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.     

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.     

The Use of Hydrophobic Adhesive Tape to Produce Miniature Wells on Microscope Slides

Hydrophobic adhesive tape was used to produce miniature wells on microscope slides for staining several sections of tissue with minimal amounts of cytochemical reagents. The wells could be tailored to individual specifications and the method allowed coverslips to be mounted close to the sections using either aqueous or xylene based mounting media. This method was especially useful for multiple immunolabelling of serial semithin cryosections.     

Immunodetection of protein glycoforms encoded by two independent genes of the self-incompatibility multigene family of brassica

Glycoprotein products of two highly homologous Brassica S gene family members were studied: SLSG (S locus-specific glycoprotein), product of an SLG gene at the S locus, and SLR1 (S locus-related) protein, product of the SLR1 gene, a gene unlinked to the S locus. A polyclonal antibody directed against a trpE-SLR1 fusion protein facilitated study of the SLR1 protein. SLR1 protein was detected in a number of crucifer species. No variation in the level of this protein was found between self-compatible and self-incompatible plants. Both SLSG and SLR1 protein occurred as glycoforms on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels. Each glycoform had several charge forms, indicated by elution patterns from a high performance liquid chromatography cation exchange column and behavior on two-dimensional gels. Deglycosylation of both SLSG and SLR1 protein caused loss of the glycoforms, which apparently arose from differences in glycosylation. Consistent with their apparent similar post-translational processing, immunolocalization showed that SLR1 protein, like SLSG, accumulated in the stigma papillae cell walls. Thus, both SLSG and SLR1 protein are present at the site of pollen-stigma interaction.     

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     

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.     

Strategies to improve the antigenicity,ultrastructure preservation and visibility of trafficking compartments in Arabidopsis tissue

Immunolabelling of (ultra)thin thawed cryosections according to Tokuyasu is one of the most reliable and efficient immunolocalisation techniques for cells and tissues. However, chemical fixation at ambient temperature, a prerequisite of this technique, can cause problems for samples, like plant tissue, because cell walls, hydrophobic surfaces and intercellular air slow down diffusion of fixative molecules into the sample. We show that a hybrid technique, based on a combination of cryofixation/freeze-substitution and Tokuyasu cryosection immunolabelling, circumvents the disadvantages associated with chemical fixation and results in an improved ultrastructure and antigenicity preservation of Tokuyasu cryosections used for light and electron microscopic immunolabelling (as shown for Myc- or mRFP-tagged proteins, KNOLLE and carbohydrate epitopes). In combination with the most sensitive particulate marker systems, like 1-nm gold or quantum dot markers, we were able to obtain a differentiated labelling pattern which allows a more detailed evaluation of plant Golgi, trans-Golgi network and multivesicular body/prevacuolar compartment markers (COPI-specific γCOP, the ADP-ribosylation factor GTPase ARF1, ARA7/RabF2b and the vacuolar sorting receptor VSR). We also discuss possibilities to improve membrane contrast, e.g., of transport vesicles like COPI, COPII and clathrin-coated vesicles, and of compartments of endosomal trafficking like the trans-Golgi network.     

A cDNA encoding an S-locus specific glycoprotein from Brassica oleracea plants containing the S5 self-incompatibility allele

Summary A cDNA sequence homologous to the Brassica self-incompatibility locus specific glycoprotein (SLSG) sequence was isolated from stigmas of B. oleracea plants homozygous for the S₅ allele. The nucleotide sequence of this cDNA was obtained and compared with the S₆ allelic form of the SLSG. Evidence is presented which indicates that this sequence does not specify the self-incompatibility response of pollen.Abbreviations SDS sodium dodecyl sulphate - PVP polyvinylpyrrolidone - BSA bovine serum albumin - SLSG self-incompatibility locus specific glycoprotein     

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

Summary 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.     

Distribution of calcium in the stigma and style of tobacco during pollen germination and tube elongation

Potassium antimonate was used to locate loosely bound calcium in the stigma and style of tobacco. The tobacco stigma is wet and covered by a thick layer of glycoprotein exudate at anthesis. The exudate contains abundant vesicles, which are densely labeled with calcium precipitates. When pollen grains arrive at the stigma, become hydrated, and as the pollen swells, Ca²⁺ precipitates accumulate at the aperture. Calcium precipitates that accumulate in pollen cytoplasm are initially concentrated within small vacuoles, but as germination proceeds these appear to fuse, forming prominent, densely labeled vesicles that preferentially accumulate near the proximal region of the growing tube. Although the stigma has abundant particles, few calcium precipitates are observed in the transmitting tissue from anthesis to 11 h after pollination. However, at 22 h after pollination, accumulation of calcium increases distally from the stigmatic interface with the transmitting tissue through the length of the style to the ovary. An examination of flowering plants with differing floral biology will be needed to understand the role of loosely bound calcium accumulation and its relationship to tissue-level changes in calcium uptake, maintenance of other calcium pools, including [Ca²⁺]_cyt, and in pollen and style maturation during the progamic phase.     

