Evidence for DNA fragmentation triggered in the self-incompatibility response in pollen of Papaver rhoeas

Studies of the molecular and biochemical basis of self-incompatibility (SI) in Papaver rhoeas have revealed much about the signalling pathways triggered in pollen early in this response. The aim of the current investigation was to begin to study downstream events in order to elucidate some of the later cellular responses involved in the SI response and identification of the mechanisms controlling the irreversible inhibition of pollen tube growth. We have used the FragEL assay to investigate if there is any evidence for DNA fragmentation stimulated in pollen of P. rhoeas in an S-specific manner. Our data clearly demonstrate that S proteins are responsible for triggering this, specifically in incompatible, and not compatible, pollen. DNA fragmentation was first detected in incompatible pollen tubes 4 h after challenge with S proteins, and continued to increase for a further 10 h. This provides the first evidence, to our knowledge, that this phenomenon is associated with the SI response. We also demonstrate that mastoparan, which increases [Ca2+]i, also triggers DNA fragmentation in these pollen tubes, thereby implicating an involvement of Ca2+ signalling in this process. Together, our data represent a significant breakthrough in understanding of the SI response in Papaver pollen.     

Signal-mediated depolymerization of actin in pollen during the self-incompatibility response

Signal perception and the integration of signals into networks that effect cellular changes is essential for all cells. The self-incompatibility (SI) response in field poppy pollen triggers a Ca(2+)-dependent signaling cascade that results in the inhibition of incompatible pollen. SI also stimulates dramatic alterations in the actin cytoskeleton. By measuring the amount of filamentous (F-) actin in pollen before and during the SI response, we demonstrate that SI stimulates a rapid and large reduction in F-actin level that is sustained for at least 1 h. This represents quantitative evidence for stimulus-mediated depolymerization of F-actin in plant cells by a defined biological stimulus. Surprisingly, there are remarkably few examples of sustained reductions in F-actin levels stimulated by a biologically relevant ligand. Actin depolymerization also was achieved in pollen by treatments that increase cytosolic free Ca(2+) artificially, providing evidence that actin is a target for the Ca(2+) signals triggered by the SI response. By determining the cellular concentrations and binding constants for native profilin from poppy pollen, we show that profilin has Ca(2+)-dependent monomeric actin-sequestering activity. Although profilin is likely to contribute to stimulus-mediated actin depolymerization, our data suggest a role for additional actin binding proteins. We propose that Ca(2+)-mediated depolymerization of F-actin may be a mechanism whereby SI-induced tip growth inhibition is achieved.     

Calcium signalling in pollen of Papaver rhoeas undergoing the self-incompatibility (SI) response

Self-incompatibility (SI) is a genetically controlled system used by many flowering plants to prevent self-pollination. We established, using calcium imaging, that the SI response in Papaver rhoeas L. (poppy) pollen involves a Ca²⁺-mediated intracellular signalling pathway. Here we review what is known about the signalling components and cascades implicated in the SI response in poppy pollen. We present some studies using calcium green (CG-1) that show SI-induced alterations in CG-1 fluorescence and localization. We have begun to examine potential sources of Ca²⁺ involved in the responses induced by SI. This work presents preliminary data showing that influx of extracellular Ca²⁺ at the ”shank” of the pollen tube is possible. This is the first evidence suggesting that influx at this localization may play a role in the SI response. We also describe preliminary studies that begin to investigate whether the phosphoinositide signalling pathway is implicated in the SI response. Received: 12 December 2000 / Revision Accepted: 22 June 2001     

Alterations in the actin cytoskeleton of pollen tubes are induced by the self-incompatibility reaction in Papaver rhoeas

Self-incompatibility (SI) is a genetically controlled process used to prevent self-pollination. In Papaver rhoeas, the induction of SI is triggered by a Ca(2)+-dependent signaling pathway that results in the rapid and S allele-specific inhibition of pollen tube tip growth. Tip growth of cells is dependent on a functioning actin cytoskeleton. We have investigated the effect of self-incompatibility (S) proteins on the actin cytoskeleton in poppy pollen tubes. Here, we report that the actin cytoskeleton of incompatible pollen tubes is rapidly and dramatically rearranged during the SI response, not only in our in vitro SI system but also in vivo. We demonstrate that nonspecific inhibition of growth does not result in similar actin rearrangements. Because the SI-induced alterations are not observed if growth stops, this clearly demonstrates that these alterations are triggered by the SI signaling cascade rather than merely resulting from the consequent inhibition of growth. We establish a detailed time course of events and discuss the mechanisms that might be involved. Our data strongly implicate a role for the actin cytoskeleton as a target for signaling pathways involved in the SI response of P. rhoeas.     

