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Journal of Experimental Botany, Vol. 65, No. 5, pp. 1331–1342, 2014
doi:10.1093/jxb/ert468 Advance Access publication 21 January, 2014
Review paper
Taking one for the team: self-recognition and cell suicide
in pollen
Katie A. Wilkins*,†, Natalie S. Poulter‡ and Vernonica E. Franklin-Tong
School of Biosciences, College of Life and Environmental Sciences, University of Birmingham, Birmingham, B15 2TT, UK
* Present address: Department of Plant Sciences, University of Cambridge, Downing Street, Cambridge, CB2 3EA, UK
† To whom correspondence should be addressed. E-mail: [email protected]
‡ Present address: Centre for Cardiovascular Sciences, Institute of Biomedical Research, The Medical School, University of Birmingham,
Birmingham, B15 2TT, UK.
Received 26 September 2013; Revised 2 December 2013; Accepted 4 December 2013
Abstract
Self-incompatibility (SI) is an important genetically controlled mechanism used by many angiosperms to prevent
self-fertilization and inbreeding. A multiallelic S-locus allows discrimination between ‘self’ (incompatible) pollen from
‘nonself’ pollen at the pistil. Interaction of matching pollen and pistil S-determinants allows ‘self’ recognition and triggers rejection of incompatible pollen. The S-determinants for Papaver rhoeas (poppy) are PrsS and PrpS. PrsS is a
small secreted protein that acts as a signalling ligand to interact with its cognate pollen S-determinant PrpS, a small
novel transmembrane protein. Interaction of PrsS with incompatible pollen stimulates increases in cytosolic free Ca2+
and involves influx of Ca2+ and K+. Data implicate involvement of reactive oxygen species and nitric oxide signalling
in the SI response. Downstream targets include the cytoskeleton, a soluble inorganic pyrophosphatase, Pr-p26.1, and
a MAP kinase, PrMPK9-1. A major focus for SI-induced signalling is to initiate programmed cell death (PCD). In this
review we provide an overview of our understanding of SI, with focus on how the signals and components are integrated, in particular, how reactive oxygen species, nitric oxide, and the actin cytoskeleton feed into a PCD network.
We also discuss our recent functional expression of PrpS in Arabidopsis thaliana pollen in the context of understanding how PCD signalling systems may have evolved.
Key words: Actin cytoskeleton, caspase, nitric oxide (NO), oxidative stress, Papaver rhoeas, pollen, programmed cell death
(PCD), reactive oxygen species (ROS), self-incompatibility (SI).
Introduction
Unwanted eukaryotic cells are often removed by programmed
cell death (PCD). In recent years, plant PCD has been a topic
of significant interest and this phenomenon has become
accepted as a mainstream event, playing a critical role in most
processes in plant development, abiotic stresses, and plant–
pathogen interactions. Several categories of plant PCD have
recently been defined according to morphological criteria (van
Doorn et al., 2011), although biochemical criteria have yet to
be dealt with in the same manner. However, not all PCD systems fit neatly into one category; a good example is the topic
of this review, the Papaver self-incompatibility (SI) response,
which exhibits hallmark features from several categories.
There are several markers for PCD that have been observed
in both animal and plant cells. However, emerging data in both
animals and plants suggest that many are also involved in normal development or necrosis, so attribution of characteristics to
establish PCD is often difficult. In addition, it is clear that PCD
in plants is quite different. Caspases play an important role in
initiating and executing mammalian cell apoptosis by cleaving
protein targets (Parrish et al., 2013). One of the key mammalian executioner caspases is caspase-3, which has a tetrapeptide
recognition motif of DEVD, so is known as a DEVDase, due
to its cleavage recognition specificity. Biochemical tools have
been developed to identify caspase-like activities using caspase
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1332 | Wilkins et al.
specific substrates and inhibitors, based on specific caspase
recognition motifs which allow the direct analysis of caspase
activities. Despite the fact that plants do not have homologues
of mammalian caspases, use of these mammalian tools has
demonstrated the involvement of caspase-like activities in several plant PCD systems (Chichkova et al., 2004; Rojo et al.,
2004; Bonneau et al., 2008).
Another important feature of many PCD networks in
plants is an oxidative burst (Gechev et al., 2006; Mittler et al.,
2011). Reactive oxygen species (ROS) play an important role
in basal resistance and has been identified as being involved in
the later stages of systemic acquired resistance and hypersensitive response, which leads to PCD (Torres et al., 2006). The
role of the oxidative burst in plant PCD is the topic of several
reviews in this special issue (Baxter et al., 2014; Demidchick,
2014;), so will not be reviewed here.
The ability to discriminate between self and nonself
(allorecognition) is usually controlled by a highly polymorphic locus and is an important feature of many organisms
(Sawada et al., 2014). Many plants are hermaphrodites, with
male and female organs in close proximity. This increases the
chances of self-fertilization and also the possibility of undesirable inbreeding depression. Many plants utilize SI to prevent inbreeding, using a cell–cell recognition system in which
pollen carrying the same haplotype as the pistil it lands on is
rejected via the initiation of PCD. This process of self-rejection prevents inbreeding; for the ‘greater good’ of the species
self-pollen is inhibited and self-fertilization does not occur.
This review focuses on the SI-induced PCD events in incompatible Papaver rhoeas pollen.
In the SI system of P. rhoeas, the female determinant,
P. rhoeas stigma S (PrsS) is a 15-kDa protein which is developmentally controlled and secreted from female tissue (Foote
et al., 1994). The male S-determinant, P. rhoeas pollen S
(PrpS) is a novel 20-kDa protein which is localized at the
plasma membrane (Wheeler et al., 2009). As PrpS is a novel
protein, it clearly is not a ‘classic’ defined/identified receptor.
