JXB Advance Access originally published online on September 14, 2007
Journal of Experimental Botany 2008 59(3):481-490; doi:10.1093/jxb/erm195
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Focus Review Paper |
Self-incompatibility in Papaver: signalling to trigger PCD in incompatible pollen
School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK
* To whom correspondence should be addressed. E-mail: M.Bosch{at}bham.ac.uk
Received 1 May 2007; Revised 18 July 2007 Accepted 24 July 2007
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Abstract |
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Top
Abstract
IntroductionSexual reproduction in higher plants uses pollination, involving interactions between pollen and pistil. Self-incompatibility (SI) prevents self-fertilization, providing an important mechanism to promote outbreeding. SI is controlled by the S-locus; discrimination occurs between incompatible pollen, which is rejected, while compatible pollen can achieve fertilization. In Papaver rhoeas, S proteins encoded by the pistil part of the S-locus interact with incompatible pollen to effect rapid inhibition of tip growth. This self-incompatible interaction triggers a Ca2+-dependent signalling cascade. SI-specific events triggered in incompatible pollen include rapid depolymerization of the actin cytoskeleton; phosphorylation of soluble inorganic pyrophosphatases, and activation of a MAPK. It has recently been shown that programmed cell death (PCD) is triggered by SI. This provides a precise mechanism for the specific destruction of ‘self’ pollen. Recent data providing evidence for SI-induced caspase-3-like protease activity, and the involvement of actin depolymerization and MAPK activation in SI-mediated PCD will be discussed. These studies not only significantly advance our understanding of the mechanisms involved in SI, but also contribute to our understanding of functional links between signalling components and initiation of PCD in a plant cell. Recent data demonstrating SI-mediated modification of soluble inorganic pyrophosphatases are also described.
Key words: Actin cytoskeleton, caspase, MAP kinase, Papaver rhoeas, pollen tube inhibition, programmed cell death (PCD), self-incompatibility
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Introduction |
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Signal perception and integration of signals is crucial for cellular responses. The self-incompatibility (SI) response in pollen of Papaver rhoeas L. (the field poppy) provides a model system to investigate signalling in plant cells. SI prevents self-fertilization, providing an important mechanism to prevent inbreeding through specific recognition and rejection of incompatible pollen. SI is controlled by the S-locus; discrimination occurs between incompatible pollen, which is rejected, while compatible pollen can achieve fertilization. Distinct molecular and genetic mechanisms have evolved that control SI in different plant species. The reader is referred to McClure and Franklin-Tong (2006) for a recent review on the genetics of SI and other SI systems. Several features of SI are of interest, including the identity and nature of the receptor–ligand interaction, the intracellular signals triggered by this interaction, and mechanisms recruited to inhibit pollen tube growth.
In Papaver, S proteins encoded by the pistil part of the S-locus interact with incompatible pollen to effect rapid inhibition of tip growth. It is assumed that these small 
15 kDa S proteins (Foote et al., 1994) act as signalling ligands by interacting with the pollen receptor in an S-specific manner. It has recently been established that programmed cell death (PCD), involving a caspase-3-like activity, is triggered by SI (Thomas and Franklin-Tong, 2004). This provides a precise mechanism for the specific destruction of ‘self’ pollen. Upon an incompatible interaction, the SI response is initiated by the S-specific triggering of a Ca2+-dependent signalling cascade in incompatible pollen (that will be inhibited and killed); compatible pollen, in contrast, does not elicit any increases in [Ca2+]i (Franklin-Tong et al., 1993) and is allowed to grow normally to achieve fertilization. In addition to the PCD signalling network, a number of SI-specific events have been identified that are triggered in incompatible pollen, including rapid depolymerization of the F-actin cytoskeleton (Geitmann et al., 2000; Snowman et al., 2002); activation of a mitogen activated protein kinase (MAPK), p56 (Rudd et al., 2003); and a rapid increase in phosphorylation of two cytosolic pollen proteins (Rudd et al., 1996), which were recently identified as soluble inorganic pyrophosphatases (sPPases), Pr-p26.1a and Pr-p26.1b (de Graaf et al., 2006).