A cascade signal pathway occurs in self-incompatibility of Pyrus pyrifolia

Pear (Pyrus pyrifolia L.) possesses an S-RNase-based gametophytic self-incompatibility (GSI) system and S-RNase, the self-incompatibility (SI) determinant in the pistil, has also been implicated in the rejection of self-pollen and genetically identical pollen. We have demonstrated that S-RNase depolymerises actin cytoskeleton, triggers mitochondrial alteration and DNA degradation in the incompatible pollen tube, which indicates programmed cell death (PCD) may occur in SI response of Pyrus pyrifolia. Recently, we have identified that S-RNase specifically disrupted tip-localized reactive oxygen species (ROS) of incompatible pollen tube via arrest of ROS formation in mitochondria and cell walls in Pyrus pyrifolia. Furthermore, tip-localized ROS disruption not only decreased the Ca²⁺ current and depolymerised the actin cytoskeleton, but it also induced nuclear DNA degradation in the pollen tube. The results mentioned above indicate that a cascade signal pathway may occur in SI of Pyrus pyrifolia and PCD is used to terminate the incompatible pollen tubes growth. In this addendum, we review the cascade signal pathway of Pyrus pyrifolia SI.Key words: S-RNase, programmed cell death, reactive oxygen species, actin cytoskeleton, Ca²⁺ current, nuclear DNA     

Increased Phosphorylation of a 26-kD Pollen Protein Is Induced by the Self-Incompatibility Response in Papaver rhoeas

We have investigated whether specific protein phosphorylation events are induced in Papaver rhoeas pollen as a consequence of the self-incompatibility (SI) response. Pollen grown in vitro in the presence of 32P-orthophosphate was challenged with biologically active recombinant S proteins, and pollen proteins were extracted and analyzed. The results provide strong evidence that the increased phosphorylation of a 26-kD protein of pl 6.2, p26, is specifically induced by the SI response. This phosphorylation event occurs in living pollen tubes and was observed specifically when pollen was challenged with S proteins that are incompatible with the S alleles carried by the pollen and not when pollen was challenged with compatible or incompatible heat-denatured S proteins. Further characterization demonstrated that p26 comprises two phosphoproteins, p26.1 and p26.2, that are found in soluble and microsomal fractions, respectively. Increased phosphorylation of p26.1 is implicated in the SI response and appears to be Ca2+ and calmodulin dependent. These data argue for the involvement of a Ca2+-dependent protein kinase requiring calmodulin-like domains, whose activation comprises an intracellular signal mediating the SI response in P. rhoeas pollen.     

The self-incompatibility response in Papaver rhoeas is mediated by cytosolic free calcium

The role of Ca²⁺ signalling during the self-incompatibility (SI) response in Papaver rhoeas L. has been investigated using Ca²⁺-sensitive dyes. Pollen tubes were micro-injected with Calcium Green-1 and cytosolic free calcium ([Ca²⁺]_i) imaged using laser scanning confocal microscopy (LSCM). Addition of incompatible stigmatic S-glycoproteins induced a transient increase in the level of [Ca²⁺]_i in pollen tubes. In contrast, no rise in [Ca²⁺]_i was detectable after addition of either compatible or heat-denatured incompatible stigmatic S-glycoproteins. The elevation of [Ca²⁺]_i was followed by the specific inhibition of pollen tube growth in incompatible reactions. It has been shown previously that gene expression in pollen tubes is switched on during an incompatible reaction. Since the [Ca²⁺]_i transient appeared to originate from the region where the nuclei are located, Ca²⁺ may be involved in locally regulating the expression of these genes. The photoactivation of caged Ca²⁺ to artificially elevate [Ca²⁺]_i resulted in the inhibition of pollen tube growth and thus mimicked the SI response. Taken together, the results provide an important link between a transient rise in [Ca²⁺]_i and the biological phenomenon of inhibition of pollen tube growth and demonstrate, for the first time, direct evidence that the SI response in P. rhoeas is mediated by [Ca²⁺]_i.     

