S-locus Glycoprotein Research Articles

The S haplotypes lacking SLG in the genome of Brassica rapa.

Self-incompatibility (SI) discriminating self- and non-self pollen is regulated by S-locus genes in Brassica. In most of the S haplotypes, a highly polymorphic S-locus glycoprotein ( SLG) gene is tightly linked to genes for the SI determinants, S-receptor kinase ( SRK) and SP11, although the precise function of SLG in SI has not been clarified. In the present study, we performed DNA gel blot analysis for S(32), S(33), and S(36) haplotypes of Brassica rapa showing normal SI phenotypes and concluded that there might be no SLG in their genome. RNA gel blot analysis of the SLG-less S haplotypes indicated the possible existence of eSRK transcripts in the stigma. These three S haplotypes are useful resources to discern the molecular mechanism of the SI reaction without SLG.

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).

Variation of prezygotic barriers in the interspecific hybridization between oilseed rape and wild radish

Escape of transgenes from genetically modified oilseed rape, Brassica napus, into wild radish, Raphanus raphanistrum, depends on sexual compatibility. The variation in prezygotic barriers of two different cultivars for interspecific hybridization with a population of wild radish was investigated by hand crossing and fluorescence microscopy of pistils. Significant differences were observed between oil seed rape cultivars in their ability to accept wild radish pollen germinating onto their stigma and the rate of fertilization of ovules. Some differences among the pollen donor plants were also detected. These results suggest that the rate of interspecific hybridization in the field would depend upon the oilseed rape cultivar and the genotype composition of the local wild populations. The implication of S-related genes, as revealed through identification by pistil tissue prints of class I and II S-types of SLG (S-Locus Glycoprotein) and SLR1 (S-Locus Related),and immuno-IEF, was not significant.

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.

Just how complex is the BrassicaS-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).

Identification and classification of S haplotypes in Raphanus sativus by PCR-RFLP of the S locus glycoprotein (SLG) gene and the S locus receptor kinase (SRK) gene.

Polymorphism of the S-locus glycoprotein (SLG) and S-locus receptor kinase (SRK) genes in Raphanus sativus was analyzed by PCR-RFLP using SLG- and SRK-specific primers. Twenty four inbred lines of R. sativus could be grouped into nine S haplotypes. DNA fragments of SLG alleles specifically amplified from five S haplotypes by PCR with Class-I SLG-specific primers showed different profiles upon polyacrylamide-gel electrophoresis after digestion with restriction endonucleases. The five R. sativus SLG alleles were determined for their nucleotide sequences of DNA fragments. Comparison of the amino-acid sequences with a reported Brassica SLG (S(6)) showed 77-84% homology. Deduced amino-acid sequences showed 12-conserved cystein residues and three hypervariable regions which are characteristic of Brassicsa SLG. A DNA fragment was also amplified by PCR from two of each S haplotype with Class-II SLG-specific primers, and showed polymorphism when cleaved with restriction endonucleases. The nucleotide sequences of amplified DNA fragments of the Class-II SLG revealed about 60% similarity with those of the Class-I SLG. It is concluded that there exist both Class I and Class II S alleles in R. sativus, as in Brassica campestris and Brassica oleracea. PCR using SRK-specific primers amplified a DNA fragment of about 1.0 kb from seven of each S haplotype out of 24 tested. These DNA fragments showed high polymorphism in polyacrylamide-gel electrophoresis after digestion with restriction endonucleases. Nucleotide sequences of the DNA fragments amplified from the seven S haplotypes showed that the fourth and the fifth exons of SRK are highly conserved, and that there is high variation in the fifth intron, the sixth intron and seventh exon of the SRK which may be responsible for the polymorphic band patterns in PCR-RFLP analysis. The PCR-RFLP method has proven useful for the identification of S alleles in inbred lines and for listing S haplotypes in R. sativus. Phylogenic analysis of the SLG and SRK sequences from Raphanus and Brassica revealed that the Raphanus SLGs and SRKs did not form an independent cluster, but were dispersed in the tree, clustering together with Brassica SLGs and SRKs. Furthermore, SLGs and SRKs from Raphanus were both grouped into Class-I or Class-II S haplotypes. Therefore, these results suggest that the diversification of the SLG and SRK alleles occurred prior to the differentiation of the two genera Brassica and Raphanus.