Apoptosis or PCD is a highly conserved process that is used to remove unwanted cells in eukaryotes. An initiation phase involves signalling cascades that prepare the cell for entry into the execution phase, bringing on an organized degradation of the cell resulting in death. In animal cells caspases, a specific family of cysteine proteases, play a critical role in PCD. These enzymes are activated rapidly during apoptosis and cleave target proteins after an aspartate residue (Riedl and Shi, 2004). Although PCD in plants is less well studied compared with animals, it is well established that PCD is triggered by biotic and abiotic stresses (Zhang and Klessig, 2001) as well as during plant development (Kuriyama and Fukuda, 2002). Many of the features characteristic of PCD have been identified in plants; (see for example, Levine et al., 1996; Lam and Pozo, 2000; Danon et al., 2004; van Doorn and Woltering, 2005) and good biochemical evidence for the activation of proteases that exhibit caspase-like activities in plants during PCD exists (see Watanabe and Lam, 2004; Woltering, 2004; Sanmartin et al., 2005, for recent reviews).
We are currently attempting to establish the function of the SI-specific events triggered in incompatible pollen, and to investigate their possible involvement in integrating a co-ordinated signalling response leading to the rapid inhibition of pollen tube growth followed by PCD. Here, the components identified as being involved in SI are reviewed, in particular focusing on the evidence for caspase-3-like activity being triggered by SI in incompatible pollen. Recent progress in establishing links between some of these components is also described. These data constitute the first steps in elucidating how SI signalling is integrated. Briefly, alteration of actin dynamics signals to initiate PCD and preliminary data suggest that the same is true for the p56-MAPK. The sPPases Pr-p26.1a/b are also connected to this signalling network; they exhibit rapid, SI-induced Ca2+-dependent hyper-phosphorylation in incompatible pollen. Since their sPPase activities are inhibited by both Ca2+ and phosphorylation, this is predicted to result in inhibition of pollen tube growth. However, whether they are directly involved in PCD is not yet known.
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SI stimulates a calcium-dependent signalling cascade in incompatible pollen |
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Cytosolic-free calcium ([Ca2+]i) is a key second messenger involved in mediating intracellular signalling in plant cells. Upon the addition of pistil S-proteins, calcium imaging revealed virtually instantaneous increases in [Ca2+]i in incompatible pollen (Fig. 1) while levels remained normal in compatible interactions (Franklin-Tong et al., 1993, 1995, 1997). This not only ascertained that S-proteins act as ligands, but also established that SI triggers a Ca2+-dependent signalling cascade in incompatible pollen. Surprisingly, the [Ca2+]i increases were localized in the ‘shank’ of the pollen tube (Franklin-Tong et al., 1993, 1997). Further studies established that these increases in [Ca2+]i involved influx of extracellular Ca2+ in incompatible pollen (Franklin-Tong et al., 2002). Furthermore, the tip-focused [Ca2+]i gradient, necessary for pollen tube growth, rapidly disappeared (Franklin-Tong et al., 1997), which coincided with cessation of pollen tube growth (Fig. 1). These changes in [Ca2+]i initiate a complex set of events within the incompatible pollen that results in its irreversible inhibition and suicide.
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SI induces programmed cell death (PCD) in incompatible pollen |
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Incompatible pollen is selectively recognized and targeted by SI interactions. Unwanted cells are often removed using cell death mechanisms such as PCD during development or in an immune or pathogen response. It was speculatively investigated whether PCD might be involved in Papaver SI because it was thought this might be a possible mechanism to kill self pollen.
