We showed that the F-box domain of PiSLF, Petunia inflata S-locus F-box protein, is essential for SI function and that a P. inflata ortholog of Antirrhinum SSK1 does not interact with PiSLF, consistent with our previous finding that PiSLF might be in a novel E3 complex.
Background and aims
Pistils of flowering plants possessing self-incompatibility (SI) can distinguish between self and non-self pollen, and only allow non-self pollen to effect fertilization. For Petunia inflata, the S-RNase gene encodes pistil specificity and multiple S-locus F-box (SLF) genes encode pollen specificity. Each SLF produced in pollen interacts with a subset of non-self S-RNases to mediate their ubiquitination and degradation by the 26S proteasome.
S-locus F-box has been proposed to function as a component of the conventional SCF (SKP1-CULLIN-F-box protein) complex, based on the finding that two SKP1-like proteins, AhSSK1 (Antirrhinum hispanicum SLF-interacting SKP1-like1) and PhSSK1 (Petunia hybrida SSK1), interact with the F-box domain of some allelic variants of SLF. However, we previously showed that PiSLF (P. inflata SLF) did not interact with any SKP1 of P. inflata or Arabidopsis thaliana, but instead interacted with a RING-finger protein, PiSBP1 (P. inflata S-RNase-Binding Protein1), which may also play the role of SKP1. Thus, the biochemical nature of the SLF-containing complex is as yet unclear.
To examine whether the F-box domain of PiSLF is required for SI function, we expressed a truncated PiSLF2 (S2 allelic variant) without this domain in S2S3 plants and showed that, unlike the full-length PiSLF2, it did not cause breakdown of SI in S3 pollen. We identified PiSSK1 (P. inflata SSK1) and found that it did not interact with PiSLF1, PiSLF2 or PiSLF3.
The finding that the truncated PiSLF2 did not cause breakdown of SI in S3 transgenic pollen suggests that the F-box domain of PiSLF2 is required for mediating degradation of S3-RNase, a non-self S-RNase, in S3 pollen, and thus is required for SI function. The finding that PiSSK1 did not interact with three allelic variants of PiSLF is consistent with our previous finding that PiSLF might not be in a conventional SCF complex.
Gametophytic self-incompatibility (GSI) in Solanaceae, Rosaceae, and Plantaginaceae is controlled by a multiallelic S-locus. The specificities of pistil and pollen are controlled by separate S-locus genes, S-RNase and SLF/SFB, respectively. Although the S-specificity is determined by the S-locus genes, factors located outside the S-locus are also required for expression of GSI. HT-B is one of the pistil non-S-factors identified in Nicotiana and Solanum, and encodes a small asparagine/aspartate-rich extracellular protein with unknown biochemical function. Here, HT-B was cloned from Petunia and characterized. The structural features and expression pattern of Petunia HT-B were very similar to those of Nicotiana and Solanum. Unlike other solanaceous species, expression of HT-B was also observed in self-compatible Petunia species. RNA interference (RNAi)-mediated suppression of Petunia HT-B resulted in partial breakdown of GSI. Quantitative analysis of the HT-B mRNA accumulation in the transgenics showed that a 100-fold reduction is not sufficient and a >1000-fold reduction is required to achieve partial breakdown of GSI.
HT-B; Petunia inflata; pistil; RNAi; self-incompatibility
S-RNase-based self-incompatibility (SI) occurs in the Solanaceae, Rosaceae and Plantaginaceae. In all three families, compatibility is controlled by a polymorphic S-locus encoding at least two genes. S-RNases determine the specificity of pollen rejection in the pistil, and S-locus F-box proteins fulfill this function in pollen. S-RNases are thought to function as S-specific cytotoxins as well as recognition proteins. Thus, incompatibility results from the cytotoxic activity of S-RNase, while compatible pollen tubes evade S-RNase cytotoxicity.
