A Mitogen-Activated Protein Kinase Signals to Programmed
Cell Death Induced by Self-Incompatibility in
Papaver Pollen1[OA]
Shutian Li2, Jozef Šamaj, and Vernonica E. Franklin-Tong*
School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom
(S.L., V.E.F.-T.); Institute of Cellular and Molecular Botany, University of Bonn, 53115 Bonn,
Germany (J.Š.); and Institute of Plant Genetics and Biotechnology, Slovak Academy of Sciences,
950 07 Nitra, Slovak Republic (J.Š.)
Self-incompatibility (SI) in higher plants is an important mechanism to prevent inbreeding and involves specific rejection of
incompatible (‘‘self’’) pollen. In field poppy (Papaver rhoeas), S proteins encoded by the stigma component of the S-locus interact
with incompatible pollen, resulting in cessation of tip growth. This ‘‘self’’ interaction triggers a Ca21-dependent signaling
network, involving programmed cell death (PCD). We previously identified p56, a mitogen-activated protein kinase (MAPK) that
is activated during the SI response in incompatible pollen. Here, we show that p56 cross-reacts with AtMPK3, but not with
AtMPK4 or salicylic acid-induced protein kinase antibodies. We provide good evidence that a MAPK is involved in initiation of
SI-induced PCD in incompatible pollen. SI rapidly reduces pollen viability and the MAPK cascade inhibitor U0126, which
prevents the SI-induced activation of p56 in incompatible pollen, ‘‘rescues’’ incompatible pollen, while its negative analog, U0124,
does not. This strongly implicates the involvement of a MAPK in SI-mediated loss of pollen viability and cell death. SI also
stimulates caspase-3-like (DEVDase) activity and later DNA fragmentation. Both these markers of PCD are significantly reduced
by pretreatment with U0126, implicating the involvement of a MAPK in signaling during early PCD. As p56 appears to be the only
MAPK activated by SI, our studies imply that p56 could be the MAPK involved in mediating SI-induced PCD.
Alterations to the phosphorylation state of proteins
via activation of protein kinases are some of the most
important molecular mechanisms whereby cells respond to extracellular signals. Mitogen-activated protein kinases (MAPKs) are highly conserved across
eukaryotes. They are Ser/Thr kinases that are activated by dual phosphorylation of Thr and Tyr residues
in a TXY motif, via specific MAPK kinases. Activated
MAPKs trigger diverse signaling cascades in response
to a variety of signals and stimuli, and are thought to
act as a universal signal transduction mechanism, regulating important cellular processes including gene
expression, cell proliferation, cell survival, and death
in eukaryotic cells (Chang and Karin, 2001). In plants,
MAPK cascades have emerged as key players in some
1
This work was supported by The Royal Society, the Biotechnology and Biological Sciences Research Council, and the Deutsche
Forschungsgemeinschaft (grant no. SA 1564/2–1 to J.Š.).
2
Present address: Department of Molecular Plant Genetics, Max
Planck Institute for Plant Breeding Research, Carl-von-Linné-Weg
10, 50829 Cologne, Germany.
* Corresponding author; e-mail [email protected].
The author responsible for distribution of materials integral to
the findings presented in this article in accordance with the policy
described in the Instructions for Authors (www.plantphysiol.org) is:
Vernonica E. Franklin-Tong ([email protected]).
[OA]
Open Access articles can be viewed online without a subscription.
www.plantphysiol.org/cgi/doi/10.1104/pp.107.101741
236
of the most essential roles in plant signaling networks.
In Arabidopsis (Arabidopsis thaliana), 20 MAPK genes
have been identified (Ichimura et al., 2002). Plant MAPK
genes belong to the extracellular signal-regulated kinase (ERK) subfamily. They have been shown to be
activated by a variety of stresses, including pathogen
infection, wounding, drought, salinity, osmolarity, UV
irradiation, ozone, and reactive oxygen species (Tena
et al., 2001; Zhang and Klessig, 2001). For example,
Arabidopsis AtMPK3 is involved in osmotic and oxidative stress, as well as in abscisic acid and pathogen
elicitor signaling (Nakagami et al., 2005).
Experimental evidence, including gain-of-function
studies, has demonstrated the involvement of MAPKs
in activation of defense responses, resulting in programmed cell death (PCD; Ligterink et al., 1997; Yang
et al., 2001; Kroj et al., 2003) and resistance to pathogens (Zhang and Klessig, 2001; Asai et al., 2002). A
complete plant MAPK cascade that is involved in
resistance to a wide range of pathogens has recently
been identified (Asai et al., 2002).
Apoptosis or PCD is a highly conserved process that
is used to remove unwanted cells in eukaryotes. An
initiation phase involves signaling cascades that prepare the cell for entry into the execution phase. In
animal cells, caspases are key proteases that are activated rapidly during apoptosis and cleave target
proteins after an Asp residue (Riedl and Shi, 2004).
Caspase-3 is the main executioner protease and is
responsible for cleavage of many cellular proteins
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A MAPK Signals to SI-Induced PCD
(Fischer et al., 2003). Various tetrapeptide inhibitors of
caspases have aided the study of caspase-dependent
apoptosis. Caspase-3 has the recognition sequence
DxxD, and the caspase-3 inhibitor DEVD is based on
this. Inhibition of protease activity using DEVD implicates the involvement of a caspase-3-like activity.
Caspases also cleave endogenous nuclease inhibitors,
which allows activation of nucleases and leads to the
fragmentation of nuclear DNA, which is a common
marker for apoptosis and PCD.