However, PrpS appears to act as a unique class of ‘receptor’
that interacts with PrsS in a very specific manner, triggering
an intracellular signalling network and resulting in a highly
specific biological response, as will be described. Analysis of
the SI-triggered events has been enabled by a unique in vitro
SI bioassay (Franklin-Tong et al., 1988; Foote et al., 1994).
This bioassay utilizes recombinant PrsS proteins expressed in
Escherichia coli, which are added to pollen growing in vitro,
and depending on the combination of S-haplotypes used,
the pollen will either be inhibited (i.e. where an incompatible
reaction is achieved) or grow (in a compatible situation). This
in vitro bioassay has led to a much better understanding of
mechanisms involved in Papaver SI than in other SI systems.
This assay can be scaled down for live-cell imaging to visualize
events in individual pollen tubes undergoing the SI response
(Bosch and Franklin-Tong, 2007; Wilkins et al., 2011) or in
fixed pollen tubes (Poulter et al., 2010) or it can be scaled up
to extract proteins for biochemical analysis (Snowman et al.,
2002; Rudd et al., 2003). Drugs can be added prior to SI being
induced to investigate the potential role of specific components
in the SI response (e.g. see Thomas and Franklin-Tong, 2004;
Li et al., 2007). Thus, the SI bioassay has proved, with the use
of relevant controls, to be a robust and adaptable system for
the analysis of events triggered specifically by SI.
Perhaps one of the most important findings relating to
events triggered by SI in Papaver pollen was the demonstration of the involvement of PCD (Thomas and FranklinTong, 2004), which provides a highly effective way to
prevent self-fertilization. SI involves a network of signalling
events and their targets, which integrate to drive incompatible pollen to commit suicide. Here we review key events
relating to SI signalling, stimulated by the S-specific interaction of recombinant pistil PrsS with pollen expressing PrpS.
The Papaver SI system has recently been found to utilize
ROS and nitric oxide (NO) to integrate signalling to PCD
in incompatible pollen. This provides a good example of a
system that straddles both these important topics that are
the focus of this Special Issue. We therefore focus attention
on the integration of these signalling components and their
cellular targets.
Mechanisms involved in SI in the
Papaver system
Calcium signalling mediates Papaver SI
Ca2+ is an important second messenger, and Ca2+-dependent
signalling networks are used to generate responses to many
stimuli in both animal and plant cells. The Papaver SI
response is thought to be initiated by an almost instantaneous increases in cytosolic free Ca2+ ([Ca2+]cyt) forming a ‘wave’
in the shank of the pollen tube (Franklin-Tong et al., 1993,
1995, 1997). These studies formed the basis of a hypothesis,
proposing that a receptor-ligand type of interaction, involving PrsS and PrpS, triggers a Ca2+-dependent signalling cascade in incompatible pollen. More recent studies, using an
electrophysiological approach, have provided evidence for
Ca2+ influx and also revealed a large influx of K+ in incompatible pollen (Wu et al., 2011). These studies suggested that
the SI-induced conductances may involve nonspecific cation
channel(s). Several signalling targets downstream of the
SI-specific increases in cytosolic Ca2+ in Papaver pollen tubes
have been identified. Ca2+ signalling often results in altered
protein kinase activity, resulting in post-translational modification such as phosphorylation.
Phosphorylation events identified in Papaver SI
MAPK cascades trigger numerous signalling networks. They
are activated by dual phosphorylation of threonine and
tyrosine residues in a TXY motif via a MAPKKK cascade.
During the SI response, a MAPK, p56, is activated specifically in incompatible, but not compatible pollen (Rudd and
Franklin-Tong, 1996; Li et al., 2007). p56 activation, which
peaks 10 min after SI induction, is temporally too late to be
involved in the inhibition of pollen tube growth; however,
studies have provided evidence that this MAPK is involved in
signalling to PCD (as will be discussed later).
Papaver self-incompatibility, ROS, NO, and PCD | 1333
SI also triggers the rapid phosphorylation of Pr-p26.1a/b,
two pollen-expressed 26-kDa soluble inorganic pyrophosphatases (sPPases) (Rudd et al., 1996; de Graaf et al., 2006).
sPPases are important enzymes involved in hydrolysis of
inorganic pyrophosphate (PPi) (Kornberg, 1962) and it
makes good sense that their activity is associated with driving
biosynthesis in these rapidly growing cells. sPPases are well
documented to have their activity inhibited by Ca2+, so this
provides a neat and potentially rapid mechanism to inhibit
incompatible pollen tube growth (de Graaf et al., 2006).
Phosphorylation of sPPases is a novel event and it may provide an additional inhibitory mechanism. However, whether
these sPPases somehow feed into the PCD network is not
known.
Papaver SI triggers alterations to the cytoskeleton
The actin cytoskeleton is a major target and effector of signalling networks (Staiger, 2000) and an intact F-actin cytoskeleton is required for pollen tube growth (Gibbon et al., 1999).
With these two pieces of information, the actin cytoskeleton
was investigated as a possible early target for SI signals, which
involve both Ca2+ signalling and inhibition of pollen tube
growth.
The normal actin cytoskeleton of Papaver pollen tubes
comprises longitudinal filament bundles of actin in the shank,
with a typical cone-shaped configuration just behind the tip
(Fig. 1A). Dramatic alterations in F-actin organization are
triggered specifically in incompatible pollen tubes by SI
induction, which involves F-actin depolymerization (Fig. 1B)
(Geitmann et al., 2000; Snowman et al., 2002). This is predicted to result in the rapid inhibition of pollen tube growth.
