Microtubules are a target for self-incompatibility signaling in Papaver pollen

Perception and integration of signals into responses is of crucial importance to cells. Both the actin and microtubule cytoskeleton are known to play a role in mediating diverse stimulus responses. Self-incompatibility (SI) is an important mechanism to prevent self-fertilization. SI in Papaver rhoeas triggers a Ca(2+)-dependent signaling network to trigger programmed cell death (PCD), providing a neat way to inhibit and destroy incompatible pollen. We previously established that SI stimulates F-actin depolymerization and that altering actin dynamics can push pollen tubes into PCD. Very little is known about the role of microtubules in pollen tubes. Here, we investigated whether the pollen tube microtubule cytoskeleton is a target for the SI signals. We show that SI triggers very rapid apparent depolymerization of cortical microtubules, which, unlike actin, does not reorganize later. Actin depolymerization can trigger microtubule depolymerization but not vice versa. Moreover, although disruption of microtubule dynamics alone does not trigger PCD, alleviation of SI-induced PCD by taxol implicates a role for microtubule depolymerization in mediating PCD. Together, our data provide good evidence that SI signals target the microtubule cytoskeleton and suggest that signal integration between microfilaments and microtubules is required for triggering of PCD.     

Self-incompatibility in Papaver: signalling to trigger PCD in incompatible pollen

Sexual 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 Ca(2+)-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.     

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.     

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.     

The Papaver self-incompatibility pollen S-determinant, PrpS, functions in Arabidopsis thaliana

Many angiosperms use specific interactions between pollen and pistil proteins as "self" recognition and/or rejection mechanisms to prevent self-fertilization. Self-incompatibility (SI) is encoded by a multiallelic S locus, comprising pollen and pistil S-determinants. In Papaver rhoeas, cognate pistil and pollen S-determinants, PrpS, a pollen-expressed transmembrane protein, and PrsS, a pistil-expressed secreted protein, interact to trigger a Ca(2+)-dependent signaling network, resulting in inhibition of pollen tube growth, cytoskeletal alterations, and programmed cell death (PCD) in incompatible pollen. We introduced the PrpS gene into Arabidopsis thaliana, a self-compatible model plant. Exposing transgenic A. thaliana pollen to recombinant Papaver PrsS protein triggered remarkably similar responses to those observed in incompatible Papaver pollen: S-specific inhibition and hallmark features of Papaver SI. Our findings demonstrate that Papaver PrpS is functional in a species with no SI system that diverged ~140 million years ago. This suggests that the Papaver SI system uses cellular targets that are, perhaps, common to all eudicots and that endogenous signaling components can be recruited to elicit a response that most likely never operated in this species. This will be of interest to biologists interested in the evolution of signaling networks in higher plants.     

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.     

The actin cytoskeleton is a target of the self-incompatibility response in Papaver rhoeas

The integration of signals received by a cell, and their transduction to targets, is essential for all cellular responses. The cytoskeleton has been identified as a major target of signalling cascades in both animal and plant cells. Self-incompatibility (SI) in Papaver rhoeas involves an allele-specific recognition between stigmatic S-proteins and pollen, resulting in the inhibition of incompatible pollen. This highly specific response triggers a Ca(2+)-dependent signalling cascade in incompatible pollen when a stigmatic S-protein interacts with it. It has been demonstrated recently that SI induces dramatic alterations in the organization of the pollen actin cytoskeleton. This implicates the actin cytoskeleton as a key target for the SI-stimulated signals. The cytological alterations to the actin cytoskeleton that are triggered in response to SI are described here and there seem to be several stages that are distinguishable temporally. Evidence was obtained that F-actin depolymerization is also stimulated. The current understanding that the actin cytoskeleton is a target for the signals triggered by the SI response is discussed. It is suggested that these F-actin alterations may be Ca(2+)-mediated and that this could be a mechanism whereby SI-induced tip growth inhibition is achieved. The potential for actin-binding proteins to act as key mediators of this response is discussed and the mechanisms that may be responsible for effecting these changes are described. In particular, the parallels between sustained actin rearrangements during SI and in apoptosis of animal cells are considered.     

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.     

