S-protein mutants indicate a functional role for SBP in the self-incompatibility reaction of Papaver rhoeas

The self-incompatibility response involves S-allele specific recognition between stigmatic S proteins and incompatible pollen, resulting in S-specific pollen inhibition. In Papaver rhoeas, the pollen S gene product is predicted to be a receptor that interacts with the stigmatic S protein in an S specific manner. We recently identified an S protein binding protein (SBP) in pollen that binds stigmatic S proteins, although apparently not in an S-allele-specific manner. In order to investigate the functional significance of the interaction between S proteins and SBP, we constructed mutant derivatives of the S1 protein and tested their SBP-binding activity and their biological activity. Here we present an evaluation of nine mutant derivatives of the S1 protein. Western ligand blotting was used to show that mutations to amino acid residues in predicted loops 2 and 6 of the S1 protein cause significant reductions in their SBP-binding activity. These same mutants show a concomitant reduction in their ability to inhibit incompatible pollen. This establishes a direct link between SBP binding and inhibition of incompatible pollen and implicates SBP as a pollen component playing a key role in the self-incompatibility reaction. We discuss the possible nature of the contribution of SBP in the S-specific rejection of incompatible pollen.     

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 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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Identification of a membrane glycoprotein in pollen of Papaver rhoeas which binds stigmatic self-incompatibility (S-) proteins

Recombinant stigmatic self-incompatibility (S-) proteins from Papaver rhoeas have previously been shown to be biologically functional, inhibiting only pollen of the same S -genotype. In an attempt to identify molecules in pollen which interact with these proteins, Western ligand blotting was used, with the recombinant S-proteins as probes followed by immunodetection of the bound S-protein. This revealed that pollen of all S -genotypes tested contained a 70–120 kDa protein which bound the S₁, S₃ and S₈ proteins in an indistinguishable manner. Binding was destroyed by pretreatment of blots with periodate, implicating a glycoprotein with activity being dependent on the glycan moiety. The activity completely partitioned into the detergent phase on condensation with Triton X-114, indicating an integral membrane protein. On aqueous two-phase partition of microsomal membrane preparations, the majority of the binding activity partitioned into the upper phase, suggesting that the molecule is located in the plasma membrane.
No equivalent binding could be detected in extracts of leaves, stems, roots or stigmas of P. rhoeas , nor in immature anthers. Identical activity was detected in pollen of some other Papaver species, but not in pollen of Brassica oleracea, Nicotiana tabacum or Petunia hybrida . The presence in mature pollen of P. rhoeas of a plasma membrane glycoprotein which binds S-proteins from the stigma of the same species, albeit in a non S -allele-specific manner, strongly suggests that this molecule has a role somewhere in the interaction of the stigma proteins with pollen. The activity is not that expected of an S-specific receptor, but by analogy with certain mammalian systems the molecule may act as an accessory receptor, or co-receptor, the presence of which may be essential for a functional interaction with an S-specific receptor.     

Genomic organization of the Papaver rhoeas self-incompatibility S(1) locus

The self-incompatibility (SI) response in Papaver rhoeas depends upon the cognate interaction between a pollen-expressed receptor and a stigmatically expressed ligand. The genes encoding these components are situated within the S-locus. In order for SI to be maintained, the genes encoded by the S-locus must be co-inherited with no recombination between them. Several hypotheses, including sequence heterogeneity and chromosomal position, have been put forward to explain the maintenance of the S-locus in the SI systems of the Brassicaceae and the Solanaceae. A region of the Papaver rhoeas genome encompassing part of the self-incompatibility S(1) locus has been cloned and sequenced. The clone contains the gene encoding the stigmatic component of the response, but does not contain a putative pollen S-gene. The sequence surrounding the S(1) gene contains several diverse repetitive DNA elements. As such, the P. rhoeas S-locus bears similarities to the S-loci of other SI systems. An attempt to localize the P. rhoeas S-locus using fluorescence in situ hybridization (FISH) has also been made. The potential relevance of the findings to mechanisms of recombination suppression is discussed.     

Involvement of extracellular calcium influx in the self-incompatibility response of Papaver rhoeas

We have previously demonstrated that increases in cytosolic free Ca2+ are triggered by the self-incompatibility (SI) response in incompatible Papaver rhoeas (the field poppy) pollen. However, one key question that has not been answered is whether extracellular Ca2+ may be involved. To address this question, we have used an ion-selective vibrating probe to measure changes in extracellular Ca2+ fluxes around poppy pollen tubes. Our data reveal several findings. First, we confirm that there is an oscillating Ca2+ influx directed at the apex of the pollen tube; we also provide evidence that Ca2+ influx also occurs at the shanks of pollen tubes. Second, upon challenge with self-incompatibility (S) proteins, there is a stimulation of Ca2+ influx along the shank of incompatible pollen tubes, approximately 50 microm behind the pollen tube tip. This demonstration of SI-induced Ca2+ influx suggests a role for influx of extracellular Ca2+ in the SI response.     

