Journal of Experimental Botany, Vol. 54, No. 380, pp. 169-174,
January 1, 2003
© 2003 Oxford University Press
Sporophytic self-incompatibility in Senecio squalidus L. (Asteraceae)—the search for S
Received 12 April 2002; Accepted 22 July 2002
1 School of Biological Sciences, University of Bristol, Woodland Road, Clifton, Bristol BS8 1UG, UK
2 Department of Plant Sciences, South Parks Road, University of Oxford, Oxford OX1 3RB, UK
3 To whom correspondence should be addressed. Fax: +44 (0)117 9257374. E-mail: simon.hiscock{at}bristol.ac.uk
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Abstract |
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Senecio squalidus (Oxford Ragwort) is being used as a model species to study the genetics and molecular genetics of self-incompatibility (SI) in the Asteraceae. S. squalidus has a strong system of sporophytic SI (SSI) and populations within the UK contain very few S alleles probably due to a population bottleneck experienced on its introduction to the UK. The genetic control of SSI in S. squalidus is complex and may involve a second locus epistatic to S. Progress towards identifying the female determinant of SSI in S. squalidus is reviewed here. Research is focused on plants carrying two defined S alleles, S1 and S2. S2 is dominant to S1 in pollen and stigma. RT-PCR was used to amplify three SRK-like cDNAs from stigmas of S1S2 heterozygotes, but the expression patterns of these cDNAs suggest that they are unlikely to be directly involved in SI or pollen–stigma interactions in contrast to SSI in the Brassicaceae. Stigma-specific proteins associated with the S1 allele and the S2 allele have been identified using isoelectric focusing and these proteins have been designated SSP1 (Stigma S-associated Protein 1) and SSP2. SSP1 and SSP2 cDNAs have been cloned by 3' and 5' RACE and shown to be allelic forms of the same gene, SSP. The expression of SSP and its linkage to the S locus are currently being investigated. Initial results show SSP to be expressed exclusively in stigmas and developmentally regulated, with maximal expression occurring at and just before anthesis when SI is fully functional, SSP expression being undetectable in immature buds. Together these data suggest that SSP is a strong candidate for a Senecio S-gene.
Key words: Asteraceae, S-gene, Senecio squalidus, sporophytic self-incompatibility, SRK-like gene.
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Introduction |
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TopAbstractIntroductionGenetics of SSI in...ConclusionsReferences |
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Self-incompatibility in the daisy family (Asteraceae) is of the sporophytic type whereby the incompatibility phenotype of the pollen is determined by the diploid genotype of its parental plant. Classical genetic studies in the 1950s first identified the sporophytic SI (SSI) system in the daisies Crepis foetida, Parthenium argenteum and Cosmos bipinnatus and showed SSI to be regulated by a single polyallelic S locus (Hughes and Babcock, 1950; Gerstel, 1950; Crowe, 1954, respectively). Subsequently SSI was also shown to operate in the Brassicaceae (Bateman, 1955). A feature of SSI is that dominance interactions are possible between different S alleles, this being a consequence of the diploid expression of the S phenotype in both pollen and stigma. Furthermore, dominance relationships between S alleles can differ in pollen and stigma leading to complex patterns of incompatibility/compatibility when progenies are intercrossed in diallels (de Nettancourt, 1977). In both the Brassicaceae and the Asteraceae incompatible pollen is inhibited at the stigma surface, generally before pollen tubes have penetrated the stigma.
In recent years molecular characterization of SSI has been directed exclusively at species from the Brassicaceae, particularly Brassica oleracea and B. campestris (syn. rapa). Three genes present at the Brassica S locus have been shown to be involved in the SSI response (see Hiscock, 2002, for a review). The S-receptor kinase (SRK) gene regulates the female (stigmatic) part of the SI response (Takasaki et al., 2000) and SCR (S cysteine-rich protein), alternative name SP11, designates the pollen S identity (Schopfer et al., 1999; Takayama et al., 2000). A third gene, SLG (S locus glycoprotein), which like SRK is expressed exclusively in the stigma, is not essential for SI, but increases the efficiency/strength of the SI response (Takasaki et al., 2000). SRK encodes a serine-threonine receptor kinase located in the plasma membrane of stigmatic papilla cells and SLG encodes a secreted glycoprotein found abundantly in papilla cell walls. For a given S haplotype, SLG and the receptor domain of SRK share around 92–95% amino acid sequence identity. SCR/SP11 encodes a small cysteine-rich protein expressed sporophytically in the anther tapetum and located in the pollen coating that acts as the ligand for SRK. Binding of SCR/SP11 and SRK induces SRK dimerization and autophosphorylation of serines and threonines in the kinase domain leading to pollen rejection; SLG is also present in this interaction complex, probably associated with SRK (Takayama et al., 2001).