PCD in plants is less studied compared with animals. However, PCD is well established to be triggered
by biotic and abiotic stresses (Zhang and Klessig,
2001), as well as during development (Kuriyama and
Fukuda, 2002). Although release of mitochondrial
cytochrome c has been identified in plants (Thomas
and Franklin-Tong, 2004; Yao et al., 2004) and biochemical evidence for caspase activation in plants during PCD is good (van Doorn and Woltering, 2005),
many of the genes encoding components of the apoptotic machinery, including caspases, have either not
yet been identified in plants or are simply not present
(Woltering et al., 2002). Nevertheless, there is no doubt
that PCD occurs in plants.
Self-incompatibility (SI) in Papaver involves interaction of pistil S-locus determinants (S proteins) with a
pollen receptor. An incompatible (‘‘self’’) interaction
triggers rapid, SI-specific increases in cytosolic free Ca21
([Ca 21]i; Franklin-Tong et al., 1993, 1997) and depolymerization of the F-actin cytoskeleton (Geitmann et al.,
2000; Snowman et al., 2002), resulting in rapid arrest of
incompatible pollen tube growth. The SI signaling cascade also triggers PCD involving a caspase-3-like activity (Thomas and Franklin-Tong, 2004), resulting in the
specific destruction of ‘‘self’’ pollen. We recently established that changes in actin filament levels or dynamics
play a functional role in initiating PCD in Papaver pollen,
triggering a caspase-3-like activity (Thomas et al., 2006),
providing evidence of a specific causal link between
actin polymerization status and PCD in pollen. In
incompatible pollen, SI also stimulates Ca21-dependent
hyperphosphorylation of Pr-p26.1a/b, two soluble inorganic pyrophosphatases (sPPases), which reduces
their sPPase activity (de Graaf et al., 2006). Since
sPPases are important for biosynthesis, this provides
a further mechanism for incompatible pollen inhibition.
Enhanced activation of a MAPK specifically in incompatible pollen originally identified p56 as being implicated in the SI-induced signaling cascade in Papaver
pollen (Rudd et al., 2003). Convincing evidence that
p56 is a MAPK included: myelin basic protein (MBP)
in-gel kinase assays; immunoprecipitation with antiphosphotyrosine antisera; immunoprecipitated proteins
exhibiting MBP in-gel kinase activity; and sensitivity
to the MAPK inhibitor apigenin (Rudd et al., 2003).
However, the timing of its activation, peaking at 10
min after SI induction, which is after initial arrest of
pollen tube inhibition, suggested that it might be involved in later events after initial arrest of pollen tube
growth, which implicated a possible role in signaling
to PCD. As MAPKs are known to be functionally
involved in regulating PCD in plants (Ligterink et al.,
1997; Yang et al., 2001; Zhang and Klessig, 2001; Ren
et al., 2002), we investigated whether p56 might be
involved in signaling to PCD in incompatible pollen.
Here, we provide data indicating that a MAPK signals
to PCD in incompatible pollen. This contributes to our
understanding of the mechanisms involved in mediating SI and integration of the SI-induced PCD signaling network.
RESULTS
The p56 MAPK Is Activated in SI in an S-Specific Manner
and Cross-Reacts with AtMPK3
Plant MAPKs have high homology to mammalian
ERK1/2 MAPKs, and ERK1/2 antisera that recognize
the dually phosphorylated forms of activated MAPKs
can be used to monitor plant MAPK activity (Samuel
et al., 2000; Lee et al., 2001; Kroj et al., 2003). We
therefore used an anti-active MAPK pTEpY antibody
(Cell Signaling Technology) that specifically recognizes activated MAPKs to confirm that p56 was phosphorylated on a pTEpY motif and was activated
specifically in incompatible interactions when SI was
induced. Figure 1A shows that p56 is activated during
SI in incompatible, but not compatible or unchallenged Papaver pollen. Quantitation revealed that p56
activity was increased 7.7- 6 0.88-fold (n 5 5) 10 min
after SI challenge with incompatible S proteins, while
compatible and biologically inactive S proteins had
only 1.1- 6 0.09-fold and 1.2- 6 0.11-fold changes (n 5
4) in MAPK activity, respectively. In addition to p56
activation, Figure 1A reveals that two other MAPKs
are activated in hydrated and growing Papaver pollen.
We have ascertained that there are at least three other
MAPKs in addition to p56 in Papaver pollen, as we
have cloned three MAPKs from Papaver pollen. Unfortunately, none of the cDNAs corresponds to p56
(K. Osman, F.C.H. Franklin, and V.E. Franklin-Tong,
unpublished data; EMBL accessions AJ784995,
AJ784996, and AJ784997). Two of these MAPKs can
be seen to be activated in growing pollen (Fig. 1A), but
their activity is clearly not stimulated by SI. The third
MAPK, which we know to be present in Papaver pollen
as we have obtained the cDNA, is apparently not
activated in hydrated or normally growing pollen (Fig.
1A); we speculate that it may be activated, like many
other MAPKs, during stress responses.
We also used antisera raised against known plant
MAPKs in an attempt to provide further evidence that
p56 is a MAPK, especially as it migrates as a rather high
molecular mass band for a MAPK on SDS-PAGE.
A peptide antibody, raised against the specific C-terminal
part of AtMPK3 (which is not found in the other 19
known MAPKs in Arabidopsis), successfully identified p56. This antibody against AtMPK3 cross-reacts
Plant Physiol. Vol. 145, 2007
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Li et al.
