1. ROS Production in Arabidopsis thaliana Leaves in Response
to Antimycin A and Monofluoroacetate Treatments
Jenifer Klabis
PBL 490  Instructor J. Yu  December 2004
Plant mitochondria are a major source of reactive oxygen species (ROS), as a response to stress.
This response causes the amplification of H2O2 formation, which can be measured using
dichloroflourscein diacetate (DCF) and 3,3-diaminobenzidine (DAB). These measurements allow the
determination of ROS production in the plant cells, which is a factor mediating the signaling for the
expression of alternative oxidase (AOX). Interruption of mitochondrial functions will not only lead to in-
expression of genes but also in basic cellular processes, such as, the Tricarboxylic Acid (TCA) cycle. The
TCA cycle is important for the production of citrate and other metabolites for the cell. Lack of these
compounds will have adverse effects on the plant mitochondria. The treatment of the leaf tissue caused an
increase in ROS production that was successfully measured and visualized. Citrate levels in the treated
and untreated tissue appeared to decrease over time.
KEYWORDS
Alternative Oxidase  Citrate  Hydrogen
Peroxide  Mitochondria  Reactive Oxygen
Species  Tricarboxylic Acid Cycle
ABBREVIATIONS
AA: Antimycin A  AOX: Alternative Oxidase 
AOX1: Nuclear gene encoding alternative
oxidase  DAB: 3,3-diaminobenzidine  DCF:
Dichloroflourscein Diacetate  DMSO: Dimethyl
Sulfoxide  MFA: Monofluoroacetate  ROS:
Reactive Oxygen Species  TCA: Tricarboxylic
Acid
INTRODUCTION
Mitochondria are a major source of
reactive oxygen species (ROS) formation,
and it is possible that this organelle could
participate in the oxidative burst in plants
(Tiwari et al., 2002). During respiration,
molecular oxygen may undergo a univalent
reduction at the sites of ROS generation in
complexes I and III of the respiratory chain,
forming superoxide, which subsequently
dismutates to hydrogen peroxide (Braidot et
al., 1999).
Mitochondria contain biochemical
pathways and components which link the
cellular processes of carbon and nitrogen
metabolism in plants. The TCA cycle links
both carbon and nitrogen metabolism by
oxidation of organic acids from glycolysis
and the export of either -ketolutarate
directly or citrate (Hodges, 2002).
Plant mitochondria are also
responsible for the signaling between itself
and the nucleus for gene expression.
Increases in cellular ROS concentrations
have been strongly implicated as a
component for stress-induced alternative
oxidase (AOX) expression (Vanlerberghe &
McIntosh, 1996; Millar et al., 2001;
Maxwell et al., 2002; Vanlerberghe et al.,
2002; Zhang et al., 2003; Norman et al.,
2004). The biological role of AOX has
remained elusive; it is currently thought that
one isozyme (AOX2) is required for normal
respiratory metabolism while the other
(AOX1) functions under stress conditions
(Gray et al., 2004).

2. A disruption in the normal function
of the mitochondria will, therefore, have
serious consequences for plant carbon
metabolism and cellular biosynthetic
reactions.
Despite extensive research on the
source of ROS, the subcellular location and
the mechanism of ROS generation has not
been unequivocally clarified (Bolwell,
1999). It is known that environmental
stresses lead to the accumulation of ROS,
such as H2O2, in plant cells (Dat et al., 2002;
Mittler, 2002; Noctor & Foyer, 1998).
Generation of ROS in plants has been
implicated in biotic and abiotic stresses.
Plants function with ROS and there
is a careful balance between ROS
production and ROS scavenging. During
environmental stress the plant can become
unbalanced. Loss of this balance often
involves a combination of increasing ROS
production and limited energy resources to
replenish defense mechanisms, such as
reductant for antioxidants, leading to these
defenses being overwhelmed and ultimately
resulting in ROS accumulation.
Once accumulation of ROS occurs,
damage to cellular components begins. This
includes direct inhibition of enzymes by
ROS, protein oxidation reactions, membrane
lipid peroxidation yielding toxic products,
and oxidative DNA and RNA damage
(Elstner, 1982).
This research sought to test the
hypothesis that cellular citrate levels are
affected by the introduction of AA and MFA
in to the plant mitochondria. Also that the
introduction of these compounds would
increase the levels of ROS production in the
cells.