The intracellular events triggered by the self-incompatibility response inPapaver rhoeas

Summary In recent years self-incompatibility (SI) has come to be recognised as an important model system for studying cell-cell interactions and signalling in flowering plants. In this article we discuss the intracellular events associated with the SI response in the field poppy,Papaver rhoeas. The SI response inP. rhoeas is known to involve a Ca²⁺-based signalling pathway, activated following molecular interactions on the surface of incompatible pollen tubes. Evidence demonstrates that, following a transient increase in the concentration of cytosolic free Ca²⁺ ([Ca²⁺];) initiated by the SI response, phosphorylation of certain cytosolic proteins occurs, followed by activation of pollen gene expression. The magnitude of this transient Ca²⁺ wave and the localisation of cytosolic [Ca²⁺]i following the SI response are discussed. We also describe the character of the proteins specifically phosphorylated in the SI response and the nature of the protein kinases involved in their phosphorylation. Finally, we consider the possibility that the end result of the SI response inP. rhoeas may be analogous to programmed-cell-death mechanisms such as those seen in developmental processes and defence responses in various plant cells.     

Growth of Pollen Tubes of Papaver rhoeas Is Regulated by a Slow-Moving Calcium Wave Propagated by Inositol 1,4,5-Trisphosphate

A signaling role for cytosolic free Ca2+ ([Ca2+]i) in regulating Papaver rhoeas pollen tube growth during the self-incompatibility response has been demonstrated previously. In this article, we investigate the involvement of the phosphoinositide signal transduction pathway in Ca2+-mediated pollen tube inhibition. We demonstrate that P. rhoeas pollen tubes have a Ca2+-dependent polyphosphoinositide-specific phospholipase C activity that is inhibited by neomycin. [Ca2+]i imaging after photolysis of caged inositol (1,4,5)-trisphosphate (Ins[1,4,5]P3) in pollen tubes demonstrated that Ins(1,4,5)P3 could induce Ca2+ release, which was inhibited by heparin and neomycin. Mastoparan, which stimulated Ins(1,4,5)P3 production, also induced a rapid increase in Ca2+, which was inhibited by neomycin. These data provide direct evidence for the involvement of a functional phosphoinositide signal-transducing system in the regulation of pollen tube growth. We suggest that the observed Ca2+ increases are mediated, at least in part, by Ins(1,4,5)P3-induced Ca2+ release. Furthermore, we provide data suggesting that Ca2+ waves, which have not previously been reported in plant cells, can be induced in pollen tubes.     

S-RNase triggers mitochondrial alteration and DNA degradation in the incompatible pollen tube of Pyrus pyrifolia in vitro

Pear ( Pyrus pyrifolia L.) has a S-RNase-based gametophytic self-incompatibility (SI) mechanism, and S-RNase has also been implicated in the rejection of self-pollen and genetically identical pollen. No studies, however, have examined the extent of organelle alterations during the SI response in Pyrus pyrifolia . Consequently, this study focused on the alterations to mitochondria and nuclear DNA in incompatible pollen tubes of the pear. Methylthiazolyldiphenyl-tetrazolium bromide was used to evaluate the viability of pollen tubes under S-RNase challenge. The results showed that the viability of the control and compatible pollen tubes decreased slightly, but that of the incompatible pollen and pollen tubes began to decline at 30 min. The mitochondrial membrane potential (Δ ψ _mit) was also tested with rhodamine 123 30 min after SI challenge, and was shown to have collapsed in the incompatible pollen tubes after exposure to S-RNase. Western blotting 2 h after SI challenge confirmed that the Δ ψ _mit collapse induced leakage of cytochrome c into the cytosol. Swollen mitochondria were detected by transmission electron microscopy as early as 1 h after SI challenge and the degradation of nuclear DNA was observed by both 4,6-diamidino-2-phenylindole and transferase-mediated dUTP nick-end labeling. These diagnostic features of programmed cell death (PCD) suggested that PCD may specifically occur in incompatible pollen tubes.     

