence ratios of asymptomatic (left) and symptomatic (right)
leaves of Nicotiana benthamiana control and PMMoV-infected plants. Averages for the whole leaf at each dpi assayed are displayed; error bars
indicate standard error. Abaxial and adaxial surfaces of the leaves are shown. Data are mean ± standard error of n = 4.
appeared at 17 dpi and only in the S leaves from PMMoV-Sinfected plants.
Fluorophore determination
Leaf extracts were analyzed by an HPLC method described
previously (11), which is able to resolve CGA, caffeic acid and
cynarin (1,3-dicaffeoylquinic acid). HPLC analysis of methanolic extracts from S leaves of N. benthamiana plants revealed
the presence of one of the major phenolic compounds,
identified as CGA by comparing its retention time and UV
absorption spectrum with that of CGA (pure standard) in
methanol. The chromatogram also revealed the presence of
other very minor unidentified compounds, which were not
further analyzed (data not shown). The concentration of CGA
increased during the viral infection (Fig. 6). Significant differences with respect to the controls appeared from 7 to 21 dpi
for PMMoV-S plants, whereas PMMoV-I-infected plants
displayed differences only at 14 dpi. Later (21 dpi) significant
differences in the CGA content between control and PMMoVI-infected plants vanished in accord with the appearance of the
recovery phase.
1056 Mónica Pineda et al.
Photochemistry and Photobiology, 2008, 84
Figure 6. Time course of CGA concentrations (mg mm)2) (·102) of
symptomatic leaves of Nicotiana benthamiana control and PMMoVinfected plants. Data are mean ± standard error of n = 4.
DISCUSSION
Although in the last few years UV-excited MCF imaging has
emerged as a sensitive and specific tool for presymptomatic
and nondestructive monitoring of changes in the physiologic
state of plants, the application of this technique has not gained
ground in the investigation of pathogen attacks, especially
viral infections. Nevertheless, Chl-FI has been broadly
employed to study such interactions (32,33,35–38,52–54).
Information about BGF imaging and the corresponding
accumulation of secondary metabolites in virus-infected plants
is available only for the HR induced by TMV (42). The present
study is the first to elucidate such changes during a systemic
infection.
Infection in N. benthamiana plants with PMMoV caused an
increase in BGF that was higher in the AB side of AS leaves
and in the case of PMMoV-S infection; the response of the
plant to PMMoV-I is delayed. This is in agreement with the
milder symptomatology induced by this tobamovirus, as well
as with the lower virulence and accumulation in the host plant
that was demonstrated in our previous studies (37,48). Areas
surrounding the veins (for N. benthamiana vein classification,
see [55]) showed the highest BGF values; viral-induced BGF
increases in the interveinal tissue are stronger for F520. This is
probably due to the higher accumulation of green fluorophores
under long-term stress (2) and the fact that F520 is less
efficiently absorbed by the Chl than F440 is (56). This is
supported by the decrease in the F440 ⁄ F520 ratio during the
experiment (Fig. 4). The increase detected in F520 and F440 in
control old leaves (corresponding to the AS in infected plants,
1057
Figs. 1 and 2) over the course of the experiment is probably the
result of accumulation of phenolic compounds and changes in
the optical properties of leaves that are associated with leaf
aging (3,17,57).
Blue-green fluorescence measurements on the AB leaf side
seem to be more adequate for following the viral infection. It is
well documented that UV-induced fluorescence signals from
the upper and lower sides of the leaf are considerably different
in dicotyledonous plants. A difference that may account for
this observation is that large amounts of Chl from packed
palisade cells of the AD side of the leaf reabsorb F440 and
F690 more efficiently than the spongy parenchyma of the AB
side (2,3).
Imaging of red fluorescence (F690) in PMMoV-infected
plants agrees well with our previous data using different ChlFI systems (37,38). From 14 (PMMoV-S, data not shown) and
17 dpi onward (PMMoV-I), Chl-F showed a heterogeneous
leaf pattern on the AD side, with increased values in the tissue
surrounding the main veins. It was demonstrated that this
pattern is directly related to the spread of the virus in AS leaves
(37,38). Fluorescence changes during pathogenesis in these
kinds of leaves could not be attributed to alterations in Chl
content, because it did not significantly change over the period
analyzed (37). The present work also supports this fact, as the
F690 ⁄ F740 ratio (inversely correlated with Chl content) was
similar (PMMoV-S) to or even lower (PMMoV-I) than the
control values (Figs. 4 and 5). The F690 ⁄ F740 ratio of the AB
surface of AS leaves of PMMoV-S-infected plants displayed
higher values than the control very late in the infection process
(17 dpi, the last day assayed), but the changes in the Chl-F
pattern started earlier (14 dpi).
