June B. Nasrallah

Self-Incompatibility in the Genus Arabidopsis: Characterization of the S Locus in the Outcrossing A. lyrata and Its Autogamous Relative A. thaliana

As a starting point for a phylogenetic study of self-incompatibility (SI) in crucifers and to elucidate the genetic basis of transitions between outcrossing and self-fertilizing mating systems in this family, we investigated the SI system of Arabidopsis lyrata. A. lyrata is an outcrossing close relative of the self-fertile A. thaliana and is thought to have diverged from A. thaliana ∼5 million years ago and from Brassica spp 15 to 20 million years ago. Analysis of two S (sterility) locus haplotypes demonstrates that the A. lyrata S locus contains tightly linked orthologs of the S locus receptor kinase (SRK) gene and the S locus cysteine-rich protein (SCR) gene, which are the determinants of SI specificity in stigma and pollen, respectively, but lacks an S locus glycoprotein gene. As described previously in Brassica, the S haplotypes of A. lyrata differ by the rearranged order of their genes and by their variable physical sizes. Comparative mapping of the A. lyrata and Brassica S loci indicates that the S locus of crucifers is a dynamic locus that has undergone several duplication events since the Arabidopsis–Brassica split and was translocated as a unit between two distant chromosomal locations during diversification of the two taxa. Furthermore, comparative analysis of the S locus region of A. lyrata and its homeolog in self-fertile A. thaliana identified orthologs of the SRK and SCR genes and demonstrated that self-compatibility in this species is associated with inactivation of SI specificity genes.

Pollen-stigma signaling in the sporophytic self-incompatibility response

Many flowering plants reproduce sexually and generate variation by the process of recombination. Because the possibility of continued evolution is dependent, at least in part, on the production of new variant combinations of genes derived from different individuals, the potential for adaptation is believed to be better under cross-pollination than under self-pollination. In fact, i f self-incompatible plants are manipulated to undergo forced self-pollination, they invariably produce progeny with marked inbreeding depression. It is therefore not surprising to find that plants have evolved a variety of mechanisms that favor outcrossing. For example, the crucifer family (Brassicaceae) has an elaborate genetic self-incompatibility (SI) system that controls mating in natural populations of nearly half of the species belonging to this family. This system acts early in pollination to arrest pollen derived from “self.” Such plants require cross-pollination to ensure maximal seed production. Radishes, kales, and cabbages are examples of crucifers that rely on outcrossing via cross-pollination by insect vectors to complete their life cycle in the wild. Genetic SI in crucifers is controlled by a complex and highly polymorphic S locus with many specificities or mating reactions. In this review, we discuss the role of the gene products encoded at the S locus in light of recent molecular genetic data derived from the analysis of three Brassica species, B. oleracea, B. campestris, and B. napus.

A highly conserved Brassica gene with homology to the S-locus-specific glycoprotein structural gene.

The S-locus-specific glycoprotein of Brassica and the gene encoding it (the SLG gene) are thought to be involved in determining self-incompatibility phenotype in this genus. It has been shown that the Brassica genome contains multiple SLG-related sequences. We report here the cloning and characterization of a Brassica oleracea gene, SLR1, which corresponds to one of these SLG-related sequences. Like the SLG gene, SLR1 is developmentally regulated. It is maximally expressed in the papillar cells of the stigma at the same stage of flower development as the onset of the incompatibility response. Unlike SLG, the SLR1 genes isolated from different S-allele homozygotes are highly conserved, and this gene, which appears to be ubiquitous in crucifers, is expressed in self-compatible strains as well as self-incompatible strains. Most importantly, we show that the SLR1 gene is not linked to the S-locus and therefore cannot be a determinant of S-allele specificity in Brassica.

The S-locus specific glycoproteins of Brassica accumulate in the cell wall of developing stigma papillae

Self-incompatibility in Brassica oleracea is now viewed as a cellular interaction between pollen and the papillar cells of the stigma surface. In this species, the inhibition of self-pollen occurs at the stigma surface under the influence of S-locus specific glycoproteins (SLSG). We used antibodies specific for a protein epitope of SLSG to study the subcellular distribution of these molecules in the stigmatic papillae. The antibodies have uncovered an interesting epitope polymorphism in SLSG encoded by subsets of S-alleles, thus providing us with useful genetic controls to directly verify the specificity of the immunolocalization data. Examination of thin sections of Brassica stigmas following indirect immunogold labeling showed that SLSG accumulate in the papillar cell wall, at the site where inhibition of self-pollen tube development has been shown to occur. In addition, the absence of gold particles over the papillar cell walls in the immature stigmas of very young buds, and the intense labeling of these walls in the stigmas of mature buds and open flowers, correlates well with the acquisition of the self-incompatibility response by the developing stigma.