The S-specificity determinants are known, but many questions remain. In this review, the genetics of SI are introduced and the characteristics of S-RNases and pollen F-box proteins are briefly described. A variety of modifier genes also required for SI are also reviewed. Mutations affecting compatibility in pollen are especially important for defining models of compatibility and incompatibility. In Solanaceae, pollen-side mutations causing breakdown in SI have been attributed to the heteroallelic pollen effect, but a mutation in Solanum chacoense may be an exception. This has been interpreted to mean that pollen incompatibility is the default condition unless the S-locus F-box protein confers resistance to S-RNase. In Prunus, however, S-locus F-box protein gene mutations clearly cause compatibility.
Two alternative mechanisms have been proposed to explain compatibility and incompatibility: compatibility is explained either as a result of either degradation of non-self S-RNase or by its compartmentalization so that it does not have access to the pollen tube cytoplasm. These models are not necessarily mutually exclusive, but each makes different predictions about whether pollen compatibility or incompatibility is the default. As more factors required for SI are identified and characterized, it will be possible to determine the role each process plays in S-RNase-based SI.
S-RNase; self-incompatibility; Solanaceae; Rosaceae; Nicotiana; Prunus
Most fruit trees in the Rosaceae exhibit self-incompatibility, which is controlled by the pistil S gene, encoding a ribonuclease (S-RNase), and the pollen S gene at the S-locus. The pollen S in Prunus is an F-box protein gene (SLF/SFB) located near the S-RNase, but it has not been identified in Pyrus and Malus. In the Japanese pear, various F-box protein genes (PpSFBB-α–γ) linked to the S-RNase are proposed as the pollen S candidate. Two bacterial artificial chromosome (BAC) contigs around the S-RNase genes of Japanese pear were constructed, and 649 kb around S4-RNase and 378 kb around S2-RNase were sequenced. Six and 10 pollen-specific F-box protein genes (designated as PpSFBB4-u1–u4, 4-d1–d2 and PpSFBB2-u1–u5, 2-d1–d5, respectively) were found, but PpSFBB4-α–γ and PpSFBB2-γ were absent. The PpSFBB4 genes showed 66.2–93.1% amino acid identity with the PpSFBB2 genes, which indicated clustering of related polymorphic F-box protein genes between haplotypes near the S-RNase of the Japanese pear. Phylogenetic analysis classified 36 F-box protein genes of Pyrus and Malus into two major groups (I and II), and also generated gene pairs of PpSFBB genes and PpSFBB/Malus F-box protein genes. Group I consisted of gene pairs with 76.3–94.9% identity, while group II consisted of gene pairs with higher identities (>92%) than group I. This grouping suggests that less polymorphic PpSFBB genes in group II are non-S pollen genes and that the pollen S candidates are included in the group I PpSFBB genes.
BAC contig; F-box protein; pollen S gene; Pyrus pyrifolia; self-incompatibility; S-locus; S-RNase
Gametophytic self-incompatibility (GSI) is controlled by a complex S locus containing the pistil determinant S-RNase and pollen determinant SFB/SLF. Tight linkage of the pistil and pollen determinants is necessary to guarantee the self-incompatibility (SI) function. However, multiple probable pollen determinants of apple and Japanese pear, SFBBs (S locus F-box brothers), exist in each S haplotype, and how these multiple genes maintain the SI function remains unclear. It is shown here by high-resolution fluorescence in situ hybridization (FISH) that SFBB genes of the apple S
9 haplotype are physically linked to the S
-RNase gene, and the S locus is located in the subtelomeric region. FISH analyses also determined the relative order of SFBB genes and S-RNase in the S
9 haplotype, and showed that gene order differs between the S
9 and S
3 haplotypes. Furthermore, it is shown that the apple S locus is located in a knob-like large heterochromatin block where DNA is highly methylated. It is proposed that interhaplotypic heterogeneity and the heterochromatic nature of the S locus help to suppress recombination at the S locus in apple.