Recent molecular studies in SSI Ipomoea trifida indicate that SRK-like and SLG-like genes are expressed in stigmas, but do not appear to be involved in SI because they are not linked to S (Kowyama et al., 2000). This observation suggests that SSI, like gametophytic SI (GSI) may operate through different molecular mechanisms in different plant families (McCubbin and Kao, 2000). To investigate the potential diversity of genes controlling SSI further, a molecular analysis of SSI has been initiated in a model member of the Asteraceae, Senecio squalidus L. (Oxford Ragwort) (Fig. 1). Here some of the initial findings of this study are discussed within the context of a brief review of SSI in Senecio.
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Genetics of SSI in S. squalidus |
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Full diallel crosses of F1 progenies derived from ‘forced’ selfings (treating stigmas with dilute saline prior to pollination overcomes SI) demonstrated that S. squalidus has a sporophytic system of SI controlled by a single S locus with potentially many S alleles (Hiscock, 2000a, b). These genetic analyses allowed the identification of individuals homozygous for defined S alleles for use in molecular studies. Interestingly, the four parental plants selected for generating the F1 progenies were found to contain just three S alleles even though the plants were obtained from different Oxford populations of S. squalidus. This suggested that British populations of S. squalidus might contain unusually low numbers of S alleles compared to other species with SSI (Hiscock, 2000a, b). Indeed a low diversity of S alleles would be predicted from the consensus of opinion that the majority of (and probably all) British populations of S. squalidus are descended from an original ‘collection’ of a small number of individuals held in the Oxford Botanic Garden during the early 1700s (Harris, 2002). Such a founder effect would have created a population bottleneck resulting in very few S alleles being represented in the original population(s). Indeed, recent population genetic studies of S alleles in S. squalidus have confirmed that there are relatively few S alleles present in UK populations (AC Brennan, SA Harris, SJ Hiscock, unpublished data), a maximum of just six S alleles was estimated for the entire Oxford population, the presumed centre of S allelic diversity in Britain (AC Brennan et al., 2002, unpublished results). Interestingly, these population genetic studies also indicate there to be a high level of dominance between S alleles such that individuals can be assigned an S genotype based on expression of just one designated S allele (Brennan et al., 2002). Higher levels of dominance interactions between S alleles have the effect of increasing mate availability when just a few S alleles are shared between individuals and is an evolutionary response to population bottlenecks predicted by theoretical models of SSI (Byers and Meagher, 1992).
Genetic studies of SSI in Senecio have also highlighted a number of crossing ‘anomalies’ that do not fit the sporophytic model of SI (Hiscock, 2000b; DA Tabah, SJ Hiscock, unpublished data). These crossing anomalies usually result in compatibility when incompatibility is predicted and suggest that other ‘modifier loci’, probably unlinked to S, can influence the outcome of certain crosses in different genetic backgrounds. One such modifier locus may be the diallelic gametophytic locus, G, identified in SSI Brassica and Raphanus by Lewis and co-workers (Lewis et al., 1988; Zuberi and Lewis, 1988). Indeed preliminary evidence suggests that a ‘gametophytic element’ may be responsible for certain anomalous crosses in Senecio and the segregation of this potential modifier locus is currently being followed in F1 and F2 progeny arrays.
The incompatibility response in S. squalidus
The inhibition of incompatible pollen occurs at the stigma surface in S. squalidus (Hiscock et al., 2002; Fig. 1). Incompatible pollen grains either fail to germinate or, if they do germinate, the emergent pollen tube is usually inhibited before it has penetrated the stigma surface. Inhibition is frequently accompanied by deposition of callose in stigmatic papillae and at the tips of incompatible pollen tubes. These features of the SI response, therefore, resemble closely those of the Brassica SI response (Hiscock et al., 2002). However, in Senecio, as compared to Brassica, many more incompatible pollen tubes appear to be able to penetrate and grow within the stigma before they are finally inhibited. Inhibition of such incompatible pollen tubes is usually accompanied by a swelling of the tube tip and deposition of callose within the tip and adjacent stigma cells (Fig. 1; Hiscock et al., 2002). Interestingly, swelling at the tip of incompatible pollen tubes has been consistently reported as a feature of the inhibition response for GSI species from the Solanaceae and Rosaceae (de Nettancourt, 1997).
