The male determinant of self-incompatibility in Brassica.

In the S locus-controlled self-incompatibility system of Brassica, recognition of self-related pollen at the surface of stigma epidermal cells leads to inhibition of pollen tube development. The female (stigmatic) determinant of this recognition reaction is a polymorphic transmembrane receptor protein kinase encoded at the S locus. Another highly polymorphic, anther-expressed gene, SCR, also encoded at the S locus, fulfills the requirements for the hypothesized pollen determinant. Loss-of-function and gain-of-function studies prove that the SCR gene product is necessary and sufficient for determining pollen self-incompatibility specificity, possibly by acting as a ligand for the stigmatic receptor.

An aquaporin-like gene required for the Brassica self-incompatibility response.

Self-incompatibility in Brassica refers to the rejection of self-related pollen and is mediated by a receptor protein kinase localized to the plasma membrane of the stigma epidermis in the flower. The recessive mutation mod eliminates self-incompatibility in the stigma. In mod mutants, self-compatibility was shown to be associated with the absence of transcripts encoded by an aquaporin-related gene. This observation suggests that a water channel is required for the self-incompatibility response of Brassica, which is consistent with the concept that regulation of water transfer from the stigma to pollen is a checkpoint in the early events of pollination in the crucifer family.

Signaling the arrest of pollen tube development in self-incompatible plants.

Self-incompatibility (SI), the cellular recognition system that limits inbreeding, has served as a paradigm for the study of cell-to-cell communication in plants since the phenomenon was first described by Darwin. Recent studies indicate that SI is achieved by diverse molecular mechanisms in different plant species. In the mustard family, the mechanism of SI shows parallels to the signaling systems found in animals that are mediated by cell-surface receptors with signal-transducing protein kinase activity.

Allele-specific receptor-ligand interactions in Brassica self-incompatibility.

Genetic self-incompatibility in Brassica is determined by alleles of the transmembrane serine-threonine kinase SRK, which functions in the stigma epidermis, and of the cysteine-rich peptide SCR, which functions in pollen. Using tagged versions of SRK and SCR as well as endogenous stigma and pollen proteins, we show that SCR binds the SRK ectodomain and that this binding is allele specific. Thus, SRK and SCR function as a receptor-ligand pair in the recognition of self pollen. Specificity in the self-incompatibility response derives from allele-specific formation of SRK-SCR complexes at the pollen-stigma interface.

Genetic Ablation of Floral Cells in Arabidopsis.

A chimeric toxic gene consisting of the diphtheria toxin A chain gene fused to a promoter previously shown to direct pistil- and anther-specific expression was used to genetically target cell killing in transgenic Arabidopsis. Flowers of Arabidopsis transformants that carried the toxic gene fusion had distinct structural defects. The papillar cells at the stigma surface were stunted and were biosynthetically inactive. Anther development was also impaired by toxic gene expression, leading to abnormalities in anther dehiscence, pollen morphology, and pollen germination. The combined defects of pistil and anther rendered transformants that carried the toxic gene fusion self-sterile. However, the transformants were cross-fertile with untransformed plants: the viable pollen of ablated plants was rescued by wild-type stigmas, and, strikingly, the ablated papillar cells allowed the growth of wild-type pollen.

Activity of an S Locus Gene Promoter in Pistils and Anthers of Transgenic Brassica.

The pollen-stigma interaction of self-incompatibility in crucifers is correlated with glycoproteins localized in the cell wall of the stigmatic papillae that are encoded by the S locus glycoprotein (SLG) gene. When fused to the [beta]-glucuronidase (GUS) reporter gene, the 5[prime] upstream regulatory region of SLG directed high level expression in the papillae of transgenic Brassica plants. Histochemical and fluorometric assays revealed that, in addition to its primary site of expression in the stigmatic papillae, the SLG-GUS fusion was also expressed in the transmitting tissue of stigma, style, and ovary, and in anthers. This conclusion was verified by the detection of transgene-encoded GUS transcripts and endogenous SLG-homologous transcripts by RNA gel blot analysis. Significantly, in anthers, the SLG promoter was active not only sporophytically in the nurse cells of the tapetum, but also in the haploid microspores. Because self-incompatibility systems exhibiting sporophytic control of pollen phenotype are thought to have evolved from systems with gametophytic control, we suggest that sporophytic control was acquired without loss of gametophytic function.

