Journal of Experimental Botany

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Journal of Experimental Botany, Vol. 54, No. 380, pp. 141-148, January 1, 2003
© 2003 Oxford University Press

Signals and targets of the self-incompatibility response in pollen of Papaver rhoeas

Received 12 April 2002; Accepted 14 June 2002

Jason J. Rudd^2, and Vernonica E. Franklin-Tong^1,

School of Biosciences, University of Birmingham, Edgbaston B15 2TT, UK

¹ To whom correspondence should be addressed. fax: +44 (0)121 414 5925. E-mail: V.E.Franklin-Tong{at}bham.ac.uk
² Present address: Institut für Pflanzenbiochemie, Weinberg 3, D-06120 Halle/Saale, Germany.

	Abstract

Top Abstract Introduction Alterations in cytosolic free... Phosphorylation cascades in the... p26: a phosphorylated inorganic... A putative MAPK is... Evidence for programmed cell... Discussion References

 
Self-incompatibility (SI) in Papaver rhoeas involves an allele-specific recognition between stigmatic S-proteins and pollen, resulting in inhibition of incompatible pollen. A picture of some of the signalling events and mechanisms involved in this specific inhibition of pollen tube growth is beginning to be built up. This highly specific response triggers a Ca²⁺-dependent signalling cascade in incompatible pollen when a stigmatic S-protein interacts with it. Rapid increases in cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) can now be attributed (at least in part) to Ca²⁺ influx. The rapid loss of the pollen apical Ca²⁺ gradient within 1–2 min is accompanied by the inhibition of pollen tube tip growth. Concomitant with this time-frame, hyper-phosphorylation of p26, a soluble pollen phosphoprotein is detected. Characterization of p26 reveals that it is a soluble inorganic pyrophosphatase, which suggests a possible direct functional role in pollen tube growth. Slightly later, a putative MAP kinase (p52) is thought to be activated. Finally, preliminary evidence that programmed cell death (PCD) may be triggered in this response is described. A key target for these signals, the actin cytoskeleton, has also been identified. In this article the current understanding of some of the components of this signalling cascade and how they are beginning to throw some light on possible mechanisms involved in this SI-induced inhibition of pollen tube growth, is discussed.

Key words: Ca²⁺ signalling, MAPK, phosphorylation, pollen, programmed cell death (PCD), self-incompatibility (SI), signal transduction.

	Introduction

Top Abstract Introduction Alterations in cytosolic free... Phosphorylation cascades in the... p26: a phosphorylated inorganic... A putative MAPK is... Evidence for programmed cell... Discussion References

 
Self-incompatibility (SI) is an extremely important genetically controlled mechanism that regulates the acceptance or rejection of pollen that lands on the stigma of the same species. It is one of the most important devices whereby higher plants prevent inbreeding. In Papaver rhoeas (the field poppy), SI is controlled by a single, multiallelic, gametophytically controlled S locus (Lawrence et al., 1978). Pollen grains carrying S alleles that are genetically identical to those carried by the pistil (incompatible) upon which it lands, are discriminated from genetically different (compatible) pollen and selectively inhibited on the stigma surface, thereby preventing self-fertilization.

Several S alleles of the stigmatic S gene have been cloned (Foote et al., 1994; Walker et al., 1996; Kurup et al., 1998). They encode small (14 kDa), highly polymorphic proteins. These S-proteins are developmentally expressed and secreted by the stigmatic papilla cells. Tip growth of incompatible pollen of P. rhoeas is generally inhibited within a few minutes of encountering the S-proteins. Both stigmatic extracts and recombinant S-proteins have been shown to have S-specific biological activity (Franklin-Tong et al., 1988; Foote et al., 1994). The available data suggest that this is a receptor-mediated response, with the stigmatic S-proteins acting as signal molecules that interact with an (unidentified) S-linked pollen S-receptor. S-proteins have been shown to bind to a pollen membrane-located proteoglycan (SBP, Hearn et al., 1996) that is essential for S-specific pollen inhibition (Jordan et al., 1999). However, whether this is the pollen S-receptor is not known.

