S-locus Specific Glycoproteins Research Articles

Stigma treatment with saline solutions: A new method to overcome self-incompatibility inBrassica oleraceaL.

SummaryIn this work we report the effect of various salt solutions in overcoming self incompatibility in Brassica oleracea L. Stigmas were treated with a drop of salt solution and 24 h after pollination the growth of the pollen tube was determined. We found that in all cases a consistent fraction of pollen germinated and pollen tubes penetrated into the ovary. On the basis of these results we propose that salt treatment alters the conformation of S-locus specific glycoproteins.

Effects of fixatives on the antigenicity ofBrassica S-locus specific glycoproteins and rapid immunolocalization by using the tissue print technique

The tissue print technique has been used in this study rapidly localize in stigma papillae the S-locus specific glycoproteins (SLSG) which are involved in the self-incompatibility response. The immunolabelling of SLSG was confirmed at the tissue level by the use of cryosections adhered on nitrocellulose. Pretreatment of both stigma prints or cryosections with buffers and fixatives used in conventional immunocytochemistry influenced immunolabelling of SLSG. The labelling was maintained following incubation in buffer alone or in buffered paraformaldehyde but was strongly decreased by addition of glutaraldehyde even at low concentrations. This experimental approach was useful to test the influence of parameters of chemical fixation on antigenic preservation of SLSG and may be applied to other plant antigens to facilitate or improve their immunocytochemical detection.

Expression of S-locus glycoprotein genes from Brassica oleracea and B. campestris in transgenic plants of self-compatible B. napus cv Westar

Self-compatible Brassica napus var ‘Westar’ was transformed with SLG, the S-locus-derived gene that encodes S-locus-specific glycoproteins (SLSG). Four allelic variants of SLG isolated from self-incompatible B. oleracea and B. campestris strains homozygous for different S alleles were used. We show that the transgenic plants synthesized SLSG with the same apparent charge, molecular weight, and antigenic properties as that produced by the corresponding self-incompatible strains from which the cloned SLG genes were isolated. In addition, transgene-encoded SLSG was detected specifically in the papillar cells of the stigma, and was correctly targeted to the papillar cell wall. However, SLSG was produced at reduced levels in transgenic plants relative to self-incompatible strains. The introduction of the SLG genes did not confer a self-incompatibility phenotype on the ‘Westar’ cultivar.

Sporophytic Self-Incompatibility Systems: Brassica S Gene Family

Incompatibility systems represent an important element of control of sexual reproduction in higher plants. By preventing self-pollination they act to promote outbreeding and therefore may exert profound effects on the genetic structures of populations and perhaps the evolution of species. Incompatibility phenotypes are genetically controlled by a single, major gene designated “ S .” Examples include the gametophytically controlled system of the Solanaceae and the sporophytic system that is characteristic of the Brassicaceae. In the species Brassica oleracea , a single highly polymorphic locus S determines both the incompatibility phenotype of pollen via expression in the diploid tapetum of the anthers and also that of the stigma papillae cells. The system would thus appear to be founded on a specialized form of cell-to-cell interaction between the surface of the pollen grain and that of the style. Any identity in expressed S alleles between these two partners results in a rapid and specific inhibition of self-pollen germination at the stigmatic surface. The high relative abundance and strict developmental and tissue-specific regulation of the Brassica S locus-specific glycoproteins (SLSGs) enable a simple strategy for the molecular cloning of the SLG -specific messages.

High pressure freezing and freeze substitution improve immunolabeling of S-locus specific glycoproteins in the stigma papillae ofBrassica

High pressure freezing and freeze substitution methods significantly improve the antigenic preservation of S-locus specific glycoproteins (SLSG). The SLSG, which are implicated in the incompatibility response, are localized over the cell wall and cytoplasm. Labeling in the cytoplasm is mainly associated with dictyosomes and rough endoplasmic reticulum. Quantitative analysis show that in cryofixed papillae the labeling was enhanced by approximately 45% over the cell wall and approximately 90% over the dictyosomes compared to chemically fixed papillae.

