Journal of Experimental Botany, Vol. 53, No. 372,
Antioxidants and Reactive Oxygen Species in Plants Special Issue,
pp. 1249–1254, May 2002
Imaging of photo-oxidative stress responses in leaves
Michael J. Fryer1, Kevin Oxborough1, Phillip M. Mullineaux2 and Neil R. Baker1,3
1
2
Department of Biological Sciences, University of Essex, Colchester, Essex CO4 3SQ, UK
John Innes Centre, Norwich Research Park, Colney, Norwich, Norfolk NR4 7UH, UK
Received 10 July 2001; Accepted 7 December 2001
Abstract
Introduction
High resolution digital imaging was used to
identify sites of photo-oxidative stress responses in
Arabidopsis leaves non-invasively, and to demonstrate the potential of using a suite of imaging
techniques for the study of oxidative metabolism
in planta. Tissue-specific photoinhibition of photosynthesis in individual chloroplasts in leaves was
imaged by chlorophyll fluorescence microscopy.
Singlet oxygen production was assessed by imaging the quenching of the fluorescence of dansyl2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrole (DanePy)
that results from its reaction with singlet oxygen.
Superoxide and hydrogen peroxide accumulation
were visualized by the reduction of nitroblue tetrazolium (NBT) to formazan deposits and by polymerization with 3,39-diaminobenzidine (DAB), respectively.
Stress-induced expression of a gene involved with
antioxidant metabolism was imaged from the bioluminescence from leaves of an Arabidopsis APX2LUC transformant, which co-expresses an ascorbate
peroxidase (APX2) with firefly luciferase. Singlet
oxygen and superoxide production were found to
be primarily located in mesophyll tissues whereas
hydrogen peroxide accumulation and APX2 gene
expression were primarily localized in the vascular
tissues.
Singlet oxygen, superoxide radical and hydrogen peroxide
are reactive oxygen species (ROS) that are generated
when plant tissues are exposed to a variety of environmental stresses (Halliwell, 1984; Asada, 1996; Fryer et al.,
1998). The extremely short life-times of ROS makes the
study of their production in planta very difficult. Damage
to proteins, lipids and DNA has often been used as an
index of oxidative stress (Halliwell and Gutteridge, 1985;
Rice-Evans et al., 1991), although this is a rather indirect
measure of the production of ROS. Spin labels have been
applied with some success for the secondary detection by
electron paramagnetic resonance (EPR) of oxygen free
radicals and organic radicals from their semi-stable
adducts (Hodgson and Raison, 1991; Heber et al., 1996;
Van Doorslaer et al., 1999), but this does not provide
information on the specific sites of ROS production in
tissues. Cell fractionation techniques have been used in
attempts to identify sites of ROS generation and the
location of ROS detoxification systems (Doulis et al.,
1997; Kingston-Smith et al., 1999), however, these involve
extensive tissue disruption which can lead to the generation of ROS. Consequently, data produced using such
destructive methods does not always result in an accurate
picture of the distribution of ROS and their detoxification
systems. Also, such in vitro studies on macerated material
cannot resolve the differences in predisposition to oxidative stress that undoubtedly exist between various tissues
and cell types within leaves. In this paper it is demonstrated how the use of ROS-specific tracer dyes in
conjunction with high resolution imaging now provides
the opportunity to identify sites of photo-oxidative stress
and ROS accumulation in leaves.
Key words: Ascorbate peroxidase, chlorophyll fluorescence,
hydrogen peroxide, singlet oxygen, superoxide.
3
To whom correspondence should be addressed. Fax: [ 44 (0)1206 873416. E-mail: [email protected]
Abbreviations: CCD, charge-coupled device; DAB, 3,39-diaminobenzidine; DanePy, dansyl-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrole; F 9,
fluorescence level at any point between F 9o and F 9m; F 9m, maximal fluorescence level from leaves in light; F 9o, minimal fluorescence level of leaves in
light; F 9q, difference in fluorescence between F 9m and F9 (F9q\F 9m–F 9); NBT, nitroblue tetrazolium; 1O2, singlet oxygen; PPFD, photosynthetically-active
photon flux density; ROS, reactive oxygen species; SOD, superoxide dismutase.
ß Society for Experimental Biology 2002
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Fryer et al.