The postulated interaction of the pollen S-receptor triggers a signalling cascade(s) in incompatible pollen tubes that results in inhibition of tip growth. A number of signalling components and targets associated with the SI response have been identified in recent years. This signalling cascade is triggered by increases in the cytosolic free Ca²⁺ concentration ([Ca²⁺]_i), which acts as a second messenger (Franklin-Tong et al., 1993). The rapid loss of the pollen apical Ca²⁺ gradient is accompanied by the inhibition of pollen tube tip growth. Concomitant with this time frame, the hyper-phosphorylation of p26, a soluble pollen phosphoprotein (Rudd et al., 1996) and dramatic alterations in the actin cytoskeleton are initiated (Geitmann et al., 2000; Snowman et al., 2000, 2002). The subject of actin as a target for SI signals is reviewed elsewhere in this issue (Staiger and Franklin-Tong, 2003). Slightly later, a putative MAP kinase (p52) is thought to be activated (JJ Rudd, K Osman, S Whitakker, FCH Franklin, VE Franklin-Tong, unpublished results). Finally, there is also preliminary evidence that programmed cell death (PCD) may be triggered quite early in the SI response (Jordan et al., 2000; ND Jordan, P Tiwari, H Ali, VE Franklin-Tong, unpublished data).

Long before molecular studies on SI were initiated, it was speculated that self-incompatibility systems may be rather similar to plant host–pathogen interactions culminating in the hypersensitive response (HR) (Bushnell, 1979; Hodgkin et al., 1988). HR is initiated by a gene-for-gene interaction between a plant encoded resistance gene and a pathogen avirulence gene, whereas the SI response depends upon the interaction of complementary stigmatic and pollen proteins. As data from molecular studies emerge for both of these systems, intriguing and striking similarities between the early signalling events triggering the SI response in P. rhoeas and the HR have been found, for example, the increases in [Ca²⁺]_i, alterations and Ca²⁺-mediated protein phosphorylation. Recent advances in understanding the components of the SI signalling cascade and some of their targets are reviewed here.

	Alterations in cytosolic free Ca2+

Top Abstract Introduction Alterations in cytosolic free... Phosphorylation cascades in the... p26: a phosphorylated inorganic... A putative MAPK is... Evidence for programmed cell... Discussion References

 
A role for cytosolic free [Ca²⁺]_i acting as a second messenger in pollen in the SI response has previously been established using Ca²⁺ imaging (Franklin-Tong et al., 1993, 1995, 1997). The addition of incompatible S-proteins from crude stigmatic extracts and from purified recombinant S-proteins induce transient increases in [Ca²⁺]_i of up to 1.5 µM in the pollen tube ‘shank’, which are triggered within seconds of SI-induction and are sustained for several minutes, peaking at 4–6 min, and then gradually declining until, by 10–12 min, the levels are close to basal levels (Franklin-Tong et al., 1993, 1997). By contrast, no increase occurred upon challenge with compatible or heat-denatured incompatible S-proteins, neither of which inhibits pollen tube growth. This is thought to initiate the SI signalling cascade, which results in a complex set of events that ultimately lead to death of the incompatible pollen. The oscillating apical high [Ca²⁺]_i that is typical of all tip-growing cells, is lost concomitantly with the arrest of tip growth. This is likely to play a role (as yet unknown) in the initial inhibition of pollen tube growth, as high apical [Ca²⁺]_i is always associated with growth. One could speculate that annexins, which are likely to play a role in vesicle fusion and which require high [Ca²⁺] to function (Battey and Blackbourn, 1993; Carroll et al., 1998) would be inactivated and that this would result in inhibition of tip growth.

The localization of the increases in [Ca²⁺]_i stimulated by the SI response was, and remains, surprising. [Ca²⁺]_i elevations are initiated in the ‘shank’ of the pollen tube, approximately 50–100 µm behind the tip, which correlates with the position of the pollen ‘nuclear complex’ (Franklin-Tong et al., 1995). This suggested that intracellular Ca²⁺ pools might be involved. Although it has been shown that both Ca²⁺ increases in pollen tubes cause pollen tube inhibition, and inhibition of pollen tube growth may also be mediated by inositol trisphosphate (Ins[1,4,5]P₃)-induced Ca²⁺ release (Franklin-Tong et al., 1996a, b), there is still have no conclusive evidence of an involvement of an inositide signalling pathway being involved in the SI response. Data have recently been obtained that indicates that there are brief, but reproducible decreases of PtdIns(4,5)P₂ in incompatible pollen (Straatman et al., 2001). This may indicate that InsP₃ might be released. However, further studies are required.