Two Ca2+-dependent actin-binding proteins (ABP), profilin
and PrABP80 (a putative gelsolin), which have strong Ca2+dependent severing activity (Huang, 2004), are implicated in
the SI-induced depolymerization. The actin then begins to
aggregate to form punctate F-actin foci and they increase in
size over time (Fig. 1C, D). Two ABPs, actin depolymerizing
factor (ADF/cofilin) and cyclase-associated protein (CAP),
rapidly colocalize to the actin foci (Poulter et al., 2010). It is
thought that both the depolymerization and the formation of
these highly stable actin structures play a role in signalling to
PCD (as will be discussed further). Compatible interactions
have no effect on the cytoskeleton (Fig. 1E). The pollen tube
microtubule cytoskeleton is also a target for the SI signals.
Cortical microtubules are rapidly depolymerized and there
is evidence that this, too, plays a role in the PCD network
(Poulter et al., 2008; see also further discussion).
It might be argued that Ca2+ signalling, actin depolymerization, and inhibition of pollen tube growth, all of which all
occur very rapidly, might simply cause inhibition of tip growth
and consequential cell death. However, studies showed that
compatible pollen tubes that stop growing ‘naturally’ around
8 h, due to limitations in the artificial growth medium, did not
exhibit DNA fragmentation (Jordan et al., 2000). This, coupled with the observation that ‘normal’ inhibition of pollen
tube growth was not associated with the very distinctive Ca2+
signature observed in incompatible pollen, helped to rule out
Fig. 1. Actin alterations during the self-incompatibility (SI)
response. SI-induced alterations to the actin cytoskeleton are
rapid and extensive. The F-actin cytoskeleton in fixed pollen tubes
was visualized using rhodamine–phalloidin. (A) Typical untreated
pollen tube with normal F-actin configuration of filaments. (B) Early
actin depolymerization, 2 min after SI; very few F-actin filaments
remain. (C) Early formation of punctate F-actin foci, 20 min after SI
induction. (D) Later formation of punctate F-actin foci, 3 h after SI,
in which the foci are much larger than earlier. (E) Compatible pollen
tube, 60 min after SI induction show F-actin configurations that
resemble those of untreated pollen tubes.
the idea that cell death is just a consequence of inhibited pollen
tube growth. It is possible that the SI-induced Ca2+ signalling
is primarily to stop the growth of the pollen tube, involving
F-actin depolymerization. In support of this, low concentrations of LatB result in pollen-tube inhibition but not DNA
fragmentation (Gibbon et al., 1999; Thomas et al., 2006).
However, both the level of SI-induced [Ca2+]cyt and the amount
of F-actin depolymerization are in huge excess of that required
simply to stop growth. Caffeine, which inhibits tip growth but
1334 | Wilkins et al.
does not affect the actin cytoskeleton of pollen tubes (Geitmann
et al., 2000; Snowman et al., 2002), has been used to show that
caffeine-inhibited pollen tubes have low DNA fragmentation
levels, not significantly different from controls (Thomas et al.,
2006). This study demonstrated that the SI-mediated DNA
fragmentation is not merely a consequence of inhibition of
growth, but is due to major changes in actin polymer levels or
assembly dynamics. The evidence supports the idea that the
SI Ca2+ signature is generated specifically to signal to trigger
excess sustained F-actin depolymerization and then aggregation and stabilization of F-actin foci, which are highly distinctive and a feature of the SI-PCD (as will be discussed).
SI-induced PCD is mediated by caspase-like activities
Temporally, one of the latest SI events is DNA fragmentation. Jordan et al. (2000) showed that DNA fragmentation occurred in an S-specific manner. DNA fragmentation
increased only in SI-induced pollen tubes, with maximal levels detected at 12–14 h after SI induction and not in compatible controls grown for the same length of time (Jordan et al.,
2000; Thomas and Franklin-Tong, 2004). Subsequently it was
shown that addition of the caspase-3 inhibitor, Ac-DEVDCHO, prior to SI induction significantly reduced SI-specific
pollen-tube inhibition as well as DNA fragmentation. In contrast, use of the caspase-1 inhibitor Ac-YVAD-CHO showed
that a caspase-1-like/YVADase activity was not involved
(Thomas and Franklin-Tong, 2004). This provided evidence that a DEVDase/caspase-like activity was involved in
SI-mediated pollen-tube inhibition and DNA fragmentation.
Subsequently, Bosch and Franklin-Tong (2007) used fluorescent AMC-based caspase substrates to verify and characterize the caspase-like activities involved in more detail.
A caspase-3-like/DEVDase and a caspase-6-like/VEIDase
activity were identified in the pollen tube cytosol in the first
1–2 h after SI induction, peaking at 5 h, suggesting that they
are involved in early PCD events (Bosch and Franklin-Tong,
2007). A caspase-4-like/LEVDase activity was also identified
in SI-induced pollen, but its activation was later, peaking at
8 h post SI induction, when DEVDase and VEIDase activity
was decreasing (Bosch and Franklin-Tong, 2007).
The identity of these ‘caspase-like’ proteins is not known;
they are certainly not caspases, as plants do not possess these
genes. Intriguingly, use of a DEVD-biotin pull-down with pollen extracts identified a vacuolar processing enzyme (VPE/
legumain) from Papaver pollen extracts as interacting with
the DEVD motif (Bosch et al., 2010). This is of interest,
even though a YVADase was thought not to be required for
the SI-induced PCD, as it hinted that a legumain/YVADase
could interact with a DEVDase-containing substrate. In
tobacco, VPE is essential for a virus-induced hypersensitive
response that involves PCD (Hatsugai, 2004). However, the
significance of the VPE in the Papaver SI-PCD system is currently unknown. With respect to the identity of the Papaver
DEVDase, an intriguing possibility is raised by the identification of PBA1, a proteasome subunit exhibiting a caspase3-like/DEVDase activity during infection of avirulent bacterial
strains in Arabidopsis thaliana which is implicated in disease
resistance and PCD (Hatsugai et al., 2009). Proteasomal activities are critical not only during PCD but during many normal
cellular processes, including pollen tube growth (Sheng et al.,
2006). The Franklin-Tong group group is currently exploring
the possibility that the SI-activated DEVDase may be due to
proteasomal activity. Although data implicate involvement
of the proteasome in modulating pollen tube growth, it is too
early to speculate about the identity of the DEVDase.