Identification of SRK-like cDNAs in S. squalidus
Given that S. squalidus has a sporophytic SI system it seemed logical to look for stigma-expressed homologues/orthologues of Brassica SRK genes as a first step towards identifying putative Senecio genes involved in SSI. Preliminary Southern blot analyses using the receptor domain of SRK63 as a probe onto Senecio genomic DNA indicated that SRK/SLG-like sequences are represented in the Senecio genome (SJ Hiscock, SM McInnis, DA Tabah, unpublished data). In order to characterize these SRK/SLG-like sequences, primers were designed to conserved regions within the receptor domain and kinase domain of Brassica SRKs (Fig. 2) and used to amplify products of the predicted size for SRK (800 bp) and SLG/receptor domain of SRK (750 bp) from mRNAs extracted from flower buds and stigmas of Senecio (Fig. 2). Similar degenerate primers designed to these same conserved domains had previously been used successfully to amplify SRK-like and SLG-like cDNAs from Ipomoea trifida (Kowyama et al., 1995; K Kakeda, personal communication). Cloned PCR products were grouped into three classes based on restriction digest patterns. Sequencing members of each class confirmed that three different cDNAs had been amplified with sequence similarity to members of the SRK multigene family (SJ Hiscock, SM McInnis, DA Tabah, unpublished data). The three classes of clone were thus designated Senecio S Receptor-Like Kinases, SSRLK-1, SSRLK-2 and SSRLK-3 and were 35–46% identical at an amino acid level to SRKs from Brassica and an S receptor-like kinase (IRK1) from Ipomoea (Fig. 3). The receptor domain of SSRLK-1 has recently been cloned from genomic DNA using a PCR-based gene walking technique (Zhang and Gurr, 2000). Sequencing this clone revealed the putative amino acid sequence to be 32% identical to the receptor domain of SRK6 from Brassica and to contain conserved cysteine residues characteristic of SRK receptor domains and SLGs.
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Southern blots of Senecio genomic DNA showed that SSRLKs are part of a multigene family in Senecio. Restriction digests of Senecio genomic DNA using infrequent (EcoRI, HindIII, Apa1) and frequent (Sau3A1, Rsa1, Hinf1) cutters failed to reveal any unique RFLPs for SSRLKs in plants carrying different S alleles suggesting that the SSRLK cDNAs so far characterized are unlikely to reside at the S locus where high levels of polymorphism are anticipated. Support for this conclusion was given when SSRLK expression was analysed in different tissue types using RT-PCR (Fig. 4). None of the three SSRLKs was expressed exclusively in stigmas, suggesting that a specific role for SSRLKs in SI or other aspects of the pollen–stigma interaction is unlikely. Nevertheless, a fourth class of SSRLK expressed in stigmas has recently been identified and this cDNA is currently the subject of expression studies and tests for S linkage.
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Identification of S-associated proteins in S. squalidus
As an alternative route to identifying putative S genes in Senecio, the protein profiles of stigmas from plants carrying defined S alleles S1 and S2 have been analysed. The S2 allele is dominant to S1 in both pollen and stigma and so facilitates backcrossing the S1S2 heterozygous plants to the S1S1 homozygous parent for segregation studies of S. Isoelectric focusing (IEF) of proteins extracted from mature stigmas of 20 plants segregating for S1 and S2 revealed basic protein bands associated with each S allele (SJ Hiscock, SM McInnis, CA Henderson, unpublished data). These bands with isoelectric points of 8.2 and 7.1 were designated Stigma S-associated Proteins (SSPs) 1 and 2, respectively. S1S1 individuals carry SSP1 and S2S2 individuals carry SSP2 while S1S2 heterozyotes carry both bands (Fig. 5) Progeny from the cross S1S1xS2S2 were incompatible among themselves and incompatible with the S2S2 parent but fully compatible with the S1S1 parent. Forced selfing of an S1S2 plant resulted in a progeny array of 15 individuals segregating for both S alleles to produce S1S1 plants carrying SSP1, S2S2 plants carrying SSP2 and S1S2 plants carrying both SSP1 and SSP2 (plants were S genotyped by test pollinations with homozygous and heterozygous stocks). These data suggest that SSP1 and SSP2 are linked to their respective S alleles.