Self-incompatibility in Papaver: A MAP kinase signals to trigger programmed cell death

Self-incompatibility (SI) in higher plants prevents inbreeding through specific recognition and rejection of incompatible (“self”) pollen. In Papaver rhoeas, S proteins encoded by the pistil component of the S-locus interact with incompatible pollen, triggering a Ca²⁺-dependent signaling network resulting in programmed cell death (PCD). We recently showed that a mitogen-activated protein kinase (MAPK) is involved in loss of pollen viability, stimulation of caspase-3-like (DEVDase) activity and later DNA fragmentation in incompatible pollen. As p56 appears to be the only MAPK activated by SI, our data suggest that p56 could be the MAPK responsible for mediating SI-induced PCD.Key words: MAPK, self-incompatibility, PCD, caspase-3-like activity, Papaver rhoeas     

Self-incompatibility in Papaver rhoeas: there is no evidence for the involvement of stigmatic ribonuclease activity

Abstract. Ribonuclease assays have revealed, in contrast to the self-incompatibility (SI) system of Nicotiana alata , there is no detectable ribonuclease activity that correlates with the presence of the functional stigmatic S-gene product in Papaver rhoeas . Thus, we have shown that the inhibition of incompatible pollen tube growth in P. rhoeas is not associated with ribonuclease activity. Furthermore, the finding that pollen from P. rhoeas , unlike that from N. alata , is insensitive to purified bovine pancreatic ribonuclease A at very high concentrations, suggests that the involvement of ribonucleases in the inhibition reaction of the SI response in P. rhoeas is unlikely. In addition, the level of ribonuclease activity in mature stigmas of P. rhoeas is very much lower than that in N. alata and significantly, the level of ribonuclease activity did not rise in conjunction with the developmental expression of SI. Therefore, as a result of these studies, we believe that SI in P. rhoeas does not involve ribonuclease activity.     

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.     

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.     

高等植物自交不亲和反应中花粉管信号传导研究进展(综述)

高等植物自交不亲和反应是由基因控制、避免发生自花授粉的一种机制。本文介绍以虞美人为主的高等植物在自交不亲和反应中肌动蛋白骨架的动态变化及Ca2 的时空变化,着重阐述花粉管生长被抑制的最初信号传导。     

nhibiting Self-Pollen： Self-Incompatibility in Papaver Involves Integration of Several Signaling Events

Cellular responses rely on signal perception and integration. A nice example of this is self incompatibility （SI）, which is an important mechanism to prevent inbreeding. It prevents self-fertilization by using a highly discriminatory cellular recognition and rejection mechanism. Most Sl systems are genetically specified by the S-locus, which has a pollen and a pistil S-component. A receptor-ligand interaction is used by Papaver rhoeas to control SI. S proteins encoded by the pistil part of the S-locus interact with incompatible pollen to achieve rapid inhibition of tip growth. The incompatible Sl interaction triggers a Ca^2＋-dependent signaling cascade. A number of Sl-specific events are triggered in incompatible pollen, including rapid depolymerization of the actin cytoskeleton; phosphorylation of soluble inorganic pyrophosphatases （SPPases）, Prp26.1; activation of a mitogen activated protein kinase, p56; programmed cell death （PCD） involving a caspase-3-1ike activity. These events contribute to prevent self-fertilizaUon. We are attempting to establish the functional significance of these events, and their possible involvement in integrating a coordinated signaling response. Here we describe the identification of these components shown to be involved in Sl, together with recent progress in identifying links between some of them. These data constitute the first steps in elucidating how SI signaling is integrated.     

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.     

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.     

Cytomechanical properties of papaver pollen tubes are altered after self-incompatibility challenge

Self-incompatibility (SI) in Papaver rhoeas triggers a ligand-mediated signal transduction cascade, resulting in the inhibition of incompatible pollen tube growth. Using a cytomechanical approach we have demonstrated that dramatic changes to the mechanical properties of incompatible pollen tubes are stimulated by SI induction. Microindentation revealed that SI resulted in a reduction of cellular stiffness and an increase in cytoplasmic viscosity. Whereas the former cellular response is likely to be the result of a drop in cellular turgor, we hypothesize that the latter is caused by as yet unidentified cross-linking events. F-actin rearrangements, a characteristic phenomenon for SI challenge in Papaver, displayed a spatiotemporal gradient along the pollen tube; this suggests that signal propagation occurs in a basipetal direction. However, unexpectedly, local application of SI inducing S-protein did not reveal any evidence for localized signal perception in the apical or subapical regions of the pollen tube. To our knowledge this represents the first mechanospatial approach to study signal propagation and cellular responses in a well-characterized plant cell system. Our data provide the first evidence for mechanical changes induced in the cytoplasm of a plant cell stimulated by a defined ligand.     

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.