Download Nitric Oxide Acts as an Antioxidant and Delays Programmed Cell

Nitric Oxide Acts as an Antioxidant and Delays
Programmed Cell Death in Barley Aleurone Layers1
Maria Veronica Beligni, Angelika Fath, Paul C. Bethke*, Lorenzo Lamattina, and Russell L. Jones
Instituto de Investigaciones Biologicas, Facultad de Ciencias Exactas y Naturales, Universidad Nacional de
Mar del Plata, 7600, Mar del Plata, Argentina (M.V.B., L.L.); and Department of Plant and Microbial Biology,
University of California, Berkeley, California 94720–3102 (A.F., P.C.B., R.L.J.)
Nitric oxide (NO) is a freely diffusible, gaseous free radical and an important signaling molecule in animals. In plants, NO
influences aspects of growth and development, and can affect plant responses to stress. In some cases, the effects of NO are
the result of its interaction with reactive oxygen species (ROS). These interactions can be cytotoxic or protective. Because
gibberellin (GA)-induced programmed cell death (PCD) in barley (Hordeum vulgare cv Himalaya) aleurone layers is mediated
by ROS, we examined the effects of NO donors on PCD and ROS-metabolizing enzymes in this system. NO donors delay
PCD in layers treated with GA, but do not inhibit metabolism in general, or the GA-induced synthesis and secretion of
␣-amylase. ␣-Amylase secretion is stimulated slightly by NO donors. The effects of NO donors are specific for NO, because
they can be blocked completely by the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide.
The antioxidant butylated hydroxy toluene also slowed PCD, and these data support our hypothesis that NO is a protective
antioxidant in aleurone cells. The amounts of CAT and SOD, two enzymes that metabolize ROS, are greatly reduced in
aleurone layers treated with GA. Treatment with GA in the presence of NO donors delays the loss of CAT and SOD. We
speculate that NO may be an endogenous modulator of PCD in barley aleurone cells.
Nitric oxide (NO) is an important second messenger in animal cells (Brunori et al., 1999; Chung et al.,
2001) and accumulating evidence suggests it is important in plant cells as well (Beligni and Lamattina,
2001; Wendehenne et al., 2001). Exogenous NO affects several aspects of plant growth and development (Ribeiro et al., 1999), and can affect the responses of plants to pathogens (Delledonne et al.,
1998; Durner et al., 1998), light (Giba et al., 1998;
Beligni and Lamattina, 2000), gravity (Pedroso and
Durzan, 2000), and oxidative stress (Beligni and Lamattina, 1999). NO may also be an endogenous maturation and senescence factor in higher plants
(Leshem et al., 1998). As a short-lived, readily diffusible free radical, NO can react with a variety of
intracellular and extracellular targets. In some cases,
these interactions are cytotoxic and result in cell
death. In macrophages, thymocytes, and tumor cells,
for example, NO has been shown to hasten apoptosis
(Cui et al., 1994; Fehsel et al., 1995; Messmer and
Bruene, 1996). NO can also paradoxically act as an
antioxidant and an antiapoptotic modulator that prevents cell death (Chung et al., 2001). These cytotoxic
and protective effects of NO are often concentration
1
This work was supported by the National Science Foundation,
by the Torrey Mesa Research Institute, San Diego, by the United
Nations Educational, Scientific, and Cultural Organization (fellowship to M.V.B.), and by Fundación Antorchas (Argentina; fellowship to M.V.B.).
* Corresponding author; e-mail [email protected];
fax 510 – 642– 4995.
Article, publication date, and citation information can be found
at www.plantphysiol.org/cgi/doi/10.1104/pp.002337.
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dependent (Wink and Mitchell, 1998). When
suspension-cultured soybean (Glycine max) cells were
infected with the bacterial pathogen Pseudomonas syringae, for example, NO increased in parallel with
other reactive oxygen species (ROS) and promoted
the hypersensitive response and programmed cell
death (PCD; Delledonne et al., 1998). In potato (Solanum tuberosum) leaves infected by the pathogen Phytophthora infestans or treated with ROS-producing
herbicides, on the other hand, NO acted as an antioxidant and prevented cell death (Laxalt et al., 1997;
Beligni and Lamattina, 1999). More recently, NO was
shown to protect wheat (Triticum aestivum) seedlings
against oxidative stress resulting from drought
(Garcia-Mata and Lamattina, 2001). This protective
role of NO may result, in part, from its interaction
with lipid hydroperoxyl radicals or highly reactive
superoxide, both of which promote lipid peroxidation (Wink and Mitchell, 1998). An additional role in
promoting stomatal closure may be equally important (Garcia-Mata and Lamattina, 2001).
Although NO can have many, often disparate, effects on plants, there is little evidence demonstrating
that endogenous NO acts as a regulator of plant
growth and development or plant responses to stress.