Molecular analysis of the conserved C4 region of the S<Subscript>11</Subscript>-RNase of <Emphasis Type="Italic">Solanum chacoense</Emphasis>

The stylar component to gametophytic self-incompatibility in Solanaceae is an S-RNase. Its primary structure has a characteristic pattern of two hypervariable regions, involved in pollen recognition, and five constant regions. Two of the latter (C2 and C3) constitute the active site, while the highly hydrophobic C1 and C5 are believed to be involved in protein stability. We analyzed the role of the C4 region by site-directed mutagenesis. A GGGG mutant, in which the four charged residues in the C4 region were replaced with glycine, did not accumulate the protein to detectable levels in styles, suggestive of a role in protein stability. A R115G mutant, in which a charged amino acid was eliminated to reduce the potential binding affinity, had no effect on the pollen rejection phenotype. This suggests the C4 does not interact with partners such as potential pollen tube receptors facilitating S-RNase uptake. Finally, a K113R mutant replaced a potential ubiquitination target with arginine. However, this RNase acted as the wild type in both incompatible and compatible crosses. The latter crosses rule out the role of the conserved C4 lysine in ubiquitination.     

芸苔属自交不亲和细胞信号转导的研究进展

在芸苔属植物的自交不亲和细胞信号转导过程中,信号分子-SCR配体是由花粉粒产生的,被柱头乳突细胞SRK受体识别后,进行细胞内信号转导。这对受体-配体是两个由S位点编码的且高度多态的蛋白质,它们决定着自交不亲和反应。配体是位于花粉粒表面的一个小的胞被蛋白,由SCR基因编码;受体是位于柱头乳突细胞原生质膜上的跨膜的蛋白质激酶,由SRK基因编码。在自交授粉过程中,配体SCR和受体SRK的相互作用激活了受体SRK,被激活的SRK通过其下游组分ARC1介导底物的泛肽化,然后泛肽化的底物在蛋白酶体/CSN中被降解,从而导致了自交不亲和性反应。这些降解的底物可能是促进花粉水合、萌发和花粉管生长的雌蕊亲和因子。主要针对芸苔属自交不亲和细胞信号转导作一综述。     

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.     

The 1.55 A resolution structure of Nicotiana alata S(F11)-RNase associated with gametophytic self-incompatibility.

The crystal structure of Nicotiana alata (ornamental tobacco) S(F11)-RNase, an S-allelic glycoprotein associated with gametophytic self-incompatibility, was determined by X-ray diffraction at 1.55 A resolution. The protein has a tertiary structure typical of members of the RNase T(2) family as it consists of a variant of the (alpha+beta) fold and has eight helices and seven strands. A heptasaccharide moiety is also present, and amino acid residues that serve as the catalytic acid and base can be assigned to His32 and His91, respectively. Two "hypervariable" regions, known as HVa and HVb, are the proposed sites of S-allele discrimination during the self-incompatibility reaction, and in the S(F11)-RNase these are well separated from the active site. HVa and HVb are composed of a long, positively charged loop followed by a part of an alpha-helix and short, negatively charged alpha-helix, respectively. The S(F11)-RNase structure shows both regions are readily accessible to the solvent and hence could participate in the process of self/non-self discrimination between the S-RNase and an unknown pollen S-gene product(s) upon pollination.     

Recombinant stigmatic self-incompatibility (S-) protein elicits a Ca2+ transient in pollen of Papaver rhoeas

Evidence for Ca²⁺ signalling in pollen during the self-incompatibility (SI) response in Papaver rhoeas L. has been presented previously. However, it was not known whether the S-protein alone could act as an elicitor of the response or whether the presence of other stigmatic components was required, since relatively crude stigmatic extracts had been used. The S ₁ gene has since been cloned and its product expressed in Escherichia coli has been shown to exhibit biological activity. In this paper it is reported that the recombinant protein (S₁e) elicits a transient rise in [Ca²⁺]_i in incompatible pollen. The Ca²⁺ signal appears indistinguishable from that elicited by S-gene products partially purified from plant extracts in terms of both its timing and spatial distribution. Pollen tube growth is arrested directly after the rise in [Ca²⁺]_i.
The results provide direct evidence that the S-protein alone acts as an elicitor which triggers the Ca²⁺ signal for the pollen SI response. In addition, it is now clear that the recombinant S-protein does not require several post-translational processing events which take place in the plant to act as an elicitor. With respect to the spatial distribution of the Ca²⁺ transient, data are presented which correlate the localized rise in intracellular Ca²⁺ ([Ca²⁺]_i) with the 'nuclear complex' and the endoplasmic reticulum which is associated with this region.     

Self-incompatibility in Petunia inflata: isolation and characterization of cDNAs encoding three S-allele-associated proteins

Summary We have identified three alleles of the S-locus controlling self-incompatibility and their associated pistil proteins in Petunia inflata, a species that displays monofactorial gametophytic self-incompatibility. These S-allele-associated proteins (S-proteins) are pistil specific, and their levels are developmentally regulated. The amino-terminal sequences determined for the three S-proteins are highly conserved and show considerable homology to those of S-proteins from Petunia hybrida, Nicotiana alata and Lycopersicon peruvianum, three other species of the Solanaceae that also exhibit gametophytic self-incompatibility. cDNA clones encoding the three S-proteins were isolated and sequenced. Comparison of their deduced amino acid sequences reveals an average homology of 75.6%, with conserved and variable residue interspersed throughout the protein. Of the 137 conserved residues, 53 are also conserved in the N. alata S-proteins studies so far; of the 64 variable residues, 29 were identified as hypervariable based on calculation of the Similarity Index. There is only one hypervariable region of significant length, and it consists of eight consecutive hypervariable residues. This region correspond approximately to the hypervariable region HV2 identified in N. alata S-proteins. Of the two classes of N. alata S-proteins previously identified, one class exhibits greater homology to the three P. inflata S-proteins reported here than to the other class of N. alata S-proteins.