Ca2+-independent phosphorylation of a 68 kDa pollen protein is stimulated by the self-incompatibility response in Papaver rhoeas

The self-incompatibility (SI) response in Papaver rhoeas involves a Ca²⁺-based signalling pathway, which mediates the SI-specific inhibition of incompatible pollen. We have previously reported the identification of p26.1, a pollen protein whose phosphorylation was increased specifically as a consequence of the SI response. We have investigated whether further specific protein phosphorylation events are induced in P. rhoeas pollen. Here we report the identification of an additional pollen protein, p68, which also responds to S proteins by an increase in its phosphorylation state. This phosphorylation event occurs in living pollen tubes grown in vitro , and can be observed specifically when pollen is challenged with biologically active S proteins that are incompatible with the S alleles carried by the pollen and not when pollen was challenged with compatible S proteins. The timing of the increase in phosphorylation of p68 is temporally later than that of p26.1, occurring between 240 sec and 400 sec after challenge. This suggests that its phosphorylation is downstream of p26.1 in the SI signalling pathway(s). Surprisingly, the kinases responsible for the phosphorylation of p68 are not Ca²⁺-dependent. This, and the later timing of the p68 response, suggests that a 'second wave' of Ca²⁺-independent signalling may follow the initial Ca²⁺-dependent SI signalling. This indicates that the SI signalling pathway(s) in pollen may be quite complex.     

Disorganization of F-actin cytoskeleton precedes vacuolar disruption in pollen tubes during the in vivo self-incompatibility response in Nicotiana alata

Background and Aims The integrity of actin filaments (F-actin) is essential for pollen-tube growth. In S-RNase-based self-incompatibility (SI), incompatible pollen tubes are inhibited in the style. Consequently, research efforts have focused on the alterations of pollen F-actin cytoskeleton during the SI response. However, so far, these studies were carried out in in vitro-grown pollen tubes. This study aimed to assess the timing of in vivo changes of pollen F-actin cytoskeleton taking place after compatible and incompatible pollinations in Nicotiana alata. To our knowledge, this is the first report of the in vivo F-actin alterations occurring during pollen rejection in the S-RNase-based SI system. Methods The F-actin cytoskeleton and the vacuolar endomembrane system were fluorescently labelled in compatibly and incompatibly pollinated pistils at different times after pollination. The alterations induced by the SI reaction in pollen tubes were visualized by confocal laser scanning microscopy. Key Results Early after pollination, about 70 % of both compatible and incompatible pollen tubes showed an organized pattern of F-actin cables along the main axis of the cell. While in compatible pollinations this percentage was unchanged until pollen tubes reached the ovary, pollen tubes of incompatible pollinations underwent gradual and progressive F-actin disorganization. Colocalization of the F-actin cytoskeleton and the vacuolar endomembrane system, where S-RNases are compartmentalized, revealed that by day 6 after incompatible pollination, when the pollen-tube growth was already arrested, about 80 % of pollen tubes showed disrupted F-actin but a similar percentage had intact vacuolar compartments. Conclusions The results indicate that during the SI response in Nicotiana, disruption of the F-actin cytoskeleton precedes vacuolar membrane breakdown. Thus, incompatible pollen tubes undergo a sequential disorganization process of major subcellular structures. Results also suggest that the large pool of S-RNases released from vacuoles acts late in pollen rejection, after significant subcellular changes in incompatible pollen tubes.     

Reactive oxygen species and nitric oxide mediate actin reorganization and programmed cell death in the self-incompatibility response of papaver

Pollen-pistil interactions are critical early events regulating pollination and fertilization. Self-incompatibility (SI) is an important mechanism to prevent self-fertilization and inbreeding in higher plants. Although data implicate the involvement of reactive oxygen species (ROS) and nitric oxide (NO) in pollen-pistil interactions and the regulation of pollen tube growth, there has been a lack of studies investigating ROS and NO signaling in pollen tubes in response to defined, physiologically relevant stimuli. We have used live-cell imaging to visualize ROS and NO in growing Papaver rhoeas pollen tubes using chloromethyl-2'7'-dichlorodihydrofluorescein diacetate acetyl ester and 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate and demonstrate that SI induces relatively rapid and transient increases in ROS and NO, with each showing a distinctive "signature" within incompatible pollen tubes. Investigating how these signals integrate with the SI responses, we show that Ca(2+) increases are upstream of ROS and NO. As ROS/NO scavengers alleviated both the formation of SI-induced actin punctate foci and also the activation of a DEVDase/caspase-3-like activity, this demonstrates that ROS and NO act upstream of these key SI markers and suggests that they signal to these SI events. These data represent, to our knowledge, the first steps in understanding ROS/NO signaling triggered by this receptor-ligand interaction in pollen tubes.     