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SSP1 and SSP2 were gel-purified using a method described in Hiscock et al. (1995) and N-terminal amino acid sequences subsequently obtained for each protein (SJ Hiscock, SM McInnis, CA Henderson, unpublished data). Despite their very different pIs, the SSP1 and SSP2 polypeptides contain an initial N-terminus of 11 identical amino acids, however, the subsequent run of six amino acids showed four differences between the two polypeptides. These observations are consistent with SSP1 and SSP2 being encoded by allelic forms of the same gene. Resolution of purified SSP1 and SSP2 by SDS–PAGE indicated that both polypeptides share the same approximate molecular weight of 35–40 kDa and a positive reaction with the Con-A-peroxidase test for N-linked glucosyl and/or mannosyl residues demonstrated that both SSPs are glycoproteins. The genes that encode SSP1 and SSP2 are currently being characterized. Full-length cDNAs were recently obtained for SSP1 and SSP2 using 3' and 5' RACE. Sequencing these cDNAs confirmed that their predicted N-terminal amino acid sequences corresponded to the N-terminal amino acid sequences obtained for SSP1 and SSP2 by Edman sequencing and both cDNAs predicted mature proteins of approximately 35 kDa (308 amino acids). SSP mRNAs have a 26 or 32 bp 5'UTR and a 131 or 161 bp 3' UTR and the putative preprotein contains a signal peptide sequence of 27 amino acids suggesting that SSPs are secreted.
Northern blots indicate that SSP1 and SSP2 are expressed exclusively in stigmas and flower buds 1–2 d before anthesis (Fig. 6). This tissue specificity and developmental regulation would be predicted for a potential female S gene and suggests that even if SSP is not ‘female S’ then it may play a role in pollen–stigma interactions in Senecio.
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Conclusions |
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TopAbstractIntroductionGenetics of SSI in...ConclusionsReferences |
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Identification of stigma-expressed SRK-like genes in Senecio was not altogether unexpected because SRKs are members of a large family of S-like receptor kinases present in many other species of flowering plant from Brassica and Arabidopsis to Ipomoea and maize (Ansaldi et al., 2000). However, the fact that none of the Senecio SRK-like genes so far characterized are expressed specifically in stigmas suggests that they are not directly involved in SI. Such a conclusion is supported by the Southern blot analyses of SSRLKs that yielded no S allele-specific RFLPs. Analyses of further SRK-like cDNAs expressed in Senecio stigmas are being continued, but initial studies suggest that the Brassica SRK-based system of SSI may not regulate SSI in Senecio and the Asteraceae. Similar conclusions have been drawn from extensive studies of SRK-like genes in the sweet potato relative Ipomoea trifida (Convolvulaceae) where, again, it seems most likely that SSI operates through a molecular pathway distinct from Brassica (Kowyama et al., 2000). Interestingly, the Convolvulaceae and Asteraceae are more closely related to each other than they are to the Brassicaceae (Chase et al., 1993), so it is perhaps more likely that they will share the same molecular mechanism of SSI than it is for either of them to share a system in common with Brassica. Such speculation can only be resolved when S genes are identified conclusively in Ipomoea and Senecio.
The identification of stigma-specific proteins associated with particular S alleles is a tried and tested route to identifying S genes (Nasrallah and Nasrallah, 1989) so this approach was followed to identify potential S-linked proteins in Senecio. Two putative S proteins, SSP1 and SSP2 were identified that appear to be the products of allelic forms of the same gene, SSP. cDNAs for SSP1 and SSP2 have been cloned and are being characterized. The expression pattern of SSP makes it a very strong candidate for a potential female S gene and initial linkage studies have shown that the SSP1 and SSP2 proteins follow their respective S alleles (S1 and S2) in small segregating populations. Ongoing work is focused on analysing the segregation of an SSP allele-specific EcoRI RFLP and SSP allele-specific SNPs (single nucleotide polymorphisms) in larger populations of individuals segregating for S1 and S2. These studies will establish whether SSP is indeed S-linked and whether SSP represents a new class of sporophytic S gene.
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Acknowledgements |
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This work was funded by the BBSRC through a David Phillips Research Fellowship and a PMS Committee Research Grant award to SJH.
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