Characterization of the SP11/SCR high-affinity binding site involved in self/nonself recognition in brassica self-incompatibility

In Brassica self-incompatibility, the recognition of self/nonself pollen grains, is controlled by the S-locus, which encodes three highly polymorphic proteins: S-locus receptor kinase (SRK), S-locus protein 11 (SP11; also designated S-locus Cys-rich protein), and S-locus glycoprotein (SLG). SP11, located in the pollen coat, determines pollen S-haplotype specificity, whereas SRK, located on the plasma membrane of stigmatic papilla cells, determines stigmatic S-haplotype specificity. SLG shares significant sequence similarity with the extracellular domain of SRK and is abundant in the stigmatic cell wall, but its function is controversial. We previously showed that SP11 binds directly to its cognate SRK with high affinity (K(d) = 0.7 nM) and induces its autophosphorylation. We also found that an SLG-like, 60-kD protein on the stigmatic membrane forms a high-affinity binding site for SP11. Here, we show that the 60-kD stigmatic membrane protein is a truncated form of SRK containing the extracellular domain, transmembrane domain, and part of the juxtamembrane domain. A transiently expressed, membrane-anchored form of SRK exhibits high-affinity binding to SP11, whereas the soluble SRK (eSRK) lacking the transmembrane domain exhibits no high-affinity binding, as is the case with SLG. The different binding affinities of the membrane-anchored SRK and soluble eSRK or SLG will be significant for the specific perception of SP11 by SRK.     

A pollen coat protein, SP11/SCR, determines the pollen S-specificity in the self-incompatibility of Brassica species

Many flowering plants have evolved self-incompatibility (SI) systems to prevent inbreeding. In the Brassicaceae, SI is genetically controlled by a single polymorphic locus, termed the S-locus. Pollen rejection occurs when stigma and pollen share the same S-haplotype. Recognition of S-haplotype specificity has recently been shown to involve at least two S-locus genes, S-receptor kinase (SRK) and S-locus protein 11 or S-locus Cys-rich (SP11/SCR). SRK encodes a polymorphic membrane-spanning protein kinase, which is the sole female determinant of the S-haplotype specificity. SP11/SCR encodes a highly polymorphic Cys-rich small basic protein specifically expressed in the anther tapetum and in pollen. In cauliflower (B. oleracea), the gain-of-function approach has demonstrated that an allele of SP11/SCR encodes the male determinant of S-specificity. Here we examined the function of two alleles of SP11/SCR of B. rapa by the same approach and further established that SP11/SCR is the sole male determinant of SI in the genus Brassica sp. Our results also suggested that the 522-bp 5'-upstream region of the S9-SP11 gene used to drive the transgene contained all the regulatory elements required for the unique sporophytic/gametophytic expression observed for the native SP11 gene. Promoter deletion analyses suggested that the highly conserved 192-bp upstream region was sufficient for driving this unique expression. Furthermore, immunohistochemical analyses revealed that the protein product of the SP11 transgene was present in the tapetum and pollen, and that in pollen of late developmental stages, the SP11 protein was mainly localized in the pollen coat, a finding consistent with its expected biological role.     

Immunohistochemical studies on translocation of pollen S-haplotype determinant in self-incompatibility of Brassica rapa

The self-incompatibility system in Brassica is controlled by the S-locus, which contains S-receptor kinase (SRK) and S-locus protein 11 (SP11). SRK and SP11 control stigma and pollen S-haplotype specificity, respectively. SP11 binding to SRK induces the autophosphorylation of SRK, which triggers the signaling cascade that results in the rejection of self-pollen. The localization of SP11 protein during pollen development and pollination, however, have never been demonstrated. In this study, we examined the localization of S(8)-SP11 protein in the anther or pollinated stigma by immuno-electron microscopy. The immunostaining suggested that S(8)-SP11 was secreted from the tapetal cell into the anther locule as a cluster and translocated to the pollen surface at the early developmental stage of the anther. During the pollination process, SP11 was translocated from the pollen surface to the papilla cell, and then penetrated the cuticle layer of the papilla cell to diffuse across the pectin cellulose layer. Furthermore, SP11 protein could only penetrate the cuticle layer of the papilla cell in the presence of pollen grains, and could not penetrate on its own. This suggests that another factor from the pollen grain is needed for SP11 protein to penetrate the papilla cell wall.     