Although imaging data seemed to implicate intracellular release of [Ca²⁺]_i, a key question is whether extracellular Ca²⁺ may be involved. Since it is well established that Ca²⁺ influx is triggered very early in the HR (Zimmermann et al., 1997; Jabs et al., 1997), this possibility has been investigated. To address this question, an ion-selective vibrating probe (Kühtreiber and Jaffe, 1990; Smith et al., 1994) was used to measure changes in extracellular Ca²⁺ fluxes around P. rhoeas pollen tubes. In contrast to commonly held assumptions, good evidence was obtained that Ca²⁺ influx also occurs at the shanks of pollen tubes (in the region 20–100 µm behind the pollen tube tip) in addition to an oscillating Ca²⁺ influx at the apex (Franklin-Tong et al., 2002). Since the shank region of the pollen tube was implicated in the increases in [Ca²⁺]_i in the SI response, it was investigated whether there might be increases in Ca²⁺ influx stimulated by the SI response. A point 50 µm behind the pollen tube tip was chosen, and readings were made before and after the SI challenge. This has provided convincing evidence for Ca²⁺ influx in incompatible pollen tubes. Challenge of incompatible pollen tubes with self-incompatibility (S)-proteins stimulated Ca²⁺ influx at the shank, as shown in Fig. 1. The mean influx was large, representing a 13.6-fold increase (P <0.001) (Franklin-Tong et al., 2002). Temporally, the influxes lasted for a time-period roughly matching that of the increases in [Ca²⁺]_i observed by imaging, suggesting that they could be responsible for triggering these increases. Although some low-level Ca²⁺ influx was detected in compatible challenges, no Ca²⁺ fluxes comparable to the levels measured in the incompatible SI response were detected, thereby demonstrating the S-specificity of the response. This strongly implicates a role for influx of extracellular Ca²⁺ in the SI response.

View larger version (20K): [in this window] [in a new window]   Fig. 1. Evidence for Ca2+ influx triggered in an incompatible SI response. A Ca2+-selective vibrating probe was used to measure Ca2+ flux temporally at the shank region of the pollen tube, 50 µm behind the pollen tube tip. The plots show net Ca2+ flux (pmol cm–2 s–1), with net influx shown as a negative reading. Measurements were made before treatment and after addition of S-proteins (indicated by arrow). (a) A typical example of the response detected in an incompatible SI response. (b) A typical compatible SI reaction. This demonstrates the S-allele specificity of the Ca2+ influxes measured, as the same S-proteins were used, and the only differences are due to different S-alleles carried by the pollen samples (adapted from Franklin-Tong et al., 2002).

 

	Phosphorylation cascades in the SI reaction

Top Abstract Introduction Alterations in cytosolic free... Phosphorylation cascades in the... p26: a phosphorylated inorganic... A putative MAPK is... Evidence for programmed cell... Discussion References

 
Investigations of pollen proteins provide further evidence for activation of signalling cascades mediated by protein kinases in the P. rhoeas SI response. Several cytosolic pollen proteins have been observed to be subject to transient increases in phosphorylation upon challenge with incompatible S-proteins (p26, p68 and p52; Rudd et al., 1996, 1997; JJ Rudd, VE Franklin-Tong, unpublished data). Since their phosphorylation is not stimulated by compatible S-proteins or heat-denatured incompatible S-proteins, this phosphorylation may be regarded as an SI-specific response. Here, recent advances in the preliminary characterization of two of these signalling components are briefly described.

	p26: a phosphorylated inorganic pyrophosphatase

Top Abstract Introduction Alterations in cytosolic free... Phosphorylation cascades in the... p26: a phosphorylated inorganic... A putative MAPK is... Evidence for programmed cell... Discussion References

 
Another candidate for the earliest downstream targets of the SI response identified is probably a calcium-dependent protein kinase (CDPK). This activity was detected by the increased phosphorylation of p26, a cytosolic protein present in pollen in incompatible pollen tubes labelled with ³²P orthophosphate. The increases were within 90 s of challenge with S-proteins (Rudd et al., 1996), so it is likely to be phosphorylated somewhat earlier than this time-point. It has been established that the phosphorylation of p26 in pollen extracts is Ca²⁺-dependent in vitro, and evidence suggests that this is most likely mediated by a CDPK (Rudd et al., 1996).