Characterization of the SI-activated caspase-1, 3, 6-like
activities revealed that they exhibit activity within a very narrow pH range, with peak activity at pH 5 and virtually no activity at pH 6 (Bosch and Franklin-Tong, 2007). This suggested
that, for these proteins to have activity in a biological context,
the cytosol would need to become acidic. Investigations using
the live-cell ratiometric pH probe, BCECF-AM, revealed that
there was, indeed, a dramatic acidification of the pollen cytosol from pH 6.9 to pH 5.5, triggered in the first 1–4 h of SI
(Bosch and Franklin-Tong, 2007; KA Wilkins, M Bosch, T
Haque, N Teng, and VE Franklin-Tong, unpublished data).
What is involved in this phenomenon is not yet clear, but it
points to major alterations to the cytosol being triggered by
SI. Thus, cytosolic acidification is likely to be an important
decision-making step, as this will allow PCD to proceed by
allowing caspase-like proteins to be activated. Surprisingly
few measurements of cytosolic pH during PCD in any system
exist, with the exception of Bosch and Franklin-Tong (2007)
and another method, based on the extinction of YFP fluorescence at low pH, which has been used to monitor pH during
PCD in onion cells, showing that loss of YFP fluorescence
could be correlated with caspase-like activity (Young et al.,
2010). More recent studies in the Papaver SI-PCD system
have shown that dramatic SI-induced acidification of the pollen cytosol occurs much earlier, with substantial acidification
occurring within 10 min (Wilkins et al., Submitted). We think
that live-cell monitoring of cytosolic pH will be an important but rather neglected area to investigate more fully in the
future to elucidate the early events of PCD in various systems.
Integrating SI-triggered events that signal
to PCD
A major focus for the SI-induced signalling network is the
activation of PCD. The interaction of the secreted pistil
S-determinant, PrsS, with the ‘self’ pollen PrpS triggers Ca2+
influx and rapid increases in [Ca2+]cyt. This kicks off several
events which ultimately lead to PCD. SI-PCD hallmarks
include: the cytoskeleton, the p56-MAPK, reactive oxygen
species (ROS), and nitric oxide (NO) (Thomas et al., 2006;
Li et al., 2007; Wilkins et al., 2011). The following discussion
and Fig. 2 summarizes data relating to our understanding of
how the signals and targets triggered by SI in incompatible
pollen tubes are integrated to mediate cell death.
Involvement of actin signalling in SI-mediated PCD
As already described, both actin depolymerization and
the formation of F-actin foci is a rapid event in Papaver SI
Papaver self-incompatibility, ROS, NO, and PCD | 1335
response. Furthermore, both actin and microtubule alterations are implicated in mediating PCD, with microtubule
depolymerization being triggered by actin depolymerization
(Thomas et al., 2006; Bosch et al., 2008; Poulter et al., 2008)
(Fig. 2). Investigations showed that both actin depolymerization and stabilization treatments (using latrunculin B and
jasplakinolide, respectively) stimulated DNA fragmentation
in Papaver pollen, which was inhibited by the DEVD-CHO
inhibitor but not the Ac-YVAD-CHO inhibitor (Thomas
et al., 2006). This provided evidence that actin alterations were placed on a pathway involving a caspase-3 like/
DEVDase activity (Fig. 2). Moreover, DNA fragmentation
correlated with the duration and extent of actin depolymerization; 50% actin depolymerization for 10 min was sufficient to induce PCD (Thomas et al., 2006). The caspase-3
substrate Ac-DEVD-AMC was also used to establish that the
DEVDase/caspase-3-like activity more directly; pollen tubes
treated with 1 μM Latrunculin B for 6 h exhibited an increase
in DEVDase activity similar to that induced by SI (Fig. 2).
These data provided evidence that major disturbance of actin
polymer dynamics could trigger a caspase-3-like/DEVDase
activity in pollen that resulted in DNA fragmentation, establishing a causal link between actin polymerization status
and initiation of PCD (Thomas et al., 2006). Furthermore,
changes in cytoskeletal dynamics have been shown to influence whether a eukaryotic cell enters into a PCD pathway (see
reviews by Franklin-Tong and Gourlay, 2008; Smertenko and
Franklin-Tong, 2011). Thus, there is evidence that both the
substantial actin depolymerization and the later aggregation
into punctate foci triggered by SI signalling play a key role in
triggering PCD, mediated by activation of a caspase-3-like/
DEVDase activity (Thomas et al., 2006; see also Fig. 2).
As mentioned earlier, actin foci are associated with the ABP
ADF/cofilin (Poulter et al., 2010). Although ADF’s name
comes from its ability to depolymerize F-actin at normal
cytosolic (~pH 7) in vitro (Carlier et al., 1997; Bamburg et al.,
1999), it has been shown that at acidic pHs ADF does not
exhibit its usual actin-filament-severing activity and instead
binds to and stabilizes F-actin (Allwood, 2002). It therefore
seems likely that SI-induced cytosolic acidification could
play an important role in altering the activity of ADF from
a depolymerizing activity to an actin-stabilizing one, which
could result in the formation of the highly stable F-actin foci
observed during SI in incompatible pollen (Fig. 1D). Thus,
cytosolic acidification, coupled with the consequent binding of ADF to the actin foci, helps to explain the strong link
between actin foci formation and PCD in incompatible pollen, and suggests that this can be used as a good hallmark
feature of PCD, at least in the SI system (Fig. 2).