Molecular mechanism for the recognition reaction in the self-incompatibility of Brassica species

Many higher plants have developed systems to prevent self-fertilization. The self-incompatibility (SI) system has been well known since the time of Darwin. In Brassica, SI is controlled by a single polymorphic locus called S. Discrimination of self and non-self pollen by the pistil should occur on the surface of the stigma. We have investigated the molecular mechanism underlying the SI system and have determined the S-gene products in pistil and pollen. The S-determinant of pollen (SP11) is a small cysteine-rich protein and is the ligand of a receptor complex in papilla cells, which is composed of S-receptor kinase (SRK) and S-locus glycoprotein (SLG). Ligand binding induces the autophosphorylation of SRK, which triggers the signaling cascade that results in the rejection of self-pollen. The ligand-receptor interaction occurs in an S-haplotype specific manner. This is the fundamental reaction of self-recognition system underlying SI in Brassica.

Direct ligand-receptor complex interaction controls Brassica self-incompatibility.

Many higher plants have evolved self-incompatibility mechanisms to prevent self-fertilization. In Brassica self-incompatibility, recognition between pollen and the stigma is controlled by the S locus, which contains three highly polymorphic genes: S-receptor kinase (SRK), S-locus protein 11 (SP11) (also called 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, and SP11 encodes a small cysteine-rich protein that determines the S-haplotype specificity of pollen. SP11 is localized in the pollen coat. It is thought that, during self-pollination, SP11 is secreted from the pollen coat and interacts with its cognate SRK in the papilla cell of the stigma to elicit the self-incompatibility response. SLG is a secreted stigma protein that is highly homologous to the SRK extracellular domain. Although it is not required for S-haplotype specificity of the stigma, SLG enhances the self-incompatibility response; however, how this is accomplished remains controversial. Here we show that a single form of SP11 of the S8 haplotype (S8-SP11) stabilized with four intramolecular disulphide bonds specifically binds the stigma membrane of the S8 haplotype to induce autophosphorylation of SRK8, and that SRK8 and SLG8 together form a high-affinity receptor complex for S8-SP11 on the stigma membrane.

Sequence comparisons among dispersed members of the Brassica S multigene family in an S9 genome.

Self-incompatibility (SI) systems prevent self-pollination and promote outbreeding. In Brassica, the SI genes SLG (for S-locus glycoprotein) and SRK (for S-receptor kinase) are members of the S multigene family, which share the SLG-like domain (S domain), which encodes a putative receptor. We have cloned members of the S multigene family from the S9 haplotype of B. campestris (syn. rapa). In addition, eight distinct genomic regions harboring 10 SLG/SRK-like genes were characterized in the present study. Sequence analysis revealed two novel SRK-like genes, BcRK3 and BcRK6 (for B. campestris receptor kinases 3 and 6, respectively). Other genes that were characterized included SFR2 (for S gene family receptor 2), SLR2 (for S locus related gene 2), and a pseudogene. Based on phylogenetic analysis of the nucleotide sequences of the S domain regions, SLG and SRK appear to be distinct from other members of the S multigene family. Linkage analysis showed that most members of the S multigene family are dispersed in the Brassica genome, and that SLR1 (S locus related gene 1) is not linked to the SLR2 in B. campestris.