Using a PCR probe designed from the amino acid sequence from p26, the gene encoding p26 has recently been cloned (JJ Rudd, EM Bell, FCH Franklin, VE Franklin-Tong, unpublished data). Database searches indicate a strong homology (80%) of p26 to soluble inorganic pyrophosphatases from angiosperms. It has therefore been tested whether the p26 recombinant protein has the expected activity of a functional soluble inorganic pyrophosphatase, using a standard assay with pyrophosphate as a substrate (Fiske and SubbaRow, 1925). As expected, shown in Fig. 2, p26 exhibits an absolute dependence on Mg²⁺ for catalytic activity, which is inhibited by Ca²⁺. These are key characteristics of other plant soluble inorganic pyrophosphatases characterized to date (du Jardin et al., 1995; Visser et al., 1998). Biochemical analysis of recombinant p26 therefore demonstrates that, as predicted from its sequence homology, p26 has the activity expected of a soluble inorganic pyrophosphatase, with activity dependent on Mg²⁺ and inhibited by high Ca²⁺. The pyrophosphatase activity in pollen cytosolic extracts has been measured under exactly the same conditions used previously to study phosphorylation of p26 in vitro (Rudd et al., 1996). These data indicate that under conditions of nM levels of Ca²⁺ that allow phosphorylation of p26, a large reduction in pyrophosphatase activity is detected (JJ Rudd, EM Bell, FCH Franklin, VE Franklin-Tong, unpublished data). Since p26 pyrophosphatase activity is inhibited at raised [Ca²⁺] that is physiologically relevant, it suggests that its activity is almost certainly altered in the SI response, where [Ca²⁺]_i is increased to the micromolar level for several minutes.

View larger version (22K): [in this window] [in a new window]   Fig. 2. Recombinant p26 has pyrophosphatase activity. The pyrophosphatase activity of the p26 recombinant protein was assayed for 30 min at 37 °C, pH 7.0 using 1 mM Na4P2O7 as a substrate, and the amount of inorganic phosphorous (Pi) released was measured according to Fiske and SubbaRow (1925). The requirement for divalent cations (Mg2+, Mn2+ and Ca2+) was tested to establish if p26 exhibited the characteristics of an inorganic pyrophosphatase. The data (four independent assays ±SD) show that the catalytic activity of p26 was strictly Mg2+-dependent, and other divalent cations (Ca2+ and Mn2+) cannot substitute for Mg2+. Furthermore, as expected, the pyrophosphatase activity is inhibited by high [Ca2+ ].

 
From what is known about the consequences of inhibition of pyrophosphatase activity in other species, it is proposed that this will almost certainly contribute to the inhibition of pollen tube tip growth. This provides the basis of a new model for what is believed to be a ‘fail-safe’ mechanism for SI in this species. The hypothesis is briefly explained here. Soluble inorganic pyrophosphatases are involved in the hydrolytic conversion of inorganic pyrophosphate to inorganic orthophosphate, and often drive cellular biosynthetic reactions (Cooperman et al., 1992). They therefore play an important role in both generating ATP and the biopolymers required for making new membranes and cell walls. It is proposed that the phosphorylation of p26 during the SI response is likely to result in the inactivation or reduction in its pyrophosphatase activity. This would almost certainly result in the depletion of biopolymers, such as long chain carbohydrates and proteins, that contribute to pollen tube membranes and cell walls, and a consequent inhibition of tip growth. However, since phosphorylation is generally reversible, it is possible that the putative consequent inhibition of tip growth may be a temporary measure to ensure the relatively rapid arrest of tip growth. However, experimental data need to be collected in order to test whether this model relating to p26 function is correct.

	A putative MAPK is activated in the SI response

Top Abstract Introduction Alterations in cytosolic free... Phosphorylation cascades in the... p26: a phosphorylated inorganic... A putative MAPK is... Evidence for programmed cell... Discussion References

 
At a simplistic level, the SI response might be thought of as a stress response. Since several stress responses, including the HR, in plants are now known to involve the activation of mitogen-activated protein kinase (MAPK) homologues (Zhang and Klessig, 1997; for recent reviews see Innes, 2001; Nürnberger and Scheel, 2001), a possible role for MAPKs in the SI response has been investigated. Data collected indicate that another target downstream of the Ca²⁺ signals triggered by the SI response is a putative MAPK, p52 from poppy pollen.