Use of a fast protein electrophoretic purification procedure for N‐terminal sequence analysis to identify S‐locus related proteins in stigmas of Brassica oleracea

In the cruciferous plant Brassica oleracea L. (cabbage), the S-locus specific glycoproteins (SLSGs) isolated only in stigmas are considered to play an important role in the normal prevention of self-fertilization. Recent molecular data have shown that the gene encoding these glycoproteins (the SLG gene) belonged to a multigenic family consisting of about 10 homologous copies among which another member is expressed, the S-locus related gene (SLR1gene). Our aim was to determine whether the SLR1-gene proteins were expressed in the stigmatic tissues. We first identified the putative SLSGs or SLR1-proteins by Con A-peroxidase detection of glycoproteins separated after isoelectric focusing in polyacrylamide gels. We describe a fast purification procedure for the glycoproteins of interest, based on analytical isoelectric focusing, electrophoresis, and electroblotting of proteins onto polyvinylidene difluoride membranes. Blotted proteins were sequenced for N-terminal amino acid determination. By comparison of the N-terminal sequences of the purified proteins with the peptide sequence predicted from the SLR1-cDNA, we demonstrate the expression of SLR1-like proteins in stigmas of B. oleracea.

Towards an in vitro bioassay for the self-incompatibility response in Brassica oleracea

Eluates of stigmas of Brassica oleracea that were known to contain S locus-specific glycoproteins (SLSG) discriminated between self and cross pollen in vitro in three different media. Discrimination was equally evident in experiments that were the in vitro equivalents of reciprocal pollinations. In a TAPS-buffered medium, self eluates depressed pollen germination in a dose-dependent manner. TAPS medium allowed a bioassay of the effects of SLSG in eluates because it optimized germination in a way that eliminated the complicating features of the stimulatory substances in the eluates. Stigma eluates affected percentage pollen germination and optimum calcium concentrations in vitro whether or not SLSG were present in the eluates, but differently in different media, and depending on whether the eluates were cross or self with respect to the pollen tested. Thus, the effect of stigma eluates on the in vitro germination of pollen in Brassica depends on the balance of stimulatory versus inhibitory substances in the eluates.

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.

A homozygous S genotype of Brassica oleracea expresses two S-like genes

The sporophytic self-incompatibility system of Brassica species is controlled by a single locus, S. Recognition of self between pollen and stigma is probably mediated by S locus-specific glycoproteins (SLSGs). We describe the isolation, from an S29 homozygote of Brassica oleracea, of two different cDNA clones for transcripts which are equally abundant in stigmas competent for self-incompatibility and each of which is homologous to previously reported SLSG sequences. Extensive DNA sequence divergence between the two clones precludes their cross-hybridisation and each acts as a gene-specific probe. All S genotypes appear to have a single copy of each gene but there are significantly different levels of polymorphism associated with each. The clear structural homology between the two indicates a gene duplication involving the S locus and, perhaps, related to the evolution of self-incompatibility.

Amino-acid sequence of glycoproteins encoded by three alleles of the S locus of Brassica oleracea

The pollen–stigma interaction of self-incompatibility in the mustard family Brassicaceae is controlled by the S locus. Nearly fifty allelic variants at this locus control the specificity of this interaction. Identity of alleles in pollen and stigma results in an incompatible reaction and the arrest of pollen development. Genetic and developmental analyses of self-incompatibility in Brassica have identified a class of stigma glycoproteins exhibiting allele-specific isoforms as being among the products of the S-locus1–3. A cDNA clone encoding one of these S-locus specific glycoproteins has been isolated from B. oleracea4, and recently, N-terminal protein sequence analysis has shown extensive similarity between three S-locus specific glycoproteins from B. campestris5. Here, we describe cDNA clones encoding such glycoproteins from three different S-allele homozygous genotypes, and report the complete amino-acid sequence of the glycoproteins based on the nucleotide sequence of the cDNA clones. We identify allele-encoded differences in the protein sequences which may be relevant to the allelic specificity of the stigma component of the pollen–stigma interaction. The sequences from the sporophytically determined incompatibility system of Brassica show no similarity to the reported S-locus-associated cDNA sequence of the gametophytic Nicotiana self-incompatibility type6.