Molecular mechanism of self-recognition in Brassica self-incompatibility

In most self-incompatible plant species, recognition of self-pollen is controlled by a single locus, termed the S-locus. In Brassica, genetic dissection of the S-locus has revealed the presence of three highly-polymorphic genes: S-receptor kinase (SRK), S-locus protein 11 (SP11) (also known as S-locus cysteine-rich protein; SCR) and S-locus glycoprotein (SLG). SRK encodes a membrane-spanning serine/threonine kinase that determines the S-haplotype specificity of the stigma. SP11 encodes a small cysteine-rich protein that determines the S-haplotype specificity of pollen. SLG encodes a secreted form of stigma protein similar to the extracellular domain of SRK. Recent biochemical studies have revealed that SP11 functions as the sole ligand for its cognate SRK receptor complex. Their interaction induces the autophosphorylation of SRK, which is expected to trigger the signalling cascade that results in the rejection of self-pollen. This so-called ligand-receptor complex interaction and receptor activation occur in an S-haplotype-specific manner, and this specificity is almost certainly the basis for self-pollen recognition.     

Brassica self-incompatibility: A glimpse below the surface

Self-incompatibility (SI) has emerged as an evolutionary strategy to enhance the genetic variability of plant species. In Brassica, it is controlled by a single multiallelic locus, the S-locus, encoding a receptor kinase (SRK) expressed in the stigma papilla cells and its ligand, a small protein (SCR) located in the pollen coat. Pollen rejection is achieved only when the receptor recognizes SCR coming from the same S-allele. If a single papilla cell is simultaneously pollinated by a self- and a cross-pollen grain, it is capable of distinguishing between the two and responding accordingly, rejecting self while accepting cross pollen. This phenomenon reveals that SI response is strictly localized and does not involve the whole papilla cell. It also suggests that the distribution of SRK inside the cell may play an important role in regulating this dual response. We have recently demonstrated that SRK is mostly intracellular, only small amounts being present in distinct domains of the plasma membrane (PM), where interaction with SCR occurs. Following ligand recognition, the receptor-ligand complex is endocytosed and degraded. Based on this, we propose a model of the significance of SRK intracellular trafficking for the functioning and specificity of SI response.Key words: self-incompatibility, S-receptor kinase, internalization, SI domains     

植物孢子体自交不亲和信号转导功能分子研究进展

孢子体自交不亲和(SSI)是许多植物采取的一种抵制近亲繁殖的重要措施,受S位点复等位基因控制。近年来,参与其信号转导的许多功能分子及它们的编码基因被分离并得到了充分研究：当自花授粉时,SPlI／SCR与SRK特异识别,造成后的Ser／Thr激酶的磷酸化,引发了一系列由SLG、ARC1及水孔蛋白等因子参与的SSI信号转导途径,最终产生自交不亲和的结果。     

芸薹属自交不亲和基因的分子生物学及进化模式

芸薹属的自交不亲和性是受单基因座、复等位基因控制的孢子体控制型。自交不亲和基因座位（S-locus）是由多个基因组成的复杂区域,称之为S多基因家族,其大多数成员分布于芸薹属的整个染色体组。目前已鉴定出100多个S等位基因,它们的起源分化始于一千万年前。S-座位上存在的多基因有3种：SRK,SLG和SCR/SP11;SRK和SLG在柱头中表达,SCR/SP11在雄蕊中表达。SRK蛋白在识别同类花粉的过程中起主要作用,而SLG蛋白增强了这种自交不亲和反应。SLG与SRK基因中编码S-结构域的核苷酸序列相似性程度高达85％～98%。基因转换可能是SLG和SRK的高度同源性能够得以保持的原因。SRK,SLG和SCR基因紧密相连,并表现出高水平的序列多样性。SRK与SLG基因间的距离很近,在20～25 kb之间。在柱头和花粉中,自交不亲和等位基因之间的共显性关系要比显性和隐性关系更加普遍,这是芸薹属自交不亲和性的一大特点。自交不亲和基因的进化模式存在两种假说：双基因进化模式和中性变异体进化模式;可能存在几种不同的进化方式,它们共同在自然群体中新的S等位基因进化过程中起作用。     