The first hint that PCD might be triggered by SI in incompatible pollen was the observation of DNA fragmentation in incompatible pollen tubes (Fig. 1) (Jordan et al., 2000). Nuclear DNA fragmentation is an important hallmark of PCD and can be measured by labelling the DNA strand breaks with a fluorescent tag. Leakage of cytochrome c (cyt c) from the mitochondria is an early marker for PCD and cell death-associated cyt c release has been observed in plants (Balk et al., 1999; Yao et al., 2004). High levels of cytosolic cyt c were observed within 1 h of SI induction (Fig. 1) (Thomas and Franklin-Tong, 2004), which provided further evidence for SI inducing PCD in incompatible Papaver pollen. The rapidity of this response indicated that PCD is initiated quite quickly.
Since strong evidence for caspase-like activities in plant cells existed (Lam and Pozo, 2000; Schaller, 2004), it was investigated whether SI triggered a caspase-like activity. Caspase-3 is the main executioner protease in mammalian systems, responsible for cleavage of many cellular proteins (Fischer et al., 2003). Tetra-peptide inhibitors are key diagnostic tools for PCD. Caspase-3 has the general recognition sequence DXXD and the caspase-3 inhibitor, DEVD-CHO, is based on this. Inhibition of protease activity using DEVD-CHO implicates the involvement of a caspase-3-like activity. Use of DEVD-CHO to pre-treat incompatible pollen tubes resulted in significantly reduced SI-induced DNA fragmentation. Furthermore, the SI-induced inhibition of pollen tube growth was overcome by this caspase-3 inhibitor and not by the caspase-1 inhibitor peptide YVAD-CHO (Thomas and Franklin-Tong, 2004). These data clearly implicate a caspase-like activity in mediating SI-induced PCD. Use of poly (ADP-ribose) polymerase (PARP), a classic substrate for caspase-3, provided further evidence for an SI-induced caspase-3-like activity. Extracts from incompatible pollen which had been SI-induced exhibited an S-specific PARP-cleavage activity, while extracts from compatible pollen did not. Generation of a 24 kDa PARP cleavage product was accompanied by a corresponding decrease in the amount of uncleaved PARP (116 kDa). Since the PARP-cleavage product was prevented by addition of DEVD-CHO and not YVAD-CHO (Thomas and Franklin-Tong, 2004), this provided compelling evidence that a caspase-3-like PARP-cleavage activity is triggered by SI. More recently, a fluorescent caspase-3 substrate, Ac-DEVD-AMC, has also been used to measure the caspase-3-like/DEVDase activity generated by SI; this activity is inhibited by DEVD-CHO (M Bosch and VE Franklin-Tong, unpublished data). This provides further evidence for a SI-induced caspase-3-like/DEVDase activity.
Together, these data demonstrate that PCD mediated by a caspase-3-like activity is triggered by SI in incompatible pollen (Fig. 1). This provides a neat mechanism to ensure that inhibited pollen does not resume growth. A link between Ca2+ signalling and PCD was provided by treating pollen tubes with mastoparan, which artificially increases [Ca2+]i in pollen tubes (Franklin-Tong et al., 1996). This also triggers DNA fragmentation (Jordan et al., 2000) and cleavage of the caspase-3 substrate, Ac-DEVD-AMC (M Bosch and VE Franklin-Tong, unpublished data), suggesting that increases in [Ca2+]i play a role in signalling to PCD activation.
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The nature of the SI-induced caspase-3-like activity |
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At present, the SI-stimulated caspase-3-like activity is being further characterized. The nature of caspase-like proteases in plant cells is currently the subject of considerable debate as plants have no caspase gene homologues (Arabidopsis Genome Initiative, 2000; Woltering et al., 2002; Sanmartin et al., 2005), although metacaspases which resemble animal caspases (Uren et al., 2000) have been identified in protozoa, fungi, and plants (Uren et al., 2000; Madeo et al., 2002). However, although there are convincing data demonstrating that plant metacaspases are involved in PCD (Madeo et al., 2002; Hoeberichts and Woltering, 2003; Suarez et al., 2004; Bozhkov et al., 2005), they are arginine/lysine-specific cysteine proteases which do not cleave caspase-specific substrates (Vercammen et al., 2004; Watanabe and Lam, 2005).