Fluorescence in situ hybridization; Malus × domestica, recombination suppression; self-incompatibility; S locus F-box brothers (SFBB); Fluorescence in situ hybridization; Fluorescence in situ hybridization
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 [1, 2]. In Papaver rhoeas, cognate pistil and pollen S-determinants, PrpS, a pollen-expressed transmembrane protein, and PrsS, a pistil-expressed secreted protein [3, 4], interact to trigger a Ca2+-dependent signaling network [5–10], resulting in inhibition of pollen tube growth, cytoskeletal alterations [11–13], and programmed cell death (PCD) [14, 15] 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 [11–15]. 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.
► PrpS, a Papaver SI determinant, functions in Arabidopsis thaliana pollen ► A “self” interaction with PrsS reveals Papaver SI hallmark features in A. thaliana ► The first evidence for transfamily functionality of an SI system (>140 my apart) ► Evidence of recruitment of signaling components for novel SI function
Higher plants produce seed through pollination, using specific interactions between pollen and pistil. Self-incompatibility (SI) is an important mechanism used in many species to prevent inbreeding, and is controlled by a multi-allelic S locus1,2. “Self” (incompatible) pollen is discriminated from “non-self” (compatible) pollen, by interaction of pollen and pistil S locus components, and is subsequently inhibited. In Papaver rhoeas, the pistil S locus product is a small protein that interacts with incompatible pollen, triggering a Ca2+-dependent signalling network, resulting in pollen inhibition and programmed cell death3-7. Here we have cloned three alleles of a highly polymorphic pollen-expressed gene, PrpS, from Papaver and provide evidence that this encodes the pollen S locus determinant. PrpS is a single copy gene linked to the pistil S gene, PrsS. Sequence analysis indicates that PrsS and PrpS are equally ancient and are likely to have co-evolved. PrpS encodes a novel ~20 kDa protein. Consistent with predictions that it is a transmembrane protein, PrpS is associated with the plasma membrane. We show that a predicted extracellular loop segment of PrpS interacts with PrsS and, using PrpS antisense oligonucleotides, we demonstrate that PrpS is involved in S-specific inhibition of incompatible pollen. Identification of PrpS represents a major advance in our understanding of the Papaver SI system. As a novel cell-cell recognition determinant it contributes to the available information concerning the origins and evolution of cell-cell recognition systems involved in discrimination between “self” and “non-self”, which also include histocompatibility systems in primitive chordates and vertebrates.
Self-incompatibility; pollen S receptor; Papaver rhoeas; pollen tube inhibition
Gametophytic self-incompatibility (GSI) is a mechanism in flowering plants, to prevent inbreeding and promote outcrossing. GSI is under the control of a specific locus, known as the S-locus, which contains at least two genes, the RNase and the SFB. Active S-RNases in the style are essential for rejection of haploid pollen, when the pollen S-allele matches one of two S-alleles of the diploid pistil. However, the nature of their mutual interactions at genetic and biochemical levels remain unclear. Thus, detailed understanding of the protein structure involved in GSI may help in discovering how the proteins involved in GSI may function and how they fulfill their biological roles. To this end, 3D models of the SC (Sf) and two SI (S8 and S23) S-RNases of almond were constructed, using comparative modeling tools. The modeled structures consisted of mixed α and β folds, with six helices and six β-strands. However, the self-compatible (Sf) RNase contained an additional extended loop between the conserved domains RC4 and C5, which may be involved in the manifestation of self-compatibility in almond.