Ectopic expression of S-RNase of <Emphasis Type="Italic">Petunia inflata</Emphasis> in pollen results in its sequestration and non-cytotoxic function

The specificity of S-RNase-based self-incompatibility (SI) is controlled by two S-locus genes, the pistil S-RNase gene and the pollen S-locus-F-box gene. S-RNase is synthesized in the transmitting cell; its signal peptide is cleaved off during secretion into the transmitting tract; and the mature “S-RNase”, the subject of this study, is taken up by growing pollen tubes via an as-yet unknown mechanism. Upon uptake, S-RNase is sequestered in a vacuolar compartment in both non-self (compatible) and self (incompatible) pollen tubes, and the subsequent disruption of this compartment in incompatible pollen tubes correlates with the onset of the SI response. How the S-RNase-containing compartment is specifically disrupted in incompatible pollen tubes, however, is unknown. Here, we circumvented the uptake step of S-RNase by directly expressing S₂-RNase, S₃-RNase and non-glycosylated S₃-RNase of Petunia inflata, with green fluorescent protein (GFP) fused at the C-terminus of each protein, in self (incompatible) and non-self (compatible) pollen of transgenic plants. We found that none of these ectopically expressed S-RNases affected the viability or the SI behavior of their self or non-self-pollen/pollen tubes. Based on GFP fluorescence of in vitro-germinated pollen tubes, all were sequestered in both self and non-self-pollen tubes. Moreover, the S-RNase-containing compartment was dynamic in living pollen tubes, with movement dependent on the actin–myosin-based molecular motor system. All these results suggest that glycosylation is not required for sequestration of S-RNase expressed in pollen tubes, and that the cytosol of pollen is the site of the cytotoxic action of S-RNase in SI.     

Turnover of diacylglycerol kinase 4 by cytoplasmic acidification induces vacuole morphological change and nuclear DNA degradation in the early stage of pear self-incompatibility response

Pear has an S-RNase-based gametophytic self-incompatibility (SI) system. Nuclear DNA degradation is a typical feature of incompatible pollen tube death, and is among the many physiological functions of vacuoles. However, the specific changes that occur in vacuoles, as well as the associated regulatory mechanism in pear SI, are currently unclear. Although research in tobacco has shown that decreased activity of diacylglycerol kinase (DGK) results in the morphological change of pollen tube vacuole, whether DGK regulates the pollen tube vacuole of tree plants and whether it occurs in SI response, is currently unclear. We found that DGK activity is essential for pear pollen tube growth, and DGK4 regulates pollen tube vacuole morphology following its high expression and deposition at the tip and shank edge of the pollen tube of pear. Specifically, incompatible S-RNase may induce cytoplasmic acidification of the pollen tube by inhibiting V-ATPase V₀ domain a1 subunit gene expression as early as 30 min after treatment, when the pollen tube is still alive. Cytoplasmic acidification induced by incompatible S-RNase results in reduced DGK4 abundance and deposition, leading to morphological change of the vacuole and fragmentation of nuclear DNA, which indicates that DGK4 is a key factor in pear SI response.     

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

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 Ca(2+)-dependent signalling cascade in incompatible pollen when a stigmatic S-protein interacts with it. Rapid increases in cytosolic free Ca(2+) concentration ([Ca(2+)](i)) can now be attributed (at least in part) to Ca(2+) influx. The rapid loss of the pollen apical Ca(2+) gradient within approximately 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.     