Despite this, the evidence for caspase-like enzymes involved in PCD in plants is good. Several non-caspase-3-like activities have been identified. For example, a plant VEIDase (caspase-6-like) is activated during embryo development in barley and pine (Bozhkov et al., 2004; Boren et al., 2006). Vacuolar processing enzymes (VPEs) exhibiting a YVADase (caspase-1-like) activity are involved in the plant hypersensitive response (HR) and have been relatively well characterized (Hatsugai et al., 2004; Rojo et al., 2004). VPE is also up-regulated during cell death in association with leaf senescence and lateral root formation (Kinoshita et al., 1999) and early stages of seed development (Nakaune et al., 2005). PCD involving VPE displayed an increase in YVADase activity (Rojo et al., 2004) and YVAD-CHO abolished tobacco mosaic virus-induced PCD. Interestingly, despite a low sequence identity, plant VPEs have several structural properties similar to human caspase-1 (Hara-Nishimura et al., 2005). Plant ‘Saspases’ (serine aspartate-specific proteases) with homology to plant subtilisin-like Ser proteases (Coffeen and Wolpert, 2004) and a TATDase (Chichkova et al., 2004) have also been identified. Thus, although these enzymes probably function in a proteolytic cascade that is functionally equivalent (but phylogenetically distinct) to animal caspases, they are clearly not caspase-3-like (DEVDase) proteases.
DEVDase/caspase-3-like activities, which are closest to that involved in the execution of animal apoptosis, have been identified in several plant systems through cleavage of a caspase-3-specific substrate, Ac-DEVD-AMC (Danon et al., 2004; Thomas et al., 2006). Use of DEVD-CHO tetra-peptide inhibitors that alleviate PCD (del Pozo and Lam, 1998; Richael et al., 2001; Thomas and Franklin-Tong, 2004), implicates this type of activity in mediating PCD in some higher plant systems. However, although there is evidence for the involvement of caspase-3-like enzymes in PCD in plant cells, the proteases involved are poorly defined and no gene encoding such a protease has been cloned to date. Furthermore, plant systems in which caspase-3-like activities have been identified, mostly also exhibited YVADase activities (del Pozo and Lam, 1998; Korthout et al., 2000; Danon et al., 2004). Therefore, the identification of an SI-induced DEVDase activity, without the presence of a YVADase activity in Papaver pollen, is of considerable interest (Thomas and Franklin-Tong, 2004).
The Papaver pollen caspase-like activities stimulated by SI have begun to be characterized using Ac-tetrapeptide-AMC derivatives. This has revealed that SI activates a DEVDase activity and, in addition, a VEIDase activity. There is also evidence that a LEVDase activity is stimulated at a later stage. No evidence was found for any activity typical of a metacaspase (M Bosch and VE Franklin-Tong, unpublished data). The SI-activated caspases are not inhibited by broad spectrum protease inhibitors, as expected (M Bosch and VE Franklin-Tong, unpublished data). The optimum pH for the pollen caspase-like activities and their sensitivity to some of the major cations has also been established. The identification of the caspase-3-like protease is currently being attempted and work has also begun to resolve the temporal features of SI-induced caspase activation in more detail.
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SI targets the pollen actin cytoskeleton |
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It is known that the plant actin cytoskeleton responds to specific stimuli by re-organization and changes in actin dynamics. For example, stomatal guard cells respond to abscisic acid or light, and root hairs respond to Nod factors from Rhizobium bacteria by actin reorganization (Eun and Lee, 1997; Cardenas et al., 1998; de Ruijter et al., 1999). It is well established that the actin cytoskeleton is a major target and effector of signalling cascades in both animal and plant cells (Schmidt and Hall, 1998; Staiger, 2000). The actin cytoskeleton is known to have a critical role in regulating pollen tube growth, which involves targeted vesicle secretion (Gibbon et al., 1999; Vidali and Hepler, 2001). As SI involves rapid inhibition of pollen tube growth, we investigated whether SI stimulated changes to the pollen actin cytoskeleton.