almond; self-(in)compatibility; 3D modeling; RNase T2
Self-incompatibility (SI) systems appeared early in plant evolution as an effective mechanism to promote outcrossing and avoid inbreeding depression. These systems prevent self-fertilization by the recognition and rejection of self-pollen and pollen from closely related individuals. The most widespread SI system is based on the action of a pistil ribonuclease, the S-RNase, which recognizes and rejects incompatible pollen. S-RNases are endocyted by pollen tubes and stored into vacuoles. By a mechanism that is still unknown, these vacuoles are selectively disrupted in incompatible pollen, releasing S-RNases into the cytoplasm and allowing degradation of pollen RNA. Recently, we have studied the timing of in vivo alterations of pollen F-actin cytoskeleton after incompatible pollinations. Besides being essential for pollen growth, F-actin cytoskeleton is a very dynamic cellular component. Changes in F-actin organization are known to be capable of transducing signaling events in many cellular processes. Early after pollination, F-actin showed a progressive disorganization in incompatible pollen tubes. However by the time the F-actin was almost completely disrupted, the large majority of vacuolar compartments were still intact. These results indicate that in incompatible pollen tubes F-actin disorganization precedes vacuolar disruption. They also suggest that F-actin may act as an early transducer of signals triggering the rejection of incompatible pollen.
F-actin; Nicotiana alata; self-incompatibility; signaling; S-RNase; pollen; vacuole
Background and Scope
Self-incompatibility (SI) in flowering plants ensures the maintenance of genetic diversity by ensuring outbreeding. Different genetic and mechanistic systems of SI among flowering plants suggest either multiple origins of SI or considerable evolutionary diversification. In the grasses, SI is based on two loci, S and Z, which are both polyallelic: an incompatible reaction occurs only if both S and Z alleles are matched in individual pollen with alleles of the pistil on which they alight. Such incompatibility is referred to as gametophytic SI (GSI). The mechanics of grass GSI is poorly understood relative to the well-characterized S-RNase-based single-locus GSI systems (Solanaceae, Rosaceae, Plantaginaceae), or the Papaver recognition system that triggers a calcium-dependent signalling network culminating in programmed cell death. There is every reason to suggest that the grass SI system represents yet another mechanism of SI. S and Z loci have been mapped using isozymes to linkage groups C1 and C2 of the Triticeae consensus maps in Secale, Phalaris and Lolium. Recently, in Lolium perenne, in order to finely map and identify S and Z, more closely spaced markers have been developed based on cDNA and repeat DNA sequences, in part from genomic regions syntenic between the grasses. Several genes tightly linked to the S and Z loci were identified, but so far no convincing candidate has emerged.
Research and Progress
From subtracted Lolium immature stigma cDNA libraries derived from S and Z genotyped individuals enriched for SI potential component genes, kinase enzyme domains, a calmodulin-dependent kinase and a peptide with several calcium (Ca2+) binding domains were identified. Preliminary findings suggest that Ca2+ signalling and phosphorylation may be involved in Lolium GSI. This is supported by the inhibition of Lolium SI by Ca2+ channel blockers lanthanum (La3+) and verapamil, and by findings of increased phosphorylation activity during an SI response.
Lolium perenne; perennial ryegrass; grasses; Poaceae; self-incompatibility; calcium inhibitors; lanthanum chloride; verapamil
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.
S-RNase; programmed cell death; reactive oxygen species; actin cytoskeleton; Ca2+ current; nuclear DNA
Background and Aims
Sexual reproduction in angiosperms involves a network of signalling and interactions between pollen and pistil. To promote out-breeding, an additional layer of interactions, involving self-incompatibility (SI), is used to prevent self-fertilization. SI is generally controlled by the S-locus, and comprises allelic pollen and pistil S-determinants. This provides the basis of recognition, and consequent rejection, of incompatible pollen. In Papaver rhoeas, SI involves interaction of pistil PrsS and pollen PrpS, triggering a Ca2+-dependent signalling network. This results in rapid and distinctive alterations to both the actin and microtubule cytoskeleton being triggered in ‘self’ pollen. Some of these alterations are implicated in mediating programmed cell death, involving activation of several caspase-like proteases.
