We have explained in the previous section that S genes should have higher polymorphism than other genes. To test this, diversity must be quantified in samples of alleles from natural populations. Genomic gel blotting (restriction fragment length polymorphism analysis) can provide some data, but shows merely that variants exist, without adequate quantification. Allele numbers depend on recombination rates and so are an unsatisfactory measure of diversity (they can be enormous for sequences with several polymorphic sites that recombine, such as MHC genes). Diversity should thus be quantified as ‘nucleotide diversity’, using measures such as the mean fraction of sites that differ between two randomly sampled alleles at a locus, π. Amino acid and nonsynonymous site diversity (πn) will be particularly high in codons directly affected by balancing selection, and regions with high πn or πn > πs (synonymous site diversity) can often help to identify these (Table 1; Richman et al., 1996). As a basis for comparison, πs in maize has been shown to range up to 3.6% across a sample of 21 loci (Tenaillon et al., 2002).
It is now understood that variability will also often be high at unselected sites close to sites where frequency-dependent selection maintains many alleles for long time-periods (Schierup et al., 2000; Takebayashi et al., 2003). If functionally different S alleles rarely recombine, their sequences will evolve like alleles in isolated populations with low ‘effective population sizes’, and hence diversity among different instances of the same functional allele will be low. Between different alleles, however, synonymous and even nonsynonymous differences can accumulate in the absence of recombination (Vekemans & Slatkin, 1994; Charlesworth et al., 2003). Given estimated plant recombination values of approx. 1 Mb per centiMorgan, the predicted diversity peaks should span only a few hundred nucleotides, which would nevertheless greatly hinder precise identification of the sites within S proteins that are involved in recognition (Charlesworth et al., 2003), a chief reason for obtaining sequences. In the MHC systems, recombination occurs and diversity is indeed largely highest at sites near known peptide-binding amino acids (Table 1).
In SI systems, maintaining coadapted combinations of pollen and pistil alleles should result in the evolution of low-recombination frequencies between the S loci. The S-locus genome region is physically too small (Fig. 1) to test this by genetic mapping with feasible family sizes, but population genetic approaches may be able to test for low recombination through detecting haplotypes of only certain pistil and pollen S-allele combinations (linkage disequilibrium). If recombination is rare, diversity may be elevated over large genomic tracts, possibly including other genes and sequences located within the S-locus region.
High diversity is a necessary condition for accepting a gene as an S locus, and candidate genes can sometimes be eliminated because of low diversity (Casselman et al., 2000; Takebayashi et al., 2003). If the S loci lie within a nonrecombining region, however, high diversity may hinder even identifying the functional S genes. Moreover, diversity data from SI plants are scarce for non-S loci, so one often cannot test whether candidate S loci have significantly elevated diversity. Transgenic experiments are thus also essential to show that candidate genes change the incompatibility reaction appropriately.
In the Brassicaceae, the pistil protein is a receptor kinase, SRK. Initial work in Brassica identified an S-domain protein, the highly polymorphic SLG (reviewed in Sato et al., 2002), but the evidence from polymorphism was misleading: SLG has no major role in recognition, but seems merely to enhance the SI response – its polymorphism probably results from close linkage to the true pistil-recognition gene, SRK (Nasrallah, 2002). SRK is anchored to the stigma membrane, presenting its extracellular S domain to ligand molecules on the pollen surface (Nasrallah, 2002). Both domains are extraordinarily polymorphic (Table 1), although the S domain is presumably the sole region subject to balancing selection. This suggests that the S locus may lie within a region of unusually low recombination frequency. As shown in Fig. 1, physical mapping shows that allelic S-locus regions are organized into haplotypes with the genes in different orders and orientations (Boyes et al., 1997). Moreover, high synonymous and also nonsynonymous site diversity in the A. lyrata SRK kinase domain (Table 1) are difficult to explain unless recombination with the S domain is infrequent. These findings are all consistent with the S-locus region containing at least two coadapted genes, the most plausible evolutionary reason for a low recombination rate. The alternative, that the region is located in a genome region, such as a centromeric region, that rarely recombines, seems not to apply in A. lyrata (Kusaba et al., 2001) whose gene locations within chromosome arms are probably similar to those in A. thaliana (Kuittinen et al. 2004).