These studies demonstrated rapid alterations in F-actin organization in incompatible, but not in compatible, pollen tubes within 1 min of SI induction. Surprisingly, these changes were not identical to those detected in pollen tubes that just stopped growing or were inhibited by some non-specific means. SI induces very striking alterations to the pollen tube actin cytoskeleton. Within a few minutes the typical longitudinal F-actin bundles largely disappear and the F-actin has a fine, speckled appearance. By 20–30 min F-actin accumulated into ‘punctate foci’ (Geitmann et al., 2000; Snowman et al., 2002). Further studies established that SI induces very rapid, large-scale, and sustained depolymerization of F-actin in incompatible pollen (Fig. 1) (Huang et al., 2004). Actin depolymerization was also induced in pollen tubes with artificially increased [Ca2+]i, suggesting that SI-induced actin depolymerization is triggered by [Ca2+]i increases. This established the actin cytoskeleton as a very early target for the SI signals in incompatible Papaver pollen.
A key mechanism to inhibit incompatible pollen tube growth is likely to be the alteration of the activity of calcium sensitive actin-binding proteins (ABPs) by SI-induced increases in [Ca2+]i. An ABP identified in Papaver pollen that is likely to mediate SI-induced actin depolymerization is PrABP80, which has the properties of a gelsolin and exhibits potent Ca2+-dependent severing activity (Huang et al., 2004). An increase in [Ca2+]i will stimulate the severing activity of PrABP80, and probably that of other ABPs involved, resulting in actin depolymerization (Fig. 2). This almost certainly causes rapid inhibition of pollen tube growth, as transport of vesicles to the tip, which is necessary for pollen tube extension, would be inhibited by actin depolymerization.
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Actin signals to PCD in incompatible pollen |
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As the large-scale, sustained depolymerization of F-actin was far in excess of that required to inhibit tip growth, it was hypothesized that it might have an additional role. Emerging evidence suggested that changes to the actin cytoskeleton can initiate apoptosis in some animal cells and, more recently, in yeast (Janmey, 1998; Korichneva and Hammerling, 1999; Rao et al., 1999; Morley et al., 2003; Gourlay and Ayscough, 2005). Since it has been established that PCD is triggered by SI in incompatible Papaver pollen (Thomas and Franklin-Tong, 2004), a possible role for actin depolymerization in signalling to PCD was investigated. Our approach was to alter the actin polymerization status in pollen tubes using the actin-depolymerizing drug, latrunculin B (LatB) or jasplakinolide (Jasp), which stabilizes actin filaments and stimulates polymerization. PCD was subsequently monitored and it was demonstrated that actin alterations play a functional role in initiating PCD in Papaver pollen.
DNA fragmentation, which was used as a marker for PCD, was stimulated by both LatB and Jasp in a concentration-dependent manner. It was tested whether this LatB- and Jasp-induced DNA fragmentation was mediated by a caspase-3 like/DEVDase activity, as it had previously been shown that SI-induced PCD involved this type of activity (Thomas and Franklin-Tong, 2004). Pollen was pre-treated with either DEVD-CHO or YVAD-CHO for 1 h prior to addition of LatB or Jasp. Pollen tubes pre-treated with DEVD-CHO had significant reductions in levels of DNA fragmentation, compared with controls without pre-treatment. Pre-treatment with YVAD-CHO had no significant effect on the levels of DNA fragmentation induced by LatB or Jasp (Thomas et al., 2006). This suggests that both actin depolymerization and stabilization can stimulate activation of a caspase-3-like enzyme upstream of DNA fragmentation and demonstrates that changes in actin filament dynamics are sufficient to induce PCD in pollen.