Manipulation of genes encoding components of antioxidant systems has facilitated the non-invasive study of
the expression of genes coding for enzymes involved in
ROS metabolism. The example presented in this paper
is the transformation of Arabidopsis with an APX2-LUC
transgene, a cytosolic ascorbate peroxidase promoter
fused to a firefly luciferase reporter gene (Karpinski
et al., 1999). APX2-LUC is silent under non-stressed conditions, but its mRNA is detectable within 15 min of the
onset of a high light stress. Induction of APX2-LUC
allows for the non-invasive bioluminescent imaging of
APX2 gene expression and the analysis of factors that
influence it.
The combination of advanced molecular genetics techniques with non-invasive digital imaging of the generation
of ROS illustrated in this paper provide extremely
powerful tools for future investigations of the development of oxidative stress in leaves and the regulation of
ROS metabolism.
Materials and methods
Plant material, probe application and stress conditions
Arabidopsis thaliana plants were germinated and raised to
mature rosette stage under controlled environmental conditions
(PPFD, 200 mmol m Y 2 s Y 1 during a 16 h photoperiod at 25 C
in a relative humidity of c. 80%). Leaves were excised at the
petiole with a razor blade and allowed transpirationally to
imbibe aqueous solutions of fluorescent probes and dyes for
90 min at the growth PPFD. Solutions were replaced with water
during the following stress period. For the majority of the
treatments, tip regions of the leaf were placed inside the chamber
of a computerized infrared gas analysis system (CIRAS,
PP Systems, Hitchin, Hertfordshire) and exposed to an air
flow rate of 347 cm3 min Y 1. Photo-oxidative stress conditions
(PPFD 650 mmol m Y 2 s Y 1 at 30 C and water vapour pressure
> 0.3 kPa) were imposed for 90 min.
The development of the Arabidopsis thaliana transformant
with an APX2-LUC trangene has been previously reported
(Karpinski et al., 1999). This transformant was grown under
the conditions described above.
High resolution, chlorophyll fluorescence imaging
High resolution imaging of chlorophyll fluorescence from
chloroplasts in intact leaves was carried out essentially as
described previously (Oxborough and Baker, 1997). F9o and F9m
define the minimal and maximal fluorescence levels from leaves
in the light, respectively. F9 is the fluorescence level at any
point between F9o and F9m. For the construction of parametized images, the specific term F9q was recently introduced
(Oxborough and Baker, 1997; Baker et al., 2001) which
denotes the difference between F9m and F9 measured immediately
before the application of a saturating pulse to measure F9m.
Under these conditions, F9q uF9m equates to the operating
quantum efficiency of PSII photochemistry. A Peltier-cooled
charge-coupled device (CCD) camera, as described earlier
(Oxborough and Baker, 1997), was used for non-invasive
imaging of ROS-fluorogenic compounds and bioluminescent
expression of APX2-LUC.
Detection of reactive oxygen species
Singlet oxygen (1O2) activity was detected by infiltrating leaves
with 40 mM DanePy (dansyl-2,2,5,5,-tetramethyl-2,5-dihydro1H-pyrrole), a dual fluorescent and spin probe. Reaction of
highly fluorescent DanePy with 1O2 yields a non-fluorescent
nitroxide radical (DanePyO) (Hideg et al., 1998, 2000, 2001).
DanePy fluorescence in leaves was excited with 345 nm
radiation produced from a UV source (ULT1004 Black Ray
LW, Scientific Laboratory Supplies, Wilford, UK) through a
345 nm band pass filter (E46-084, Edmund Scientific, York,
UK) and imaged using the CCD camera (described above)
protected with a 532 nm band pass filter (E43-122, Edmund
Scientific, York, UK).
Infiltration of leaves with 6 mM nitroblue tetrazolium (NBT)
allowed the detection of superoxide. When the pale yellow NBT
reacts with superoxide a dark blue insoluble formazan compound is produced (Flohe and Otting, 1984; Beyer and
Fridovich, 1987). Superoxide is thought to be the major
oxidant species responsible for reducing NBT to formazan
(Maly et al., 1989). Chlorophyll was removed from the leaves
prior to imaging by infiltrating them with lacto-glycerol-ethanol
(1 : 1 : 4 by vol) and boiling in water 5 min. The location of
formazan deposits was visualized by subtracting background
(non-formazan) pixels from the leaf image.
Infiltration of leaves with 5 mM 3,39-diaminobenzidine
(DAB) at pH 3.8 allows the detection of hydrogen peroxide
in leaves (Thordal-Christensen et al., 1997; Orozoco-Cardenas
and Ryan, 1999). DAB forms a deep brown polymerization
product upon reaction with H2O2 in the presence of peroxidase
(Thordal-Christensen et al., 1997), which can be imaged after
removal of chlorophyll from the leaf, as described above.