Involvement of MAPK signalling in SI-mediated PCD
MAPKs are well-known key players in plant signalling networks and are activated under both abiotic and biotic stress
responses and during PCD (Colcombet and Hirt, 2008;
Meng and Zhang, 2013). This places them as key mediators of PCD signalling networks. As aforementioned, SI
activates the MAPK p56 (Li et al., 2007; Rudd et al., 2003).
The MAPK cascade inhibitor U0126, which blocks MAPK
activity in both animals and plants, prevented the activation of the SI-stimulated p56-MAPK and alleviated several
SI-induced events, including DNA fragmentation, caspase3-like/DEVDase activity, and loss of viability (Li et al., 2007);
see Fig. 2. As p56 appears to be the only MAPK activated
by SI, this provides evidence that p56 is implicated in signalling upstream of a caspase-3/DEVDase-like activity (Rudd
et al., 2003; Li et al., 2007). The activation of the MAPK p56
peaked 10 min after the induction of SI (which is considered
the initiation phase of SI, up to which time point any changes
induced are reversible by washing out of PrsS; P Wright
and VE Franklin-Tong, unpublished data). This potentially
places p56, and other events around 10 min post SI, as key
turning point events that may help form the decision to enter
into irreversible PCD.
A role for ROS and NO signalling in SI-mediated PCD
ROS and NO have been shown to often participate in PCD
and various stresses in plant cells (see Baxter et al., 2014;
Passaia et al., 2014, in this issue). The Franklin-Tong group
has investigated whether ROS and NO play a role in mediating PCD in the SI response. Live-cell imaging of ROS and
NO in growing Papaver pollen tubes, using chloromethyl2′7′-dichlorodihydrofluorescein diacetate acetyl ester and
4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate,
respectively, showed that SI induces relatively rapid and
transient increases in ROS and NO (Fig. 3A, B), with distinct temporal ‘signatures’ (Wilkins et al., 2011). SI-induced
increases in ROS were documented as early at 2 min, lasting
up to 15 min in some pollen (Fig. 3A) and increases in NO
occurred ~15 min after SI induction, lasting for up to ~40 min
(Fig. 3B). The integration of SI-PCD responses with these
putative signals were investigated with the ROS and NO scavengers diphenyliodonium, tempol, and c-PTIO. This revealed
the alleviation of the formation of the distinctive SI-induced
actin punctate foci and the activation of a DEVDase/caspase-3-like activity when using these drugs combined (Fig. 3;
Wilkins et al., 2011). These data provided evidence that ROS
and NO increases are not only upstream of these key SI markers, but contribute to signalling to activation of a DEVDase
(Wilkins et al., 2011; see also Fig. 2). These data represent
the first steps in understanding ROS/NO signalling, providing an important biological context and improving the understanding of how several key components of the SI response
are integrated to bring about PCD in Papaver pollen tubes.
A possible role of the p56-MAPK may be in stimulating NO.
Although this link between MAPK activation and NO generation in SI is speculative at present, this link has been shown
in plant pathogen signalling (Asai et al., 2008).
Because diphenyliodonium, which is a NADPH oxidase
inhibitor, resulted in inhibition of SI-induced increases in
ROS, this suggests that a respiratory burst oxidase homologue (RBOH) might be responsible for some of these
increases in ROS. RBOHs, which are present on the
plasma membrane, are key contributors of the oxidative
burst in stress responses (Torres et al., 2005). Imaging of
1336 | Wilkins et al.
Fig. 2. Cartoon showing a model of the integrated self-incompatibility (SI) programmed cell death (PCD) signalling network in Papaver
rhoeas pollen. Interaction of a pistil S-determinant PrsS with its cognate pollen S-determinant, PrpS, in a S haplotype-specific
manner (e.g. PrsS1 with PrpS1) rapidly triggers the SI signalling network, resulting in the rapid inhibition of pollen tube tip growth and
culminating with DNA fragmentation and death of incompatible pollen. Different coloured boxes indicate key events triggered by SI;
grey arrow indicates approximate timings; yellow arrows show SI-induced events; red arrows indicate agonist drugs used to stimulate
events; black symbols show drugs used to inhibit events; pink vertical line represents the cell wall; double green line represents the
plasma membrane. One of the earliest events include rapid influx of Ca2+ and K+, and almost instantaneous increases in cytosolic
free Ca2+ (blue box) [Ca2+]cyt increases are required for many SI events, including phosphorylation and inactivation of soluble inorganic
pyrophosphatases (sPPases) Pr-26.1a/b, actin depolymerization, and transient increases in reactive oxygen species (ROS) and nitric
oxide (NO). Moreover, mastoparan or the Ca2+ ionophore A23187 (red arrows) can mimic SI-induced increases in [Ca2+]cyt. Lanthanum
(La3+) has been used extensively to block SI-induced Ca2+ events and has demonstrated a requirement for Ca2+ as a key mediator of
SI signalling to death. This places Ca2+ signalling as one of the first SI events, upstream of many of the targets. The end focus of the
SI-induced network appears to be DNA fragmentation and cell death (red box), which are inhibited by pretreatment with the tetrapeptide
inhibitor Ac-DEVD-CHO, but not Ac-YVAD-CHO. The inferred DEVDase activity has been more directly measured using the tetrapeptide
substrate, Ac-DEVD-AMC, which is cleaved by SI-induced pollen extracts. This approach revealed three activities (green box):
DEVDase and VEIDase increase between 1–5 h post SI and LEVDase increases later (still increasing at 8 h). DEVDase activity peaks
at 5 h and has been shown to be required for DNA fragmentation, which can be prevented by the DEVDase inhibitor Ac-DEVD-CHO.