Ablation of Papillar Cell Function in Brassica Flowers Results in the Loss of Stigma Receptivity to Pollination.

Plant reproduction in crucifers is dependent on interactions that occur at the stigma surface between the male gametophyte (pollen and pollen tube) and papillar cells. To dissect these complex interactions, papillar cells were genetically ablated by targeting the expression of a toxin to appropriate cells of the flower with a flower-specific and developmentally regulated promoter. In transgenic Brassica plants that expressed the toxic gene fusion, flower morphology was normal except for aberrant papillar cell development and partial pollen sterility. Microscopic, biochemical, and functional analyses, mainly focused on papillar cell responses, revealed that papillar cells lost their ability to elongate, to synthesize cell-specific proteins, and to support pollen germination after self- or cross-pollination. This loss of stigma receptivity to pollination was mimicked by treating pistils with protein phosphatase inhibitors. Differences in the effects of genetic and chemical ablation on the pollination responses of Brassica and Arabidopsis flowers are discussed and are ascribed in part to a requirement for phosphorylation/dephosphorylation events in Brassica but not in Arabidopsis.

Brassica S-Proteins Accumulate in the Intercellular Matrix along the Path of Pollen Tubes in Transgenic Tobacco Pistils.

A tobacco plant transformed with a Brassica oleracea SLG-22 gene was analyzed by immunocytochemical methods to determine the localization of the transgene-encoded protein product. Immunolabeling was observed in the pistil along the path followed by pollen tubes after pollination. S-antigen accumulated in the intercellular matrix of the transmitting tissue of the style and its continuation in the basal portion of the stigma and outside a few special cells of the placental epidermis of the ovary. This pattern of S-antigen distribution closely resembles that described for the S-associated glycoproteins of self-incompatible Nicotiana alata and differs from its distribution in B. oleracea.

Genetic control of quantitative variation in self-incompatibility proteins detected by immunodiffusion.

Single radial immunodiffusion was used to study the self-incompatibility (S) proteins present in stigmas of two inbred lines of Brassica oleracea: a self-incompatible line and a self-compatible mutant derived from it. The genetic basis of observed quantitative differences in S proteins between the two inbreds was shown to be determined by a single gene with dosage effects. Self-pollination of individual plants with high, intermediate and low levels of S protein in the stigmas, respectively, resulted in low, intermediate and high seed set.

Effects of Inhibitors of Protein Serine/Threonine Phosphatases on Pollination in Brassica.

We have examined the effect of the protein phosphatase inhibitors okadaic acid and microcystin on pollen-pistil interactions in Brassica. Inhibitor-treated flowers or floral buds were pollinated with untreated pollen and examined for pollen tube growth by fluorescence microscopy. Our results show that type 1 or type 2A serine/threonine phosphatases play a crucial role in the pollination responses of Brassica. We observed two distinct effects of protein phosphatase inhibitors on pollination: (a) the inhibition of pollen tube growth during cross-pollination in flowers, and (b) the break-down of self-incompatibility or promotion of pollen tube growth during self-pollination in flower buds just prior to anthesis. Thus, treatment of flower pistils with protein phosphatase inhibitors resulted in the inhibition of pollen tube growth at the surface of the papillar cells of the stigma in crosses between different self-incompatible Brassica oleracea strains, in an interspecific cross between B. oleracea and Brassica campestris, and in self-pollinations of a self-fertile Brassica napus cultivar. With four different self-incompatibility genotypes, treatment of mature flowers with protein phosphatase inhibitors had no effect on self-pollination response. In contrast, treatment of flower buds just prior to the anthesis stage allowed self-pollen tube invasion of papillar cells. However, the magnitude of this effect was genotype dependent, being most pronounced in the S22 genotype. The data support the conclusion that pollinations in Brassica are controlled in part by the presence of phosphorylated proteins in the papillar cells of the stigma, and that the quantity of these proteins or their levels of phosphorylation changes during stigma development.