S-RNases in apple are expressed in the pistil along the pollen tube growth path

 The molecular bases of self-incompatibility have been intensively studied in a restricted number of model species, but for most families the expression and distribution of S-proteins is unknown. In this work, pistil cryosections from apple were used for in situ detection of S-proteins. Two specific antibodies, one against the S3-protein and another against all apple S-proteins were used. S-proteins were shown to be localised in the intercellular space of the transmitting tissue, both in the stigma and style, which agrees with the proposed mechanism of action for S-RNases in gametophytic self-incompatibility. Some intracellular labelling was also observed in all ovary sections, confined to one layer of the nucellus surrounding the embryo sac, but this labelling was found to be non-S-allele-specific. Nevertheless, the signal in the ovary was tissue-specific, which may indicate that some component not encoded by the S-locus but similar to S-proteins was detected. To the best of our knowledge this is the first report on the precise distribution of S-RNases in a rosaceous species. Received: 15 July 1998 / Revision accepted: 29 December 1998     

β-1,3-glucanase activity in the stigma of healthy petunia flowers

β-1,3-Glucanase activity was detected in extracts of different tissues of healthy mature petunia flowers except the filament. The stigma was studied further as it had the highest enzyme activity and there is a paucity of information on the occurrence of this enzyme in this tissue. Specific activity of the enzyme was found to increase within the stigmatic tissue from early development until just before anthesis. Following non-denaturing polyacrylamide gel electrophoresis at pH 8.8, extracts of dehiscent stigma seem to contain three acidic isoforms of β-1,3-glucanase. Crude extracts of stigma was passed through a pachyman affinity column. A fraction of affinity-purified active β-1,3-glucanase enzyme was found to have no antifungal activity against Trichoderma viride, Phloma clematidina and Cladosporium fulvum.     

黄瓜雌花发育过程中柱头的腺特征(英文)

利用透射电镜技术研究了黄瓜（Ｃｕｃｕｍｉｓ　ｓａｔｉｖｕｓ　Ｌ．）雌花柱头发育过程中传递组织、分泌组织和乳突细胞的超微结构。在整个发育过程中,乳突细胞和分泌组织细胞的细胞质内密布很多管状及槽库膨大的内质网,产生很多分泌囊泡;在成熟柱头的传递组织和分泌组织细胞间观察到大量的胞间连丝;乳突细胞和分泌细胞高度液泡化,质膜内折;在柱头发育过程中分泌组织细胞的核周腔扩大形成裂瓣状核,到柱头成熟阶段裂瓣状核更加明显。进一步的研究显示,在成熟柱头的不同组织细胞中,　ＡＴＰａｓｅ的活性呈现在质膜和液泡膜上,随着柱头的发育,ＰＭ－Ｈ＋－ＡＴＰａｓｅ的比活性明显增强。结果表明,黄瓜雌花柱头的腺特征随发育进程而趋于显著。     

Attachment of the Flavescence doree pathogen (MLO) to leafhopper vectors and other insects

Flavescence doree (FD) is an important yellows disease of grapevine, caused by mycoplasma-like-organism (MLO) and is transmitted in the field by the leafhopper Scaphoideus titanus Ball. It can be transmitted in the laboratory between Vicia faba test plants by the leafhopper, Euscelidius variegatus Kbm. A technique to identify a specific attachment system between the MLO and the leafhopper vectors was developed. In this method, called “Double Dot”, extracts of macerated healthy whole insects or organs applied to a support membrane or cryosections of healthy whole leafhoppers, are incubated with a MLO-enriched extract from FD-infected V. faba or FD-infected E. variegatus. Attached MLO cells were identified by immunolabelling using FD-MLO specific monoclonal antibodies. Attachment of MLO cells was obtained on extracts of healthy S. titanus and E. variegatus and on tissues such as salivary glands, hemolymph and alimentary tract. On cryosections, MLO attachment was obtained on acini IV and V of the salivary glands and on some acini III, on the ventriculus of the alimentary tract, and on the abdomen fat bodies. “Double dot” experiments were done using other insect species, and MLO cells attachment was obtained on most MLO-vector insects but also on insects from a few non-vector species.     