A breakthrough in establishing the existence of separate pollen S genes was the identification of the Brassica and Arabidopsis pollen SI determinant. The candidate gene for the ligand in Brassicaceae is a small, cysteine-rich pollen-coat protein, SCR (also called SP11; Schopfer et al., 1999; Takayama et al., 2000). Its sequence diversity in Brassica is high (Watanabe et al., 2000; Shiba et al., 2002); however, a full picture of variability is not yet available because alleles in dominant haplotypes differ so greatly that not all genetically detectable alleles can be sequenced. In A. lyrata, with only two alleles compared to date, many differences were found in this short protein sequence (Kusaba et al., 2001).
Direct evidence from transgenic experiments shows that SCR(SP11) is indeed the pollen S gene. Transfer of SRK and SCR alleles of a particular A. lyrata haplotype causes SI in A. thaliana buds, though only transiently in many strains (Nasrallah et al., 2004), and transferring Raphanus SCR alleles into B. campestris (rapa) caused the recipients’ pollen to be rejected by Brassica plants with a particular incompatibility type (Sato et al., 2004), showing that SCR determines the pollen incompatibility type and also that certain alleles in the two species have the same incompatibility type (despite several sequence differences).
Even with this detailed knowledge from Brassicaceae, unexpected properties of S genes continue to emerge, notably expression differences of SCR(SP11) alleles. In heterozygotes with alleles of differing dominance, only the more dominant allele is expressed (Kusaba et al., 2002; Shiba et al., 2002). This mono-allelic expression is like that in other chemoreceptor systems, including mammalian odorant receptors (Goldmit & Bergman, 2004), and ensures that each pollen grain has a single SI type. Unlike other receptor systems, expression choice is deterministic: dominant alleles are expressed in both the anther tapetum and, gametophytically, in pollen, whereas recessive alleles are not expressed in pollen. Expression is presumably controlled by different upstream sequences, which suggests that the haplotype structure of S-locus regions may embrace those regions also. New alleles may thus be constrained to have the same dominance as their progenitors. This has not yet been incorporated into evolutionary models, but it may explain the clustering of Brassica allele sequences according to their dominance.
Solanaceae, Anthirrhinum and Rosaceae
Unlike the SI of Brassicaceae, where inhibition occurs on the stigma surface, in these species incompatible pollen tubes are inhibited in the stylar transmitting tract. Consistent with this, the pistil S-RNase protein is expressed in style tissue. It is taken up by growing pollen tubes, degrading RNAs within incompatible pollen tubes and causing subsequent arrest of growth in the style (Kao & Tsukamoto, 2004). Like SRK, S-RNases are highly polymorphic (Table 1), although most sequence data come from the reverse transcription–polymerase chain reaction amplification of pistil cDNA. Synonymous and nonsynonymous diversity are both high (e.g. Richman et al., 1996). Only rarely are segregation tests carried out to verify that sequences are allelic and that different incompatibility classes in families correspond with different sequences. Natural population studies generally simply assume that different sequences represent different alleles (presumably treating cases with few differences as sequencing errors).
The pollen determinant of SI has now been identified in species of all three families with S-RNase systems (Solanaceae, Rosaceae and Antirrhinum), and the findings suggest a mechanism for these systems, and may explain some of their odd features. Genes encoding F-box proteins have been candidates for the gene controlling pollen recognition since the discovery of S-linked F-box (SFB) genes in A. hispanicum (Lai et al. 2002) and Prunus mume (Rosaceae, Entani et al., 2003). However, formal proof was needed that an SFB gene determines pollen specificity, as this is a very large gene family (694 estimated genes in A. thaliana; Gagne et al., 2002), and several such genes have been found in the S-locus region of some species (Entani et al., 2003; Kao & Tsukamoto, 2004). Transformation experiments now confirm F-box genes as the pollen S of both P. inflata (Sijacic et al., 2004) and A. hispanicum (Qiao et al. 2004).