Biosynthesis of glycoproteins involved in the pollen-stigma interaction of incompatibility in developing flowers of Brassica oleracea L.

De-novo synthesis of the S-allele-specific glycoproteins of Brassica oleracea is demonstrated in stigmas at different developmental stages. Excised stigmas incorporate (14)C-labeled amino acids into their S-glycoproteins early in development and before the self-incompatibility response is acquired, but the rate of synthesis accelerates prior to anthesis, resulting in the accumulation of high levels of the S-glycoproteins in the stigma and coinciding with the acquisition of the pollen-stigma incompatibility response. Since the self-compatible and self-incompatible zones of developing inflorescences are very sharply delineated, a threshold quantity of S-glycoproteins appears to be critical for the onset of self-incompatibility. Incorporation experiments in which [(35S)methionine was applied to intact stigma surfaces indicate that the papillae are the main sites of synthesis of the S-specific glycoproteins.

Isolation of genes abundantly expressed in rice anthers at the microspore stage.

A cDNA library of rice (Oryza sativa L.) has been constructed from anthers at an early stage of pollen development. By differential screening of the library, we have isolated cDNAs of two genes, designated as Osc4 and Osc6, that are abundantly expressed in anthers containing tetrads and uninucleate microspores, but are not expressed in leaves or roots. Expression of Osc4 is absent in mature anthers, while Osc6 is present although the expression decays during pollen maturation. A comparison of the nucleotide and deduced amino acid sequences with those in data banks has not shown significant homology to known molecules.

Molecular genetic analysis of the candidate gene for MOD, a locus required for self-incompatibility in Brassica rapa.

The MIP-MOD (for MOD-locus associated Major Intrinsic Protein) gene encodes an aquaporin-like product, and has been reported to be a candidate for the MOD gene which is required for the self-incompatibility response in Brassica rapa. In an antisense suppression experiment designed to investigate the role of MIP-MOD, we found that levels of MIP-MOD mRNA in the stigmas of fourteen antisense transgenics, as well as in the self-incompatible cultivar Osome (Osm), were much lower than in the stigmas of the self-incompatible S8 homozygous (S8) strain. Therefore, we analyzed the molecular structure of the MIP-MOD gene in three B. rapa strains: S8, Osm, and the self-compatible var. Yellow Sarson (YS). Nucleotide sequence analysis of the MIP-MOD genes isolated from the three strains revealed that all three encode the same amino acid sequence and that YS and Osm contain the same MIP-MOD allele, designated MIP-MOD(YS). Analysis of other self-incompatible B. rapa strains that are homozygous for the MIP-MOD(YS) allele indicated that high levels of MIP-MOD transcripts are not essential for the self-incompatibility response. Furthermore, a MOD mutant generated by gamma-irradiation was found to contain a wild-type MIP-MOD gene that is expressed at normal levels. These data suggest that MIP-MOD is not MOD itself. We suggest that this gene should be renamed MLM (for MIP gene linked to MOD).

Self-incompatibility genes of Brassica oleracea: Expression, isolation, and structure.

We demonstrate by in situ hybridization the cell-type-specific expression of transcripts encoded by the self-incompatibility (S) locus of Brassica oleracea. These transcripts are not detected early in stigma development, their expression is switched on in the papillar cells of the stigma surface, and their levels increase in these cells in correlation with the acquisition by the stigma of the self-incompatibility response. By using a probe derived from the untranslated sequences at the 3' end of S cDNA, an S-gene copy expressed in the papillar cells has been isolated from among the multiple S-related copies that occur in the Brassica genome. Structural analysis of this gene shows that it lacks introns. In light of the strict spatial and temporal regulation of S-gene expression in precisely the cells that constitute the barrier to self-pollination, the self-incompatibility response may be viewed as a cell-cell interaction between one pollen grain and one papillar cell.