It is clear that plants synthesize NO, and the production of NO from NO2⫺ in a reaction catalyzed by
nitrate reductase (NR) is well characterized (Yamasaki, 2000). Other sources of NO are likely, though
controversy exists regarding the synthesis of NO in
plants by NO synthase (NOS). Genes encoding NOS
have not been identified in the sequenced genomes of
plants (Yamasaki, 2000) or their ancestors (Mallick et
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Nitric Oxide Delays Aleurone Cell Death
al., 2000), and NOS genes appear absent from the
genomes of bacteria and fungi. Yet NOS activity has
been reported in plants (Ninnemann and Maier, 1996;
Delledonne et al., 1998; Durner et al., 1998; Leshem et
al., 1998), and inhibitors of NOS have been shown to
reduce this activity (Ninnemann and Maier, 1996;
Delledonne et al., 1998).
ROS are key players in the hormone-induced PCD
of cells in the barley (Hordeum vulgare) aleurone
layer. The barley aleurone layer is a secretory tissue
that surrounds the starchy endosperm and embryo of
barley grain. Gibberellins (GAs) and abscisic acid
(ABA), two plant hormones, control the function of
the aleurone cells that make up the layer. GAs initiate
signaling cascades that result in the synthesis and
secretion of ␣-amylase and other hydrolytic enzymes.
These degrade the storage reserves in the starchy
endosperm and provide the growing embryo with
nutrients. Following GA-induced enzyme secretion,
the aleurone layer is not necessary for seedling establishment and dies (Bethke et al., 1999; Fath et al.,
2001). ABA, on the other hand, inhibits hydrolase
production and secretion, and ABA-treated aleurone
cells do not die (Bethke et al., 1999; Fath et al., 2001).
GA-induced PCD in barley aleurone layers is preceded by a decline in the activity of enzymes that
metabolize ROS (Fath et al., 2001) and this leads to an
increase in the susceptibility of aleurone cells to ROS
(Bethke and Jones, 2001). In this paper, we show that
NO prolongs the life of barley aleurone cells incubated in GA. The effects of NO can be mimicked by
the antioxidant butylated hydroxy toluene (BHT),
indicating that NO may act as an antioxidant in
aleurone cells. We provide evidence that aleurone
cells synthesize NO, and we suggest that NO may be
an endogenous modulator of aleurone cell viability.
RESULTS
NO Donors Delay PCD in Barley Aleurone Layers
PCD in intact barley aleurone layers is prevented
by ABA and stimulated by GA (Bethke et al., 1999;
Fath et al., 2001). ABA can also delay death of aleurone cells that have been treated with GA. In this
report, we extend our observations on PCD to show
that NO can selectively delay the death of GA-treated
aleurone cells. To monitor death and viability of barley aleurone cells, aleurone layers were incubated in
5 ␮m GA or 5 ␮m ABA for up to 48 h and were then
stained with the fluorescent dyes fluorescein diacetate (FDA) and N-(3-triethylammoniumpropyl)-4(6-[4-(diethylamino) phenyl] hexatrienyl) pyridinium
dibromide (FM 4-64) to detect living (green fluorescence) and dead (red fluorescence) cells, respectively
(Fig. 1). PCD in GA-treated layers had already begun
24 h after incubation in GA, when approximately
20% of the cells were dead, and was well under way
after 36 h in GA, when about 60% of the cells were
dead (Fig. 1A). Two days after hormone treatment,
Plant Physiol. Vol. 129, 2002
87% of cells in GA-treated aleurone layers were dead
(Fig. 1A), but virtually no cells were dead in ABAtreated aleurone layers (Fig. 1D).
We used net O2 uptake as an additional measure of
cell viability in barley aleurone layers and as an
indicator of overall metabolic activity (Fath et al.,
2001). O2 uptake data for layers from the experiment
described above (Fig. 1) are presented in Figure 2. O2
consumption changed in parallel with cell viability as
measured by FDA and FM 4–64 staining (Fig. 1).
Whereas O2 consumption by ABA-treated layers was
relatively constant for 48 h, O2 consumption by GAtreated layers decreased to almost zero between 24
and 48 h of incubation (Fig. 2). The rapid decline in
O2 consumption by GA-treated layers between 24
and 48 h of incubation occurs at the time when cells
are undergoing PCD (Fig. 1A).
We also measured O2 consumption by barley aleurone layers to gauge the effect of the NO donors
sodium nitroprusside (SNP) and S-nitroso-Nacetylpenicillamine (SNAP) on cell death. ABA- or
GA-treated aleurone layers were incubated for 48 h
with NO donors at various concentrations, and relative rates of O2 consumption were determined (Fig.