These activities are optimal at pH 5. The caspase-like activities, which do not have any activity at normal cytosolic pH, need the pollen
cytosol to acidify; this has been shown to occur in the first few hours of SI (rainbow box). Several components appear to be integrated
in signalling to SI-mediated cell death. Central to this is the cytoskeleton (yellow boxes). SI-stimulates actin depolymerization, which
causes inhibition of pollen tube growth. Low concentrations of Latrunculin B (Lat B) also inhibits pollen tube tip growth (orange box).
SI triggers much higher levels of depolymerization than that required to inhibit pollen tube growth and higher concentrations of LatB
that mimic this triggers activation of a DEVDase activity (measured with the Ac-DEVD-AMC substrate) and DNA fragmentation that is
prevented by pretreatment with Ac-DEVD-CHO and not Ac-YVAD-CHO. Moreover, low concentrations of actin stabilizing Jasplakinolide
(Jasp) can counteract and alleviate SI- or Lat B-induced DNA fragmentation, presumably by lowering the level of actin depolymerization.
Subsequently, in SI, F-actin aggregates form highly stable punctate F-actin foci that are resistant to depolymerization and are associated
with the actin-binding proteins (ABPs) actin-depolymerizing factor (ADF), and cyclase-associated protein (CAP; yellow box). High
concentrations of Jasp can also trigger DNA fragmentation which is alleviated by pretreatment with Ac-DEVD-CHO and not Ac-YVADCHO. The formation of stable F-actin foci may be analogous to the stabilization using Jasp. Caffeine, which inhibits pollen tube growth
without affecting the cytoskeleton, does not trigger DNA fragmentation. Together, these data implicate actin dynamics in modulating
the DEVDase activity. SI also triggers microtubule depolymerization. This can be mimicked by use of the microtubule depolymerizer,
oryzalin. Actin depolymerization using LatB triggers microtubule depolymerization but not vice versa, suggesting that SI-induced
F-actin depolymerization signals to microtubule depolymerization. The tubulin-stabilizing drug, taxol, alleviates SI-induced Ac-DEVDAMC cleavage, implicating a requirement for tubulin depolymerization for the DEVDase activation. Disrupting microtubule dynamics
alone using oryzalin does not trigger increased cleavage of the DEVDase substrate Ac-DEVD-AMC: this suggests that both actin
and microtubule depolymerization are required for DEVDase-mediated SI-induced PCD. Evidence for involvement of a SI-activated
Papaver self-incompatibility, ROS, NO, and PCD | 1337
Fig. 3. SI stimulates increases in reactive oxygen species (ROS) and nitric oxide (NO) in incompatible pollen tubes. (A) ROS detected
in pollen tubes by the live-cell probe chloromethyl-2′7′-dichlorodihydrofluorescein diacetate acetyl ester at time points 0, 7, 9, 12, 14,
20 min after SI induction. (B) NO detected in pollen tubes by the live-cell probe DAF2-DA at time points 0, 23, 27, 31, 53 and 63 h after
SI induction. Bars = 10 μm. Reproduced from Wilkins KA, Bancroft J, Bosch M, Ings J, Smirnoff N, Franklin-Tong VE. 2011. Reactive
oxygen species and nitric oxide mediate actin reorganization and programmed cell death in the self-incompatibility response of Papaver.
Plant Physiology 156, 404–416, by kind permission of the American Society of Plant Biologists (www.plantphysiol.org).
the SI-induced ROS signatures showed that the increases in
ROS as a wave in the shank of the pollen tube and that the
source of the ROS appeared to be intracellular organelles,
as ‘hot spots’ (Fig. 3A). This suggests that at least some of
the SI-induced ROS may originate from organelles. Thus,
there might be several sources of SI-induced ROS. There is
good evidence that the intracellular sites of ROS production influence its signalling role (Laloi et al., 2004; Foyer
and Noctor, 2005). SI results in inhibition of tip growth
(and presumably tip-ROS is lost during SI, although this
has not been directly ascertained) and stimulates increases
in ROS in the ‘shank’ of the pollen tube. This is a very different response to that observed in root hairs in response to
NOD factors that occur at the tip (Cárdenas et al., 2008).
This supports the concept that distinct spatial-temporal
‘signatures’ can generate signal specificity and (Foyer and
Noctor, 2005) implicates different sources for ROS and
NO, as well as different enzymes for PCD and tip growth
inhibition. This provides an interesting area to pursue in
the future, which would help achieve a better understanding of how the growth ROS/NO signals and the SI-induced
ROS/NO signals involved in PCD achieve their very different biological outputs in a single cell.
How is the pollen tube cellular disassembly
achieved?
One area that has been neglected to date is the question
of how the pollen tube is disassembled during SI-induced
death. Electron microscopy studies have shown that organelles are reorganized within 1 h of SI induction (Geitmann
et al., 2004). In contrast to control pollen grains (Fig. 5A),
SI-induced pollen grains had striking curved endoplasmic
reticulum cisternae that were wrapped around empty structures that may be the vacuole (Fig. 5B) and mitochondria
p56-MAPK comes from use of the MEK inhibitor U0126 and its negative analogue U0124. Although U0126 inhibits pollen tube growth
and p56-MAPK activity, it does not affect pollen viability. These drugs provided evidence that p56-MAPK activation is required for the
activation of a DEVDase, DNA fragmentation, and cell death. SI stimulates transient increases in ROS and later NO. Pretreatment of
pollen tubes with either the NADPH oxidase inhibitor diphenyliodonium or the NO scavenger cPTIO and then stimulating SI showed
that both were required to reduce the SI-induced DEVDase activity. This suggests that ROS and NO act in concert or tandem to
signal to activate SI-induced PCD through the activation of a DEVDase activity. Moreover, these drugs alleviated the formation of the
characteristic punctate F-actin foci. This implicates ROS and NO signalling for the formation of these F-actin foci.
