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.     

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.     

Loss of callose in the stigma papillae does not affect the Brassica self-incompatibility phenotype

As part of the Brassicaceae self-incompatibility response, callose is deposited in the stigma papillar cells. To determine if callose plays an important role in the rejection of incompatible pollen by the stigma, transgenic Brassica napus. L. plants were produced which express the tobacco β-1,3-glucanase cDNA (the enzyme which degrades callose) in the stigma papillae. Using aniline blue fluorescence, little or no callose was detected in the papillar cells of transgenic stigmas. However, the self-incompatibility system appeared to be unaffected based on the lack of pollen tube growth and the subsequent lack of seed set. The transgene had no effect on compatible pollinations. Thus, while callose deposition is associated with the B. napus self-incompatibility response, it is not required for the rejection of incompatible pollen. Received: 14 March 1997 / Accepted: 15 April 1997     

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.     

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.     

Functional analysis of a Brassica oleracea SLR1 gene promoter.

The Brassica oleracea S-locus-related gene 1 (SLR1) is expressed in the papillar cells of Brassica stigmas from a few days before anthesis. We have previously shown that a 1500-bp fragment of the SLR1 gene promoter is sufficient to direct high-level, temporally regulated expression of the beta-glucuronidase reporter gene in the pistils of transgenic tobacco. We have carried out a deletion analysis of the SLR1 promoter and found that elements required for pistil expression are located between -258 and -327 bp (relative to the translation start site). Furthermore, specific binding of pistil nuclear factors to sequences within this region was demonstrated by gel retardation analysis. Sequences between -1350 and -1500 were found to be required for high-level expression.     

Expression of a polygalacturonase enzyme in germinating pollen of <Emphasis Type="Italic">Brassica napus</Emphasis>

Penetration of pollen tubes through stigmatic tissues in Brassica napus L. may involve the release of cell wall modifying enzymes from the pollen tube tip. We examined the expression of a pectin-degrading polygalacturonase (PG) enzyme in unpollinated and early and late pollinated stigmas via immunoblotting and immuno-light microscopy using a PG polyclonal antibody. Immunoblotting analysis indicated that PG enzyme was present at low levels in unpollinated stigmas and at high levels in pollinated stigmas. The level of PG did not detectably increase between early and late pollinated stigmas. Immuno-light microscopy demonstrated that PG enzyme was present in ungerminated pollen grains, stigmatic papillae and in the tip of pollen tubes growing into the papillar wall. This latter evidence suggests that PG enzyme may play an important role in papillar cell wall penetration during pollination although other interpretations of the role of pollen PG should not be discounted. Received: 9 November 2000 / Accepted: 7 December 2000     

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

Summary 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.     

Identification of gene expression profile of neural crest-derived cells isolated from submandibular glands of adult mice

Neural crest cells in the embryo migrate to reach target sites as neural crest-derived cells (NCDCs) where they differentiate into a variety of derivatives. Some NCDCs are maintained in an undifferentiated state throughout the life of the animal and are considered to be a useful cell source for regenerative medicine. However, no established method to obtain NCDCs sufficient for regenerative medicine from adults with high purity has been presented, since their distribution in adult tissues is not fully understood. It is critical to identify reliable markers for NCDCs in adults, as the expressions of P0 and Wnt1, the most reliable NCDC markers, are shut off in the embryonic stage. To analyze the characteristics of NCDCs in adult tissues, we utilized a double transgenic mouse strain, P0-Cre/CAG-CAT-EGFP transgenic mice (P0 mice), in which NCDCs were shown to express EGFP and we were able to recognize GFP-positive cells in those. We focused on the submandibular glands (SMGs), which are known to be derived from the neural crest. GFP-positive cells were shown to be scattered like islands in the SMGs of adult P0 mice. We surgically removed SMGs from adult mice and digested samples into single cell suspensions. GFP-positive cells separated using flow cytometry expressed a high level of Sox10, a marker of embryonic neural crest cells, suggesting successful isolation of NCDCs. To identify candidate marker genes in isolated NCDCs, we performed DNA microarray analyses and real-time PCR analysis of GFP-positive and -negative cells isolated from P0 mice, then selected genes showing differential gene expression patterns. As compared to GFP-negative cells, GFP-positive cells expressed Gpr4 and Ednrb at higher levels, whereas Pdgfra and Pdgfrb were expressed at lower levels. Furthermore, DNA microarray analysis showed that GFP-positive cells were positive for aquaporin 5, a marker for acinar cells. Together, our results indicate that NCDCs in adult SMGs have characteristic gene expression profiles specially their cell surface molecules. Cell sorting using a combination of these specific cell surface proteins would be a useful strategy for isolation of NCDCs from SMGs with high purity.     