3). There was virtually no net O2 uptake by aleurone
layers incubated in GA for 48 h, but addition of SNP
(100 ␮m) or SNAP (300 ␮m to 2 mm) resulted in O2
consumption at rates comparable with those for
ABA-treated layers (Fig. 3). SNP and SNAP were
most effective at maintaining O2 consumption at 100
␮m and 1 mm respectively. O2 consumption declined
more slowly between 24 and 48 h when layers were
incubated in GA plus 100 ␮m SNP (Fig. 2A) or GA
plus 300 ␮m SNAP (Fig. 2B) compared with layers
incubated in GA alone. This indicates that these NO
donors delayed PCD in GA-treated aleurone layers.
The O2 consumption data also establish that the delay in PCD resulting from incubation with NO donors does not result from a general inhibition of
metabolism. O2 consumption by layers treated with
GA and NO donors is not lower than that for layers
treated with GA alone (Fig. 2).
Incubation of GA-treated aleurone layers in SNAP
or SNP also dramatically reduced the rate of PCD as
measured by fluorescence microscopy (Fig. 1, B and
C). Virtually no cells were dead 24 h after incubation
in GA plus 100 ␮m SNP (Fig. 1B) or 300 ␮m SNAP (Fig.
1C), and only 28% (SNP) or 35% (SNAP) of cells were
dead at 36 h compared with 60% of the cells in layers
incubated in GA alone. After 48 h with NO donors,
61% of cells in GA ⫹ SNP and 52% of the cells in GA
⫹ SNAP remained alive. We added the NO scavenger
2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1oxyl-3-oxide (cPTIO) to aleurone layers incubated
with GA and SNAP to confirm that the effect of SNAP
on PCD was mediated specifically by NO (Fig. 1E).
Virtually all cells were dead after 48 h with cPTIO and
SNAP, and cell death at 24 h in the presence of SNAP
and the NO scavenger was higher than in cells incu-
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Beligni et al.
Figure 1. PCD in barley aleurone layers is delayed by the NO donors SNP and SNAP. Digital images of fluorescently labeled
barley aleurone cells are shown in A through E. Layers were incubated in FDA (green, live cells) and FM 4-64 (red, dead
cells) prior to image capture. Layers were treated with 5 ␮M GA (A–C and E) alone (A) or with 100 ␮M SNP (B), 300 ␮M SNAP
(C), 300 ␮M SNAP plus 300 ␮M cPTIO (E), or with 5 ␮M ABA alone (D) for the times indicated.
bated in GA alone (compare Fig. 1, A with E). When
cPTIO was added to cells incubated in GA alone, cell
death was also accelerated (data not shown). This
latter result suggests that endogenous sources of NO
may play a role in delaying PCD in aleurone layers.
cPTIO added to non-hormone-treated aleurone layers
had no effect on cell death, and this makes it unlikely
that the NO scavenger exerts a toxic effect on its own
(data not shown).
NO Donors Do Not Inhibit GA-Stimulated
␣-Amylase Synthesis
NO donors delayed PCD in barley aleurone layers,
but did not inhibit overall metabolism as measured
by net O2 consumption. To determine if delayed PCD
resulted from a global inhibition of GA-induced responses, we carried out experiments to establish
whether the GA-stimulated synthesis and secretion
of ␣-amylase was affected by these treatments. To do
this, we measured the activity of ␣-amylase secreted
to the incubation medium and the steady-state
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amounts of ␣-amylase mRNA in aleurone layers that
were incubated in the presence and absence of ABA,
GA, and NO donors. As has been reported frequently, GA stimulates and ABA inhibits the synthesis and secretion of ␣-amylase (Fig. 4). When GAtreated aleurone layers were incubated with NO
donors, the rate of ␣-amylase secretion and the total
amount of ␣-amylase produced were not less than
that from layers treated with GA alone. ␣-Amylase
accumulation in the medium was enhanced slightly
by NO donors (Fig. 4). Whereas the accumulation of
␣-amylase activity in the incubation medium ceased
24 h after incubation in GA alone, incubation of
layers in 100 ␮m SNP or 300 ␮m SNAP prolonged the
period of ␣-amylase synthesis and secretion. This
resulted in a significantly greater amount of
␣-amylase activity in the medium surrounding layers
treated with SNAP or SNP (Fig. 4).