In-gel kinase assays, using myelin basic protein (MBP), a classic substrate for MAPK activity, have been used to identify stress-induced MAPKs from plants, such as SIPK (Zhang and Klessig, 1997). Using this type of assay, preliminary evidence for activation of a putative MAPK activity in incompatible pollen undergoing the SI response has been obtained (JJ Rudd, K Osman, S Whitakker, FCH Franklin, VE Franklin-Tong, unpublished results). A 52 kDa pollen protein, which has been named p52, is activated in a SI-specific manner. When pollen is challenged with incompatible stigmatic S-proteins, an increase in this putative MAPK activity is detected, as shown in Fig. 3. This is not detected when pollen tubes are challenged with biologically inactive S-proteins or with compatible S-proteins (data not shown). Activation of p52 has also been detected in incompatible in vivo pollinations, which gives a further direct link between the functional biology of the SI system and the activation of this p52 protein kinase.

View larger version (42K): [in this window] [in a new window]   Fig. 3. A putative MAPK activity is stimulated in incompatible pollen. Protein extracts were made from pollen undergoing an incompatible SI reaction in vitro. The times at which samples were taken (between 0 and 30 min) is indicated. The samples were used for in-gel protein kinase assays using myelin basic protein (MBP) as a substrate for putative MAP kinase activity. The area of the autoradiograph showing p52 phosphorylation is shown. A marked increase in activity of the p52 putative MBP-kinase is detected, peaking at 10 min.

 
Several pieces of biochemical evidence suggest that p52 is a putative MAPK homologue (JJ Rudd, K Osman, S Whitakker, FCH Franklin, VE Franklin-Tong, unpublished results) and several MAPKs present in poppy pollen are currently being cloned (K Osman, FCH Franklin, VE Franklin-Tong, unpublished data). However, although there is evidence that p52 is downstream of the Ca²⁺ signals triggered by the SI response, at present there are no data that indicate what the exact role of this pathway is, or what the ultimate target of the MAPK signal is. However, the activation of p52 appears to peak at 10 min after SI induction (JJ Rudd, K Osman, S Whitakker, FCH Franklin, VE Franklin-Tong, unpublished results), which is after the initial inhibition of pollen tube growth. This suggests that it does not play a direct role in early inhibition events, but instead may play a role in later, downstream events. Interestingly, recent data implicate a possible role for activation of MAPKs in the induction of programmed cell death (PCD). A stress-induced MAPK (SIPK) is strongly implicated in PCD since activation of SIPK alone has been demonstrated to be sufficient to induce HR-like cell death PCD in tobacco leaves (Yang et al., 2001; Zhang and Liu, 2001). Further evidence comes from demonstrations that the activation of endogenous Arabidopsis MAPKs by the induction of AtMEK4 and AtMEK5, two Arabidopsis MAPKKs, results in HR-like cell death (Ren et al., 2002).

	Evidence for programmed cell death (PCD)

Top Abstract Introduction Alterations in cytosolic free... Phosphorylation cascades in the... p26: a phosphorylated inorganic... A putative MAPK is... Evidence for programmed cell... Discussion References

 
A well-established feature of the HR is the triggering of PCD (Greenberg et al., 1994; Pennell and Lamb, 1997; Heath, 2000). Studies to examine if there are further parallels between the HR and the SI response in poppy pollen have investigated if PCD might play a role in irreversible inhibition of pollen tube growth. One of the key effector proteins involved in the apoptotic signalling cascade is caspase-3, which is activated by cleavage by caspase-9. One of the key targets for caspase activity is nuclear DNA, which is cleaved at regular intervals to produce oligosomal DNA fragments. This is considered a key characteristic of PCD, and Fragment End Labelling (FragEL), which is widely used as a diagnostic tool for PCD has provided good evidence that nuclear DNA degradation occurs in plant cells undergoing the HR (Ryerson and Heath, 1996; Wang et al., 1996).