Blue-green fluorescence changes preceded (10 dpi PMMoVS; 12 dpi PMMoV-I) the detection of the virus by tissue
printing (14 and 17 dpi, respectively, for the two viral strains;
[37]) or northern blotting (58) in AS leaves, suggesting an
earlier activation of the secondary plant metabolism and the
subsequent accumulation of fluorophores.
Increases in the BGF emission of PMMoV-infected plants
are also reflected in the higher F440 ⁄ F690 and F440 ⁄ F740
ratios, even on the AD leaf side (Fig. 4, Table 2). Similar
increases were reported after point measurements in Chinese
cabbage infected with Turnip yellow mosaic virus (41). These
ratios have been demonstrated to be the best stress indicators
(21). It was possible to detect virus-induced stress earlier (5 dpi
AD side and 7 dpi AB side) with the F440 ⁄ F690 ratio than
with other fluorescence parameters analyzed in the case of
PMMoV-I infection. F440 ⁄ F520 was not as efficient as
BGF ⁄ Chl-F in following the PMMoV infection.
The recovery phase of PMMoV-I-infected plants (21–
28 dpi) also seems to be evident in the old leaves, as
fluorescence values (Fig. 1 left panel, Table 1) and ratios
(Fig. 4 left panel, Table 2) of either AD or AB surfaces from
AS leaves of these plants are close to the control values.
An analysis of the fluorescence yield along a broader line
across the leaf area is possible (22,59). Some authors (20) have
used horizontal transects to demonstrate that BGF and Chl-F
Figure 5. Images of F440 ⁄ F520, F440 ⁄ F690, F440 ⁄ F740 and F690 ⁄ F740 fluorescence ratios of asymptomatic (a) and symptomatic (b) leaves of
Nicotiana benthamiana control and PMMoV-infected plants. Abaxial and adaxial surfaces of the leaves are shown. Images obtained before (5 dpi)
and after (17 dpi) fluorescence changes occurred are displayed. The color scale applied is shown for each panel. Different sizes of asymptomatic
(old) and symptomatic (young) leaves are due to leaf age.
1058 Mónica Pineda et al.
have a negative contrast in variegated leaves. This relationship
is also evident in the vein area of AS leaves from N.
benthamiana infected with PMMoV (Fig. 3). Profiles display
the heterogeneous accumulation of blue-green fluorophores,
but the Chl-F heterogeneity is poorly represented by these
transects, once again establishing that images offer more
information about changes triggered by PMMoV infection
(37,38).
In order to focus on the S leaves, we first need to summarize
some data about viral movement in the host plant. It is well
known that tobamoviruses, such as PMMoV, move rapidly
from the inoculated basal leaves to the plant apex (60) via
phloem (55,61,62). Tissue prints taken from 7 to 28 dpi of the
young leaves showed a quick viral accumulation in the whole
leaf in the early stages of infection (37). Recently (data not
shown) we detected the entrance of PMMoV-S in the leaf at
4 dpi and later (6 dpi) for the less virulent strain PMMoV-I.
The rapid viral movement to the S leaves compared with the
AS leaves (37), could explain the absence of a dynamic imaging
Chl-F pattern in S leaves (Fig. 2b). However, average wholeleaf Chl-F values could distinguish S leaves of infected leaves
from their corresponding controls, but not in a presymptomatic manner (Fig. 1 right panel, Table 1). These changes in
Chl-F emission preceded the decrease in Chl content (37), as
shown by the F690 ⁄ F740 ratio (Fig. 4 right panel, Table 2).
Either PMMoV detection in the S leaf preceded alterations
in BGF emission or the two processes took place concomitantly (F520 of AB surface from S leaves of PMMoV-Sinfected plants, Table 1). The changes in fluorescence emission
on the AB surface of S leaves were more significant in the case
of AS leaves for the same reasons described above. The
accumulation of phenolic compounds in S leaves induced by
PMMoV, contrary to AS ones, did not precede virus detection.
The decrease in BGF values in the S leaves of PMMoV-Iinfected plants between 21 and 28 dpi, compared with previous
points of measurement, was indicative of the symptomrecovery phase affecting young leaves.
To identify the major compounds responsible for the
emission of BGF in N. benthamiana plants, HPLC analyses
of leaf extracts were performed. Wolfbeis (63) reviewed a series
of natural compounds which are known to be present in leaves
and which emit BGF upon UV excitation. The two main types
of BGF emitters are HCAs and nicotinamide and flavine
nucleotides (3,16,64). In the methanolic extracts of control and
S leaves from PMMoV-infected N. benthamiana plants, CGA
was the only HCA detected in high concentrations. A boost in
the CGA concentration in S leaves was correlated with the
virulence of the viral strain analyzed—PMMoV-S induced the
most dramatic changes; and HCA values for the PMMoV-Iinfected plants decreased to levels close to that of the controls
during the recovery phase. In addition, the CGA concentrations in S leaves concur well with the BGF values (F440, F520)
and in both cases increased during the infection with both viral
strains. The characteristics of the UV-induced absorption and
excitation spectra that we have recorded previously (65) in leaf
pieces from PMMoV-infected plants match those of the CGA.