RNA blots probed with an ␣-amylase cDNA are
consistent with the observation that NO donors do
not inhibit the synthesis of ␣-amylase (Fig. 5). GA
stimulates the accumulation of ␣-amylase mRNA in
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Plant Physiol. Vol. 129, 2002
Nitric Oxide Delays Aleurone Cell Death
Figure 2. Barley aleurone layers treated with GA and NO donors
remain viable longer than layers treated with GA alone. Layers were
incubated in 5 ␮M GA alone (A and B) or in the presence of 100 ␮M
SNP (A) or 300 ␮M SNAP (B). Relative rates of O2 consumption were
measured at various times of incubation using an O2 electrode. For
comparison, O2 consumption rates for ABA-treated (5 ␮M) layers are
shown in A and B. Data are means ⫹ SD of at least four individual
layers.
of barley Cat2 mRNA was reduced within 3 h of
exposure to GA, and by 6 h in GA, Cat2 mRNA was
not detectable. Incubation in GA plus SNAP slowed
the loss of Cat2 mRNA by up to 6 h (Fig. 6A), but did
not result in a dramatic, sustained increase as is seen
for layers incubated in ABA (Fig. 6A). Protein blotting
shows that GA treatment also caused a rapid decline
in the amount of CAT protein, and this decline was
delayed by 3 h in GA-treated cells by the presence of
SNAP (Fig. 6B). The amount of CAT protein did not
decline in ABA-treated tissue (Fig. 6B).
We also determined the steady-state amounts of
Sod4 mRNA and SOD protein in aleurone layers exposed to GA and NO donors (Fig. 7). Using a maize
Sod4 probe, we found that Sod4 mRNA declined in
layers incubated in GA or GA plus SNAP. In both
cases, after 9 h of incubation, Sod4 mRNA was barely
detectable. In contrast, the amount of Sod4 mRNA in
ABA-treated layers was greater after 24 h of incubation than in freshly prepared layers (Fig. 7A). Protein
blotting shows that the amount of SOD protein declined in aleurone cells exposed to GA, and that this
decline was less rapid in layers treated with GA plus
SNP or GA plus SNAP (Fig. 7B).
barley aleurone layers, and in these experiments,
␣-amylase mRNA abundance peaked between 9 and
18 h of incubation. By 24 h of incubation in GA, the
amount of ␣-amylase mRNA remaining was a small
fraction of that present at 9 to 18 h (Fig. 5). When
layers were treated with SNP or SNAP, on the other
hand, the amount of ␣-amylase mRNA remained
high through 24 h of incubation in GA (Fig. 5). This
effect of GA and NO donors contrasts with that of
ABA, which inhibits the accumulation of ␣-amylase
mRNA in aleurone layers (Fig. 5).
NO Donors and Enzymes of ROS Metabolism
We showed previously that steady-state amounts
of mRNAs encoding the ROS metabolizing enzymes
catalase (CAT) and superoxide dismutase (SOD), as
well as CAT and SOD protein and enzyme activity,
are strongly correlated with GA-induced PCD (Fath
et al., 2001). GA treatment reduced the amounts of
these mRNAs and proteins and promoted cell death,
whereas ABA brought about an increase in their
abundance and prevented cell death (Fath et al.,
2001). We confirmed and extended these observations in this study to determine if NO donors prolonged the life of cells in GA-treated layers by increasing their capacity to metabolize ROS (Figs. 6 and
7). RNA blotting shows that the steady-state amount
Plant Physiol. Vol. 129, 2002
Figure 3. NO donors act in a concentration-dependent manner on
GA-treated layers, but do not affect O2 consumption by ABA-treated
barley aleurone layers. GA or ABA layers were incubated for 48 h
with SNP (A) or SNAP (B) at the concentrations shown, and relative
rates of O2 consumption were determined using an O2 electrode.
Data are means ⫾ SD of at least four independent layers.
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1645
Beligni et al.
the rate of PCD decreased (Fig. 8, A and C). In this
experiment, 76% of cells were dead after 36 h in GA
alone, and after 48 h in GA, almost all (91%) cells
were dead. In aleurone layers treated with GA and 1
mm BHT, only 25% of cells were dead at 36 h, and
more than 50% of cells remained alive after 48 h (Fig.
8C). As a positive control for these experiments, cell
viability of ABA-treated layers was determined, and
all cells were alive 48 h after ABA treatment in the
presence or absence of BHT (Fig. 8B). BHT did not
reduce the rate or extent of ␣-amylase secretion by
aleurone layers (Fig. 8D).
Detection of NO in Barley Aleurone Protoplasts with
Fluorescent Probes
Figure 4. NO donors increase the amount of ␣-amylase secreted by
GA-treated barley aleurone layers. ␣-Amylase activity in the medium
surrounding barley aleurone layers treated with ABA alone (5 ␮M; A
and B) or GA (5 ␮M) in the absence (A and B) or presence of 300 ␮M
SNAP (A) or 100 ␮M SNP (B) is shown at incubation times between
zero and 56 h. Data are means ⫾ SD for three flasks of layers.