S-HAPLOTYPE POLYMORPHISM IN BRASSICA OLERACEA

Self-incompatibility (SI) promotes outcrossing in Brassica species and has been exploited for producing Fl hybrids in cole crops. The SI locus (S-locus) is very polymorphic and cross-compatibility between plants depends on the allelic forms (S-haplotypes) of genes at the S-locus. Among these genes, SLG (S-Locus Glycoprotein gene) is specifically expressed in the stigma. Two classes (class I and class II) of SLG genes have been described and antibodies allowing a specific identification of class I or class II SLG are available. In this paper, we report the polymorphism in Brassica oleracea cultivars (cauliflower, broccoli, romanesco, cabbage, kale and wild forms of the Normandy coast) as assessed by using an isoelectric focusing and immunostaining combined analysis. Plants from an S-allele collection obtained from Dr D. Ockendon were also introduced in the analysis to allow the identification of S-haplotypes according to his nomenclature. Using the two class-specific-SLG antibodies, approximately 80 different immunostaining patterns were identified out of the 950 plants analysed. However, we were unable to find specific immunostaining patterns for about 10% of the analysed plants. Among these plants, we identified 9 S-haplotypes by crossing experiments with lines of known S-haplotype. We also observed that three S-haplotypes (S2, S29, S63) gave variable immunostaining patterns, where SLG bands were either clearly detectable, faintly detectable or not present. This indicates that, in these particular S-haplotypes, the amount of SLG is highly dependent on the genetic background of the plants. All but one of the S-' haplotypes described in the wild forms were also found in cultivated forms. The three class II haplotypes (i.e., S2, S5, S15) were detected in wild Brassica but only S15 was common to all cultivated as well as wild Brassica plants. Moreover, S15 was the most frequent S-haplotype detected in the species.

Homology-dependent Suppression of Stigma Phenotype by an Antisense S-locus Glycoprotein (SLG) Gene in Brassica rapa L.

Self-incompatibility (SI) in Brassicaceae is sporophytically controlled by haplotypes of the polymorphic S locus complex. Two tightly linked polymorphic genes at the S locus, S-locus glycoprotein (SLG) and S-receptor kinase (SRK) genes, are specifically expressed in the stigma. S-haplotypes have been classified into class I and class II types based on the sequence similarity of their SLGs, and their SRKs. To investigate the effect of an antisense SLG gene on the class divergency of the endogenous SLG and SRK genes, we introduced an antisense class I SLG43 cDNA into a cultivar Osome in Brassica rapa which was heterozygous for class I S52 haplotype and class II S60. SLG43 is more similar to the endogenous SLG52 (87.8% identity) than to SLG60 (74.8% identity), Out of ten primary transformants analyzed, two were completely self-compatible; the SI phenotype of stigma was altered from S52S60 to S60, but that of pollen was not. In these two plants, the expression levels of mRNA and protein of SLG52 were reduced, whereas those of SLG60 were not. We suggest that an antisense class I SLG-transgene causes homology-dependent suppression, which leads to breakdown of the class I S-haplotype specificity in stigma but not the class II S-haplotype.

Characterization of Brassica S-haplotypes lacking S-locus glycoprotein

Self-incompatibility (SI) in Brassica is regulated by a single multi-allelic locus, S, which contains highly polymorphic stigma-expressed genes, SLG and SRK. While SRK is shown to be the determinant of female SI specificity, SLG is thought to assist the function of SRK. Here we report that the SLG genes of self-incompatible S 18 and S 60 homozygotes of Brassica oleracea have an in-frame stop codon and a 23 bp deletion resulting in a frame-shift, respectively. The finding that these SLG genes do not encode functional SLG proteins suggests that SLG is not essential for SI. The possible role of SLG in SI was discussed.

Post-transcriptional maturation of the S receptor kinase of Brassica correlates with co-expression of the S-locus glycoprotein in the stigmas of two Brassica strains and in transgenic tobacco plants.

The S-locus-encoded S receptor kinase (SRK) is an intrinsic plasma membrane protein that is viewed as the primary stigma determinant of specificity in the self-incompatibility response of Brassica spp. We analyzed two self-compatible mutant strains that express low levels of the S-locus glycoprotein (SLG), a cell wall-localized protein also encoded at the S locus that is coordinately expressed with SRK. We found that mutant stigmas synthesized wild-type levels of SRK transcripts but failed to produce SRK protein at any of the developmental stages analyzed. Furthermore, SRK was shown to form aberrant high-molecular mass aggregates when expressed alone in transgenic tobacco (Nicotiana tabacum) plants. This aggregation was prevented in tobacco plants that co-expressed SRK and SLG, but not in tobacco plants that co-expressed SRK and SLR1, an SLG-related secreted protein not encoded at the S locus. In analyses of protein extracts under reducing and non-reducing conditions, evidence of intermolecular association was obtained only for SLG, a fraction of which formed disulfide-linked oligomers and was membrane associated. The data indicate that, at least in plants carrying the S haplotypes we analyzed, SRK is an inherently unstable protein and that SLG facilitates its accumulation to physiologically relevant levels in Brassica stigmas.