Cell-cell communication in plants: self-incompatibility in flower development.

Self-incompatibility, a mechanism that prevents self-fertilization in plants, is based on the ability of the pistil to discern the presence of self-pollen and on the female tissue's capacity to inhibit the growth or germination of self-related, but not of genetically unrelated, pollen. As a self-recognition system, self-incompatibility responds to specific cellular products and signals and thus offers a unique system in which to study the components of cellular communication in plants. The cytological manifestations of self-incompatibility have been well studied, and, with the cloning of cDNAs for several proteins associated with this recognition process, a detailed molecular view of self-incompatibility is emerging.

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.

An alternative transcript of the S locus glycoprotein gene in a class II pollen-recessive self-incompatibility haplotype of Brassica oleracea encodes a membrane-anchored protein.

Recent reports have shown that SLG, one of two genes linked to the S locus of Brassica, encodes a secreted glycoprotein. We have used RNA gel blot analysis, genomic and cDNA clone analysis, expression in transgenic plants, and immunodetection to characterize SLG2, the SLG gene derived from the S2 haplotype. This haplotype belongs to the class II group of S haplotypes that exhibit a weak incompatibility phenotype and are pollen recessive. We showed that SLG2 produces two transcript forms: the expected 1.6-kb transcript that predicts a secreted glycoprotein and an alternative 1.8-kb transcript that predicts a membrane-anchored protein. Stigmas of the S2 haplotype and pistils of transgenic tobacco plants transformed with the SLG2 gene produce a membrane-associated 62-kD protein as well as soluble 57- and 58-kD glycoforms. Because of the sequence similarity between SLG2 and the extracellular domain of the S Locus Receptor Kinase (SRK2) gene, the membrane-anchored form of SLG2 may be viewed as a naturally occurring truncated form of the receptor that lacks the kinase catalytic domain. The occurrence of this protein has potential implications for the activity of the full-length receptor. Furthermore, the underlying structure of the SLG2 gene suggests the evolution of SLG from an ancestral SRK-like gene.

A Brassica S locus gene promoter directs sporophytic expression in the anther tapetum of transgenic Arabidopsis.

The S locus glycoprotein (SLG) gene of Brassica encodes stigmatic glycoproteins that are implicated in the pollen-stigma interaction of self-incompatibility. We have transformed the related plant Arabidopsis thaliana with a chimaeric gene consisting of the promoter region of an SLG gene fused to the reporter gene beta-glucuronidase (GUS). In transgenic plants the gene was expressed in two cell types of the flower. In stigmas, the timing and distribution of GUS activity was similar to that previously described for SLG expression in Brassica. In anthers, expression was detected at an earlier stage of flower development with GUS activity restricted to the tapetal cell layer. The novel finding of SLG-promoter activity in the anther supports the hypothesis that sporophytic control of self-incompatibility is a result of SLG-gene expression in the tapetum.

Transformation of Brassica oleracea with an S-locus gene from B. campestris changes the self-incompatibility phenotype.

An SLG gene derived from the S-locus and encoding and S-locus-specific glycoprotein of Brassica campestris L. was introduced via Agrobacterium-mediated transformation into B. oleracea L. A self-incompatible hybrid and another with partial self-compatibility were used as recipients. The transgenic plants were altered in their pollen-stigma interaction and were fully compatible upon self-pollination. Reciprocal crosses between the transgenic plants and untransformed control plants indicated that the stigma reaction was changed in one recipient strain while the pollen reaction was altered in the other. Due to interspecific incompatibility, we could not demonstrate whether or not the introduced SLG gene confers a new allelic specificity in the transgenic plants. Our results show that the introduced SLG gene perturbs the self-incompatibility phenotype of stigma and pollen.