The Antioxidant BHT Mimics the Effects of NO
Donors on Aleurone Cell Viability
NO has been shown to act synergistically with ROS
in plant and animal cells to promote cell death (Delledonne et al., 1998; Wendehenne et al., 2001), but NO
has also been shown to act as an antioxidant and to
prevent death (Beligni and Lamattina, 1999; Beligni
and Lamattina, 2001; Wendehenne et al., 2001). We
tested whether the antioxidant BHT could mimic the
effect of NO donors and slow down GA-induced
PCD in barley aleurone layers. When aleurone layers
were incubated in GA in the presence of 1 mm BHT,
We used NO-sensitive fluorescent probes to establish whether barley aleurone cells produce NO. A
method for detecting NO production in live cells was
developed by Heiduschka and Thanos (1998). This
method uses 1,2-diaminonanthraquinone (DAQ) as
an NO-sensitive fluorophore. Aleurone protoplasts
accumulate DAQ and become fluorescent when incubated with this probe (Fig. 9A). These observations
support the hypothesis that aleurone cells produce
NO. We used 1-aminonanthraquinone (1-AQ) as a
negative control for DAQ (Fig. 9B). 1-AQ lacks one of
the two amino groups that make up the NO-reactive
site in DAQ. Therefore, 1-AQ is a useful control to
assess the extent that enzymatic activities, independent of NO, might result in the production of a
fluorescent compound from DAQ. When aleurone
cells were incubated in 1-AQ, fluorescence from these
cells was much less intense than that from cells incubated in DAQ (Fig. 9, A and B), though still greater
than that from autofluorescence alone (Fig. 9C).
These data suggest that the conversion of DAQ from
a nonfluorescent to fluorescent molecule was largely
dependent on NO. There was no pronounced effect
of ABA or GA treatment on the intensity of fluorescence from aleurone protoplasts incubated in DAQ.
4,5-Diaminofluorescein diacetate (DAF-2 DA) is
another cell-permeable probe that becomes fluorescent when it reacts with NO (Nakatsubo et al., 1998),
and DAF-2 DA has been used previously to demon-
Figure 5. The NO donors SNP and SNAP do not inhibit the accumulation of ␣-amylase mRNA. Barley aleurone layers were
treated with GA alone, GA plus SNAP, GA plus SNP, or ABA alone, and total RNA was isolated at the times indicated. Northern
blots were probed with a cDNA for ␣-amylase. An rRNA probe was used as a loading control. Data are from one experiment
and are representative of two independent experiments.
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Plant Physiol. Vol. 129, 2002
Nitric Oxide Delays Aleurone Cell Death
Figure 6. The NO donor SNAP delays the GA-induced loss of CAT
mRNA and protein. The amount of barley Cat2 mRNA (A) was
determined by probing northern blots with a Cat2 cDNA probe. The
amount of CAT protein (B) was determined by probing protein blots
with an antibody against maize (Zea mays) CAT. Total RNA and
protein were extracted at the times indicated from barley aleurone
layers treated with GA, GA plus SNAP, or ABA. Data are from a
single experiment and are representative of at least two replicates.
strate NO production by plant cells (Foissner et al.,
2000; Pedroso and Durzan, 2000). Figure 9, D and G,
shows a field of aleurone protoplasts loaded with
DAF-2 DA and viewed by differential interference
contrast (Fig. 9G) or fluorescence microscopy (Fig.
9D). Damaged cells (Fig. 9, D and G, arrows) are
fluorescent, but undamaged cells are not. To see if
DAF-2 DA was reporting NO production by injured
cells that was not observed with DAQ, we used
4-aminofluorescein diacetate (4-AF DA) as a negative
control. Like 1-AQ, 4-AF DA lacks one of the amino
groups that make up the NO-reactive site in DAF-2
DA. In contrast to the low-intensity labeling seen
with 1-AQ, all aleurone protoplasts accumulated the
negative control 4-AF DA and showed bright fluorescence (Fig. 9E). These data indicate that DAF-2 DA
is not a suitable probe for monitoring NO production
in barley aleurone cells.
to protect the cell from oxidative stress. However,
unlike NO, BHT does not prolong the period of
␣-amylase secretion (compare Fig. 4 with Fig. 8D).
These data suggest that NO may play an additional
role in barley aleurone cells, perhaps as a signaling
molecule. Our fluorescence microscopy data indicate
that NO is synthesized by aleurone cells.
Cytotoxic and protective effects of NO in animals
and plants have been reported (Wink and Mitchell,
1998; Beligni and Lamattina, 2001). The simplest explanation for our data showing that NO donors delay
ROS-mediated PCD in barley aleurone layers (Figs. 1
and 2) is that NO acts as an antioxidant in this
system. Our results with barley aleurone layers are in
line with data showing, for example, that NO can
delay senescence (Mallick et al., 2000), prevent ROSinduced cytotoxicity (Beligni and Lamattina, 1999),
and slow chlorophyll loss resulting from pathogen
infection (Laxalt et al., 1997).