Imaging of gene expression
Expression of APX2-LUC was monitored in leaves after
spraying with 1 mM luciferin (Promega). The treated leaves
were then imaged for 60 min with the Peltier-cooled CCD
camera (described above) in order to generate an image of the
bioluminescence produced.
Results and discussion
Photoinhibition of photosynthesis results in changes in
the quantum efficiency of photosynthetic electron transport, which can be estimated from the fluorescence parameter, F9quF9m. Examples of images of the chlorophyll
fluorescence parameter F9 from chloroplasts in the bundle
sheath (Fig. 1A) and mesophyll (Fig. 1D) cells of an intact
leaf were used to generate images of F9quF9m (Fig. 1B, E)
from which the individual chloroplasts can be isolated
and their F9quF9m values determined (Fig. 1C, F). The
chloroplasts in the bundle sheath and mesophyll cells of
the leaf prior to the photoinhibitory treatment of 60 min
at a PPFD of 600 mmol m Y 2 s Y 1 had F9q uF9m values of 0.67
and 0.65, respectively (data not shown), which decreased
to 0.25 and 0.39, respectively during the photo-oxidative
stress (Fig. 1C, F) demonstrating a large decrease in the
operating efficiency of non-cyclic photosynthetic electron
transport.
The accumulation of 1O2 during this photo-oxidative
stress treatment is shown in Fig. 2. An Arabidopsis leaf
Imaging photo-oxidative stress
1251
Fig. 1. Imaging of chlorophyll fluorescence parameters from an Arabidopsis leaf after 60 min of exposure to high light. The leaf was grown under a
PPFD of 200 mmol m Y 2 s Y 1 and then exposed to a light stress of 600 mmol m Y 2 s Y 1 for 60 min. Images of F9 (A, D) from bundle sheath (A) and
mesophyll (D) cells. Images of F9q uF9mwere calculated from images of F9 and F9m for the bundle sheath (B) and mesophyll (E) cells, and presented in false
colour. Using Fluorchart software the chloroplasts within the two cell types (bundle sheath in B and mesophyll in E) were isolated and an average value
of F9q uF9m calculated (C, F, shown above the chloroplast images). Data have been mapped to the colour palette shown between (B) and (E).
Fig. 2. An Arabidopsis leaf was infiltrated with DanePy and then an area of the leaf tip was exposed to a PPFD of 600 mmol m Y 2 s Y 1 for 60 min,
which is indicated by the white oval in the reflected light image (A). The images of fluorescence emission from the DanePy before (B) and after (C) the
high light treatment show that quenching of the fluorescence occurs within the area of the leaf tip exposed to the high light. This fluorescence quenching
results from formation of non-fluorescent DanePyO when 1O2 reacts with DanePy.
was infiltrated with DanePy and then a region of the leaf
tip was irradiated with a PPFD of 600 mmol m Y 2 s Y 1.
Images of the fluorescence of the DanePy show that there
is a marked decrease in the fluorescence emission from
the area of the leaf exposed to the high light treatment
(Fig. 2), indicating the production of the nitroxide radical
DanePyO produced from the reaction of DanePy with
1
O2. It has been previously demonstrated that DanePy
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Fryer et al.
penetrates leaf tissues well and enters cells and chloroplasts (Hideg et al., 2001). It appears that 1O2 is
formed primarily in mesophyll tissue (Fig. 2). This would
be expected since the majority of the leaf ’s photosynthetic
apparatus is contained in mesophyll chloroplasts and
1
O2 will be produced by photosensitized energy transfer
reactions between excited triplet state chlorophyll and
ground state molecular oxygen whenever excess excitation
energy cannot be efficiently dissipated as photochemistry
or heat from chlorophylls (Halliwell, 1984; Wise and
Naylor, 1987). 1O2 is suspected to be involved in damage
to the D1 reaction centre protein of PSII (Vass et al.,
1992; Aro et al., 1993), has also been implicated in lipid
peroxidation by direct reaction with polyunsaturated
fatty acyl residues in membranes and in general oxidative
degradation of proteins (Halliwell, 1984).
Imaging of purple formazan deposits, which result
from the reaction of NBT with superoxide, identifies the
regions of superoxide formation in a leaf. The tip region
of the leaf that has been exposed to the high light treatment clearly shows more intense staining than the nonstressed area, with the majority of the staining being
associated with mesophyll tissue (Fig. 3). Numerous small,
localized ‘hotspots’ were observed at the distal points of
the microvasculature (Fig. 3). The petiole exhibited very
heavy staining which, presumably, is associated with
superoxide accumulation resulting from NADPH oxidase
activity, a major effector of the oxidative burst in the
wound response (Mehdy et al., 1996; Wojtaszek, 1997).
