Modification of the Pollen-Stigma Interaction in Brassica oleracea by Water

The presence of a film of distilled water on the stigma surfaceof freshly opened flowers results in complete inhibition ofpollen following both incompatible and compatible pollinationsin self-incompatible (SI) genotypes of Brassica oleracea, SIgenotypes of B. campestris and one self-compatible (SC) genotypeof B. campestris. The application of water to the stigmas afterpollination also resulted in a marked reduction in pollen germinationand tube penetration. An increase in the time intervals betweenthe application of pollen onto the stigma and the water treatmentprogressively reduced this inhibition. Pollen germination wasalso completely inhibited when stigmas from freshly-opened flowersof SI B. campestris and B. oleracea genotypes were washed inwater, dried and pollinated with pollen grains of either compatibility.The ability of stigmas to induce pollen germination and tubegrowth was restored over a period, the length of which was dependenton the incompatibility (S) genotype. Stigmas of B. napus (SC)and SC mutants of SI B. campestris were found to be affectedby washing, but stigmas of a SC variety of B. campestris andthe immature stigmas from buds of B. oleracea were found tobe considerably less affected. Microscopic examination of pollenplaced on washed stigmas reveals that grains, irrespective oftheir compatibility, fail to hydrate normally. When inducedto hydrate by raising atmospheric humidity, pollen grains onwashed stigmas did germinate, but most of the tubes failed topenetrate the papillar wall and very few entered the style.It is proposed that the water treatment mobilises componentsof the pellicle which reorganize to block the activity of molecules,present in both SC and SI individuals, responsible for establishingfull contact between the pellicle and pollen grain coating. Brassica, pellicle, pollen, recognition, self-incompatibility     

Self-compatibility of two apricot selections is associated with two pollen-part mutations of different nature

Loss of pollen-S function in Prunus self-compatible mutants has recently been associated with deletions or insertions in S-haplotype-specific F-box (SFB) genes. We have studied two self-compatible cultivars of apricot (Prunus armeniaca), Currot (S(C)S(C)) and Canino (S(2)S(C)), sharing the naturally occurring self-compatible (S(C))-haplotype. Sequence analysis showed that whereas the S(C)-RNase is unaltered, a 358-bp insertion is found in the SFB(C) gene, resulting in the expression of a truncated protein. The alteration of this gene is associated with self-incompatibility (SI) breakdown, supporting previous evidence that points to SFB being the pollen-S gene of the Prunus SI S-locus. On the other hand, PCR analysis of progenies derived from Canino showed that pollen grains carrying the S(2)-haplotype were also able to overcome the incompatibility barrier. However, alterations in the SFB(2) gene or evidence of pollen-S duplications were not detected. A new class of F-box genes encoding a previously uncharacterized protein with high sequence similarity (approximately 62%) to Prunus SFB proteins was identified in this work, but the available data rules them out of producing S-heteroallelic pollen and thus the cause of the pollen-part mutation. These results suggest that cv Canino has an additional mutation, not linked to the S-locus, which causes a loss of pollen-S activity when present in pollen. As a whole, these findings support the proposal that the S-locus products besides other S-locus independent factors are required for gametophytic SI in Prunus.     

Dry stigmas,water and self-incompatibility in Brassica

The evolution of dry stigmas has been accompanied by the development — in the pollen — of mechanisms for accessing water from the stigmatic epidermis. Development of self- and cross-pollen on the stigmatic surface has been examined in Brassica oleracea, focusing on the hydration of the grains. Unlike self-compatible (SC) Arabidopsis thaliana, pollen hydration of self-incompatible (SI) Brassica oleracea is preceded by a latent period of between 30–90 min, which is significantly shortened by inhibition of protein synthesis in the stigma. Physiological experiments, some with isolated pollen coatings, indicate that during the latent period signals passing from the pollen to the sigma are responsible for readying the stigmatic surface for penetration and — after self-pollination — activation of the SI system. The changes at the stigma surface include the expansion of the outer layer of the cell wall beneath the grain. This expansion does not occur following self-pollination, when coating-derived signals stimulate a stigmatic response which interrupts hydration and arrests grain development. Cell manipulation studies suggest that self grains are not inhibited metabolically, but are physiologically isolated from the subjacent stigmatic papilla. This focusing of the SI response at the pollen-stigma interface ensures that a single papilla can simultaneously accept cross-pollen and reject self-grains. The evolution of this highly efficient SI system is disussed in the perspective of pathogen-defence mechanisms known also to be located in epidermal cells.     

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.     