We have shown that ROS play a central role in
promoting PCD in barley aleurone cells (Bethke and
Jones, 2001; Fath et al., 2001), and ROS are likely
targets of NO and BHT in aleurone cells. Aleurone
cells store large amounts of triglycerides (approximately 25% of cell volume) and these are converted to
sugars through ␤-oxidation and the glyoxylate cycle.
The first step in ␤-oxidation is catalyzed by fatty acyl
coenzyme A, a flavin-containing enzyme that generates superoxide and hydrogen peroxide (Bethke and
Jones, 2001; Fath et al., 2001). Hence, glyoxysomes,
along with mitochondria, are likely to be significant
sites of ROS synthesis. We have proposed that peroxidation of membrane lipids and rupture of the plasma
membrane occurs when the rate of ROS production
exceeds the cell’s capacity for ROS metabolism. NO
has been shown to protect membranes and lipoproteins from oxidation directly, by interacting with lipid
peroxyl radicals, or indirectly, by inhibiting lipoxy-
DISCUSSION
In this paper, we show that NO delays the onset of
GA-induced PCD of barley aleurone cells. Whether
determined by vital staining with FDA and FM 4-64
(Fig. 1) or by monitoring net O2 consumption (Fig. 2),
NO delays PCD by 12 h or more in GA-treated layers
when supplied to aleurone cells using the NO donors
SNP or SNAP. The effects of SNP and SNAP are
specific for NO because the NO scavenger cPTIO
reverses the effects of NO donors on cell viability
(Fig. 1E) and because aleurone cells incubated with
SNP or SNAP remain fully responsive to GA, as
shown by the synthesis and secretion of ␣-amylase
(Fig. 4). NO’s effect on cell viability can be mimicked
by the antioxidant BHT, indicating that NO may act
Plant Physiol. Vol. 129, 2002
Figure 7. The NO donor SNAP delays the GA-induced loss of SOD
mRNA and protein. The amount of barley Sod4 mRNA (A) was
determined by probing northern blots with a maize Sod4 cDNA
probe. The amount of SOD protein (B) was determined by probing
protein blots with an antibody against maize SOD4. Total RNA and
protein were extracted at the times indicated from barley aleurone
layers treated with GA, GA plus SNAP, or ABA, and total protein was
also extracted from layers treated with GA plus SNP at the times
indicated. Data are from a single experiment and are representative
of two replicates.
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Beligni et al.
Figure 8. BHT delays GA-induced PCD in barley aleurone layers. Aleurone layers were incubated with GA alone or with
GA plus 1 mM BHT, and were then loaded with the fluorescent probes FDA and FM 4-64 at 24, 36, and 48 h. Epifluorescence
images (A) of live (green) and dead (red) cells were quantified as shown in C. ␣-Amylase secretion by layers from the same
experiment is quantified in D. Note that cells in layers treated with ABA alone or ABA plus BHT do not die (B).
genase activity (Patel et al., 2000). At present, our data
do not allow us to specify the exact mechanism by
which NO exerts its protective effect.
Aleurone cells contain a suite of enzymes that metabolize ROS. The synthesis of these enzymes is tightly
regulated by ABA and GA (Figs. 6 and 7, and Fath et
al., 2001). Freshly isolated aleurone layers and those
incubated in ABA have high amounts of mRNAs encoding CAT and SOD, and corresponding high
amount of these proteins. These mRNAs and proteins
decline in GA-treated layers (Figs. 6 and 7, and Fath et
al., 2001), and there is a strong correlation between the
amounts of CAT and SOD mRNA, protein, and enzyme activity and the effects of GA on PCD (Figs. 1
and 2, and Fath et al., 2001). CAT and SOD proteins
decline in amount less rapidly in tissue incubated in
GA plus NO donors than in tissue incubated in GA
alone (Figs. 6 and 7). For CAT at least, this delayed
loss of protein is not a consequence of delayed cell
death. CAT loss occurs at a time when all cells in
layers treated with GA alone or GA plus NO donors
are still alive. An increased ability to metabolize ROS
may contribute somewhat to the extended viability of
cells treated with NO donors by further reducing the
degree of oxidative stress. It is interesting that Yamasaki et al. (2001) reported that NO inhibits cytochrome c oxidase and oxidative phosphorylation in
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mitochondria isolated from mung bean (Vigna radiata),
but that the alternative oxidase pathway is resistant to
NO. A greater dependence on the alternative oxidase
pathway in the presence of NO may lower the
respiration-dependent production of ROS in mitochondria, and thus may be protective by reducing the
overall rate of ROS synthesis (Yamasaki et al., 2001).
