Self-incompatibility in the Brassicaceae: Regulation and mechanism of self-recognition.

Self-incompatibility is one of the most common mechanisms used by plants to prevent self-fertilization. In the Brassicaceae, the inhibition of self-pollen is triggered right at the stigma surface by interaction of two highly polymorphic self-recognition proteins that are encoded by tightly linked genes of the S-locus haplotype: a receptor protein kinase displayed at the surface of stigma epidermal cells and its small diffusible ligand that is localized in the outer coat of pollen grains. It is the specific interaction between receptor and ligand encoded in the same S haplotype that determines specificity in the rejection of self-pollen. The chapter reviews recent results that have shed light on the genetic control, cell biology, and regulation of the self-recognition molecules, as well as the structural basis of ligand recognition. Models that aim to explain how diversification of the self-recognition repertoire can occur in this two-gene self-recognition system are discussed.

Evolution of interspecies unilateral incompatibility in the relatives of Arabidopsis thaliana.

The evolutionary concurrence of intraspecies self-incompatibility (SI) and explosive angiosperm radiation in the Cretaceous have led to the hypothesis that SI was one of the predominant drivers of rapid speciation in angiosperms. Interspecies unilateral incompatibility (UI) usually occurs when pollen from a self-compatible (SC) species is rejected by the pistils of a SI species, while the reciprocal pollination is compatible (UC). Although this SI × SC type UI is most prevalent and viewed as a prezygotic isolation barrier to promote incipient speciation of angiosperms, comparative evidence to support such a role is lacking. We show that SI × SI type UI in SI species pairs is also common in the well-characterized accessions representing the four major lineages of the Arabidopsis genus and is developmentally regulated. This allowed us to reveal a strong correlation between UI strength and species divergence in these representative accessions. In addition, analyses of a SC accession and the pseudo-self-compatible (psc) spontaneous mutant of Arabidopsis lyrata indicate that UI shares, at least, common pollen rejection pathway with SI. Furthermore, genetic and genomic analyses of SI × SI type UI in A. lyrata × A. arenosa species pair showed that two major-effect quantitative trait loci are the stigma and pollen-side determinant of UI, respectively, which could be involved in heterospecies pollen discrimination. By revealing a close link between UI and SI pathway, particularly between UI and species divergence in these representative accessions, our findings establish a connection between SI and speciation. Thus, the pre-existence of SI system would have facilitated the evolution of UI and accordingly promote speciation.

Plant mating systems: self-incompatibility and evolutionary transitions to self-fertility in the mustard family.

Flowering plants have evolved diverse mechanisms that promote outcrossing. The most widespread of these outbreeding devices are self-incompatibility systems, the highly selective prefertilization mating barriers that prevent self-fertilization by disrupting pollen-pistil interactions. Despite the advantages of outcrossing, loss of self-incompatibility has occurred repeatedly in many plant families. In the mustard family, the highly polymorphic receptors and ligands that mediate the recognition and inhibition of self-pollen in self-incompatibility have been characterized and the 3D structure of the receptor-ligand complex has been solved. Sequence analyses and empirical studies in self-incompatible and self-compatible species are elucidating the genetic basis of switches from the outcrossing to selfing modes of mating and beginning to provide clues to the diversification of the self recognition repertoire.

Ligand-Mediated cis-Inhibition of Receptor Signaling in the Self-Incompatibility Response of the Brassicaceae.

The inhibition of self-pollination in self-incompatible Brassicaceae is based on allele-specific trans-activation of the highly polymorphic S-locus receptor kinase (SRK), which is displayed at the surface of stigma epidermal cells, by its even more polymorphic pollen coat-localized ligand, the S-locus cysteine-rich (SCR) protein. In an attempt to achieve constitutive activation of SRK and thus facilitate analysis of self-incompatibility (SI) signaling, we coexpressed an Arabidopsis lyrata SCR variant with its cognate SRK receptor in the stigma epidermal cells of Arabidopsis (Arabidopsis thaliana) plants belonging to the C24 accession, in which expression of SRK and SCR had been shown to exhibit a robust SI response. Contrary to expectation, however, coexpression of SRK and SCR was found to inhibit SRK-mediated signaling and to disrupt the SI response. This phenomenon, called cis-inhibition, is well documented in metazoans but has not as yet been reported for plant receptor kinases. We demonstrate that cis-inhibition of SRK, like its trans-activation, is based on allele-specific interaction between receptor and ligand. We also show that stigma-expressed SCR causes entrapment of its SRK receptor in the endoplasmic reticulum, thus disrupting the proper targeting of SRK to the plasma membrane, where the receptor would be available for productive interaction with its pollen coat-derived SCR ligand. Although based on an artificial cis-inhibition system, the results suggest novel strategies of pollination control for the generation of hybrid cultivars and large-scale seed production from hybrid plants in Brassicaceae seed crops and, more generally, for inhibiting cell surface receptor function and manipulating signaling pathways in plants.