A mitogen-activated protein kinase signals to programmed cell death induced by self-incompatibility in Papaver pollen

Self-incompatibility (SI) in higher plants is an important mechanism to prevent inbreeding and involves specific rejection of incompatible ("self") pollen. In field poppy (Papaver rhoeas), S proteins encoded by the stigma component of the S-locus interact with incompatible pollen, resulting in cessation of tip growth. This "self" interaction triggers a Ca(2+)-dependent signaling network, involving programmed cell death (PCD). We previously identified p56, a mitogen-activated protein kinase (MAPK) that is activated during the SI response in incompatible pollen. Here, we show that p56 cross-reacts with AtMPK3, but not with AtMPK4 or salicylic acid-induced protein kinase antibodies. We provide good evidence that a MAPK is involved in initiation of SI-induced PCD in incompatible pollen. SI rapidly reduces pollen viability and the MAPK cascade inhibitor U0126, which prevents the SI-induced activation of p56 in incompatible pollen, "rescues" incompatible pollen, while its negative analog, U0124, does not. This strongly implicates the involvement of a MAPK in SI-mediated loss of pollen viability and cell death. SI also stimulates caspase-3-like (DEVDase) activity and later DNA fragmentation. Both these markers of PCD are significantly reduced by pretreatment with U0126, implicating the involvement of a MAPK in signaling during early PCD. As p56 appears to be the only MAPK activated by SI, our studies imply that p56 could be the MAPK involved in mediating SI-induced PCD.     

Ratio-imaging of Ca2+i in the self-incompatibility response in pollen tubes of Papaver rhoeas

The data presented here describe ratio-imaging of in intracellular free calcium (Ca²⁺_i) during the self-incompatibility (SI) response in pollen. Use of the ratiometric indicator, fura-2 dextran, in pollen tubes of Papaver rhoeas has provided new, detailed information about the spatial-temporal alterations in Ca²⁺_i, and has permitted calibration of alterations in the concentration of intracellular free calcium ([Ca²⁺]_i) in the SI response. Ratio images demonstrate that, like other pollen tubes, normally growing P. rhoeas pollen tubes exhibit a tip-focused gradient of Ca^2+bf_i, with levels reaching 1–2 μM at the extreme apex of the pollen tube. Non-growing pollen tubes did not exhibit this tip-focused gradient. Basal levels of Ca²⁺_i in the shank of the pollen tube were fairly consistent and had a mean value of 210 nM, with low-level fluctuations +/? 50 nM observed. Challenge with incompatible S proteins resulted in S-specific, rapid and dramatic alterations in [Ca²⁺]_i within a few seconds of challenge. Increases in [Ca²⁺]_i were visualized in the subapical/shank regions of the pollen tube and alterations in [Ca²⁺]_i in this region subsequently increased for several minutes, reaching> 1.5 μM. At the pollen tube tip, a diminution of the tip-focused gradient was observed, which following some fluctuation, was reduced to basal levels within ～1 min. Our data suggest that some of these alterations in [Ca²⁺]_i might be interpreted as a calcium wave, as the changes are not global. Although the increases in [Ca²⁺]_i in the subapical/shank region are very rapid, because tip [Ca²⁺]_i oscillates during normal growth, it is difficult to ascertain whether the increases in the shank of the pollen tube precede the decreases in [Ca²⁺]_i at the pollen tube tip.     

Investigating mechanisms involved in the self-incompatibility response in Papaver rhoeas

Sexual reproduction in flowering plants is controlled by recognition mechanisms involving the male gametophyte (the pollen) and the female sporophyte (the pistil). Self-incompatibility (SI) involves the recognition and rejection of self- or incompatible pollen by the pistil. In Papaver rhoeas, SI uses a Ca(2+)-based signalling cascade triggered by the S-protein, which is encoded by the stigmatic component of the S-locus. This results in the rapid inhibition of incompatible pollen tube growth. We have identified several targets of the SI signalling cascade, including protein kinases, the actin cytoskeleton and nuclear DNA. Here, we summarize progress made on currently funded projects in our laboratory investigating some of the components targeted by SI, comprising (i) the characterization of a pollen phosphoprotein (p26) that is rapidly phosphorylated upon an incompatible SI response; (ii) the identification and characterization of a pollen mitogen-activated protein kinase (p56), which exhibits enhanced activation during SI; (iii) characterizing components involved in the reorganization and depolymerization of the actin cytoskeleton during the SI response; and (iv) investigating whether the SI response involves a programmed cell death signalling cascade.