We measured the production of NO in aleurone
cells using two NO-sensitive probes, DAQ (Heiduschka and Thanos, 1998) and DAF-2 DA (Nakatsubo et al., 1998 and Fig. 9). Although others have
used DAF-2 DA to monitor NO production in plant
cells, DAF-2 DA proved unsuitable for this purpose
in barley aleurone cells, even though we could detect
a fluorescent product when aleurone protoplasts
were incubated with the probe. Increases in fluorescence from DAQ were more specific for NO than
those seen with DAF-2 DA because the signal from
protoplasts incubated in the NO-insensitive probe
1-AQ was much less intense than that from protoplasts incubated in DAQ. Our data suggest that DAQ
is interacting with endogenous NO, and they support
the hypothesis that aleurone cells synthesize NO. We
did not observe a difference in the intensity of the
fluorescent signal between GA- and ABA-treated
cells, but these data do not allow us to conclude that
NO production is equivalent regardless of hormone
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Copyright © 2002 American Society of Plant Biologists. All rights reserved.
Plant Physiol. Vol. 129, 2002
Nitric Oxide Delays Aleurone Cell Death
ported (Ferrari and Varner, 1970). Using mass spectroscopy as an independent measurement technique,
we have confirmed that barley aleurone layers synthesize NO from NO2⫺ (P. Bethke, M. Badger, and R.
Jones, unpublished data).
MATERIALS AND METHODS
Plant Material and Chemicals
Aleurone layers were prepared from deembryonated barley (Hordeum
vulgare cv Himalaya, 1991 harvest, Washington State University, Pullman)
grains as described previously (Fath et al., 2001). In brief, the embryo and
distal end of the grain were removed, and the resulting half-grains were
surface sterilized and imbibed in water for 4 d. Aleurone layers were
isolated by removing the starchy endosperm and were incubated in a
medium containing 20 mm CaCl2 and 5 ␮m GA or 5 ␮m ABA. Incubation
was performed in the absence or presence of an NO donor: SNAP (Molecular Probes, Eugene, OR) at concentrations ranging from 1 ␮m to 2 mm, or
SNP (Merck, Darmstadt, Germany) at concentration ranging from 1 ␮m to 1
mm. The potassium salt of the NO scavenger cPTIO (Molecular Probes) was
used as a control for NO action. BHT (Sigma, St. Louis) was added to freshly
isolated aleurone layers to a final concentration of 1 mm in the presence or
absence of 5 ␮m ABA or 5 ␮m GA in 20 mm CaCl2, and layers were
incubated for the indicated time.
O2 Consumption
Figure 9. Fluorescent probes report the presence of NO in barley
aleurone protoplasts. Epifluorescence (A–F) and differential interference contrast (G–I) images of barley aleurone protoplasts incubated
with DAQ (A), 1-AQ (B), DAF-2 DA (D and G), 1-AF DA (E and H),
or no probe (C, F, and I). Arrows point to damaged cells.
treatment. Fluorescence from an NO-reactive probe
depends on the rate of NO production and on the
rate that NO reacts with molecules other than the
probe. ABA-treated cells have a much higher capacity for the metabolism of ROS than GA-treated cells.
If endogenous molecules that react with NO are also
more abundant in ABA-treated cells than in GAtreated cells, their interaction with NO would lessen
the intensity of fluorescence from the cell. As a consequence, fluorescence intensity would underreport
the extent of NO production in ABA-treated cells
relative to GA-treated cells.
Although we have not explored the possibility that
aleurone cells synthesize NO via a NOS, we hypothesize that NO is likely to be synthesized enzymatically
via NR or nonenzymatically in the apoplast (Yamasaki, 2000). NR is known to generate NO in plants,
and this enzyme is present in barley aleurone layers
and can be induced by nitrate (Ferrari and Varner,
1970). The apoplast may also be a source of NO in
aleurone layers. NO can by synthesized from NO2⫺ at
an acidic pH in the presence of a reductant (Yamasaki,
2000). We have shown that aleurone layers release
reduced ascorbate into the apoplast (J. Sung, P. Bethke, and R. Jones, unpublished data) and that apoplastic pH is 3 to 4 (Drozdowicz and Jones, 1995).
Therefore, nitrate released into the apoplast could be
converted rapidly into NO, and GA-dependent release
of NO2⫺ from barley aleurone layers has been rePlant Physiol. Vol. 129, 2002
O2 consumption was measured using an O2-sensitive electrode (model
10, Digital Oxygen System; Rank Brothers, Cambridge, UK). Layers were
transferred to a measuring chamber containing 3 mL of sterile distilled
water at least 20 min prior to determining the rate of O2 consumption. The
data presented are means from at least four aleurone layers per treatment.