In vivo imaging of the S-locus receptor kinase, the female specificity determinant of self-incompatibility, in transgenic self-incompatible Arabidopsis thaliana.

BACKGROUND AND AIMS: The S-locus receptor kinase (SRK), which is expressed in stigma epidermal cells, is responsible for the recognition and inhibition of 'self' pollen in the self-incompatibility (SI) response of the Brassicaceae. The allele-specific interaction of SRK with its cognate pollen coat-localized ligand, the S-locus cysteine-rich (SCR) protein, is thought to trigger a signalling cascade within the stigma epidermal cell that leads to the arrest of 'self' pollen at the stigma surface. In addition to the full-length signalling SRK receptor, stigma epidermal cells express two other SRK protein species that lack the kinase domain and whose role in the SI response is not understood: a soluble version of the SRK ectodomain designated eSRK and a membrane-tethered form designated tSRK. The goal of this study was to describe the sub-cellular distribution of the various SRK protein species in stigma epidermal cells as a prelude to visualizing receptor dynamics in response to SCR binding. METHODS: The Arabidopsis lyrata SRKb variant was tagged with the Citrine variant of yellow fluorescent protein (cYFP) and expressed in A. thaliana plants of the C24 accession, which had been shown to exhibit a robust SI response upon transformation with the SRKb-SCRb gene pair. The transgenes used in this study were designed for differential production and visualization of the three SRK protein species in stigma epidermal cells. Transgenic stigmas were analysed by pollination assays and confocal microscopy. KEY RESULTS AND CONCLUSIONS: Pollination assays demonstrated that the cYFP-tagged SRK proteins are functional and that the eSRK is not required for SI. Confocal microscopic analysis of cYFP-tagged SRK proteins in live stigma epidermal cells revealed the differential sub-cellular localization of the three SRK protein species but showed no evidence for redistribution of these proteins subsequent to incompatible pollination.

Site-specific N-glycosylation of the S-locus receptor kinase and its role in the self-incompatibility response of the brassicaceae.

The S-locus receptor kinase SRK is a highly polymorphic transmembrane kinase of the stigma epidermis. Through allele-specific interaction with its pollen coat-localized ligand, the S-locus cysteine-rich protein SCR, SRK is responsible for recognition and inhibition of self pollen in the self-incompatibility response of the Brassicaceae. The SRK extracellular ligand binding domain contains several potential N-glycosylation sites that exhibit varying degrees of conservation among SRK variants. However, the glycosylation status and functional importance of these sites are currently unclear. We investigated this issue in transgenic Arabidopsis thaliana stigmas that express the Arabidopsis lyrata SRKb variant and exhibit an incompatible response toward SCRb-expressing pollen. Analysis of single- and multiple-glycosylation site mutations of SRKb demonstrated that, although five of six potential N-glycosylation sites in SRKb are glycosylated in stigmas, N-glycosylation is not important for SCRb-dependent activation of SRKb. Rather, N-glycosylation functions primarily to ensure the proper and efficient subcellular trafficking of SRK to the plasma membrane. The study provides insight into the function of a receptor that regulates a critical phase of the plant life cycle and represents a valuable addition to the limited information available on the contribution of N-glycosylation to the subcellular trafficking and function of plant receptor kinases.

S-locus receptor kinase signalling.

SRK (S-locus receptor kinase) is the receptor that allows stigma epidermal cells to discriminate between genetically related ('self') and genetically unrelated ('non-self') pollen in the self-incompatibility response of the Brassicaceae. SRK and its ligand, the pollen coat-localized SCR (S-locus cysteine-rich protein), are highly polymorphic, and their allele-specific interaction explains specificity in the self-incompatibility response. The present article reviews current knowledge of the role of SRK in the recognition and response phases of self-incompatibility, and highlights the new insights provided by analysis of a transgenic self-incompatible Arabidopsis thaliana model.

Exploring the role of a stigma-expressed plant U-box gene in the pollination responses of transgenic self-incompatible Arabidopsis thaliana.

Recognition of "self" pollen in the self-incompatibility (SI) response of the Brassicaceae is determined by allele-specific interaction between the S-locus receptor kinase (SRK), a transmembrane protein of the stigma epidermis, and its ligand, the pollen coat-localized S-locus cysteine-rich (SCR) protein. The current model for SRK-mediated signaling proposes a central role for the plant U-box (PUB) Armadillo repeat-containing protein ARC1, an E3 ligase that interacts with, and is phosphorylated by, the kinase domain of SRK. According to the model, activated ARC1 causes the degradation of factors required for successful pollen tube growth. However, Arabidopsis thaliana plants transformed with functional SRK and SCR genes isolated from self-incompatible A. lyrata can express an intense SI response despite lacking a functional ARC1 gene. Here, we tested the possibility that a different member of the A. thaliana PUB protein family might have assumed the role of ARC1 in SI. Toward this end, we analyzed the AtPUB2 gene, which is annotated as being highly expressed in stigmas. Our functional analysis of a T-DNA insertion pub2 allele, together with yeast two-hybrid interaction assays and reporter analysis of AtPUB2 promoter activity, demonstrates that AtPUB2 does not function in SI. The results leave open the question of whether the proposed model of ARC1-mediated signaling applies to transgenic SRK-SCR self-incompatible A. thaliana plants.