Pollen-pistil interactions inBrassica oleracea: Cell calcium in self and cross pollen grains

Summary The levels of calcium in pollen grains on the stigma, after self vs. cross pollinations, were compared inBrassica oleracea, a species showing sporophytic self-incompatibility. Self pollen was characterized by higher levels of chlorotetracycline fluorescence and by higher calcium signals in energy-dispersive analysis of X-rays than cross pollen. Cellular integrity of pollen grains was maintained after rejection, and self pollen could be rescued from the stigma to germinate 4 h after pollination, suggesting that the rejection response was not irreversible.abbreviations CTC chlorotetracycline - EDAX energy dispersive analysis of x-rays - FDA fluorescein diacetate - RH relative humidity - SSI sporophytic self-incompatibility - SLSG S locus-specific glycoproteins     

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.     

植物自交不亲和基因研究进展

自交不亲和性的研究是植物生殖生物学和分子生物学研究的热点之一,对自交不亲和基因和蛋白质的深入研究是解析自交不亲和性机理的关键.对控制孢子体自交不亲和性和配子体自交不亲和性的S基因及其蛋白质产物的分子生物学研究进展进行了综述.孢子体自交不亲和性植物S位点上至少存在3个基因,即SLG、SRK和SCR基因.其中SLG、SRK基因控制雌蕊自交不亲和性,而SCR控制花粉自交不亲和性.配子体自交不亲和植物雌蕊S基因产物为S-RNase,具有核酸酶活性;配子体自交不亲和植物花粉S基因产物尚未找到.     

Studies on heteromorphic self-incompatibility systems: Physiological aspects of the incompatibility system of Primula obconica

Summary In Primula obconica, a species with a heteromorphic self-incompatibility system, the distinction between compatible and incompatible pollen tubes takes place on the stigma surface in thrum flowers, self tubes growing randomly over the papillar cells. No differences were seen between self and cross tube behaviour on the pin stigma surface, but self tubes were inhibited within the stigmatic tissue with differences in tube length evident after 24 h. The stigma surface bears a proteinaceous pellicle and binds the lectin Concanavalin A. Removal of the stigma removes the incompatibility barrier in mature gynoecia. Bud pollination shows that pollen tubes cannot grow in a normal manner on immature stigmas; the random growth of tubes over the stigma surface resembles that of mature thrum selfs. Fewer compatible tubes reach the style base of young gynoecia and smaller numbers of seeds are set than in mature flowers. Pin and thrum pollen grains germinate and grow in aqueous media, thrum tubes growing longer than pin. The presence of H₃BO₄ and CaCl₂ in the growth medium promotes tube elongation and lengths equivalent to compatible styles can be obtained. The pollen grains have proteinaceous materials in their walls which diffuse out on moistening. Prolonged washing in aqueous media removes these materials but the incompatibility reaction remains unchanged. Thus the incompatibility reaction is between pollen tubes and stigmatic tissue and differs from the homomorphic, sporophytic system where pollen wall proteins elicit the incompatibility response.     

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.     

The Skp1‐like protein SSK1 is required for cross‐pollen compatibility in S‐RNase‐based self‐incompatibility

The self‐incompatibility (SI) response occurs widely in flowering plants as a means of preventing self‐fertilization. In these self/non‐self discrimination systems, plant pistils reject self or genetically related pollen. In the Solanaceae, Plantaginaceae and Rosaceae, pistil‐secreted S‐RNases enter the pollen tube and function as cytotoxins to specifically arrest self‐pollen tube growth. Recent studies have revealed that the S‐locus F‐box (SLF) protein controls the pollen expression of SI in these families. However, the precise role of SLF remains largely unknown. Here we report that PhSSK1 (Petunia hybrida SLF‐interacting Skp1‐like1), an equivalent of AhSSK1 of Antirrhinum hispanicum, is expressed specifically in pollen and acts as an adaptor in an SCF(Skp1‐Cullin1‐F‐box)^SLF complex, indicating that this pollen‐specific SSK1‐SLF interaction occurs in both Petunia and Antirrhinum, two species from the Solanaceae and Plantaginaceae, respectively. Substantial reduction of PhSSK1 in pollen reduced cross‐pollen compatibility (CPC) in the S‐RNase‐based SI response, suggesting that the pollen S determinant contributes to inhibiting rather than protecting the S‐RNase activity, at least in solanaceous plants. Furthermore, our results provide an example that a specific Skp1‐like protein other than the known conserved ones can be recruited into a canonical SCF complex as an adaptor.     