The FragEL assay has recently been used to establish that nuclear DNA fragmentation occurs in an S-specific manner (Jordan et al., 2000). Figure 4 shows a comparison between the level of DNA in compatible and incompatible pollen challenged with S-proteins, clearly demonstrating that this hallmark feature of PCD is triggered specifically by SI induction in incompatible pollen tubes. Nuclear DNA fragmentation is considered to be one of the later events of PCD, and observations confirm that it is only detectable several hours after SI induction. It has also been demonstrated that treatment of pollen with mastoparan (which increases [Ca²⁺]_i in pollen tubes; Franklin-Tong et al., 1996b) results in DNA fragmentation (Jordan et al., 2000). Since there is good evidence for Ca²⁺ signalling being involved in the induction of PCD in the HR in plant cells (Levine et al., 1997), it suggests that the Ca²⁺ signals induced by SI might also trigger PCD in pollen. Although these data suggest that PCD might be induced in incompatible pollen, it is also possible that the DNA degradation may be indicating necrosis, which is a very different phenomenon. Therefore, other pieces of evidence must be collected before it can definitively be claimed that PCD is triggered in the SI response.

View larger version (42K): [in this window] [in a new window]   Fig. 4. S-specific DNA fragmentation occurs in incompatible pollen. S-proteins were added to growing pollen tubes to give compatible and incompatible SI responses. Pollen tubes were fixed at the times indicated and assayed for nuclear DNA fragmentation using FragEL. The histograms show that the percentage of FragEL-positive nuclei detected in incompatible pollen (white bars) increased over time to over 70% at 14 h after SI induction. There were virtually no FragEL-positive nuclei detected in compatible pollen (black bars) after the same time period. This clearly shows the S-specificity of the DNA fragmentation induced (from Jordan et al., 2000).

 
One problem with the current understanding of PCD in plant cells is that although several key features of PCD have been identified, some of the key regulatory genes do not appear to be present in plants, despite the complete DNA sequence for Arabidopsis being available (Arabidopsis Genome Initiative, 2000; Lam et al., 2001). For example, DNA fragmentation should theoretically involve a caspase-3-like activity, but no caspase homologues have been identified in plants. This means that either these very highly conserved genes are not present, or that analogous genes may operate in plants. Despite this, the accumulated evidence for PCD in plant cells is good and, somewhat surprisingly, there is evidence for a caspase-like activity being activated in the HR (D’Silva et al., 1998; Richael et al., 2001).

Preliminary data have recently been obtained that provide convincing evidence for a possible role for PCD in the SI response in P. rhoeas. Tetra-peptide caspase inhibitors, such as Ac-DEVD-CHO, are commonly used to block PCD in animal cells and, more recently, have been demonstrated to inhibit pathogenesis in plant cells (Richael et al., 2001). Ac-DEVD-CHO was used successfully to block the DNA fragmentation normally found in incompatible pollen tubes (ND Jordan, P Tiwari, H Ali, VE Franklin-Tong, unpublished data). This suggests that SI-induced DNA fragmentation could be a consequence of a caspase-like/DEVDase activity. This implicates the presence of not only a caspase-like activity triggered by SI in incompatible poppy pollen, but also a target for this activity, which must have sufficient homology to be recognized by the DEVD motif for this peptide inhibitor to be effective. It also suggests that a caspase-like activity plays an active role in the SI response. Furthermore, since incompatible pollen tubes pre-treated with DEVD achieved approximately the same length as compatible pollen tubes (ND Jordan, P Tiwari, H Ali, VE Franklin-Tong, unpublished data), the activation of this caspase-like activity must be quite early in the signalling cascade for the inhibition effect of self-incompatibility to be reversed. These data therefore suggest that PCD is triggered by the SI response in incompatible pollen.

	Discussion

Top Abstract Introduction Alterations in cytosolic free... Phosphorylation cascades in the... p26: a phosphorylated inorganic... A putative MAPK is... Evidence for programmed cell... Discussion References

 
A model for self-incompatibility in P. rhoeas
A working model for SI in P. rhoeas, based on current data, is described here (Fig. 5). It is proposed that stigmatic S-proteins act as signalling molecules, interacting with at least one receptor molecule on the surface of pollen tubes. SBP has previously been proposed potentially to act as an accessory receptor to an S-allele-specific receptor (Hearn et al., 1996). However, it may be the S-receptor, though there is no firm evidence for this. An interaction between the S-receptor and its matching S-protein stimulates an incompatible reaction. This triggers an intracellular signalling cascade(s) involving Ca²⁺ acting as a second messenger (Franklin-Tong et al., 1993).