It was also established that transient changes in actin filament dynamics could initiate PCD, and that sustained alterations were not required. ‘Washout’ experiments were conducted using LatB treatments to mimic SI-mediated depolymerization. Pollen tubes were treated with LatB for various time points, after which the drug was washed out. The pollen tubes were then left for 8 h, after which the incidence of DNA fragmentation was assessed. F-actin levels were quantified and shown to be reduced in a concentration- and time-dependent manner (Thomas et al., 2006). Importantly, after washouts, F-actin levels returned to similar levels as found in untreated pollen, demonstrating that F-actin was only transiently depolymerized. DNA fragmentation correlated with the duration and extent of actin depolymerization. Reducing F-actin levels to 50% for 10 min resulted in high levels of DNA fragmentation, even though F-actin levels returned to normal after washing (Thomas et al., 2006). This indicates that a 50% reduction in F-actin within this brief time period is sufficient for an irreversible ‘decision-making’ step to be made, pushing pollen into PCD. The levels of DNA fragmentation achieved were very similar to those induced by 8 h LatB. Interestingly, there is good correspondence between this threshold level of actin depolymerization for initiating PCD with LatB and that induced 10 min after SI induction (Snowman et al., 2002). This suggests that PCD is initiated within 10 min of SI. Together, these data provide strong evidence that even quite transient LatB-induced actin depolymerization triggers a caspase 3-like/DEVDase activity upstream of DNA fragmentation (Thomas et al., 2006). Since the activation of caspases is considered to commit the cell to die, this implicates alterations in actin depolymerization in playing a functional role in the PCD cascade in pollen.
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Jasplakinolide counteracts and alleviates Lat B- and SI-induced PCD |
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Having shown that altering actin dynamics stimulates PCD, there was a need to establish whether SI-stimulated PCD is triggered by actin depolymerization. Transient induction of SI for 30 min, followed by a washout and then leaving pollen for 8 h, gave high levels of DNA fragmentation, which was only slightly lower than that induced by SI for 8 h. Thus, interaction of incompatible pollen with S proteins for 30 min was sufficient to trigger PCD. Transient treatments were used to see whether Jasp might ‘rescue’ SI-induced pollen from PCD by counteracting the actin depolymerization. As 10 min SI stimulated a 69% reduction in F-actin levels (Snowman et al., 2002), SI was allowed to progress for 10 min to ensure that a significant level of actin depolymerization had been initiated and then Jasp was added for 20 min in an attempt to counteract the SI-induced depolymerization. This treatment significantly reduced the SI-induced DNA fragmentation, demonstrating that Jasp can alleviate SI-induced PCD (Thomas et al., 2006). This suggests that the actin depolymerization induced by SI plays a functional role in the initiation phase of PCD in pollen tubes. A similar experiment using Jasp to counteract the effect of LatB-induced depolymerization demonstrated that Jasp can counteract the actin depolymerization induced by LatB, and thereby ‘rescues’ pollen from entry into PCD. This clearly demonstrates the involvement of actin polymerization status in initiating PCD in pollen.
Together, these data suggest a key role for the actin cytoskeleton as a sensor of cellular stress in plant cells. Relatively transient, but substantial, F-actin depolymerization can trigger PCD which is mediated by a caspase-3-like activity (Fig. 2). Establishing a causal link between actin polymerization status and initiation of PCD in a plant cell may provide a significant advance in our understanding of how PCD can be triggered in plant cells in general. It also throws light on the mechanisms involved in early SI. Thus, the SI-induced actin depolymerization not only inhibits pollen tube tip growth, but also functions in a PCD signalling network that causes incompatible pollen to commit suicide.
Links between actin dynamics and PCD have previously been reported for animal cells and yeast (Janmey, 1998; Korichneva and Hammerling, 1999; Rao et al., 1999; Morley et al., 2003; Gourlay et al., 2004), but it is not always actin depolymerization that triggers apoptosis. For example, in yeast, actin stabilization triggers apoptosis (Morley et al., 2003; Gourlay et al., 2004). Therefore, some of the mechanisms involved must be fundamentally different. Because of these differences, it has been proposed that it may be the dynamics of actin polymerization that modulates apoptotic signalling cascades. However, how this is achieved is not yet established in any cell type.