Here we review and discuss our current understanding of the cytoskeletal alterations induced in incompatible pollen during SI and their relationship with programmed cell death. We focus on data relating to the formation of F-actin punctate foci, which have, to date, not been well characterized. The identification of two actin-binding proteins that interact with these structures are reviewed. Using an approach that enriched for F-actin from SI-induced pollen tubes using affinity purification followed by mass spectrometry, further proteins were identified as putative interactors with the F-actin foci in an SI situation.
Previously two important actin-binding proteins, CAP and ADF, had been identified whose localization altered with SI, both showing co-localization with the F-actin punctate foci based on immunolocalization studies. Further analysis has identified differences between proteins associated with F-actin from SI-induced pollen samples and those associated with F-actin in untreated pollen. This provides candidate proteins implicated in either the formation or stabilization of the punctate actin structures formed during SI.
This review brings together for the first time, our current understanding of proteins and events involved in SI-induced signalling to the actin cytoskeleton in incompatible Papaver pollen.
Actin cytoskeleton; actin-binding proteins; mass spectrometry; Papaver rhoeas; pollen; self-incompatibility; signalling
Self-incompatibility (SI) is widespread in the angiosperms, but identifying the biochemical components of SI mechanisms has proven to be difficult in most lineages. Coffea (coffee; Rubiaceae) is a genus of old-world tropical understory trees in which the vast majority of diploid species utilize a mechanism of gametophytic self-incompatibility (GSI). The S-RNase GSI system was one of the first SI mechanisms to be biochemically characterized, and likely represents the ancestral Eudicot condition as evidenced by its functional characterization in both asterid (Solanaceae, Plantaginaceae) and rosid (Rosaceae) lineages. The S-RNase GSI mechanism employs the activity of class III RNase T2 proteins to terminate the growth of “self” pollen tubes. Here, we investigate the mechanism of Coffea GSI and specifically examine the potential for homology to S-RNase GSI by sequencing class III RNase T2 genes in populations of 14 African and Madagascan Coffea species and the closely related self-compatible species Psilanthus ebracteolatus. Phylogenetic analyses of these sequences aligned to a diverse sample of plant RNase T2 genes show that the Coffea genome contains at least three class III RNase T2 genes. Patterns of tissue-specific gene expression identify one of these RNase T2 genes as the putative Coffea S-RNase gene. We show that populations of SI Coffea are remarkably polymorphic for putative S-RNase alleles, and exhibit a persistent pattern of trans-specific polymorphism characteristic of all S-RNase genes previously isolated from GSI Eudicot lineages. We thus conclude that Coffea GSI is most likely homologous to the classic Eudicot S-RNase system, which was retained since the divergence of the Rubiaceae lineage from an ancient SI Eudicot ancestor, nearly 90 million years ago.
Plants have many ways to regulate the type of pollen that arrives on the stigma surface. Once there, further control mechanisms regulate compatibility. The latter controls are largely based on biochemical interactions that support compatible pollination and prevent incompatible matings. S-RNase-based self-incompatibility (SI) systems are the most phylogenetically widespread mechanisms for controlling pollination. Studies of Nicotiana establish a firm link between SI and unilateral interspecific incompatibility. Although implicated in both inter- and intraspecific compatibility, S-RNase operates through at least three distinct genetic mechanisms that differ in their dependence on non-S-RNase factors. Identification and characterization of these non-S-RNase factors is currently an area of active research. Searching for genetic and biochemical interactions with S-RNase can identify candidate non-S-RNase factors. HT-protein is one factor that is required for S-allele-specific pollen rejection in the Solanaceae. Major style arabinogalactan proteins such as TTS interact biochemically with S-RNase. These glycoproteins are known to interact with compatible pollen tubes and have long been suggested as possible recognition molecules. Their binding to S-RNase implies a link between stylar systems for compatibility and incompatibility. Thus, genetic and biochemical studies suggest a highly networked picture of pollen-pistil interactions.
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.
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.