利用氨基酸分析快速测定甘蓝自交不亲和性

利用氨基酸分析方法,对甘蓝（ＢｒａｓｉｃａｏｌｅｒａｃｅａＬ．ｖａｒ．ｃａｐｉｔａｔａ）自交不亲和系与亲和系的柱头和花粉蛋白质进行了研究。结果表明,自交不亲和系与亲和系的柱头和花粉氨基酸组分有显著差别。柱头中苏氨酸和酪氨酸含量以及花粉中甘氨酸和丙氨酸含量可以作为评价自交不亲和性的指标。鉴定标准：自交不亲和系柱头中苏氨酸含量高于０．２２３％,酪氨酸含量高于０．３８５％;花粉中甘氨酸含量高于１．５９３％,丙氨酸含量高于１．４６４％。亲和系柱头中苏氨酸含量低于０．１８５％,酪氨酸含量低于０．１６４％;花粉中甘氨酸含量低于１４７０％,丙氨酸含量低于１００６％     

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

Summary 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.     

Identification of differentially accumulating pistil proteins associated with self‐incompatibility of non‐heading Chinese cabbage

Non‐heading Chinese cabbage (Brassica campestris L. ssp. chinensis Makino), an important vegetable crop in China, exhibits a typical sporophytic self‐incompatibility (SI) system. To better understand the mechanism of SI response and identify potential candidate proteins involved in the SI system of this vegetable crop, the proteomic approach was taken to identify differential accumulating pistil proteins. Pistils were collected at 0 h and 2 h after self‐pollination at anthesis in self‐incompatible and compatible lines of non‐heading Chinese cabbage, and total proteins were extracted and separated by two‐dimensional gel electrophoresis (2‐DE). A total of 25 protein spots that displayed differential abundance were identified by matrix‐assisted laser desorption/ionisation‐time of flight mass spectrometry (MALDI–TOF/TOF MS) and peptide mass fingerprinting (PMF). Among them, 22 protein spots were confidently established. The mRNA levels of the corresponding genes were detected by quantitative RT‐PCR. The 22 identified protein spots are involved in energy metabolism (four), protein biosynthesis (three), photosynthesis (six), stress response and defence (five), and protein degradation (four). Among these potential candidate proteins, UDP‐sugar pyrophosphorylase could be involved in sucrose degradation to influence pollen germination and growth. Glutathione S–transferases could be involved in pollen maturation, and affect pollen fertility. Senescence‐associated cysteine protease, which is related to programmed cell death, could be mainly related to self pollen recognition of non‐heading Chinese cabbage. The study will contribute to further investigations of molecular mechanism of sporophytic SI in Brassicaceae.     

Identification of the self-incompatibility locus F-box protein-containing complex in Petunia inflata

The polymorphic S-locus regulating self-incompatibility (SI) in Petunia contains the S-RNase gene and a number of S-locus F-box (SLF) genes. While penetrating the style through the stigma, a pollen tube takes up all S-RNases, but only self S-RNase inhibits pollen tube growth. Recent evidence suggests that SLFs produced by pollen collectively interact with and detoxify non-self S-RNases, but none can interact with self S-RNase. An SLF may be the F-box protein component of an SCF complex (containing Cullin1, Skp1 and Rbx1), which mediates ubiquitination of protein substrates for degradation by the 26S proteasome. However, the precise nature of the complex is unknown. We used pollen extracts of a transgenic plant over-expressing GFP-fused S₂-SLF1 (SLF1 of S ₂-haplotype) for co-immunoprecipitation (Co-IP) followed by mass spectrometry (MS). We identified PiCUL1-P (a pollen-specific Cullin1), PiSSK1 (a pollen-specific Skp1-like protein) and PiRBX1 (an Rbx1). To validate the results, we raised transgenic plants over-expressing PiSSK1:FLAG:GFP and used pollen extracts for Co-IP–MS. The results confirmed the presence of PiCUL1-P and PiRBX1 in the complex and identified two different SLFs as the F-box protein component. Thus, all but Rbx1 of the complex may have evolved in SI, and all SLFs may be the F-box component of similar complexes.     

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).     

Genomic organization of the S locus: Identification and characterization of genes in SLG/SRK region of S(9) haplotype of Brassica campestris (syn. rapa).