It is well known that biotic and abiotic stress factors induce
the phenylpropanoid metabolism (66,67). An increase in
phenolic compounds has been observed in plants infected with
Wheat streak mosaic virus (68), as well as during a combined
infection with TMV and the Potato virus X (PVX) (69).
Solanaceae, such as the genus Nicotiana, accumulate CGA
as the major phenolic compound (70). This HCA was found to
be most abundant in tobacco plants (13), and was also present
in these plants under different environmental stress conditions,
as UV light or mineral deficiencies ([12] and references
therein).
In the past few years, the antioxidant function of CGA, one
of the main products of the phenylpropanoid pathway, has
been emphasized. In addition, it has been proposed that the
involvement of CGA in an alternative pathway for photochemical energy dissipation under stress has the added benefit
of enhancing the antioxidant capacity of the cell (70).
Different authors have suggested that CGA functions as a
preformed agent against biotic challenges and operates
through its antioxidant activity. Tomato plants with elevated
levels of CGA showed a slower progression and reduced levels
of infection by Pseudomonas syringae. Similar results were
reported for tobacco with different levels of CGA, which were
infected with either the fungus Cercospora nicotianae or
P. syringae var. tomato ([71] and references therein). The
induction of the synthesis of phenolic compounds such as
CGA for biologic control of the phytopathogenic fungus
Phytophora nicotianae has been also reported (72). However,
little information obtained from BGF imaging is available
about the leaf distribution of phenolic compounds that are
produced during pathogenesis, as existing data are only
concerned with the HR (42).
It is noteworthy that we found similarities between the
images of AS leaves of PMMoV-infected plants with a defined
spatial pattern of BGF emission at 10–12 dpi (data not shown)
and those of the leaf thermal pattern (37) at 7–10 dpi (viral
strain dependent) as well as with those of the nonphotochemical quenching pattern at 17 dpi (PMMoV-I) (38) from our
previous studies. In addition, the images were in good
agreement with later viral immunolocalization on tissue prints
at 14–17 dpi. As the thermal changes in these leaves (the
temperature increase was due to stomatal closure) were the
earliest ones during pathogenesis and prior to the viral
detection, some systemic plant factors are presumably
involved. In our system, greater BGF emission corresponding
to activation of the secondary metabolism occurred 3 days
later than thermal changes for every viral strain analyzed.
Alterations in the Chl-F pattern were evident when the virus
was already present in the leaf. Some of these changes must
play a part in the defense against the pathogen, because
photoinhibition indicating photosystem II damage was only
evident in PMMoV-infected plants in the final infection steps
(38,48).
In conclusion, the BGF imaging of the AB side of the leaf
was able to distinguish between the two viral strains; and the
magnitude of the changes correlated with their virulence. The
response was also delayed in the less virulent strain. A
symptom-recovery phase in PMMoV-I-infected plants, affecting only the young S leaves, in the last infection steps was also
detected by these measurements. BGF emission also differs
between S and AS leaves. BGF imaging could detect virally
induced metabolic changes in the absence of symptoms in AS
leaves preceding even viral detection. The situation in S leaves,
where the virus arrives earliest, is more complex, due to the
quick invasion by the pathogen; viral spread and changes in
BGF emission take place concomitantly. CGA was
Photochemistry and Photobiology, 2008, 84
demonstrated to be the main contributor to BGF emission in
N. benthamiana plants. In addition, there was a correlation
between its levels in developing leaves and the virulence of the
tobamovirus. Taken together, these results indicate that
UV-induced fluorescence is a promising tool for visualizing a
systemic infection before the appearance of visible symptoms
and before the pathogen is present.
16.
17.
18.
Acknowledgements—The authors are very grateful to Ruth Sagardoy
for assistance with the HPLC analyses. This research and M.P.’s
contract were supported by grants from the Spanish Ministry of
Science and Education (MEC-FEDER, grant BIO2004-04968-C02-02
to M.B.A.). Ruth Sagardoy was supported by the University of
Valencia ⁄ EEAD-CSIC contract—within the ESA ESRIN contract
19187 ⁄ 05 ⁄ I-EC—to F.M. The travel associated with this work was
supported by the Spanish-Hungarian Intergovernmental Scientific and
Technological Fund (E-19 ⁄ 04 in Hungary) and Acciones Integradas
Programme (HH2004-0032 in Spain).
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