Functional test of Brassica self-incompatibility modifiers in Arabidopsis thaliana.

The self-incompatibility (SI) system of the Brassicaceae is based on allele-specific interactions among haplotypes of the S locus. In all tested self-incompatible Brassicaceae, the S haplotype encompasses two linked genes, one encoding the S-locus receptor kinase (SRK), a transmembrane kinase displayed at the surface of stigma epidermal cells, and the other encoding its ligand, the S-locus cysteine-rich (SCR) protein, which is localized in the pollen coat. Transfer of the two genes to self-fertile Arabidopsis thaliana allowed the establishment of robust SI in several accessions, indicating that the signaling cascade triggered by this receptor-ligand interaction and the resulting inhibition of "self" pollen by the stigma have been maintained in extant A. thaliana. Based on studies in Brassica species, the membrane-tethered kinase MLPK, the ARM repeat-containing U-box protein ARC1, and the exocyst subunit Exo70A1 have been proposed to function as components of an SI signaling cascade. Here we tested the role of these molecules in the SI response of A. thaliana SRK-SCR plants. We show that the A. thaliana ARC1 ortholog is a highly decayed pseudogene. We also show that, unlike reports in Brassica, inactivation of the MLPK ortholog AtAPK1b and overexpression of Exo70A1 neither abolish nor weaken SI in A. thaliana SRK-SCR plants. These results do not support a role for these molecules in the SI response of A. thaliana.

Self-incompatibility systems: barriers to self-fertilization in flowering plants.

Flowering plants (angiosperms) are the most prevalent and evolutionarily advanced group of plants. Success of these plants is owed to several unique evolutionary adaptations that aid in reproduction: the flower, the closed carpel, double fertilization, and the ultimate products of fertilization, seeds enclosed in the fruit. Angiosperms exhibit a vast array of reproductive strategies, including both asexual and sexual, the latter of which includes both self-fertilization and cross-fertilization. Asexual reproduction and self-fertilization are important reproductive strategies in a variety of situations, such as when mates are scarce or when the environment remains relatively stable. However, reproductive strategies promoting cross-fertilization are critical to angiosperm success, since they contribute to the creation of genetically diverse populations, which increase the probability that at least one individual in a population will survive given changing environmental conditions. The evolution of several physical and genetic barriers to self-fertilization or fertilization among closely related individuals is thus widespread in angiosperms. A major genetic barrier to self-fertilization is self-incompatibility (SI), which allows female reproductive cells to discriminate between "self" and "non-self" pollen, and specifically reject self pollen. Evidence for the importance of SI in angiosperm evolution lies in the highly diverse set of mechanisms used by various angiosperm families for recognition of self pollen tube development and preventing self-fertilization.

Specificity determinants and diversification of the Brassica self-incompatibility pollen ligand.

Self-incompatibility in crucifers is effected by allele-specific interactions between the highly polymorphic stigmatic S locus receptor kinase (SRK) and its pollen ligand, the S locus cysteine-rich protein (SCR). Here we show that specificity in SCR function is determined by four contiguous amino acids in one variant, indicating that the minimum sequence requirement for gaining a new specificity can be low. We also provide evidence for an extraordinarily high degree of evolutionary flexibility in SCR, whereby SCR can tolerate extensive amino acid changes within the limits of maintaining the same predicted overall structure. This remarkable adaptability suggests a hypothesis for generation of new self-incompatibility specificities by gradual modification of SRK-SCR affinities and, more generally, for functional specialization within families of homologous ligands and receptors.

Generation of self-incompatible Arabidopsis thaliana by transfer of two S locus genes from A. lyrata.

Transitions from cross-fertilizing to self-fertilizing mating systems have occurred frequently in natural and domesticated plant populations, but the underlying genetic causes are unknown. We show that gene transfer of the stigma receptor kinase SRK and its pollen-borne ligand SCR from one S-locus haplotype of the self-incompatible and cross-fertilizing Arabidopsis lyrata is sufficient to impart self-incompatibility phenotype in self-fertile Arabidopsis thaliana, which lacks functional orthologs of these genes. This successful complementation demonstrates that the signaling cascade leading to inhibition of self-related pollen was maintained in A. thaliana. Analysis of self-incompatibility will be facilitated by the tools available in this species.

Recognition and rejection of self in plant reproduction.

Plant self-incompatibility (SI) systems are unique among self/nonself recognition systems in being based on the recognition of self rather than nonself. SI in crucifer species is controlled by highly polymorphic and co-evolving genes linked in a complex. Self recognition is based on allele-specific interactions between stigma receptors and pollen ligands that result in the arrest of pollen tube development. Commonalities and differences between SI and other self/nonself discrimination systems are discussed.