Radial glial cell‐specific ablation in the adult Zebrafish brain

The zebrafish brain can continue to produce new neurons in widespread neurogenic brain regions throughout life. In contrast, neurogenesis in the adult mammalian brain is restricted to the subventricular zone (SVZ) and dentate gyrus (DG). In neurogenic regions in the adult brain, radial glial cells (RGCs) are considered to function as neural stem cells (NSCs). We generated a Tg(gfap:Gal4FF) transgenic zebrafish line, which enabled us to express specific genes in RGCs. To study the function of RGCs in neurogenesis in the adult zebrafish brain, we also generated a Tg(gfap: Gal4FF; UAS:nfsB‐mcherry) transgenic zebrafish line, which allowed us to induce cell death exclusively within RGCs upon addition of metronidazole (Mtz) to the media. RGCs expressing nitroreductase were specifically ablated by the Mtz treatment, decreasing the number of proliferative RGCs. Using the Tg(gfap:Gal4FF; UAS:nfsB‐mcherry) transgenic zebrafish line, we found that RGCs were specifically ablated in the adult zebrafish telencephalon. The Tg(gfap:Gal4FF) line could be useful to study the function of RGCs. genesis 53:431–439, 2015. © 2015 Wiley Periodicals, Inc.     

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     

Recovery of transgenic plants by pollen-mediated transformation in <Emphasis Type="Italic">Brassica juncea</Emphasis>

The aroA-M1 encoding the mutant of 5-enolpyruvyl-shikimate-3-phosphate synthase (EPSPS) was introduced into the Brassica juncea genome by sonication-assisted, pollen-mediated transformation. The plasmid DNA and collected pollen grains were mixed in 0.3 mol/L sucrose solution and treated with mild ultrasonication. The treated pollen was then pollinated onto the oilseed stigmas after the stamens were removed artificially. Putative transgenic plants were obtained by screening germinating seeds on a medium containing glyphosate. Southern blot analysis of glyphosate-resistant plants indicated that the aroA-M1 gene had been integrated into the oilseed genome. Western blot analysis further confirmed that the EPSPS coded by aroA-M1 gene was expressed in transgenic plants. The transgenic plants exhibited increased resistance to glyphosate compared to untransformed plants. Some of those transgenic plants had considerably high resistance to glyphosate. The genetic analysis of T₁ progeny further confirmed that the inheritance of the introduced genes followed the Mendelian rules. The results indicated that foreign genes can be transferred by pollen-mediated transformation combined with mild ultrasonication.     

The Anatomy, Histochemistry and Ultrastructure of Stigmas and Styles in Commelinaceae

The anatomy and ultrastructure of stigmas in 37 species of 13genera of Commelinaceae are described. The stigmas are papillate,papillae forming a dense fringe of cells around the mouth ofthe stylar canal in most species. The papillar cell wall iscovered by an unstructured cuticle of variable thickness andis of variable thickness because of small wall ingrowths. Thecuticle and the external surface of the papillar cell wall arevariably disrupted, particularly in the mid and basal regionsof the cell. This was not found in species of the genus Aploleiaor Callisia. The cell cytoplasm possesses all major organellesexcept chloroplasts and each cell is vacuolate. In all species except Aploleia mulitiflora the style comprisesan epidermis, a cortex and a hollow, tripartite canal whichis continuous into the ovary cavity. The three vascular strandsare positioned at the apex of each canal lobe. The canal cellsare elongate and tabular and the wall abutting the canal hasingrowths. The style in Aploleia is solid and the transmittingtissue comprises cells whose walls are electron opaque. Thecytoplasms of both types of cell are similar in content althoughthere is a single, large vacuole in canal cells and many smallvacuoles in transmitting tissue. The morphology, position and histochemistry of stigmatic andstylar exudate was similar in all ‘wet’ stigmas.Most of the exudate originates from the stylar canal althoughsignificant contributions are made by the papillae in stigmasof Coleotrype, Dichorisandra and Thyrsanthemum. There is no apparent relationship between stigma structure andthe presence of self-incompatibility. Stigma papillae, stylar canal, transmitting tissue, Commelinaceae     