The Petunia study made elegant use of the ‘competition interaction’ phenomenon, which creates difficulties for transgenic experiments with pollen S genes, but provides a characteristic specific for the male SI determinant. In many known gametophytic systems, pollen grains heterozygous for S alleles may be compatible with plants carrying both alleles (Lewis, 1947). Thus, self-compatible plants in species with S-RNase systems commonly arise by tetraploidy or duplication of the S locus, making some of the pollen effectively heterozygous for the pollen S gene. This does not occur in the Brassicaceae: tetraploids maintain normal incompatibility reactions (Mable et al., 2004). The pollen SFB protein belongs to a protein family involved in ubiquitin-mediated protein degradation. F-box proteins often play crucial roles, delivering appropriate targets to the ubiquitin–protein ligase complex (Gagne et al., 2002). The ‘inhibitor model’ for SI proposes that interactions of pollen-recognition proteins with nonself S-RNases do not allow RNase activity, whereas activity does occur after interaction with the cognate S-RNase (Entani et al., 2003; Kao & Tsukamoto, 2004). Unexpectedly, the new results offer a simpler model and can explain the competition phenomenon. Competition suggests active destruction of RNase activity by any pollen S-protein not recognized as the cognate one, for example compatibility resulting from degradation of all nonself S-RNase proteins (Fig. 2).
Figure 2. Diagram showing a possible mechanism of self-incompatibility in S-RNase systems (a), and the competitive interaction phenomenon (b). In this model, SI is caused by failure of each pollen S-linked F-box (SFB) allele product to destroy its cognate pistil S-RNase. (a) A pollen tube from a haploid pollen grain carrying the S2SFB allele is shown, in the self-pollination of a plant with genotype S1S2. Pistil S-RNases encoded by both alleles enter the tube, and that from the S1 haplotype is degraded while the S2 S-RNase is not. RNA in the pollen tube is then digested by the S2 S-RNase (i.e. the pollen is incompatible). (b) In pollen containing both S1 and S2SFB alleles, both S-RNases are degraded, leading to compatibility. Other interactions between alleles in diploid pollen (Lewis, 1947, 1960) can also be explained on this model. Weakening of SI suggests that the pollen SFB proteins must interact, probably forming dimers (or a higher-order complex) that fail to eliminate all RNase, even though each homodimer still leads to destruction of some of its noncognate RNase, as with ‘competing’SFB alleles; as heterodimers should represent half of the SFB protein present, sufficient active RNAse might be present to give weak incompatibility. If heterodimers fail to cause destruction of either of their cognate RNases (even though homodimers lead to degradation), some RNase activity of both allelic types might remain (coexpressed alleles). Finally, if heterodimers affect only one RNase (say, the S2 product), some of the other – S1– RNase might remain active and digest RNA; if even a small amount of active RNase causes incompatibility, the pollen S1 allele would behave as dominant.
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Surprisingly, expression of the P. inflata PiSLF gene peaks in immature bicellular pollen in the anthers, not during pollen tube growth. Diversity data and duplications in self-compatible strains (Tsukamoto et al., 2005), however, support the conclusion that the true pollen S has been found, although further data are needed. An approximate quantification for PiSLF, using a few alleles, suggests extremely high amino acid diversity (Sijacic et al., 2004), consistent with the extremely high S-RNase πs and πn values compared with other plant loci (Table 1). However, in some Prunus and Petunia species with S-RNase systems, the S loci may lie close to centromeric regions (with low recombination). Until linkage has been quantified, and diversity estimated for nearby loci, evidence from diversity of candidate genes is incomplete.
In Papaveraceae, only the pistil S protein has been characterized to date. In Papaver rhoeas, it is the pistil S protein that is a small extracellular signal molecule (see Thomas & Franklin-Tong, 2004), presumably interacting with a receptor on pollen tube surfaces. Despite the pollen determinant still being hypothetical, the cellular mechanism of SI is known in considerable detail. Self-stigma S proteins elicit a rapid increase in Ca2+ within pollen tubes growing in vitro, suggesting the involvement of programmed cell death (PCD) processes in the SI response. Indeed, inhibiting caspase-3-protease, a key PCD enzyme, abolishes endonuclease activity and prevents DNA fragmentation and pollen-tube growth inhibition that occur in normal incompatibility (Thomas & Franklin-Tong, 2004).