The Evolution of Selfing in Arabidopsis thaliana

Unlike most of its close relatives, Arabidopsis thaliana is capable of self-pollination. In other members of the mustard family, outcrossing is ensured by the complex self-incompatibility (S) locus,which harbors multiple diverged specificity haplotypes that effectively prevent selfing. We investigated the role of the S locus in the evolution of and transition to selfing in A. thaliana. We found that the S locus of A. thaliana harbored considerable diversity, which is an apparent remnant of polymorphism in the outcrossing ancestor. Thus, the fixation of a single inactivated S-locus allele cannot have been a key step in the transition to selfing. An analysis of the genome-wide pattern of linkage disequilibrium suggests that selfing most likely evolved roughly a million years ago or more.

Physical linkage of the SLG and SRK genes at the self-incompatibility locus of Brassica oleracea.

SummaryIn Brassica oleracea, the pollen-stigma interaction of self-incompatibility is controlled by a single genetically defined locus designated S. Molecular studies have identified two genes that are tightly linked to the classically defined S locus: The S-Locus Glycoprotein (SLG) gene and the S-Receptor Kinase (SRK) gene. In previous RFLP linkage analyses with probes specific for SLG and SRK, we were unable to identify any recombination events between SLG, SRK, and self-incompatibility phenotype. In this paper, we use pulsed-field gel electrophoresis (PFGE) in conjunction with DNA blot analysis to characterize the S-locus region from two highly divergent self-incompatibility genotypes, S2 and S6. We establish the physical linkage of SLG and SRK in each genotype, and demonstrate that the two genes are separated by a maximum distance of 220 kb in the S6 genotype and 350 kb in the S2 genotype. Furthermore, a comparison of the data from the two genotypes reveals that a high level of polymorphism exists across the entire S-locus region.

DNA sequences of self-incompatibility genes from Brassica campestris and B. oleracea: polymorphism predating speciation.

Self-incompatibility describes the rejection of selfpollen and/or the prevention of self-fertilization in some plants. In Brassica, self-incompatibility occurs at the initial site of pollination, the stigma, where pollen germination either does not commence or is aborted before invasion of the papillar cell wall by the pollen tube. Genetic analysis has shown this trait to be governed by a single locus, the S-locus with over 50 S-locus alleles reported. Self-incompatibility occurs when the same S-locus allele is active in pollen and papillar cells. Analysis of self-incompatibility in Brassica has focused on the study of a class of stigma glycoproteins, the S-locus specific glycoproteins (SLSG). SLSG extracted from Brassica oleracea and Brassica campestris stigmas with different S alleles have been analyzed [reviewed in 5]. An increase in the level of SLSG corresponds with the onset of self-incompatibility and the polymorphisms of these molecules correlate with S-allele polymorphism and segregation in controlled genetic crosses, cDNAs and S-locus glycoprotein (SLG) genes coding for the SLSG ofB. oleracea

Genome-Wide Identification of Genes Expressed in Arabidopsis Pistils Specifically along the Path of Pollen Tube Growth

6, S13, 514 , S22 and 529 alleles have been isolated [3, 4, 10]. Nucleic acid sequences derived from the

Transformation of Brassica oleracea with an S-locus gene from B. campestris changes the self-incompatibility phenotype

6 and S29 alleles have been reported, but for the other alleles only deduced amino acid sequences have been published. In B. campestris, partial amino-acid sequences are available and have been derived by direct protein sequencing of SLSG associated with the

An alternative transcript of the S locus glycoprotein gene in a class II pollen-recessive self-incompatibility haplotype of Brassica oleracea encodes a membrane-anchored protein.

8, S 9 and 512 alleles [1, 9]. These nucleic acid and protein sequence analyses have revealed the presence of a signal peptide, and by immunolocalization studies we have established that SLSG is secreted to the papillar cell wall [2]. The sub-cellular location of SLSG is in keeping with their proposed function in the cell-cell recognition between pollen and stigma necessary to effect the self-incompatibility response. Support for this role has been discovered recently. A gene, ZmPK1, was isolated from Zea mays and shown to encode a putative receptor protein kinase with a cytoplasmic domain exhibiting homology to serine/threonine protein kinases and an SLG-homologous extracellular domain speculated to be involved in ligand binding [ 11 ]. The function of the corn receptor protein is unknown. However, it is reasonable to hypothesize that in Brassica flowers plant receptor protein kinases trigger the physiological processes involved in pollination and that these processes can be specifically modulated by the secretion of ligand binding glycoproteins, the SLSG, resulting in the self-incompatibility response [6]. In this report, we present two previously unreported SLG nucleic acid sequences: SLG-8o, a genomic sequence derived from a B. campestris