A light and electron microscopic study of stigmas inAneilema andCommelina species (Commelinaceae)

Summary The stigmas of species inAneilema andCommelina are trifid and comprise elongate papillae. Progressive degeneration of papular cells is observed in stigmas from open flowers and at anthesis papillae may be moribund and collapsed. Fluid emanating from the hollow style flows onto the surface through ruptures in the cuticle at the interpapillar junctions into the interstices at maturity. This secretion stains positively for protein. Stigmas are of the wet type.The cuticle overlying the papillar cells is ridged and at the final stages prior to flowering this cuticle becomes detached from the underlying cellulosic wall. The sub-cuticular space so formed is filled with secretion. InAneilema species detachment of cuticle is at the papillar tip and along the lateral walls. InCommelina species the anticlinal walls of adjacent papillae are strongly attached for much of their length and thus detachment of cuticle is restricted to the papillar tip. The cell wall at the tip in both genera may proliferate forming a rudimentary transfer-cell type wall. The secretion is considered to be produced by the papillar cells. It is PAS positive but fails to stain for protein and in both the light and electron microscopes appears heterogenous.Pollen attachment, hydration, germination and early tube growth are very rapid following self-pollination, the pollen tubes entering the neck of the style within ten minutes of attachment.A unique character combination involving pollen and stigmas in these genera indicates a monophyletic origin.     

Oleosin-like proteins are not present on the surface of tobacco pollen

Pollen-stigma interactions on wet- and dry-type stigmas involve similar processes: the hydration of the pollen, followed by pollen tube growth and penetration of the stigma. Furthermore, in some species, identical molecules, namely lipids, are used to achieve this. In addition to lipids, oleosin-like proteins of the pollen coat of dry-type stigma plants have been shown to be involved in pollen-stigma interactions. However, little information is present about the proteins on the surface of pollen of wet-type stigma plants, in particular that of the Solanaceae. To analyze proteins from the surface of pollen of Nicotiana tabacum (tobacco), a solanaceous plant, we used an antiserum raised against Brassica pollen coat, a dry-type stigma plant of the Brassicaceae. In addition we used a molecular approach to identify tobacco homologues of oleosin-like genes. Our results show that no proteins similar to Brassica oleracea pollen coat proteins are present on the surface of tobacco pollen, and that oleosin-like genes are not expressed in tobacco anthers or stigmas.     

Identification of the Genes Specifically Expressed in Orally Tolerized T Cells

Oral tolerance is the systemic immunological unresponsiveness that occurs after feeding protein antigens. Its physiological role is thought to be the prevention of hypersensitivity to food antigens, and its therapeutic use to treat inflammatory diseases has been suggested. Although it has been shown that CD4⁺ T cells mediate oral tolerance, the precise molecular mechanisms remain unclear. In the present study, we employed suppression subtractive hybridization and identified 10 genes specifically expressed in orally tolerized T cells. These included genes that were interesting in terms of their putative functions in the negative regulation of T cell activation, e.g. Culin 1, LAX, and Zfhx1b, as well as four genes that encoded unknown proteins. We further investigated the expression of these genes in hyporesponsive T cells induced in vitro (in vitro anergized T cells). We found that six of the 10 genes were highly expressed in these cells, and kinetic studies suggested that one was associated with the induction of anergy, while the other five were associated with the maintenance of anergy. The remaining 4 genes that were not expressed in in vitro anergized T cells are also of interest as they may play a specific role in in vivo T cell tolerance. Functional analysis of these genes should help to understand the complex mechanisms underlying the induction and maintenance of oral tolerance, and moreover, in vivo immune tolerance in general.