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.
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.
Confocal microscopy; F-actin cytoskeleton; Nicotiana alata; pollen tube; self-incompatibility; S-RNase; vacuolar system
Plants have evolved many systems to prevent undesirable fertilization. Among these, incompatibility is a well-organized system in which pollen germination or pollen tube growth is inhibited in pistils. We previously found that a novel one-way pollen–stigma incompatibility response [unilateral incompatibility (UI)] occurred between two self-incompatible Brassica rapa plants, a Turkish line, and a Japanese cultivated hybrid variety, “Osome.” Pollen from the Turkish line is rejected on the stigma of the Osome line, but the reverse cross is compatible; such a UI phenotype closely resembles self-incompatibility (SI). The pollen factor of this UI has been genetically explained by a single locus which is different from the S-locus. In this study, we performed further genetic analyses of this intraspecies UI and showed that the stigma factor was also controlled by a single locus, and we named the loci corresponding to the stigma and pollen factors of the intraspecies UI, stigmatic unilateral incompatibility (SUI), and pollen unilateral incompatibility (PUI) loci, respectively. Interestingly, segregation analyses of SUI and PUI indicated that they are closely linked to each other and behave as a single unit. To investigate the effect of an SI-related gene, MLPK in this UI, we produced segregation lines for SUI and mlpk. A distorted segregation ratio of SUI phenotype in an mlpk background indicated involvement of MLPK in SUI, suggesting the existence of an MLPK-dependent novel pollen–stigma recognition mechanism.
Brassica rapa L; unilateral incompatibility; self-incompatibility; pollen-stigma recognition
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 self-incompatibility mechanism that reduces inbreeding in many plants of the Rosaceae is attributed to a multi-allelic S locus which, in the Prunoideae and Maloideae subfamilies, comprises two complementary genes, a stylar-expressed S-RNase and a pollen-expressed SFB. To elucidate incompatibility in the subfamily Rosoideae, stylar-specific RNases and self-(in)compatibility status were analysed in various diploid strawberries, especially Fragaria nubicola and F. viridis, both self-incompatible, and F. vesca, self-compatible, and in various progenies derived from them. Unexpectedly, two unlinked RNase loci, S and T, were found, encoding peptides distinct from Prunoideae and Maloideae S-RNases; the presence of a single active allele at either is sufficient to confer self-incompatibility. By contrast, in diploid Maloideae and Prunoideae a single locus encodes S-RNases that share several conserved regions and two active alleles are required for self-incompatibility. Our evidence implicates the S locus in unilateral inter-specific incompatibility and shows that S and T RNases can, remarkably, confer not only allele-specific rejection of cognate pollen but also unspecific rejection of Sn Tn pollen, where n indicates a null allele, consistent with the the presence of the pollen component, SFB, activating the cognitive function of these RNases. Comparison of relevant linkage groups between Fragaria and Prunus suggests that Prunus S-RNases, unique in having two introns, may have resulted from gene conversion in an ancestor of Prunus. In addition, it is shown that there is a non-S locus that is essential for self-incompatibility in diploid Fragaria.