Figure 1. Evidence that p56 is a MAPK. A, SI stimulates p56 activity in
an S-specific manner. SI-induced incompatible pollen protein extracts
(Inc) display p56 activity (black arrow), measured using a pTEpY
antibody. Controls comprising pollen mock-challenged with GM,
compatible S proteins (Comp), and heat-denatured incompatible S
proteins (HD Inc) do not. Two other MAPK activities are detected in
controls (white arrows), but their activity is not SI activated. B, p56
cross-reacts with AtMPK3 antibody. Pollen protein extracts from control
(GM) and calyculin-treated (CA) pollen contain a protein corresponding to p56 (black arrowhead) that cross-reacts strongly with AtMPK3
antibody. C, p56 does not cross-react with SIPK antibody. Pollen protein
extracts, GM and Inc as described, contain a protein migrating to
approximately 48 kD that cross-reacts with SIPK antisera (white arrowhead). This does not correspond to p56. D, p56 does not cross-react
with AtMPK4 antibody. Pollen protein extracts, GM and CA as
described, contain a protein migrating to approximately 43 kD that
cross-reacts with AtMPK4 antibody (white arrowhead) but does not
correspond to p56.
with a protein that migrates to the same position as
p56 (Fig. 1B), thus further confirming that p56 is
indeed a MAPK. This recognition of p56 by antiAtMPK3 antibody in poppy (Papaver rhoeas) extracts is
specific, as we have found that p56 does not cross-react
with tobacco (Nicotiana tabacum) salicylic acid-induced
protein kinase (SIPK) antibody (Fig. 1C); SIPK is an
ortholog of AtMPK6. p56 also is not recognized by a
peptide antibody that specifically recognizes AtMPK4,
which is partially homologous to AtMPK3 (Fig. 1D).
AtMPK3, AtMPK4, and SIPK recognize MAPKs regardless of phosphorylation state (indicated by use of
calyculin, which prevents dephosphorylation). Thus,
p56 cross-reacts with the pTEpY antibody when activated by SI, and also specifically with AtMPK3. These
data, together with previously published evidence
(Rudd et al., 2003), provide good evidence that p56 is
an authentic MAPK.
Furthermore, SI stimulates only one MAPK activity
in pollen that is detected by MBP in-gel kinase assays
(Rudd et al., 2003) and the TEY antibody here, implicating that this MAPK plays a role in signaling to
SI-induced events. Thus, p56 is a candidate for the
involvement in SI signaling networks in incompatible
pollen.
The MAPK Inhibitor U0126 Inhibits Pollen Tube Growth
and p56 Activity But Does Not Affect Pollen Viability
To establish whether a MAPK activity was involved
in triggering SI-induced PCD, we used an inhibitor of
MAPK cascades, U0126. This drug is a potent and
specific inhibitor and has been shown to block the
MAPK cascades in cell-based assays in both animal
and plant cells (Lee et al., 2001). In tobacco, elicitor activation of SIPK was inhibited by 100 mM U0126 (Lee
et al., 2001); MAPK phosphorylation in response to
lipopolysaccharides was also inhibited by U0126 (Piater
et al., 2004). However, while the use of this drug can
serve as useful preliminary data suggestive of MAPK
involvement, we cannot rule out the possibility that
U0126 may have other targets.
We examined whether U0126 inhibited p56 activity
using anti-phospho-MAPK immunoblotting. We pretreated pollen with 100 mM of either U0126 or its negative analog, U0124, and then induced SI. The p56
activity was stimulated by SI and strongly inhibited by
U0126, while the U0124 pretreatment did not affect
p56 activity (Fig. 2). Although p56 was not obviously
activated in normally growing pollen tubes, we found
that U0126 inhibited normal pollen tube growth, while
U0124, which is a negative analog of U0126 (Favata
et al., 1998), did not (Fig. 3). These data (assuming that
the specificity of U0126 is as expected) suggest that
MAPK activity is required for normal pollen tube
growth, which to our knowledge has not previously
been demonstrated. We have identified three other
MAPK sequences in Papaver pollen (K. Osman, F.C.H.
Franklin, and V.E. Franklin-Tong, unpublished data)
that do not correspond to p56; two of these MAPKs are
activated in hydrated and normally growing pollen,
but are not specifically activated by SI (Fig. 1A). This
suggests that these MAPKs may play a functional role
in signaling to pollen tube growth. Examination of the
viability of pollen treated with U0126 and U0124,
using fluorescein diacetate (FDA), revealed that viabilities for untreated pollen and pollen treated with
U0124 or U0126 for 8 h were 95.3% 6 0.88%, 94.0% 6
2.65% (P 5 0.830, N.S., n 5 3), and 94.7% 6 1.2% (P 5
0.658, N.S., n 5 3), respectively (Table I). These data
established that U0126 does not affect pollen viability
and is a suitable inhibitor to investigate the effects of
inhibiting p56 activity.
Figure 2. The MAPK inhibitor U0126 inhibits p56 activity. p56 activity
was detected using a pTEpY antibody. No p56 activity was detected in
controls: GM, DMSO, U0124, or U0126 treatment alone. Activity was
detected in incompatible SI-induced samples (Inc) and SI-induced
samples pretreated with DMSO (Inc/DMSO). The p56 activity was
inhibited in extracts from SI-induced pollen pretreated with 100 mM
U0126 (Inc/U0126), but not inhibited in extracts from pollen pretreated
with 100 mM U0124 (Inc/U0124). p56 activity was not detected in
compatible SI-induced samples (Comp).
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A MAPK Signals to SI-Induced PCD
U0124 had low levels of DNA fragmentation (13.25% 6
1.2% and 7.18% 6 0.82%, respectively, n 5 3), with no
significant difference in levels of DNA fragmentation
compared to untreated controls (P 5 0.102 and 0.054,
respectively). SI induced high levels of DNA fragmentation in incompatible pollen tubes (76.42% 6 2.49%,
n 5 6; Fig. 4). Importantly, pollen tubes pretreated with
U0126 and then SI induced had reduced levels of DNA
fragmentation (41.64% 6 0.82%, n 5 3), significantly
different from SI induced alone (P , 0.001, ***). In
contrast, the negative analog U0124 did not inhibit the
SI-induced DNA fragmentation, and levels were
75.91% 6 3.71% and not significantly different from
SI treatments alone (Fig. 4; P 5 0.915, N.S.). These data
indicate that inhibiting MAPK activation prevents
DNA fragmentation.