Cell Viability and Death
The number of live and dead cells was determined by double staining
with the fluorescent probes FDA and FM 4-64 (Molecular Probes; Bethke
and Jones, 2001). Aleurone layers were incubated in FDA (2 ␮g mL⫺1 in 20
mm CaCl2) for 30 min, briefly rinsed with 20 mm CaCl2, and then incubated
in FM 4-64 (1 ␮g mL⫺1 in 20 mm CaCl2) for 3 min. Layers were briefly
washed with 20 mm CaCl2 and were mounted on microscope slides. Aleurone cells were observed with a fluorescent microscope (Axiophot; Zeiss,
Thornwood, NJ) using a 20⫻ objective. Images of the fluorescent signal were
captured using a digital camera. Randomly selected fields from at least three
different aleurone layers per treatment were counted to determine the
percentage of live cells.
Enzyme Assays
␣-Amylase activity secreted into the medium by aleurone layers was
assayed as described by Bush et al. (1986).
RNA Isolation and Northern Blotting
Aleurone layers (10–15) were ground to powder with liquid N2. Total
RNA was isolated using the plant RNeasy kit (Qiagen, Valencia, CA)
according to the manufacturer’s instructions. RNAs (5 ␮g lane⫺1) were
separated on denaturing 1.2% (w/v) agarose gels and then blotted onto a
nylon membrane (Hybond-N; Amersham Biosciences, Piscataway, NJ).
␣-Amylase, Cat2, and Sod4 cDNA probes were labeled with [32P]dCTP (ICN
Biomedicals, Costa Mesa, CA) using the Prime-it RmT random prime labeling kit (Stratagene, La Jolla, CA). Membranes were hybridized at 65°C in 7%
(w/v) SDS, 0.5 m Na2PO4 pH 7.2, 1 mm EDTA, and 0.1 mg mL⫺1 sonicated
herring sperm DNA. Blots were washed at 65°C in 2⫻ SSC and 1% (w/v)
SDS for 15 min, then in 0.5⫻ SSC and 1% (w/v) SDS for 20 min, and finally
in 0.1⫻ SSC and 1% (w/v) SDS for 10 min. The blots were reprobed with an
rRNA probe to verify equal loading. The amount of 32P-labeled cDNA probe
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Copyright © 2002 American Society of Plant Biologists. All rights reserved.
1649
Beligni et al.
hybridizing to specific mRNAs was quantified by laser densitometry using
a PhosphorImager (Molecular Dynamics, Sunnyvale, CA).
Protein Isolation and Immunoblotting
Aleurone layers (11) were ground to a fine powder in liquid N2 and were
extracted in 300 ␮L of buffer (0.1 m Tris-HCl, pH 7.5, 0.5 m NaCl, 50 mm
EDTA, 20 ␮m leupeptin, 20 ␮m pepstatin A, and 25 ␮m E64) at 4°C. The
homogenates were centrifuged at 4,6000 rpm in a table-top centrifuge for 15
min at 4°C, and equal-volume aliquots of the supernatants were separated
by SDS-PAGE (12.5%, w/v). After electrophoresis, proteins were transferred
to nitrocellulose membranes (Schleicher & Schuell, Keene, NH). Protein
blots were blocked with phosphate-buffered saline containing 3% (w/v)
skim milk powder, and antibodies were incubated overnight in the same
medium. Secondary antibodies (goat anti-rabbit immunoglobulin Gs) coupled to horseradish peroxidase (Sigma) were incubated in phosphatebuffered saline for 1 h and visualized chromogenically.
Detection of NO Using Fluorescent Probes
Barley aleurone protoplasts were prepared as described previously
(Bethke and Jones, 2001). GA- or ABA-treated protoplasts were washed at
least twice with fresh incubation medium, incubated with or without probe,
and examined using epifluorescence microscopy. The concentration of the
probe stock solution (in dimethyl sulfoxide), the dilution used, and the
incubation times were: DAQ (Calbiochem, San Diego) 10 mm, 1:500, 10 to 60
min; 1-AQ (Aldrich, Milwaukee, WI), 10 mm, 1:500, 10 to 60 min; DAF-2 DA
(Calbiochem), 4 mm, 1:500, 1 h; and 4-aminofluorescein diacetate (Calbiochem), 4 mm, 1:500, 1 h.
ACKNOWLEDGMENTS
We thank Dr. Ron Skadsen (U.S. Department of Agriculture-Agricultural
Research Service, Cereal Crops Research Unit, Madison, WI) for providing
the barley Cat2 cDNA and Dr. John Scandalios (North Carolina State University, Raleigh) for the gift of the maize Sod4 cDNA and the anti-maize
SOD4 and anti-maize-CAT polyclonal antibodies.
Received December 24, 2001; returned for revision February 19, 2002; accepted April 30, 2002.
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