Plant reproductive development is dependent on successful pollen-pistil interactions. In crucifers, the pollen tube must breach the stigma surface and burrow through the extracellular matrix of the stigma epidermal cells and transmitting tract cells before reaching its ovule targets. The high degree of specificity in pollen-pistil interactions and the precision of directional pollen tube growth suggest that signals are continually being exchanged between pollen/pollen tubes and cells of the pistil that line their path. However, with few exceptions, little is known about the genes that control these interactions. The specialized functions of stigma epidermal cells and transmitting tract cells are likely to depend on the activity of genes expressed specifically in these cells. In order to identify these genes, we used the Arabidopsis (Arabidopsis thaliana) ATH1 microarray to compare the whole-genome transcriptional profiles of stigmas and ovaries isolated from wild-type Arabidopsis and from transgenic plants in which cells of the stigma epidermis and transmitting tract were specifically ablated by expression of a cellular toxin. Among the 23,000 genes represented on the array, we identified 115 and 34 genes predicted to be expressed specifically in the stigma epidermis and transmitting tract, respectively. Both gene sets were significantly enriched in predicted secreted proteins, including potential signaling components and proteins that might contribute to reinforcing, modifying, or remodeling the structure of the extracellular matrix during pollination. The possible role of these genes in compatible and incompatible pollen-pistil interactions is discussed.

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 ‫ف‬ 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. INTRODUCTIONSelf-incompatibility (SI) is the major outcrossing mechanism in the family Brassicaceae (de Nettancourt, 1977). Species in this family have been grouped into 19 tribes on the basis of morphological criteria (Schultz, 1936), and SI has been described in all tribes analyzed to date. When Bateman (1955) surveyed 182 species distributed in 11 tribes, he found that approximately half of these species included selfincompatible accessions. In a survey of 59 taxa in the subtribe Brassicineae of the tribe Brassiceae (which includes Brassica and Raphanus ), 50 taxa were self-incompatible (Takahata and Hinata, 1980). In all cases analyzed, SI has been shown to be controlled sporophytically by a single S (sterility) locus, with multiple alleles or variants and complex dominance relationships between alleles (Bateman, 1954(Bateman, , 1955Thompson and Taylor, 1966): in self-incompatible plants, pollen will not develop on a stigma that expresses the same S alleles as the pollen parent.Molecular analysis of the Brassica S locus region has shown that this mendelian locus is a gene complex consisting of distinct stigma-expressed and anther-expressed genes that determine SI specificity in stigma and pollen, respectively (reviewed in Nasrallah, 2000). The SRK (for S locus receptor kinase) gene (Stein et al., 1991) encodes a plasma membrane-spanning receptor serine/threonine kinase specific to the stigma epidermis (Stein et al., 1996) and is the determinant of SI specificity in the stigma (Takasaki et al., 2000). ...

The interplay of balancing selection within a species and rapid gene evolution between species can confound our ability to determine the functional equivalence of interspecific and intergeneric pairs of alleles underlying reproduction. In crucifer plants, mating specificity in the barrier to self-fertilization called self-incompatibility (SI) is controlled by allele-specific interactions between two highly polymorphic and co-evolving proteins, the S-locus receptor kinase (SRK) and its S-locus cysteine rich (SCR) ligand. These proteins have diversified both within and between species such that it is often difficult to determine from sequence information alone if they encode the same or different SI specificity. The self-fertile Arabidopsis thaliana was derived from an obligate outbreeding ancestor by loss of self-incompatibility, often in conjunction with inactivation of SRK or SCR. Nevertheless, some accessions of A. thaliana can express self-incompatibility upon transformation with an SRK-SCR gene pair isolated from its self-incompatible close relative A. lyrata. Here we show that several additional and highly diverged SRK/SCR genes from A. lyrata and another crucifer plant, Capsella grandiflora, confer self-incompatibility in A. thaliana, either as intact genes isolated from genomic libraries or after manipulation to generate chimeric fusions. We describe how the use of this newly developed chimeric protein strategy has allowed us to test the functional equivalence of SRK/SCR gene pairs from different taxa and to assay the functionality of endogenous A. thaliana SRK and SCR sequences.

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 Slocus and therefore cannot be a determinant of S-allele specificity in Brassica.

Six sequences that are closely related to the S gene family of the largely self-incompatible Brassica species have been identified in self-fertilizing Arabidopsis. The sequences define four genomic regions that map to chromosomes 1 and 3. Of the four functional genes identified, only the previously reported Arabidopsis AtS1 gene was expressed specifically in papillar cells and may function in pollination. The remaining three genes, including two novel genes designated ARK2 and ARK3, encode putative receptor-like serine/threonine protein kinases that are expressed predominantly in vegetative tissues. ARK2 promoter activity was detected exclusively in above-ground tissues, specifically in cotyledons, leaves, and sepals, in correlation with the maturation of these structures. ARK3 promoter activity was detected in roots as well as above-ground tissues but was limited to small groups of cells in the root-hypocotyl transition zone and at the base of lateral roots, axillary buds, and pedicels. The nonoverlapping patterns of expression of the ARK genes and the divergence of their sequences, particularly in their predicted extracellular domains, suggest that these genes perform nonredundant functions in specific aspects of development or growth of the plant body.

Genetic and molecular analysis of the self-incompatibility locus (S-locus) of the crucifer Brassica has led to the characterization of a multigene family involved in pollen-stigma interactions. While the crucifer Arabidopsis thaliana does not have a self-incompatibility system, S-related sequence were detected in this species by cross-hybridization with Brassica DNA probes. In this paper, we show that an A. thaliana S-related sequence, designated AtS1, is expressed specifically in flower buds. Sequence analysis suggests that AtS1 encodes a secreted glycoprotein that is most similar to the Brassica S-locus related protein SLR1. As has been proposed for SLR1, this gene may be involved in determining some fundamental aspect of pollen-stigma interactions during pollination. The molecular and genetic advantages of the Arabidopsis system will provide many avenues for testing this hypothesis.

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Summary The self-incompatibility response of crucifers is a barrier to fertilization in which arrest of pollen tube development is mediated by allele-specific interactions between polymorphic receptors and ligands encoded by the S-locus haplotype. The binding of the stigma-expressed S-locus receptor kinase (SRK) [1] to the pollen coat-localized S-locus cysteine-rich (SCR) ligand [2-5], activation of the receptor, and pollen rejection occurs only if receptor and ligand are encoded by the same S haplotype [4, 6-8]. To identify residues within the extracellular domain of SRK (eSRK) that are required for its ligand-selective activation, we assayed chimeric receptors and receptor variants containing substitutions at polymorphic sites in transgenic Arabidopsis thaliana [9, 10]. We show that only a small number of the ∼100 polymorphic residues in eSRK are required for ligand-specific activation of self-incompatibility in vivo. These essential residues occur in two non-contiguous clusters located at equivalent positions in the two variants tested. They also correspond to sites showing elevated levels of substitutions in other SRKs, suggesting that these amino acids could define SI specificity in most SRKs. The results demonstrate that the majority of eSRK residues that show signals of positive selection and previously surmised to function as specificity determinants are not essential for specificity in the SRK-SCR interaction.