Since U0126 prevented both p56 activation and DNA
fragmentation normally induced by SI, our data implicate the functional involvement of a MAPK in
initiating the signaling cascade leading to SI-induced
DNA fragmentation. Assuming that the specificity of
U0126 is as expected, our data implicate MAPK signaling involvement in SI-mediated PCD. Although
U0126 will not target p56 activity specifically, as p56
appears to be the only MAPK activated by SI, our
studies imply that p56 could be the MAPK involved in
mediating SI-induced PCD.
Figure 3. The MAPK inhibitor U0126 inhibits pollen tube growth.
Pollen tube lengths in untreated pollen tubes (white bar), those treated
with 100 mM U0126 (black bar), and those treated with 100 mM U0124
(shaded bar), after 1 h, are presented. Growth was inhibited by U0126
but not by U0124. Data are means 6 SE of three independent experiments. One hundred pollen tubes were measured for each experiment.
The MAPK Inhibitor U0126 Inhibits DNA Fragmentation
To investigate if p56 was involved in signaling to
SI-induced PCD, we used one of the hallmark features
of PCD, DNA fragmentation. We previously demonstrated that this is triggered by SI in an S-specific
manner and involves a caspase-3-like (DEVDase) activity (Thomas and Franklin-Tong, 2004). We pretreated
growing pollen with 100 mM U0126 or U0124 for 1 h
and then induced SI, and subsequently measured
DNA fragmentation after 8 h as a marker for PCD
using TUNEL labeling (Fig. 4). In cells that had undergone PCD, TUNEL labeling was detected coincident with nuclear DNA. Levels of DNA fragmentation
in untreated and control pollen tubes were low (9.99% 6
0.64%, n 5 6), as were levels in pollen tubes that had
undergone a compatible SI interaction (7.52% 6 0.68%,
n 5 3). Pollen tubes treated with 100 mM U0126 or
SI Stimulates Loss of Viability of Pollen Tubes and
Pretreatment with U0126 ‘‘Rescues’’ Pollen Tube Viability
after SI Challenge
We also examined viability of pollen tubes during
these treatments, using FDA staining (Heslop-Harrison
and Heslop-Harrison, 1970; Heslop-Harrison et al.,
1984; Table I). Untreated pollen, those treated with the
drugs, and SI-challenged compatible pollen tubes
retained their viability over the 8-h experimental period. However, growth was inhibited by 100 mM
U0126, suggesting that MAPKs may be involved in
regulating pollen tube growth. There was no significant difference in viability between untreated pollen
Table I. SI stimulates loss of viability of pollen tubes and pretreatment with U0126 ‘‘rescues’’ pollen
tube viability after SI
Pollen tube viability was measured using FDA at different time intervals (h) for different treatments. Inc SI,
Incompatible pollen; Comp SI, compatible pollen; Inc SI 1 U0126, pollen pretreated with 100 mM U0126
prior to SI challenge; Inc SI 1 U0124, pollen pretreated with 100 mM U0124 prior to SI challenge. Controls
comprised untreated pollen (UT) and pollen treated with drugs alone (U0126, U0124). One hundred
pollen tubes were counted for each treatment and percentage viability determined from a total of three
independent experiments.
Treatment
UT
U0126
U0124
Comp SI
Inc SI
Inc SI 1 U0126
Inc SI 1 U0124
0h
98.33
98.67
98.0
98.0
98.0
98.67
98.0
6
6
6
6
6
6
6
1h
0.88
0.33
0.58
1.15
1.0
0.33
1.0
97.33
97.67
98.0
97.0
58.33
97.67
55.0
6
6
6
6
6
6
6
3h
1.76
0.33
0.58
1.73
2.03
0.58
2.08
95.00
97.33
95.67
96.33
18.33
97.33
20.33
6
6
6
6
6
6
6
8h
2.65
0.67
0.33
1.45
0.88
0.67
0.33
94.0 6 2.65
94.67 6 1.2
95.33 6 0.88
93.33 6 2.68
060
94.67 6 2.08
060
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Li et al.
Figure 4. The MAPK inhibitor U0126 inhibits DNA fragmentation.
SI-induced DNA fragmentation, measured using TUNEL after 8 h, was
low in controls (all shown as white bars): untreated (UT) pollen, pollen
treated with 100 mM U0126 and U0124 alone, and compatible SIinduced samples (Comp). Incompatible SI-induced samples (Inc; black
bar) had high levels of DNA fragmentation induced, which was alleviated by pretreatment with 100 mM U0126 (Inc-U0126; dark shaded bar)
but not alleviated by pretreatment with 100 mM U0124 (Inc-U0124;
speckled bar). For UT and SI treatments, 50 pollen tubes were scored in
each of six independent experiments; for all other treatments, 50 pollen
tubes were scored in each of three independent experiments.
tubes and pollen tubes treated with U0126 and U0124
at 8 h (P 5 0.830 and P 5 0.658, respectively). In
contrast, SI-challenged incompatible pollen tubes
showed significant loss of viability within 1 h (P ,
0.001, ***) and had completely lost viability by 8 h.