Heterostyly inPrimula. 1. Fine-structural and cytochemical features of the stigma and style inPrimula vulgaris huds

Summary The stigmas of the heterostylous genusPrimula are of the dry type without a free-flowing surface secretion. The papillae of the stigma surface cells of the two morphs, in pin (stigma exserted) and thrum (stamens exserted), bear a thin proteinaceous surface pellicle, overlying a discontinuous cuticle. The vacuoles of the papillate cells contain tannins, and tannin cells extend in files through the stigma heads and form a loose sheath surrounding the pollen-tube transmitting tract in the styles. The cells of the transmitting tissue in the stigma heads have a normal complement of organelles, and abundant ribosomal endoplasmic reticulum. The intercellular spaces contain an internal secretion which reacts cytochemically for both carbohydrate and protein. The transmitting tract in the styles forms a central core surrounded by several vascular strands. The cells are elongated, and the intercellular spaces here also have a carbohydrate-protein content. In a compatible pollination, thrum pollen tubes enter the stigma by penetrating the cuticle at the tip or on the flank of the pin papilla. Pin tubes on the thrum stigma enter between adjacent papillae, penetrating the thin cuticle at the base. The tubes grow through the transmitting tracts in the intercellular material.     

黄瓜雌花发育过程中柱头的腺特征

利用透射电镜技术研究了黄瓜(Cucumis sativus L.)雌花柱头发育过程中传递组织、分泌组织和乳突细胞的超微结构.在整个发育过程中,乳突细胞和分泌组织细胞的细胞质内密布很多管状及槽库膨大的内质网,产生很多分泌囊泡;在成熟柱头的传递组织和分泌组织细胞间观察到大量的胞间连丝;乳突细胞和分泌细胞高度液泡化,质膜内折;在柱头发育过程中分泌组织细胞的核周腔扩大形成裂瓣状核,到柱头成熟阶段裂瓣状核更加明显.进一步的研究显示,在成熟柱头的不同组织细胞中,ATPase的活性呈现在质膜和液泡膜上,随着柱头的发育,PM-H -ATPase的比活性明显增强.结果表明,黄瓜雌花柱头的腺特征随发育进程而趋于显著.     

Immunocytochemical localization of S100A1 in mitochondria on cryosections of the rat heart

Immunocytochemical localization studies of S100A1 in muscle cells have so far yielded variable and conflicting results mainly due to different sample preparation techniques for immunoelectron microscopy. To minimize denaturation by fixation and embedding, cryofixation and cryosectioning followed by immunolabelling were used in the present study. Rat hearts were gently prefixed in a mixture of paraformaldehyde and glutaraldehyde. Samples from left and right ventricles and left and right atria were cryoprotected by sucrose and shock-frozen in liquid nitrogen. Ultrathin cryosections were labelled with rabbit polyclonal antiserum against S100A1. The sections were then incubated with secondary antibody conjugated to FITC (for fluorescence microscopy) or with protein A conjugated to 5 nm gold particles (for electron microscopy). The most prominent sites immunolabelled for S100A1 were mitochondria. In the fluorescence microscope the labelling of mitochondria was intense, suppressing the labelling in other compartments. In accordance with previous studies labelling of sarcoplasmic reticulum, Z-lines, actin and myosin filaments could also be detected in the electron microscope.     

Changes of pH in <Emphasis Type="Italic">Petunia hybrida</Emphasis> (Hort.) styles induced by pollination and influence of proton pump and ion channels on its regulation

Ionselective microelectrode method was used to study changes of pH in transmitting tissue of style in Petunia hybrida (Hort.). Effect of pollination and pollen tube growth were examined. Subsequently solutions of ions and various stimulators or blockers of ion channels were applied on pollinated styles to examine the possible role of ion channels in pH stabilisation. It was confirmed in the present study that: (1) there is a pH gradient in the transmitting tissue of a petunia unpollinated style with the stigma region being more acidic; (2) pollination causes further acidification of transmitting tissue: (3) the gradient of pH first vanishes at 24 h after pollination then is reversed up to 72 h after pollination; (4) active transport of ions plays an important role in pH regulation in transmitting tissue. The presented results confirm the role of pH changes and Ca²⁺ as a mediator in controlling proton influx into the apoplast of the transmitting tissue during pollen tube growth.