Multiple S Gene Family Members Including Natural Antisense Transcripts Are Differentially Expressed during Development of Maize Flowers

Within the large Brassica S gene family, SLG (S locus glycoprotein) and SRK (S locus receptor kinase) participate to the control of pollen-stigma self-incompatibility. In the self-compatible species maize, S gene family members are predominantly expressed in vegetative organs but are also expressed to a lesser extent in the stigma (silk). To determine if the expression of any S gene family members correlates with female receptivity, we analyzed their expression in developing maize silks. We show that a large family of maize S transcripts is expressed in developing silks. Surprisingly, we isolated a cDNA complementary to a large portion of the antisense strand of the maize receptor kinase S domain. Rapid amplification of cDNA ends (RACE)-polymerase chain reaction, RNase protection, and Northern hybridization with single-stranded riboprobes confirmed that natural antisense S transcripts exist in leaves and seedling shoots and in all sexual tissues tested except mature pollen. These natural antisense S transcripts co-exist with several less abundant sense S transcripts. The accumulation of sense and antisense S transcripts is differentially regulated during pollen and silk development. Thus, these results support a role for S gene family members in sexual tissue development and/or compatible pollination and reveal a new level of complexity in the regulation and function of the S gene family in maize.

Physical distances between S-locus genes in various S haplotypes of Brassica rapa and B. oleracea

In Brassica, self-incompatibility genes SLG (for S-locus glycoprotein) and SRK (for S-receptor kinase) are located in the S-locus complex region with several other S-linked genes. The S locus is a highly polymorphic region: polymorphism has been observed not only in sequences of SLG and SRK but also in the location of the S-locus genes. In order to compare the physical location of the S-locus genes in various S haplotypes, we used six class-I S haplotypes of B. rapa and seven class-I S haplotypes of B. oleracea in this study. DNA gel blot analysis using pulsed-field gel electrophoresis (PFGE) showed that the physical distances between SLG and SRK in B. rapa are significantly shorter than those in B. oleracea and that the sizes of MluI and BssHII fragments harboring SLG and SRK are less variable within B. rapa than within B. oleracea. We concluded that several large genomic fragments might have been inserted into the S-locus region of B. oleracea after allelic differentiation of S-locus genes.

Self-pollen rejection in Brassica

Evidence has accumulated for the Brassica self-incompatibility system, indicating that the cell wall-localized S locus glycoprotein (SLG) and the plasma membrane-anchored S receptor kinase (SRK) are necessary for the stigma to inhibit self-pollen. By introducing 55 kb of DNA-containing SLG, SRK and an anther-expressed gene into a self-compatible Brassica napus line, SLG and SRK have been shown to be sufficient for the female side but not for the male side of the self-incompatibility phenotype in Brassica. This suggests that there must be an independent pollen S factor encoded outside the cloned region. Proc. Natl. Acad. Sci. U. S. A. (2000) 97, 3713–3717.

The S locus glycoprotein and the S receptor kinase are sufficient for self-pollen rejection in Brassica.

Self-incompatibility (SI) is one of several mechanisms that have evolved to prevent inbreeding in plants. SI in Brassica is controlled by the polymorphic S locus complex. Two S locus-encoded proteins are coordinately expressed in the stigma epidermis: the cell wall-localized S locus glycoprotein (SLG) and the plasma membrane-anchored S receptor kinase (SRK). These proteins are thought to recognize a pollen factor that leads to the rejection of self-pollen. Evidence has accumulated that indicates that both proteins are necessary for the ability of the stigma to inhibit self-pollen. However, it has not been possible to prove this necessity definitively or to demonstrate that these genes are sufficient for this phenotype, because previous attempts to transfer this phenotype via transformation have not been successful. In this study, two overlapping S locus genomic clones, which cover approximately 55 kilobases of DNA and contain the SLG, SRK, and an anther-expressed gene in the region common to the two, were introduced into a self-compatible Brassica napus line. The resulting transgenic plants were shown to carry the female part of the SI phenotype, rejecting pollen in a haplotype-specific manner. However, the pollen SI phenotype was not found in any of the transgenic plants. These data show that the SLG and SRK are sufficient for the female side but not the male side of the SI phenotype in Brassica and that there must be an independent pollen S factor encoded outside the cloned region.