Notably, pretreatment with U0126 prior to SI induction
prevented loss of viability; there was a highly significant difference between SI-induced samples and
those pretreated with U0126 (P , 0.001, ***) at 1 and
3 h, while pretreatment with U0124 did not have an
effect on SI-induced death (P 5 0.315 [N.S.] and 0.101
[N.S.], respectively; Table I). Thus, pretreatment with
U0126 and prevention of p56 activation prior to SI
‘‘rescues’’ incompatible pollen from death. Together
with the DNA fragmentation data, this clearly implicates the involvement of MAPK activation in playing a
functional role in mediating PCD.
showed that incompatible SI-induced pollen extracts
cleave PARP, producing a 24-kD PARP cleavage fragment, which was prevented by addition of the caspase-3
tetrapeptide inhibitor Ac-DEVD-CHO (Thomas and
Franklin-Tong, 2004). To establish if a MAPK was implicated in signaling to caspase-3 activation, we examined if inhibition of MAPK activation by U0126
prevented the PARP cleavage activity exhibited by
SI-induced pollen. SI-induced incompatible pollen
protein extracts have a caspase-3-like activity that
cleaves bovine PARP in vitro (Thomas and FranklinTong, 2004; Fig. 5). A doublet at approximately 24 kD
was consistently detected only in PARP samples incubated with incompatible SI-induced pollen extracts
(n 5 3). The detection of a PARP cleavage fragment,
coupled with a corresponding decrease in the amount
of uncleaved PARP at approximately 116 kD, provides
compelling evidence for a SI-induced PARP cleavage
activity. The S specificity of this PARP cleavage activity
was demonstrated because SI-induced compatible pollen, untreated pollen, or pollen treated with the U0124
and U0126 drugs alone did not show a disappearance
of the 116-kD intact PARP fragment or the appearance
of a 24-kD PARP fragment (Fig. 5). Notably, in the samples pretreated with U0126 prior to SI induction, the
116-kD intact PARP fragment was still present and the
24-kD SI-specific doublet was much less intense in
the U0126-pretreated samples (Fig. 5). The samples
pretreated with U0124 prior to SI induction were indistinguishable from those without pretreatment, and
SI-Mediated Poly (ADP-Rib) Polymerase Cleavage Is
Inhibited by U0126
SI-induced PCD in Papaver involves a caspase-3-like
(DEVDase-like) activity (Thomas and Franklin-Tong,
2004). We previously demonstrated that SI-induced
pollen protein extracts exhibited a caspase-3-like cleavage activity in vitro, using bovine poly (ADP-Rib)
polymerase (PARP) as a substrate. The nuclear DNA
repair protein PARP is a classic substrate for caspase-3
activity in animal cells (Lazebnik et al., 1994), and its
cleavage is often used as evidence for apoptosis. We
Figure 5. SI-mediated PARP cleavage is inhibited by U0126. Uncleaved
PARP (PARP) comprises a major band at 116 kD (white arrow). Extracts
from untreated pollen (UT), compatible SI-induced pollen (Comp), and
pollen treated with 100 mM drugs alone (U0124, U0126) show nonspecific proteolysis of PARP. Extracts from incompatible SI-induced pollen
(Inc) showed an S-specific cleavage pattern: disappearance of the 116kD band (white arrow) and appearance of a doublet at 24 kD (double
arrow). Pretreatment with 100 mM U0126 before SI induction (IncU0126) retained the 116-kD band and had reduced levels of the 24-kD
bands; pretreatment with 100 mM U0124 had no effect.
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A MAPK Signals to SI-Induced PCD
showed loss of the 116-kD intact PARP fragment and a
strong 24-kD SI-specific doublet (Fig. 5). The alleviation of SI-mediated PARP cleavage by U0126, but not
U0124, suggests that inhibiting MAPK activity prevents
activation of caspase-3-like activity. These data implicate MAPK signaling to PCD in incompatible pollen.
The SI-Induced Caspase-3-Like (DEVDase) Activity Is
Inhibited by Pretreatment with U0126
We also used the fluorescent caspase-3 substrate AcDEVD-AMC to measure the caspase-3-like (DEVDase)
activity generated by SI and to test whether U0126
inhibited this activity. Extracts from untreated pollen,
compatible SI-treated pollen, and those treated with
U0126 and U0124 alone gave low levels of DEVDase
activity (Fig. 6). SI induced high levels of DEVDase
activity. The tetrapeptide Ac-DEVD-CHO (DEVD), a
caspase-3 inhibitor, was used to provide evidence for
a caspase-3 activity in apoptotic animal cells and in
plant cells undergoing PCD (Garcia-Calvo et al., 1998;
Richael et al., 2001; Danon et al., 2004; Thomas and
Franklin-Tong, 2004). Addition of Ac-DEVD-CHO
inhibited this activity, while addition of the Ac-YVADCHO (YVAD) caspase-1 inhibitor did not. Pretreatment
of pollen with U0126 prior to SI induction resulted in
a significant alleviation of the DEVDase activity (P 5
0.003, **, n 5 3), while pretreatment with U0124 did
not (P 5 0.949, N.S., n 5 3).
These data clearly demonstrate that SI-mediated
caspase-3-like (DEVDase) activation is inhibited by a
MAPK cascade inhibitor. Assuming the specificity of
U0126, this implicates that a MAPK is activated during
early PCD and functions to signal to PCD. Since p56 is
apparently the only MAPK activated by SI, our data
suggest that p56 activation is necessary for progression of PCD in pollen. As sequence information is not
yet available for p56, reverse genetic studies cannot
currently be carried out to provide definitive evidence
that it is p56 that is involved.
SI-mediated loss of pollen viability and cell death,
though we cannot rule out the possibility that U0126
may also act on other targets. SI also stimulates DNA
fragmentation, which is significantly alleviated by pretreatment with U0126, implicating the functional involvement of a MAPK in initiating signaling upstream
of SI-induced DNA fragmentation. SI stimulates a
caspase-3-like (DEVDase) activity, and this activity
is inhibited by U0126. Our demonstration that the
SI-mediated caspase-3-like (DEVDase) activity is inhibited by a MAPK cascade inhibitor strongly implicates a MAPK being involved in signaling to activate a
caspase-3-like (DEVDase) activity involved in PCD.