Evolution of the self-incompatibility system in the Brassicaceae: identification of S-locus receptor kinase (SRK) in self-incompatible Capsella grandiflora

Self-incompatibility (SI) has been well studied in the genera Brassica and Arabidopsis, which have become models for investigation into the SI system. To understand the evolution of the SI system in the Brassicaceae, comparative analyses of the S-locus in genera other than Brassica and Arabidopsis are necessary. We report the identification of six putative S-locus receptor kinase genes (SRK) in natural populations of Capsella grandiflora, an SI species from a genus which is closely related to Arabidopsis. These S-alleles display striking similarities to the Arabidopsis lyrata SRK alleles in sequence and structure. Our phylogenetic analysis supports the scenario of differing SI evolution along the two lineages (The Brassica lineage and Arabidopsis/Capsella lineage). Our results also argue that the ancestral S-locus lacked the SLG gene (S-locus glycoprotein) and that the diversification of S-alleles predates the separation of Arabidopsis and Capsella.     

SRK, the stigma-specific S locus receptor kinase of Brassica, is targeted to the plasma membrane in transgenic tobacco.

The S locus receptor kinase (SRK) gene is one of two S locus genes required for the self-incompatibility response in Brassica. We have identified the product of the SRK6 gene in B. oleracea stigmas and have shown that it has characteristics of an integral membrane protein. When expressed in transgenic tobacco, SRK6 is glycosylated and targeted to the plasma membrane. These results provide definitive biochemical evidence for the existence in plants of a plasma membrane-localized transmembrane protein kinase with a known cell-cell recognition function. The timing of SRK expression in stigmas follows a time course similar to that previously described for another S locus-linked gene, the S locus glycoprotein (SLG) gene, and correlates with the ability of stigmas to mount a self-incompatibility response. Based on SRK6 promoter studies, the site of gene expression overlaps with that of SLG and exhibits predominant expression in the stigmatic papillar cells. Although reporter gene studies indicated that the SRK promoter was active in pollen, SRK protein was not detected in pollen, suggesting that SRK functions as a cell surface receptor exclusively in the papillar cells of the stigma.     

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.     

Molecular aspects of self-incompatibility in Brassica species.

Many flowering plants possess self-incompatibility (SI) systems to prevent inbreeding. SI in Brassica species is controlled by a single S locus with multiple alleles. In recent years, much progress has been made in determining the male and female S determinant in Brassica species. In the female, a gain-of-function experiment clearly demonstrated that SRK was the sole S determinant, and that SLG enhanced the SI recognition process. By contrast, the male S determinant (termed SP11/SCR) was identified in the course of genome analysis of S locus to be a small cysteine-rich protein, which was classified as a pollen coat protein. This SP11/SCR may function as a ligand for the S domain of SRK in the SI recognition reaction of Brassica species.     

Just how complex is the Brassica S-receptor complex?

Of the plant self-incompatibility (SI) systems investigated to date, that possessed by members of the Brassicaceae is currently the best understood. Whilst the recent demonstrations of interactions between the male determinant (S-locus cysteine rich protein, SCR) and the female determinant (S-locus receptor kinase, SRK) indicate the minimal requirement for SI in Brassica, no consensus exists as to the nature of these molecules in vivo and the potential involvement of accessory molecules in establishing the active S-receptor complex. Variation between S haplotypes appears to be present in the molecular composition of the receptor complex, the regulation of downstream signalling and the requirement for accessory molecules. This review discusses what constitutes an active receptor complex and highlights potential differences between haplotypes. The role of accessory molecules, in particular SLG (S-locus glycoprotein) and low molecular weight pollen coat proteins (PCPs), in pollination are discussed, as is the link between SI and unilateral incompatibility (UI).     