The purpose of this experiment is to
determine the effects of treatments on
mitochondrial signaling pathways and the
expression of AOX. DAB and DCF
measurements were taken to examine the
effect on the ROS dependant pathway and
the TCA cycle intermediate, citrate, was
used to determine the effects on the ROS
independent pathway.
MATERIALS AND METHODS
Plant Growth and Chemicals
Arabidopsis thaliana was grown in a
basic soil mixture and maintained under 12 hr of
light (75 Em-2
*s-1
) and 12 hr of dark at 20C.
For all experiments 4 to 5 week old plants were
used. Antimycin A (AA), Monofluoroacetate
(MFA), Menadione, and 3,3-diaminobenzidine
(DAB) were purchased from Sigma.
Dichloroflourscein Diacetate (DCF) was
purchased from Molecular Probes. H2O2 was
purchased through J.T. Baker. 1000-fold stocks
of AA, MFA, Menadione, and DCF were
dissolved in appropriate solvents of 2-Propanol,
H2O, EtOH respectively. H2O2 was diluted to the
desired concentration. DAB stocks were
prepared by dissolving the DAB powder in
100L Dimethyl Sulfoxide (DMSO), 1/10 MS
Salts was then added and was pH to 3.8.Tween
20 was used in all incubation mixes at a
concentration of 0.1% to act as a surfactant.
H2O2 Detection by the ‘DAB-uptake
method’
By using 3,3-diaminobenzidine (DAB)
as a substrate, H2O2 was visually detected
(Thordal-Christensen et al., 1997). Leaves were
excised at the base with a sharp razor blade. The
leaves were then pre-incubated in a 1mg/ml
solution of DAB, pH 3.8, for 1 hr in the dark at
room temperature. Then AA and Menadione
were then added to the incubation mixes and the
leaves returned to incubation for another 8 hr.
The experiments were terminated by the
immersion of the leaves in boiling 95% ethanol
for 10 min. This treatment decolorized the
leaves except for the brown polymerization
product produced by the reaction on DAB with
H2O2. The samples were then stored and
examined in 95% ethanol at room temperature
and photographed.
ROS Detection by Spectrofluorometry

3. Intracellular production of ROS was
measured using Dichloroflourscein (DCF). This
compound is initially nonfluorescent and is
rapidly oxidized into a highly fluorescent
reactant by H2O2 and other cellular peroxides
(Maxwell et al., 1999). Fluorescence was
visualized by using the Kodak Image Station
2000mm Camera with excitation and emission
wavelengths set at 488 nm and 525 nm,
respectively. Results were normalized to control
values, which were arbitrarily set as one.
Quantification was done by reading the media
using Molecular Devices’ Spectramax M2 with
the excitation set at 480 nm and the emission set
at 525 nm. These values were then averaged to
produce a graph.
Citrate Assay
Samples were frozen in liquid nitrogen,
thoroughly crushed or homogenized then
lyophilized over night. Samples were then
extracted using 20% Percloric Acid then
neutralized using 1M TEA 5NKOH. A citric
acid kit, supplied by R-Biopharm, was then used
and followed procedures as laid out in the kit.
Samples were then measured at 340nm using a
Beckman DU 7400 Spectrophotometer. Data
was then compiled and calculated against a
standard curve.
RESULTS
All results presented in this publication are preliminary
results only. Further work is still being done to validate the
data and material presented here.
40
80
120
160
200
2 Hrs 4 Hrs 6 Hrs 8 Hrs
Time ( hrs)
Control
1mM M FA
10uM AA
200
250
300
350
400
450
500
550
0 2 4 6 8
Time (hrs)
umolesCitrate/gDW
Control
MFA
AA
Fig. 2 Citrate assay of Arabidopsis thaliana tissue,
with MFA and AA over an 8 hr period. Data was run
in triplicate and shown is the average and standard
deviation.
The chemical probe DCF has been
used as a noninvasive, in vivo measure of
intracellular ROS in tobacco and
Arabidopsis thaliana cultured cells
(Maxwell et al., 1999). DCF was used here
to determine the effects of MFA and AA on
the production of ROS in plant leaf tissue.