DISCUSSION
We previously established that PCD is triggered
in incompatible pollen (Thomas and Franklin-Tong,
2004), which will ensure that pollen tube growth does
not resume and provides a neat way of disposing of
unwanted incompatible pollen. Here, we provide evidence that MAPK activation is likely to be required
for SI-induced PCD. Our data, assuming the lack of
side effects of U0126, strongly implicate a MAPK in
signaling to early events initiating PCD in poppy
pollen. To our knowledge, this represents the first
evidence for involvement of MAPK signaling in PCD
p56 Activation Is Upstream of the SI-Induced
DEVDase Stimulation
We also investigated whether the caspase-3 inhibitor
DEVD might inhibit p56 activity in order to establish if
p56 activation is upstream of the SI-induced caspase3-like activation. We pretreated pollen with DEVD for
1 h prior to the induction of SI and pollen extracts were
examined for p56 activation. Pretreatment of pollen
with DEVD prior to SI induction did not alter p56 activation (Fig. 7), indicating that the SI-induced MAPK
activation is upstream of caspase-3-like activity triggered by SI.
In summary, we have shown that SI induces p56
activation in incompatible pollen and that the MAPK
cascade inhibitor U0126 prevents its activation. SI rapidly reduces pollen viability and U0126 ‘‘rescues’’ incompatible pollen, while its negative analog does not.
These data implicate the involvement of a MAPK in
Figure 6. The SI-induced caspase-3-like (DEVDase) activity is inhibited
by pretreatment with U0126. SI induces a caspase-3-like (DEVDase)
activity that is alleviated by U0126 pretreatment. Controls comprising
untreated pollen (UT) and pollen treated with 100 mM U0126 or U0124
alone (white bars) gave low levels of caspase-3-like activity. Incompatible SI-induced pollen (Inc; black bar) had high levels of caspase3-like activity. Incompatible SI-induced pollen pretreated with 100 mM
U0126 (Inc-U0126; black bar) had reduced levels of caspase-3-like
activity, while incompatible SI-induced pollen pretreated with 100 mM
U0124 (Inc-U0124; gray bar) did not. Incubation of incompatible
SI-induced pollen extracts with DEVD (Inc-DEVD; cross-hatched bar)
inhibited the caspase-3-like activity while incubation of incompatible
SI-induced pollen extracts with YVAD (Inc-YVAD; cross-hatched bar)
did not, verifying the caspase-3-like specific activity. Caspase-3-like
activity was measured using Ac-DEVD-AMC, and data are means 6 SE
of three independent experiments.
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Li et al.
Figure 7. p56 stimulation is upstream of the SI-induced DEVDase
activation. p56 activity was detected using a pTEpY antibody. No p56
activity was detected in controls of untreated pollen or pollen treated
with drugs alone (GM, DMSO, U0124, U0126, DEVD, YVAD). p56
activation was detected in incompatible SI-induced samples (Inc) and
SI-induced samples mock-challenged by pretreatment with DMSO
(Inc-DMSO) and not compatible SI-induced samples (Comp), demonstrating S specificity. This p56 activity was not inhibited in extracts from
SI-induced pollen pretreated with DEVD (Inc-DEVD) or YVAD (IncYVAD), indicating that the caspase-3-like activation is downstream of
MAPK activation.
triggered by SI. SI appears to stimulate a single MAPK
activity: p56 (Rudd et al., 2003; this study). As activation of p56 peaks 10 min after SI induction, it cannot be
involved in the rapid arrest of pollen tube growth.
Here, we provide evidence implicating a MAPK as
being involved in the initiation phase of PCD in incompatible pollen, which provides a significant step
forward in our understanding of SI-induced events.
Inhibitors of MAPK cascades have been extremely
useful for identifying some of the physiological roles
of the cell signaling pathways that they inhibit. U0126
is recognized as a highly specific inhibitor of MAPK
cascades (Davies et al., 2000). There is good evidence
for U0126 preventing MAPK activation in plants. For
example, pathogen response gene expression in tobacco cells was inhibited by U0126 at similar concentrations to those we used (Lee et al., 2001), providing
what was considered good evidence of a functional
involvement of a MAPK in plant defense activation.
Our data therefore strongly suggest the involvement
for a MAPK(s) playing a role in PCD. We cannot definitively say that p56 is involved, as we have not
obtained the cDNA corresponding to it, so cannot
currently use an antisense approach to explore this.
However, because p56 appears to be the only MAPK
stimulated by SI, our data strongly implicate the
involvement of p56 in initiating PCD. Furthermore,
we provide evidence that the SI-activated MAPK
signals to activate a caspase-3-like (DEVDase) activity.
This is confirmed by our finding that although the
tetrapeptide inhibitor Ac-DEVD-CHO inhibits PCD, it
does not inhibit p56 activation, suggesting that the
caspase-3-like (DEVDase) activity is downstream of
p56 in the signaling cascade. Together, our data provide important information about the integration of
the SI signaling network, indicating that we have a
complex network of events to stop pollen tube growth.
MAPK cascades have emerged as key players in
some of the most essential roles in eukaryotic signaling
networks. Plant MAPKs have been shown to be activated by a variety of stresses (Tena et al., 2001; Zhang
and Klessig, 2001), and the involvement of MAPKs in
activation of defense responses resulting in PCD
(Ligterink et al., 1997; Yang et al., 2001; Kroj et al.,
2003) and resistance to pathogens (Zhang and Klessig,
2001; Asai et al., 2002) is well established in plants.
Surprisingly, although MAPKs have been demonstrated to play a role in PCD in plants, apart from an
involvement in pollen hydration (Wilson et al., 1993),
virtually nothing is known about MAPK signaling in
mature pollen, though they are known to be implicated in pollen development (Prestamo et al., 1999).