Fragaria; Rosaceae; Rosoideae; self-incompatibility; S/T RNases; unilateral incompatibility
Tetraploid sour cherry (Prunus cerasus L.) exhibits gametophytic self-incompatibility (GSI) whereby the specificity of self-pollen rejection is controlled by alleles of the stylar and pollen specificity genes, S-RNase and SFB (S haplotype-specific F-box protein gene), respectively. As sour cherry selections can be either self-compatible (SC) or self-incompatible (SI), polyploidy per se does not result in SC. Instead the genotype-dependent loss of SI in sour cherry is due to the accumulation of non-functional S-haplotypes. The presence of two or more non-functional S-haplotypes within sour cherry 2x pollen renders that pollen SC. Two new S-haplotypes from sour cherry, S33 and S34, that are presumed to be contributed by the P. fruticosa species parent, the complete S-RNase and SFB sequences of a third S-haplotype, S35, plus the presence of two previously identified sweet cherry S-haplotypes, S14 and S16 are described here. Genetic segregation data demonstrated that the S16-, S33-, S34-, and S35-haplotypes present in sour cherry are fully functional. This result is consistent with our previous finding that ‘hetero-allelic’ pollen is incompatible in sour cherry. Phylogenetic analyses of the SFB and S-RNase sequences from available Prunus species reveal that the relationships among S-haplotypes show no correspondence to known organismal relationships at any taxonomic level within Prunus, indicating that polymorphisms at the S-locus have been maintained throughout the evolution of the genus. Furthermore, the phylogenetic relationships among SFB sequences are generally incongruent with those among S-RNase sequences for the same S-haplotypes. Hypotheses compatible with these results are discussed.
Prunus cerasus; self-incompatibility; SFB; S-RNase
Self-incompatibility (SI) involves the recognition and rejection of self or genetically identical pollen. Gametophytic SI is probably the most widespread of the SI systems and, so far, two completely different SI mechanisms, which appear to have evolved separately, have been identified. One mechanism is the RNase system, which is found in the Solanaceae, Rosaceae and Scrophulariaceae. The other is a complex system, so far found only in the Papaveraceae, which involves the triggering of signal transduction cascade(s) that result in rapid pollen tube inhibition and cell death. Here, we present an overview of what is currently known about the mechanisms involved in controlling pollen tube inhibition in these two systems.
Intraspecific mate selectivity often is enforced by self-incompatibility (SI), a barrier to self-pollination that inhibits productive pollen-pistil interactions. In the Brassicaceae, SI specificity is determined by two highly-polymorphic proteins: the stigmatic S-locus receptor kinase (SRK) and its pollen coat-localized ligand, the S-locus cysteine-rich protein (SCR). Arabidopsis thaliana is self fertile, but several of its accessions can be made to express SI, albeit to various degrees, by transformation with functional SRK-SCR gene pairs isolated from its close self-incompatible relative, Arabidopsis lyrata. Here, we use a newly identified induced mutation that suppresses the SI phenotype in stigmas of SRK-SCR transformants of the Col-0 accession to investigate the regulation of SI and the SRK transgene. This mutation disrupts NRPD1a, a gene that encodes a plant-specific nuclear RNA polymerase required for genomic methylation and production of some types of silencing RNAs. We show that NRPD1a, along with the RNA-dependent RNA polymerase RDR2, is required for SI in some A. thaliana accessions. We also show that Col-0 nrpd1a mutants exhibit decreased accumulation of SRK transcripts in stigmas, which is not, however, responsible for loss of SI in these plants. Together, our analysis of the nrpd1a mutation and of SRK promoter activity in various accessions reveals that the SRK transgene is subject to several levels of regulation, which vary substantially by tissue type and by accession. This study thus helps explain the well-documented differences in expression of SI exhibited by SRK-SCR transformants of different A. thaliana accessions.
self-incompatibility; S-locus receptor kinase; NRPD1a; Arabidopsis thaliana
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.