View larger version (52K): [in this window] [in a new window]   Fig. 5. Current model of signals and targets involved in the SI response in P. rhoeas. Induction of an incompatible SI signalling cascade in pollen that interacts with a matching stigmatic S-protein triggers several rapid events, most of which are likely to contribute to the inhibition of pollen tube growth. These include: increases in [Ca2+]i in the ‘shank’ region and loss of the apical high [Ca2+]i gradient; alterations to the actin cytoskeleton; the phosphorylation of soluble pollen proteins including p26, with a postulated resultant reduction in pyrophosphatase activity. Most of these early events are likely to contribute to the rapid arrest of incompatible pollen tube growth. Later, further changes in the actin cytoskeleton are detected and a putative MAPK is activated; their roles are not known, but MAPKs are known to be involved in stress-induced responses. DNA fragmentation and other more recent data suggest that a PCD signalling pathway is triggered, suggesting that later events may ‘commit’ incompatible pollen to die. Thus, it appears that there are several ‘fail-safe’ mechanisms involved in pollen tube inhibition (adapted from Franklin-Tong et al., 2002).

 
A transient increase in cytosolic [Ca²⁺]_i, involving Ca²⁺ influx is generated in the pollen tube shank, accompanied by a loss of the apical gradient of Ca²⁺ and the rapid arrest of pollen tube growth (Franklin-Tong et al., 2002). The increases in [Ca²⁺]_i leads to rapid phosphorylation of p26 via a CDPK (Rudd et al., 1996). It is suggested that, since p26 has homology to an inorganic pyrophosphatase, it is highly likely to contribute to an arrest (perhaps temporary) of biosynthesis of cell membrane and wall material essential for growth. This provides a sensible (although so far, unproven) method to prevent pollen tube synthesis and therefore growth.

Dramatic alterations in the actin cytoskeleton are also stimulated very early (Geitmann et al., 2000) and recent data indicate that this involves F-actin depolymerization (Snowman et al., 2002; Staiger and Franklin-Tong, 2003). Tip growth in general is highly dependent upon the actin cytoskeleton and analysis of the effects of latrunculin B, an actin depolymerizing agent, demonstrate that only small amounts of actin depolymerization are sufficient to perturb tip growth (Gibbon et al., 1999). The actin SI response appears to be rapid enough to cause the arrest of growth. However, the level of depolymerization is surprisingly large and it is sustained for several hours. This hints that other events, subsequent to the initial rapid inhibition of tip growth, may be stimulated during SI. This is discussed further in Staiger and Franklin-Tong (2003).

A MAP kinase (p52) is also activated downstream of the Ca²⁺ signals; however, its function is unknown. It is activated after pollen tube arrest has been effected, which suggests that it probably has another function than tip growth inhibition per se. Thus, although pollen tube tip growth is arrested very rapidly, since several SI-specific events occur after this, it suggests that further events are required to make this inhibition irreversible. Furthermore, a PCD-like mechanism appears to be triggered quite early in the SI response, and could be responsible for making sure that growth does not resume. It is proposed that a critical ‘decision-making’ phase is required, after which tip growth becomes irreversibly inhibited. In support of this, ‘wash-out’ experiments (removing S-proteins at various time-points after challenge) result in only temporary inhibition of pollen tube growth if carried out up to 9 min after SI induction (G Davidson, VE Franklin-Tong, unpublished data). Exactly which signals and components are involved in this ‘belt and braces’ approach to ensure that incompatible pollen is stopped irreversibly is not yet known, and further studies are required to see if the MAPK activation and proposed PCD events function in this way. However, it seems that a complex set of controls has been utilized to make sure that incompatible pollen does not start to grow again.

	Acknowledgement

 
The Biotechnology and Biological Research Council (BBSRC) supported this research.

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Top Abstract Introduction Alterations in cytosolic free... Phosphorylation cascades in the... p26: a phosphorylated inorganic... A putative MAPK is... Evidence for programmed cell... Discussion References

 
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