As shown in Fig. 1, the level of DCF
fluorescence in the AA treated
samples were approximately twice as great
as those untreated and almost three times as
great as those treated with MFA. AA is a
known suppressor of respiratory cellular
functions in plants (Vanlerberghe &
McIntosh, 1992).
Addition of AA to the tissue samples
resulted in an increase in intracellular ROS
as measured by DCF fluorescence in all
treated samples. The ROS production was
decreased, compared to the control, with the
addition of MFA.
Over the 8 hr incubation the citrate
levels dropped, as can be seen in Fig. 2. The
AA treated tissue experiences the greatest
drop in citrate levels, while the MFA treated
tissue had the highest levels of citrate at the
end of the incubation. It is unknown why the
levels of citrate continued to decrease over
the incubation period.Fig. 1 DCF Fluorescence of Arabidopsis thaliana
tissue, with MFA and AA over an 8 hr period. Data
is taken from a single experiment.

4. Experiment 39
0
50
100
150
200
250
300
350
8 Hrs
Time
DCFFlourescence
Control
25uM AA
100uM Menadione
500uM Menadione
Fig. 3 DCF fluorescence at 8 hr with AA and
Menadione treatments. Data was run in triplicate and
shown is the average and standard deviation.
DCF fluorescence readings from the
Specrtomax M2, Fig. 3, coincide with the
images taken by the Kodak camera, Fig. 4.
The visualization of the DAB method, as
can be seen in Fig. 4, also follows the same
trend. Menadione resulted in a much higher
concentration of ROS production then that
of AA. Menadione was used to show the
range of the ROS measurement techniques,
due to the fact that Menadione produces
very large amount of ROS (Fig. 3).
The concentration of added H2O2
needed in order to obtain a clear DAB
staining reaction in detached epidermal
tissue is of the magnitude of 1 mM. DAB
can detect H2O2 in leaves at levels as low as
0.1 M concentrations, but strong color
develops only at higher concentrations of
about 1-10 M (Thordal-Christensen et al.,
1997).
DISCUSSION
In order to localize H2O2 in intact
plant tissue, we have to take advantage of
the immediate peroxidase-dependant
polymerization of DAB upon contact with
H2O2. So by allowing living leaves to uptake
DAB, the instability if H2O2 is overcome by
the high stability if the DAB polymer.
Fig. 4 DCF fluorescence and DAB visualization at 8
hr with AA and Menadione treatments.
DAB polymerizes and turns deep
brown in the presence of H2O2, and the
intensity of the coloration and its
localization can be qualitatively assessed
and photographed. The development of the
DAB- H2O2 reaction product in Arabidopsis
thaliana tissue in response to AA and
Menadione treatments is detectable
as early as 8 hr after introduction into the
media. The color initially is visible at the
wound site on the cut stem, and then
appeared in major and minor veins
throughout the leaves.
We wanted to look at the
amplification of ROS produced solely by the
introduction of our treatments. Our
experiments did not use an external source
of H2O2 so that we could measure the
natural occurrence of its production caused
by stress.
Known limitations of out ROS
measurement techniques are changes in
temperature and high light; due to that DCF
is degraded in high light. These variables
were not tested in this series of experiments
and the data is not presented here. The DAB
procedure has a very pronounce limitation
that measurements can only be take once on
one leaf; this is not true with DCF. Even
though these techniques are still the most
effect way to measure in vivo ROS levels
Control 25µM AA 100µM Mena 500µM Mena
DCFDAB

5. and our result show that both methods give
the same results (Fig. 4).
An increase in the TCA cycle
intermediate citrate, after inhibition of
aconitase by MFA, was accompanied by a
rapid and dramatic increase in the level of
AOX1 (Vanlerberghe & McIntosh, 1996).
The decrease of citrate levels in our tissue
does not concure with that data presented in
Vanlerberghe and McIntosh’s work. This
could be due to that they used tobacco
suspension cultures and we chose to use
Arabidopsis thaliana leaves. So our results
reflect the stress response of multiple types
of cells in the tissue, whereas theirs only
shows the results of one specific type of cell.
The results present here demonstrate
that ROS production is linked with exposure
to oxidative stresses. DCF is an effect way
to measure ROS levels in tissue and not just
cell cultures, and that DCF and DAB
measurement coincide with each other.
Future work will continue to focus on citrate
levels in leaves, until it is clear as to why
there is a lack of accumulation of TCA cycle
intermediates.
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