In Brassica, two self-incompatibility genes, encoding SLG (S locus glycoprotein) and SRK (S-receptor kinase), are located at the S locus and expressed in the stigma. Recent molecular analysis has revealed that the S locus is highly polymorphic and contains several genes, i.e., SLG, SRK, the as-yet-unidentified pollen S gene(s), and other linked genes. In the present study, we searched for expressed sequences in a 76-kb SLG/SRK region of the S(9) haplotype of Brassica campestris (syn. rapa) and identified 10 genes in addition to the four previously identified (SLG(9), SRK(9), SAE1, and SLL2) in this haplotype. This gene density (1 gene/5.4 kb) suggests that the S locus is embedded in a gene-rich region of the genome. The average G + C content in this region is 32.6%. An En/Spm-type transposon-like element was found downstream of SLG(9). Among the genes we identified that had not previously been found to be linked to the S locus were genes encoding a small cysteine-rich protein, a J-domain protein, and an antisilencing protein (ASF1) homologue. The small cysteine-rich protein was similar to a pollen coat protein, named PCP-A1, which had previously been shown to bind SLG.     

Linear dominance relationship among four class-II S haplotypes in pollen is determined by the expression of SP11 in Brassica self-incompatibility

Self-incompatibility (SI) prevents self-fertilization by rejecting pollen from plants with the same S phenotype. The Brassica SI system is controlled sporophytically by multiple alleles at the single locus, S, and dominance relationships among S haplotypes are observed in both stigma and pollen. We have identified previously five different class-II S haplotypes in Brassica campestris. Here, we performed test-crosses between S heterozygotes and their respective parental S homozygotes for four of these class-II S haplotypes, and observed a linear dominance relationship on the pollen side. To determine how this relationship is controlled, we performed RNA gel blot analyses for six S heterozygotes and their respective parental S homozygotes using the corresponding SP11 clone as a probe. In all six S heterozygotes, SP11 derived from a dominant haplotype was predominantly expressed, and SP11 derived from a recessive haplotype was repressed. Thus, the linear dominance relationship of the SI phenotype on the pollen side is regulated by the expression of SP11.     

Cellular Pathways Regulating Responses to Compatible and Self-Incompatible Pollen in Brassica and Arabidopsis Stigmas Intersect at Exo70A1, a Putative Component of the Exocyst Complex

In the Brassicaceae, compatible pollen–pistil interactions result in pollen adhesion to the stigma, while pollen grains from unrelated plant species are largely ignored. There can also be an additional layer of recognition to prevent self-fertilization, the self-incompatibility response, whereby self pollen grains are distinguished from nonself pollen grains and rejected. This pathway is activated in the stigma and involves the ARM repeat–containing 1 (ARC1) protein, an E3 ubiquitin ligase. In a screen for ARC1-interacting proteins, we have identified Brassica napus Exo70A1, a putative component of the exocyst complex that is known to regulate polarized secretion. We show through transgenic studies that loss of Exo70A1 in Brassica and Arabidopsis thaliana stigmas leads to the rejection of compatible pollen at the same stage as the self-incompatibility response. A red fluorescent protein:Exo70A1 fusion rescues this stigmatic defect in Arabidopsis and is found to be mobilized to the plasma membrane concomitant with flowers opening. By contrast, increased expression of Exo70A1 in self-incompatible Brassica partially overcomes the self pollen rejection response. Thus, our data show that the Exo70A1 protein functions at the intersection of two cellular pathways, where it is required in the stigma for the acceptance of compatible pollen in both Brassica and Arabidopsis and is negatively regulated by Brassica self-incompatibility.     

The mutated S1-haplotype in sour cherry has an altered S-haplotype-specific F-box protein gene

Gametophytic self-incompatibility (GSI) is an outcrossing mechanism in flowering plants that is genetically controlled by 2 separate genes located at the highly polymorphic S-locus, termed S-haplotype. This study characterizes a pollen part mutant of the S(1)-haplotype present in sour cherry (Rosaceae, Prunus cerasus L.) that contributes to the loss of GSI. Inheritance of S-haplotypes from reciprocal interspecific crosses between the self-compatible sour cherry cultivar Ujfehértói Fürt?s carrying the mutated S(1)-haplotype (S(1)'S(4)S(d)S(null)) and the self-incompatible sweet cherry (Prunus avium L.) cultivars carrying the wild-type S(1)-haplotype revealed that the mutated S(1)-haplotype confers unilateral incompatibility with a functional pistil component and a nonfunctional pollen component. The altered sour cherry S(1)-haplotype pollen part mutant, termed S(1)', contains a 615-bp Ds-like element within the S(1)-haplotype-specific F-box protein gene (SFB(1)'). This insertion generates a premature in-frame stop codon that would result in a putative truncated SFB(1) containing only 75 of the 375 amino acids present in the wild-type SFB(1). S(1)' along with 2 other previously characterized Prunus S-haplotype mutants, S(f) and S(6m), illustrate that mobile element insertion is an evolutionary force contributing to the breakdown of GSI.