Furthermore, since U0126 inhibits pollen tube growth,
it implicates MAPKs as playing a role in regulating
pollen tube growth. Interestingly, it has recently been
demonstrated that SIMK (stress-induced MAPK) is
involved in regulating root hair tip growth (Samaj
et al., 2002). Our findings, therefore, not only provide a
significant advance in our understanding of mechanisms involved in mediating the SI response in
Papaver, but also shed some light on the role of MAPKs
in pollen. Thus, to our knowledge, these data provide
the first evidence for MAPK signaling in growing
pollen tubes.
SI involves modification of several cellular components in incompatible pollen. Figure 8A outlines a
model of the current state of play in our understanding
of this signaling network. Figure 8B shows the new
data described here, implicating MAPK involvement.
PCD involves release of cytochrome c into the cytosol
and later DNA fragmentation, which is mediated by a
caspase-3-like (DEVDase) activity (Thomas and FranklinTong, 2004). One of the first SI-induced events is rapid
Ca21 influx (Franklin-Tong et al., 2002), accompanied
by SI-specific increases in [Ca21]i (Franklin-Tong et al.,
1993, 1997; Fig. 8A); this appears to initiate the SI signaling network, causing rapid inhibition of pollen tube
growth. We have evidence that the [Ca21]i increases
are required for PCD (Thomas and Franklin-Tong, 2004).
SI also triggers depolymerization of the F-actin cytoskeleton (Geitmann et al., 2000; Snowman et al., 2002),
resulting in rapid arrest of incompatible pollen tube
growth. Evidence that artificially raising [Ca21]i also
results in actin depolymerization suggests that this SIinduced event is on the same signaling pathway (Fig.
8A). We have recently shown that actin depolymerization is involved in initiating PCD (Thomas et al., 2006),
providing the first piece of evidence integrating some
of the SI-stimulated components.
Here, we have provided important evidence linking
a second SI-stimulated component, activation of a
MAPK, to this PCD network (Fig. 8B). We previously
showed that p56 activation is inhibited by La31 (Rudd
et al., 2003), a Ca21 channel blocker, indicating that the
SI-induced MAPK activation is downstream of the
initiating Ca21 signals. Since p56 activation is 10 min
after SI induction, this event must be downstream
of actin depolymerization. Although we previously
showed that altering actin dynamics using latrunculin
B and jasplakinolide can trigger PCD in pollen tubes
(Thomas et al., 2006), preliminary data suggest that
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A MAPK Signals to SI-Induced PCD
ing of SI and signaling to regulate incompatible pollen
tube growth. We propose that this represents the
‘‘gateway’’ through which incompatible pollen must
pass to become irreversibly inhibited.
MATERIALS AND METHODS
SI Induction and Pollen Treatments
Figure 8. A model of events triggered by SI in incompatible pollen. A,
Current overall understanding of events involved. Incompatible stigmatic S proteins trigger a number of events in incompatible pollen. This
starts with influx of Ca21 and increases in [Ca21]i (assumed to be
triggered by the interaction of S proteins with their pollen receptors).
This results in phosphorylation of two sPPases, Pr-p26.1a and Pr-p26.1b;
their sPPase activity is inhibited by phosphorylation. Large-scale actin
depolymerization occurs, which plays a role in triggering a caspase-3like activity and PCD. p56-MAPK activation is also triggered by SI; here,
we show that it is likely to be involved in mediating PCD (see B). B,
Data described here that contributes to the model. SI triggers activation
of a MAPK, p56. This activity is inhibited by U0126. Viability tests show
that pretreatment with U0126 can ‘‘rescue’’ incompatible pollen that
would normally rapidly die. Both SI-induced caspase-3-like activity
and DNA fragmentation are dramatically reduced by pretreatment with
U0126. Together, these data implicate the involvement of MAPK
activation in regulating SI-induced PCD.
actin depolymerization does not directly signal to p56
activation, as latrunculin B and jasplakinolide do not
activate p56 (S. Li and V.E. Franklin-Tong, unpublished
data). Therefore, SI-induced p56 activation, although
it signals to PCD, may not be on the same signaling
pathway as the actin alterations. Interestingly, we recently established that SI also stimulates Ca21-dependent
hyperphosphorylation of Pr-p26.1a/b, which are sPPases
in incompatible pollen (de Graaf et al., 2006; Fig. 8A).
Since these enzymes are involved in biosynthesis, this
modification reduces their sPPase activity and is also
predicted to result in inhibition of pollen tube growth
(de Graaf et al., 2006). Thus, SI triggers complex signaling networks in incompatible pollen.
In conclusion, this is the first evidence (to our knowledge) suggesting a specific causal link between MAPK
activation and initiation of PCD in SI. Our findings
implicate a fundamentally important role for MAPK
signaling in SI, showing that it most likely participates
in initiating the early PCD signaling cascade, committing the pollen tube to die. Although these data fall
short of providing definitive evidence, they nevertheless provide a significant advance in our understand-
Pollen of Papaver rhoeas, the field poppy, was hydrated at 25°C for 45 min,
then germinated and grown in vitro in liquid germination medium [GM;
0.01% H3BO3, 0.01% KNO3, 0.01% Mg(NO3)26H2O, 0.036% CaCl22H2O, 13.5%
Suc], as described by Snowman et al. (2002), at 25°C for 45 min before any
treatments were applied.