The S locus glycoprotein and the S receptor kinase are sufficient for self-pollen rejection in Brassica

Self-incompatibility (SI) is one of several mechanisms that have evolved to prevent inbreeding in plants. SI in Brassica is controlled by the polymorphic S locus complex. Two S locus-encoded proteins are coordinately expressed in the stigma epidermis: the cell wall-localized S locus glycoprotein (SLG) and the plasma membrane-anchored S receptor kinase (SRK). These proteins are thought to recognize a pollen factor that leads to the rejection of self-pollen. Evidence has accumulated that indicates that both proteins are necessary for the ability of the stigma to inhibit self-pollen. However, it has not been possible to prove this necessity definitively or to demonstrate that these genes are sufficient for this phenotype, because previous attempts to transfer this phenotype via transformation have not been successful. In this study, two overlapping S locus genomic clones, which cover approximately 55 kilobases of DNA and contain the SLG, SRK, and an anther-expressed gene in the region common to the two, were introduced into a self-compatible Brassica napus line. The resulting transgenic plants were shown to carry the female part of the SI phenotype, rejecting pollen in a haplotype-specific manner. However, the pollen SI phenotype was not found in any of the transgenic plants. These data show that the SLG and SRK are sufficient for the female side but not the male side of the SI phenotype in Brassica and that there must be an independent pollen S factor encoded outside the cloned region.

Isolation and characterization of pollen coat proteins of Brassica campestris that interact with S locus-related glycoprotein 1 involved in pollen-stigma adhesion

Adhesion of pollen grains to the stigmatic surface is a critical step during sexual reproduction in plants. In Brassica, S locus-related glycoprotein 1 (SLR1), a stigma-specific protein belonging to the S gene family of proteins, has been shown to be involved in this step. However, the identity of the interacting counterpart in pollen and the molecular mechanism of this interaction have not been determined. Using an optical biosensor immobilized with S gene family proteins, we detected strong SLR1-binding activity in pollen coat extracts of Brassica campestris. Two SLR1-binding proteins, named SLR1-BP1 and SLR1-BP2, were identified and purified by the combination of SLR1 affinity column chromatography and reverse-phase HPLC. Sequence analyses revealed that these two proteins (i) differ only in that a proline residue near the N terminus is hydroxylated in SLR1-BP1 but not in SLR1-BP2, and (ii) are members of the class A pollen coat protein (PCP) family, which includes PCP-A1, an SLG (S locus glycoprotein)-binding protein isolated from Brassica oleracea. Kinetic analysis showed that SLR1-BP1 and SLR1-BP2 specifically bound SLR1 with high affinity (K(d) = 5.6 and 4.4 nM, respectively). The SLR1-BP gene was specifically expressed in pollen at late stages of development, and its sequence is highly conserved in Brassica species with the A genome.

Isolation and characterization of pollen coat proteins of Brassica campestris that interact with S locus-related glycoprotein 1 involved in pollen-stigma adhesion.

Adhesion of pollen grains to the stigmatic surface is a critical step during sexual reproduction in plants. In Brassica, S locus-related glycoprotein 1 (SLR1), a stigma-specific protein belonging to the S gene family of proteins, has been shown to be involved in this step. However, the identity of the interacting counterpart in pollen and the molecular mechanism of this interaction have not been determined. Using an optical biosensor immobilized with S gene family proteins, we detected strong SLR1-binding activity in pollen coat extracts of Brassica campestris. Two SLR1-binding proteins, named SLR1-BP1 and SLR1-BP2, were identified and purified by the combination of SLR1 affinity column chromatography and reverse-phase HPLC. Sequence analyses revealed that these two proteins (i) differ only in that a proline residue near the N terminus is hydroxylated in SLR1-BP1 but not in SLR1-BP2, and (ii) are members of the class A pollen coat protein (PCP) family, which includes PCP-A1, an SLG (S locus glycoprotein)-binding protein isolated from Brassica oleracea. Kinetic analysis showed that SLR1-BP1 and SLR1-BP2 specifically bound SLR1 with high affinity (K(d) = 5.6 and 4.4 nM, respectively). The SLR1-BP gene was specifically expressed in pollen at late stages of development, and its sequence is highly conserved in Brassica species with the A genome.