1338 | Wilkins et al.
Fig. 4. SI-induced reactive oxygen species (ROS) and nitric oxide (NO) are upstream of caspase-3-like activities and actin foci
formation. (A) DEVDase activity in Papaver pollen extracts was measured using Ac-DEVD-AMC and is shown as a percentage of
untreated pollen tube activities (white bar). SI induction (black bar) stimulated activity. Pollen tubes treated with an NADPH inhibitor,
diphenyliodonium, and/or the NO scavenger c-PTIO (dotted bars) had slightly increased DEVDase activity, while pollen tubes which had
these as a pretreatment before SI induction (crosshatched bars) had reduced DEVDase activity. Values are mean ± SE (n = 5). (B) Fixed
pollen tubes were stained with rhodamine–phalloidin to examine actin organization. Pollen was treated with tempol (a ROS scavenger)
and/or the NO scavenger c-PTIO, with PrsS (SI) or combined. Pollen actin cytoskeleton morphology was scored into one of three
categories; filaments, foci, or intermediate (combination of both foci and filaments; Fig. 1). Values are % (n = 70 of three independent
replicates). Pollen treated with ROS and NO scavengers prior to SI induction resulted in a significant reduction in actin foci formation.
Reproduced from Wilkins KA, Bancroft J, Bosch M, Ings J, Smirnoff N, Franklin-Tong VE. 2011. Reactive oxygen species and nitric
oxide mediate actin reorganization and programmed cell death in the self-incompatibility response of Papaver. Plant Physiology 156,
404–416, by kind permission of the American Society of Plant Biologists (www.plantphysiol.org).
were greatly altered (Fig. 5C). By 3–4 h, the organelles were
almost indistinguishable but there did not seem to be wholesale lysis (Fig. 5D; Geitmann et al., 2004), even though cytosolic pH was 5.5 by this time. Subsequent studies showed
that, within 30 min of SI induction, the endoplasmic
reticulum configuration had altered and it appeared to be
wrapped around other organelles and vacuoles. Organelles
appeared to be engulfed in what appeared to be vacuolar
compartments within 3 h of SI induction (NS Poulter and
VE Franklin-Tong, unpublished data; Fig. 5D). These studies hint at the possibility of autophagy being involved.
Recruitment of signalling for SI events in
other species
Recent studies have involved introduction of PrpS, the
Papaver pollen S-determinant into self-compatible A. thaliana, a model plant. Exposing transgenic A. thaliana pollen
expressing PrpS-GFP to recombinant Papaver PrsS protein
resulted in the hallmark features of incompatible Papaver
pollen being triggered, including inhibition of pollen tube
growth, formation of F-actin foci, and increases in caspase3-like/DEVDase activity after addition of recombinant PrsS
(de Graaf et al., 2012). This demonstrated PrpS functions
as an S-determinant and is capable of triggering PCD when
transferred into a self-compatible species from a distantly
related genus. It is well established that these two SI systems
evolved independently (Allen and Hiscock, 2008) and all the
available evidence indicates that Arabidopsis does not possess
the P. rhoeas S-determinants, so it is unlikely that A. thaliana
possesses genes that have operated in an ancestral form of
Papaver-like SI, although formally this cannot be discounted.
This finding is notable because the evolutionary distance
between the Ranunculales and Brassicales is very large, estimated at ~140 million years (Bell et al., 2010), and because
Papaver and Arabidopsis lack a common SI system. Despite
this, transgenic A. thaliana pollen was not only rejected,
but also displayed hallmark features of the Papaver PCD
responses in incompatible pollen (de Graaf et al., 2012). Our
data provide good evidence that A. thaliana possesses proteins that can be recruited to form new PCD signalling networks and targets that are used for a function that does not
normally operate in this species. Indeed, as the features of a
‘Papaver-like’ SI-induced PCD response, which have not been
observed in the Brassica-type SI response, this suggests that
the PrpS–PrsS interaction is sufficient to specify a particular
downstream signalling network to obtain this PCD outcome.
Summary and future perspectives
ROS and NO signalling form an integral part of SI-PCD
(see this issue). Here, we have described evidence that ROS
and NO signalling is central to SI-induced alterations in the
actin cytoskeleton and the activation of a caspase-3-like/
DEVDase activity, which suggests that they play a key role
in SI-induced PCD. We have begun to tease out the relationship between ROS and NO and their role in triggering
Papaver self-incompatibility, ROS, NO, and PCD | 1339
Fig. 5. Electron microscopy reveals major changes in Papaver
rhoeas pollen after SI induction. (A) Typical control pollen grain
after 1 h in vitro; electron-dense mitochondria (arrow heads) have
densely arranged cristae, vacuoles, and dense vesicular bodies
(arrows); bar = 2 μm. (B) Pollen grain 1 h post SI induction;
organelles are reorganized and striking curved endoplasmic
reticulum cisternae are wrapped around empty structures that
may be vacuoles; bar = 0.5 μm. (C) Pollen grain 1 h post SI
induction; mitochondria (arrow) are swollen and have reduced
cristae; the darker, distinctive structures may be dilated Golgi
bodies (arrowhead); bar = 0.5 μm. (D) Pollen grain 3 h post SI
induction; multiple organelles are engulfed; bar = 0.5 μm. Images
A, B, and C are reproduced from Geitmann A, Franklin-Tong VE,
Emons AC. 2004. The self-incompatibility response in Papaver
rhoeas pollen causes early and striking alterations to organelles.