For SI treatments, recombinant proteins were produced by cloning the
nucleotide sequences specifying the mature peptide of the S1, S3, and S8 alleles
of the S gene (pPRS100, pPRS300, and pPRS800) into the expression vector
pMS119 as described previously (Foote et al., 1994). Expression and purification of the proteins were as described (Kakeda et al., 1998). SI was induced by
adding recombinant S proteins, S1e and S8e (final concentration 10 mg mL21),
to pollen from S1S8 (incompatible) and S2S6 (compatible) plants, growing in
vitro, as described (Snowman et al., 2002). Heat-denatured S proteins boiled
for 30 min prior to use were used as additional controls.
The inhibitor U0126 [1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene; Calbiochem] was used at a concentration of 100 mM to inhibit p56
activation. Its inactive analog, U0124 [1,4-diamino-2,3-dicyano-1,4-bis(methylthio)butadiene; Calbiochem], was used as a control and did not inhibit
MAPK cascades even at concentrations of 100 mM. The drugs were added to
pollen growing in vitro as a pretreatment for 1 h prior to experiments.
Controls comprised addition of dimethyl sulfoxide (DMSO), which was used
to solubilize the inhibitors, at a final concentration of 0.1% (v/v).
MAPK Activity Assays and Immunoblots
Cytosolic proteins were extracted from pollen according to Rudd et al.
(1996). Pollen was collected by centrifugation at 2,000 rpm for 5 min and then
extracted into IP buffer (Rudd et al., 2003) in a ground-glass homogenizer.
Protein concentration was assayed using a Bio-Rad protein assay kit. Pollen
protein extracts were separated using SDS-PAGE and then western blotted.
Blots were probed with anti-ACTIVE MAP kinase polyclonal Ab (pTEpY; Cell
Signaling Technology), which recognizes activated MAPKs. For testing the
cross-reaction of p56 with AtMPK3, immunoblots were probed with a 1:1,000
dilution of the polyclonal anti-AtMPK3 antibody raised against the specific
peptide corresponding to amino acids 359 to 370 at the C terminus of Arabidopsis (Arabidopsis thaliana) MPK3 (M8318; Sigma). This sequence is specific to
AtMPK3 because it is not found in the other 19 known MAPKs in Arabidopsis.
Subsequently, blots were washed and incubated with an anti-rabbit horseradish peroxidase secondary antibody (A-0545; Sigma) and detected using an
ECL kit (RPN2209; Amersham). As controls, tobacco (Nicotiana tabacum) SIPK
antibody (generous gift from Shuqun Zhang, University of Missouri), which is
an ortholog of Arabidopsis MPK6, and AtMPK4 antibody (A6979; Sigma)
raised against synthetic peptide corresponding to the C terminus of Arabidopsis MPK4 (amino acids 365–376; this sequence is specific to AtMPK4 and it
is not found in the other 19 known Arabidopsis MAPKs) were also used.
Measurement of Pollen Tube Lengths
Pollen tubes were treated as described above, but incubated at 25°C for 1 h,
and then fixed in 4% paraformaldehyde and mounted in Tris-buffered saline,
pH 7.6. Pollen tubes were imaged and pollen tube lengths measured using a
Quips PathVysion image analysis system (Applied Imaging International) and
with IPlab software. One hundred pollen tubes were measured for each
treatment and mean length determined from a total of three independent
experiments.
Viability Assays
Pollen viability was assessed using the fluorochromatic test procedure as
described (Heslop-Harrison and Heslop-Harrison, 1970; Heslop-Harrison
et al., 1984). Pollen was incubated in 5 mg mL21 FDA in GM for 5 min at
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Li et al.
room temperature. FDA is an indicator of cellular metabolic activity; viable
pollen can cleave FDA, so is fluorescent; unviable pollen is not. Images were
captured with a Nikon T300 fluorescence microscope, using a Quips PathVysion
image analysis system. One hundred pollen tubes were counted for each
treatment and percentage viability determined from a total of three independent experiments.
We thank Shuqun Zhang for kindly providing the SIPK antibody. S.L. also
thanks Kim Osman, Chris Franklin, Barend de Graaf, Steve Thomas, and
Maurice Bosch for helpful comments and assistance.
DNA Fragmentation Assays
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Capture and analysis of images was made at 20°C using a Photometrics
SenSys camera (model KAF1400-G2) and a Quips PathVysion image analysis
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buffer (50 mM HEPES, pH 7.4, 10 mM NaCl, 0.1% CHAPS, 10 mM dithiothreitol,
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Caspase-3-Like Activity Measurements
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to establish the caspase-3-like activity directly. Protein extracts were made
from treated pollen tubes by grinding in extraction buffer (50 mM sodium
acetate, 10 mM L-Cys, pH 6.0). Ten micrograms of total protein was incubated
with 50 mM Ac-DEVD-AMC at 27°C, and fluorescence was monitored at 460
nm using a FLUOstar Optima 413-1183 time-resolved fluorescence plate
reader (BMG-LabTech; 380 excitation, 460 emission) as described by Thomas
et al. (2006). Fluorescence at 480 nm indicated cleavage of the substrate by a
DEVDase activity. Relative fluorescence units were expressed as percentage
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Caspase Inhibitor Treatments
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caspase substrate experiments using Ac-DEVD-AMC. To test if MAPK activation was upstream of caspase activation, pollen was grown as described and
pretreated by addition of 100 mM Ac-DEVD-CHO or Ac-YVAD-CHO (Calbiochem) for 1 h; SI was then induced. Extracts for MAPK activity were made at
10 min and western blotted with anti-ACTIVE MAP kinase polyclonal Ab
(pTEpY; Promega; see above). At least three independent experiments were
carried out.
Sequence data from this article can be found in the GenBank/EMBL data
libraries under accession numbers AJ784995, AJ784996, and AJ784997.
ACKNOWLEDGMENTS
We thank the horticultural staff for growing the plants and helping
harvest material. S.L. was supported by a Royal Society China Fellowship.
Received May 9, 2007; accepted July 17, 2007; published July 27, 2007.
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