MAPK; self-incompatibility; PCD; caspase-3-like activity; Papaver rhoeas
‘Jin Zhui’ is a spontaneous self-compatible mutant of ‘Ya Li’ (Pyrus bretschneideri Rehd. S21S34), the latter displaying a typical S-RNase-based gametophytic self-incompatibility (GSI). The pollen-part mutation (PPM) of ‘Jin Zhui’ might be due to a natural mutation in the pollen-S gene (S34 haplotype). However, the molecular mechanisms behind these phenotypic changes are still unclear. In this study, we identified five SLF (S-Locus F-box) genes in ‘Ya Li’, while no nucleotide differences were found in the SLF genes of ‘Jin Zhui’. Further genetic analysis by S-RNase PCR-typing of selfed progeny of ‘Jin Zhui’ and ‘Ya Li’ × ‘Jin Zhui’ progeny showed three progeny classes (S21S21, S21S34 and S34S34) as opposed to the two classes reported previously (S21S34 and S34S34), indicating that the pollen gametes of ‘Jin Zhui’, bearing either the S21- or S34-haplotype, were able to overcome self-incompatibility (SI) barriers. Moreover, no evidence of pollen-S duplication was found. These findings support the hypothesis that loss of function of S-locus unlinked PPM expressed in pollen leads to SI breakdown in ‘Jin Zhui’, rather than natural mutation in the pollen-S gene (S34 haplotype). Furthermore, abnormal meiosis was observed in a number of pollen mother cells (PMCs) in ‘Jin Zhui’, but not in ‘Ya Li’. These and other interesting findings are discussed.
Pollen–pistil interactions are an essential prelude to fertilization in angiosperms and determine compatibility/incompatibility. Pollen–pistil interactions have been studied at a molecular and cellular level in relatively few families. Self-incompatibility (SI) is the best understood pollen–pistil interaction at a molecular level where three different molecular mechanisms have been identified in just five families. Here we review studies of pollen–pistil interactions and SI in the Asteraceae, an important family that has been relatively understudied in these areas of reproductive biology.
We begin by describing the historical literature which first identified sporophytic SI (SSI) in species of Asteraceae, the SI system later identified and characterized at a molecular level in the Brassicaceae. Early structural and cytological studies in these two families suggested that pollen–pistil interactions and SSI were similar, if not the same. Recent cellular and molecular studies in Senecio squalidus (Oxford ragwort) have challenged this belief by revealing that despite sharing the same genetic system of SSI, the Brassicaceae and Asteraceae molecular mechanisms are different. Key cellular differences have also been highlighted in pollen–stigma interactions, which may arise as a consequence of the Asteraceae possessing a ‘semi-dry’ stigma, rather than the ‘dry’ stigma typical of the Brassicaceae. The review concludes with a summary of recent transcriptomic analyses aimed at identifying proteins regulating pollen–pistil interactions and SI in S. squalidus, and by implication the Asteraceae. The Senecio pistil transcriptome contains many novel pistil-specific genes, but also pistil-specific genes previously shown to play a role in pollen–pistil interactions in other species.
Studies in S. squalidus have shown that stigma structure and the molecular mechanism of SSI in the Asteraceae and Brassicaceae are different. The availability of a pool of pistil-specific genes for S. squalidus offers an opportunity to elucidate the molecular mechanisms of pollen–pistil interactions and SI in the Asteraceae.
Asteraceae; Senecio; pistil; stigma; pollen; pollen–pistil interactions; self-incompatibility; transcriptome
Programmed cell death (PCD) has been found to be induced after pollination both in papillar cells and in self-incompatible pollen in the olive (Olea europaea L.). Reactive oxygen species (ROS) and nitric oxide (NO) are known to be produced in the pistil and pollen during pollination but their contribution to PCD has so far remained elusive. The possible role of ROS and NO was investigated in olive pollen–pistil interaction during free and controlled pollination and it was found that bidirectional interaction appears to exist between the pollen and the stigma, which seems to regulate ROS and NO production. Biochemical evidence strongly suggesting that both O2˙− and NO are essential for triggering PCD in self-incompatibility processes was also obtained. It was observed for the first time that peroxynitrite, a powerful oxidizing and nitrating agent generated during a rapid reaction between O2˙− and NO, is produced during pollination and that this is related to an increase in protein nitration which, in turn, is strongly associated with PCD. It may be concluded that peroxynitrite mediates PCD during pollen–pistil interaction in Olea europaea L. both in self-incompatible pollen and papillar cells.
Nitric oxide; Olea europaea L.; peroxynitrite; programmed cell death; pollen–pistil interaction; reactive oxygen species