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MAPK involvement in SI |
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Ca2+ signals are frequently transduced by protein phosphorylation and mitogen activated protein kinase (MAPK) cascades are thought to act as universal signal transduction mechanisms, connecting diverse signalling cascades in response to a variety of signals and stimuli (Chang and Karin, 2001). MAPKs are serine/threonine kinases that are activated by dual phosphorylation of threonine and tyrosine residues in a TXY motif, via a MAPKKK cascade. MAPK cascades have emerged as key players in plant signalling networks. Arabidopsis has 20 MAPK genes (Ichimura et al., 2002) and plant MAPKs are activated by a variety of stresses (Tena et al., 2001; Zhang and Klessig, 2001). Moreover, an involvement of MAPKs in the activation of defence 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.
It was recently shown that SI induces enhanced activation of a MAPK, which was named p56, as it migrates to 56 kDa on SDS–PAGE (Rudd et al., 2003). This S-specific activation of a MAPK suggested a possible role for MAPK signalling in Papaver SI. Thus, p56 is a good candidate to function as a transducer and co-ordinator of the SI response. There are several lines of evidence showing that p56 is a bona fide MAPK. Immunoprecipitation of p56 using anti-phospho-tyrosine antibodies was specific for SI-induced pollen, and it was demonstrated that the immunoprecipitated pollen proteins have MAPK activity using myelin basic protein, a kinase substrate, in-gel kinase assays (Rudd et al., 2003). It was also shown that activated p56 was sensitive to the MAPK inhibitor, apigenin (Rudd et al., 2003). Furthermore, TEY antibodies, that reliably detect activated MAPKs, only detected activated p56 in incompatible pollen undergoing SI. No p56 activity was detected in extracts from untreated pollen, pollen challenged with compatible S-proteins, or incompatible pollen challenged with heat-denatured (biologically inactive) incompatible S-proteins using either assay.
The rather late timing of the activation of the p56-MAPK, peaking at 10 min after SI induction (Fig. 1), which is after initial arrest of pollen tube growth, suggested that it might be involved in later events. 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), it was investigated whether the p56-MAPK might be involved in signalling to PCD in incompatible pollen. Preliminary evidence was recently obtained for this, using the potent and specific inhibitor of MAPK cascades, U0126, as a tool. This drug has been shown to block MAPK cascades in cell-based assays in both animal and plant cells. For example, elicitor-activation of the salicylic acid-induced protein kinase (SIPK) MAPK in tobacco was inhibited by 100 µM U0126 (Lee et al., 2001). Our preliminary data indicate that U0126 (and not its inactive analogue), prevents activation of the SI-stimulated p56-MAPK and also alleviates SI-induced DNA fragmentation, inhibits SI-induced PARP cleavage and SI-induced caspase-3-like/DEVDase activity, and ‘rescues’ incompatible pollen from loss of viability induced by SI (Li et al., 2007). These data strongly implicate the involvement of a MAPK in SI-mediated loss of pollen viability and signalling to PCD. Since the p56-MAPK is the only MAPK activated by SI, this strongly suggests that it plays a key functional role in signalling to the SI-induced PCD.
Thus, there are preliminary data for a causal link between MAPK activation and initiation of PCD in SI. These new data, together with previously published data (Rudd et al., 2003; Thomas and Franklin-Tong, 2004), suggest that the p56-MAPK activated during SI participates in initiating the early PCD signalling cascade (Fig. 2), committing the pollen tube to die. This may represent a ‘gateway’ through which incompatible pollen must pass to become irreversibly inhibited. As there is evidence that MAPK signalling is also connected to alterations in actin organization (Samaj et al., 2004), it is of considerable interest to investigate whether there is also cross-talk between p56-MAPK and SI-induced actin alterations.