SRK-SCR转基因拟南芥自交不亲和性的研究进展

十字花科植物自交不亲和性（SI）受墨位点（S-locus）编码的sRK和sCR控制,它们分别是柱头和花粉中的sI特异识别因子。野生型拟南芥不具有sI,而近来通过转基因手段将外源艘K—scR基因转入野生型拟南芥可以使其表现sI,由此建立了一个可用于十字花科sI研究的新型模式植物。本文综述了利用这种转基因拟南芥在SI机制及进化方面取得的进展,包括sI新基因的挖掘、候选基因功能分析和拟南芥生殖模式的转变等。     

S cysteine-rich (SCR) binding domain analysis of the Brassica self-incompatibility S-locus receptor kinase

Brassica self-incompatibility, a highly discriminating outbreeding mechanism, has become a paradigm for the study of plant cell-cell communications. When self-pollen lands on a stigma, the male ligand S cysteine-rich (SCR), which is present in the pollen coat, is transmitted to the female receptor, S-locus receptor kinase (SRK). SRK is a membrane-spanning serine/threonine receptor kinase present in the stigmatic papillar cell membrane. Haplotype-specific binding of SCR to SRK brings about pollen rejection. The extracellular receptor domain of SRK (eSRK) is responsible for binding SCR. Based on sequence homology, eSRK can be divided into three subdomains: B lectin-like, hypervariable, and PAN. Biochemical analysis of these subdomains showed that the hypervariable subdomain is responsible for most of the SCR binding capacity of eSRK, whereas the B lectin-like and PAN domains have little, if any, affinity for SCR. Fine mapping of the SCR binding region of SRK using a peptide array revealed a region of the hypervariable subdomain that plays a key role in binding the SCR molecule. We show that residues within the hypervariable subdomain define SRK binding and are likely to be involved in defining haplotype specificity.     

Recent Loss of Self-Incompatibility by Degradation of the Male Component in Allotetraploid Arabidopsis kamchatica

The evolutionary transition from outcrossing to self-fertilization (selfing) through the loss of self-incompatibility (SI) is one of the most prevalent events in flowering plants, and its genetic basis has been a major focus in evolutionary biology. In the Brassicaceae, the SI system consists of male and female specificity genes at the S-locus and of genes involved in the female downstream signaling pathway. During recent decades, much attention has been paid in particular to clarifying the genes responsible for the loss of SI. Here, we investigated the pattern of polymorphism and functionality of the female specificity gene, the S-locus receptor kinase (SRK), in allotetraploid Arabidopsis kamchatica. While its parental species, A. lyrata and A. halleri, are reported to be diploid and mainly self-incompatible, A. kamchatica is self-compatible. We identified five highly diverged SRK haplogroups, found their disomic inheritance and, for the first time in a wild allotetraploid species, surveyed the geographic distribution of SRK at the two homeologous S-loci across the species range. We found intact full-length SRK sequences in many accessions. Through interspecific crosses with the self-incompatible and diploid congener A. halleri, we found that the female components of the SI system, including SRK and the female downstream signaling pathway, are still functional in these accessions. Given the tight linkage and very rare recombination of the male and female components on the S-locus, this result suggests that the degradation of male components was responsible for the loss of SI in A. kamchatica. Recent extensive studies in multiple Brassicaceae species demonstrate that the loss of SI is often derived from mutations in the male component in wild populations, in contrast to cultivated populations. This is consistent with theoretical predictions that mutations disabling male specificity are expected to be more strongly selected than mutations disabling female specificity, or the female downstream signaling pathway.     

Evolution of the S-locus region in Arabidopsis relatives

The S locus, a single polymorphic locus, is responsible for self-incompatibility (SI) in the Brassicaceae family and many related plant families. Despite its importance, our knowledge of S-locus evolution is largely restricted to the causal genes encoding the S-locus receptor kinase (SRK) receptor and S-locus cysteine-rich protein (SCR) ligand of the SI system. Here, we present high-quality sequences of the genomic region of six S-locus haplotypes: Arabidopsis (Arabidopsis thaliana; one haplotype), Arabidopsis lyrata (four haplotypes), and Capsella rubella (one haplotype). We compared these with reference S-locus haplotypes of the self-compatible Arabidopsis and its SI congener A. lyrata. We subsequently reconstructed the likely genomic organization of the S locus in the most recent common ancestor of Arabidopsis and Capsella. As previously reported, the two SI-determining genes, SCR and SRK, showed a pattern of coevolution. In addition, consistent with previous studies, we found that duplication, gene conversion, and positive selection have been important factors in the evolution of these two genes and appear to contribute to the generation of new recognition specificities. Intriguingly, the inactive pseudo-S-locus haplotype in the self-compatible species C. rubella is likely to be an old S-locus haplotype that only very recently became fixed when C. rubella split off from its SI ancestor, Capsella grandiflora.