Staining in the petiole was most intense in veinal tissue
close to where the leaf was excised from the plant and
decreased with distance away from the wound; presumably this demonstrates that a systemic wounding signal
that stimulates production of superoxide has been
propagated along the veins from the wound. Although
NBT also reacts with ascorbate, large changes in the
ascorbate pool would not be expected in leaf tissues
during the period of photo-oxidative stress used and,
consequently, imaging of formazan production can be
considered to indicate superoxide accumulation; large
increases in the rate of superoxide production would
be expected during the photoinhibitory stress. Areas of
formazan deposits indicate cells in which the rate of superoxide production has become significantly greater than
the rate of detoxification.
The production of H2O2 was imaged in leaves infiltrated with DAB, which reacts with H2O2 in the presence
of peroxidases to produce a brown polymerization product. Considerably more H2O2 was detected in the tip
region of the leaf that had been exposed to the high light
treatment, with it being primarily associated with the
vascular tissue (Fig. 4). The ascorbate–glutathione cycle
has been proposed for the removal of H2O2 in order to
prevent its conversion to the extremely reactive and
oxidizing hydroxyl radical within leaves (Halliwell, 1984).
Ascorbate peroxidase (APX) converts H2O2 to monodehydroascorbate. Using the APX2-LUC Arabidopsis
transgenote (Karpinski et al., 1999) it is possible to
image the expression of the gene for a cytosolic form of
ascorbate peroxidase when leaves are exposed to the high
light treatment. Clearly there is widespread APX2 gene
expression in the light-treated area of the leaf, however,
Fig. 3. Image of an Arabidopsis leaf infiltrated with NBT. The top half of the leaf was exposed to a high light treatment of 600 mmol m Y 2 s Y 1 for
60 min. The purple coloration indicates the formation of insoluble formazan deposits that are produced when NBT reacts with superoxide.
Imaging photo-oxidative stress
1253
Fig. 4. Image of an Arabidopsis leaf infiltrated with DAB. The top half of the leaf was exposed to a high light treatment of 600 mmol m Y 2 s Y 1 for
60 min. The brown staining indicates the formation of a brown polymerization product when H2O2 reacts with DAB.
Fig. 5. False colour image of the luminescence from a leaf of the APX2LUC Arabidopsis transformant after exposure to the high light treatment
of 600 mmol m Y 2 s Y 1 for 60 min and spraying with luciferin. The
luminescence indicates the expression of the LUC and APX2 genes.
some expression was also observed around the vascular
tissue in the regions of the leaf that were kept in the dark
(Fig. 5) demonstrating the systemic signalling system which
induces APX2 expression (Karpinski et al., 1999).
Imaging the accumulation of 1O2, superoxide and H2O2
in intact leaves in parallel with fluorescence imaging of
the operating efficiencies of electron transport in specific
tissues can clearly provide some interesting and novel
insights into the spatial organization of oxidant biochemistry occurring in response to photo-inhibitory stresses.
In the leaves examined in this study accumulation of 1O2
(Fig. 2) and superoxide (Fig. 3) appeared to be principally
located in mesophyll tissues during photo-oxidative
stress, whereas H2O2 accumulation was more associated
with the vascular tissue (Fig. 4). These observations
suggest that excess light results in a larger increase in
superoxide production in the mesophyll chloroplasts
compared to that in the choroplasts of the vascular
tissue, while hydrogen peroxide preferentially accumulates in the vascular tissues. A similar specific localization
of H2O2 within vascular tissue has been described in
wounded leaves and is thought to be associated with
the systemic octadecanoid signalling pathway (OrozocoCardenas and Ryan, 1999). Presumably the accumulation
of ROS in photosynthetic tissues is likely to be involved
in the signalling process associated with the switching
on of APX2 gene expression in response to the high light
stress.
Clearly, imaging of photoinhibition of photosynthesis,
ROS production and the expression of genes coding
for enzymes involved in detoxification of ROS in intact
leaves has great future potential for resolving the
heterogeneity and nature of the responses of leaf tissues
to photo-oxidative stress.
Acknowledgements
These studies were supported by a grant from the Biotechnology
and Biological Sciences Research Council to NRB and PMM.
The authors are grateful to Christine Edwards for synthesizing
the DanePy.
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