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Another mechanism is involved in SI |
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Surprisingly, there is yet another mechanism employed by Papaver SI to ensure that incompatible pollen is inhibited. This involves calcium-dependent phosphorylation of two soluble inorganic pyrophosphatases (PPases), Pr-p26.1a and Pr-p26.1b (de Graaf et al., 2006). These sPPases exhibit S-specific increases in phosphorylation within 90 s of SI-induction (Fig. 1) (Rudd et al., 1996). It was recently established that both calcium and phosphorylation reduce their sPPase activity (de Graaf et al., 2006). This provides a further novel mechanism to inhibit pollen tube growth, since sPPases are important enzymes involved in regulating biosynthesis in general (Cooperman, 1992; Kornberg, 1962). Pollen tube growth requires extensive biosynthesis of membrane and cell wall components to enable it to grow. Inhibition of sPPase activity causes arrest of pollen tube growth and inorganic pyrophosphate (PPi) levels were increased after SI induction, as predicted if sPPase activity is reduced (de Graaf et al., 2006). Thus, the SI-mediated increases in [Ca2+]i and phosphorylation of the sPPases Pr-p26.1a/b play a key role in regulating pollen tube growth. It is assumed that these effects on wall synthesis mediated by changes in sPPase activity, act synergistically with changes to the actin cytoskeleton, which would directly affect transport of material to and from the pollen tube tip and thus affect growth. However, why this is employed in addition to PCD is a mystery.
In summary, it has been shown that Papaver has a complex network of signalling events which are integrated to contribute to SI-mediated inhibition and death of incompatible pollen tubes through activation of caspase-like activities (Fig. 2). The temporal sequence of events is shown in Fig. 1. It appears that several fundamental processes required for pollen tube growth can be inhibited by SI. Each of these, individually, could stop pollen tube growth and therefore prevent self-fertilization. However, several of these ‘failsafe’ mechanisms are used, presumably to ensure inhibition. A major challenge for the future is to identify and characterize the caspase-3-like protease involved in mediating SI-induced PCD, as well as to investigate some of the key early events mediating SI-induced PCD in more detail. Ultimately it will be important to establish what ‘cross-talk’ between the various components is used during SI signalling to PCD.
Figure 2 summarizes what is known so far about SI signalling and its targets in Papaver pollen. It is assumed that SI involves an interaction of the secreted stigma S-protein with its cognate pollen S-receptor (as yet unidentified). This interaction causes rapid elevation of [Ca2+]i and an influx of extracellular Ca2+ in the pollen tube. This triggers a signalling network to stop pollen tube growth, which is inhibited within a few minutes (comprising ‘phase 1’ of SI; see Fig. 1). The SI signal is transduced by [Ca2+]i, which triggers several downstream events. Inhibition of sPPases by Ca2+-dependent phosphorylation is predicted to result in the inhibition of pollen tube biosynthesis, which will contribute to the inhibition of tip growth. Rapid actin depolymerization is triggered, probably mediated through the co-operative action of several Ca2+-regulated ABPs including PrABP80, resulting in the rapid inhibition of pollen tube growth. Actin depolymerization also acts to initiate PCD, committing the inhibited incompatible pollen to die. PCD comprises ‘phase 2’ of SI (see Fig. 1) and involves cyt c leakage, activation of a caspase-like activities, and, eventually, DNA fragmentation. Activation of the p56-MAPK occurs after tip growth inhibition and there is good preliminary evidence that it signals to PCD.
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Acknowledgements |
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Work in the laboratory of VEF-T is funded by the Biotechnology and Biological Sciences Research Council (BBSRC). We are indebted to the many people involved in generating these data and contributing to our research effort over the years. VEF-T would like to acknowledge her long-term collaborators Chris Staiger and Chris Franklin.
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Abbreviations |
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SI, self-incompatibility; MAPK, mitogen activated protein kinase; PCD, programmed cell death; PARP, poly (ADP-ribose) polymerase; cyt c, cytochrome c; Lat B, Latrunculin B; Jasp, Jasplakinolide; [Ca2+], cytosolic-free calcium concentration.
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