Cell Death and Differentiation 11, 812–822, by kind permission of
Nature Publishing Group (www.nature.com/cdd).
and executing PCD in incompatible pollen. The finding that
Papaver SI can be induced by Papaver PrsS–PrpS interaction in transgenic A. thaliana carrying PrpS suggests that
the Papaver SI system either can recruit ‘common’ endogenous signalling components in A. thaliana pollen and
utilize them to signal using a Papaver-like PCD response,
via actin and caspase-3-like/DEVDase activities, or that it
activates an existing PCD pathway in A. thaliana. The latter
is supported by the fact that different cell death processes
are known to share signalling pathways (van Doorn et al.,
2011). However, it is surprising that it produces a Papaverlike PCD response that has not (so far) been described for
any Arabidopsis PCD response. We believe that this may
work because the signals (e.g. Ca2+, phosphorylation, ROS,
NO) and targets for Papaver SI (e.g. the actin cytoskeleton)
are ‘universal’, ancient, and perhaps common to all eudicots. This has implications for our perspective of evolution
of signalling networks both between different SI systems
and, more generally, potentially in PCD and other signalling responses.
The finding that Papaver SI-PCD can be reproduced by
stimulating interaction of PrpS-GFP in transgenic A. thaliana pollen expressing PrpS-GFP opens up new approaches
for the future. Use of the Arabidopsis system expressing
PrpS-GFP may allow use of the toolbox of analytical tools
and resources available for Arabidopsis, including genetic
approaches, with the availability of knockout lines, to analysing how Papaver SI-induced PCD is integrated. Postulated
parallels between SI and other well-characterized PCD
responses, such as plant–pathogen resistance (Hodgkin et al.,
1988; Sanabria et al., 2008), which also use ROS in an oxidative burst, have been made. Like the SI response, hypersensitive response PCD has been associated with increases
in [Ca2+]cyt, ROS, and microtubule depolymerization (Levine
et al., 1997; Lecourieux et al., 2002; Rentel et al., 2004) and
use of Arabidopsis knockout lines could provide an approach
to analysing this possibility.
Another area to investigate in the future is to understand
how the source and location of intracellular ROS influences
its signalling role (Laloi et al., 2004; Foyer and Noctor, 2005)
and establish the differences between ROS signals in normal
cells and during PCD. This could help to tease out the role
of specific ROS ‘signatures’. At present, the source of ROS
during SI-PCD is unknown, although both NADPH oxidase and an organelle-based source has been implicated. To
mediate downstream responses, ROS and NO must presumably alter target proteins; these types of studies in plant cells
are in their infancy. However, it is known that NO-mediated
S-nitrosylation of proteins, particularly cysteine residues of
redox-sensitive proteins (Wang et al., 2006; Moreau et al.,
2010), can affect protein activity and so has the potential to
play a key role in regulating cellular events (Lindermayr et al.,
2005). A possible target of interest is actin, as this is a major
target of SI-PCD signalling which utilizes NO/ROS signals
(Wilkins et al., 2011). Although there are virtually no data on
NO signalling to actin in plant cells, actin has been identified
as being both S-nitrosylated and Y-nitrated in Arabidopsis
(Lindermayr et al., 2005; Lozano-Juste et al., 2011). Moreover,
nitrosylation of a single cysteine residue on mammalian actin
can alter actin dynamics (Dalle-Donne et al., 2000) and this
can affect the outcome of NO-induced apoptosis in animal
cells. Despite the known sensitivity of the actin cytoskeleton
to ROS and NO, how they alter the dynamics and organization of F-actin is far from clear, even in animal cell systems. Preliminary investigations aimed at identifying pollen
proteins that are modified by S-nitrosylation, nitration, and
oxidation as a result of SI signalling to PCD are currently
being undertaken.
Looking further into the future, in a translational context,
transfer of the Papaver PrpS and PrsS S-determinants could
possibly, in the longer term, perhaps provide a tractable SI
system for some crop plants, which has long been the goal
of plant breeders. Many self-compatible crop plants could
benefit from improved yields that could be obtained from
generating F1 hybrid plants (which are generally superior,
but expensive to produce). If SI could be harnessed, these
could potentially be made more easily and cheaply. To date,
focus has been on SI relatives of crop plants or species
closely related to crops, as it was thought that transfer of
SI would be limited to close relatives. So far, the only movement of SI systems has been within species or closely related
1340 | Wilkins et al.
species in the same family that share an ancestral SI system.
Our demonstration that interaction of PrsS with A. thaliana
PrpS-GFP pollen to elicit a ‘SI’ response suggests there is
scope for transfer of the Papaver SI system to completely
unrelated crop species. This has implications for helping to
solve food security issues, by allowing breeding of superior
F1 hybrid plants more easily and cheaply. We are currently
exploring whether PrpS-GFP functions in a self-compatible
monocot crop, barley, as a case study to see if transfer of this
SI-PCD system can operate in even more distantly related
angiosperms. Furthermore, assuming that PrpS can be readily functionally expressed in other cell types, our findings
open up the possibility of developing an inducible PCD system in a variety of cell types. If these were tissue-specifically
expressed, this might open up the possibility to be able to
control the death of particular cell types. We are starting to
explore this possibility by seeing if PrpS-GFP can operate a
PCD response in A. thaliana leaf protoplasts, as a test system
to investigate if this SI-PCD system might work in cells other
than pollen and we hope to see how far we can extend this
system. This approach may also provide some answers relating to evolutionary aspects of how this PCD system might
have evolved.
Acknowledgements
Work in the lab of VEF-T has been funded long term by the
Biotechnology and Biological Sciences Research Council
(BBSRC). KAW and N.S.P. were funded by BBSRC PhD
studentships. Other sources of funding include: China
Scholarship Council, Royal Society China Fellowship, and
Wellcome Trust. This work would not have been possible
without the help of long-term collaborators Chris Staiger
and Chris Franklin and the many members of the FranklinTong lab who were involved in these studies.
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