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 ¹⁴C-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 [^35Smethionine was applied to intact stigma surfaces indicate that the papillae are the main sites of synthesis of the S-specific glycoproteins.Abbreviations IEF isoelectric focusing - SC self-compatibility - SDS sodium dodecyl sulfate - SI self-incompatibility     

Self-incompatibility of Zinnia angustifolia HBK (compositae)

Summary Visible light and UV epifluorescence microscopy were used to assess self-incompatibility (SI) in Zinnia angustifolia clones. Pistils were fixed 24 h after pollination and stained either with aniline blue in lactophenol (visible light microscopy) or decolorized aniline blue (fluorescence microscopy). Percentage of florets with embryos 21 days following pollination (% embryo set) was used as a control. Embryo set following self- or incompatible cross-pollinations ranged from 0% to 9.9%, whereas compatible crosses yielded 55.5%–87.1% embryo set. Observations using visible light microscopy indicated pollen load and number of germinated grains were significantly higher for compatible compared to incompatible crosses, and both variables were positively correlated (r = 0.89–0.96) to % embryo set. Examinations with UV epifluorescence microscopy revealed pollen load was higher and little or no callose accumulated in stigmatic papillae following compatible crosses, whereas for incompatible crosses, pollen load was low and callose lenticules were deposited in stigmatic papillae; the correlation between pollen load and % embryo set was 0.89. The intensity of callose fluorescence of the pollen tube-papillae attachment sites was quantitatively measured via micro spectrofluorometry. Callose fluorescence intensity ranged from 47.9% to 62.6% for incompatible and from 6.4% to 9.9% for compatible crosses, and was negatively correlated (r= — 0.95) with % embryo set. Microscopal techniques permit rapid assessment of SI and may be used routinely when each observed or measured parameter is highly correlated to the incompatibility response.Publication no. 2898 of the Massachusetts Agricultural Experiment Station.     

Cytoskeleton,cell surface and the development of invasive plasmodial tapetum inTradescantia virginiana L.

Summary The anther tapetum inTradescantia virginiana L. is of the invasive plasmodial type: the cells lose their walls during early spore meiosis and develop long invasion processes which invade the loculus to penetrate spaces between the sporogenous cells. Fusion to form a syncytium is delayed and conventional ultra-thin sections and the Thiéry reaction reveal the presence of a loose fibrillar extracellular cell coat on the free surfaces of tapetal cells and their invasion processes. Cell fusion involves formation of apposition areas characterized by an absence of cell coat and the local appearance of microtubular arrays. Conspicuous membrane sacs, associated closely with microtubules, were found to migrate to and accumulate at the plasma membranes near the fusion sites and sporogenous cells. Microtubules are always present in the cortical regions of the tapetal cells and their invasion processes. It is surmised that microtubules are not responsible either for initiating or guiding tapetal invasion of the loculus; instead they may help to sustain the form of the invasion processes, help in the migration of membrane sacs, and participate in cell fusion. The cell coat disappears with syncytium formation towards the end of meiosis, and the developing spore cells become surrounded by a perispore membrane, which, derived from the original plasma membranes and augmented by membrane sacs, forms labyrinthine membrane reservoirs that are described further in the accompanying paper.     

Self-incompatibility in Petunia inflata: isolation and characterization of cDNAs encoding three S-allele-associated proteins

Summary We have identified three alleles of the S-locus controlling self-incompatibility and their associated pistil proteins in Petunia inflata, a species that displays monofactorial gametophytic self-incompatibility. These S-allele-associated proteins (S-proteins) are pistil specific, and their levels are developmentally regulated. The amino-terminal sequences determined for the three S-proteins are highly conserved and show considerable homology to those of S-proteins from Petunia hybrida, Nicotiana alata and Lycopersicon peruvianum, three other species of the Solanaceae that also exhibit gametophytic self-incompatibility. cDNA clones encoding the three S-proteins were isolated and sequenced. Comparison of their deduced amino acid sequences reveals an average homology of 75.6%, with conserved and variable residue interspersed throughout the protein. Of the 137 conserved residues, 53 are also conserved in the N. alata S-proteins studies so far; of the 64 variable residues, 29 were identified as hypervariable based on calculation of the Similarity Index. There is only one hypervariable region of significant length, and it consists of eight consecutive hypervariable residues. This region correspond approximately to the hypervariable region HV2 identified in N. alata S-proteins. Of the two classes of N. alata S-proteins previously identified, one class exhibits greater homology to the three P. inflata S-proteins reported here than to the other class of N. alata S-proteins.     

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.     

Self-incompatibility alleles control a low molecular weight,basic protein in pistils of Petunia hybrida

Summary Proteins extracted from the pistils of several clones of Petunia hybrida carrying differing pairs of S alleles were examined by gel electrophoresis. The major protein of pistils, a basic glycoprotein of relatively low molecular weight, showed properties which varied in a simple manner with the S genotype. For each S allele we were able to assign a specific molecular weight (ranging from 27,000 to 33,000) and isoelectric point (in the range 8.3 to 8.7) for this putative S protein. Pistils homozygous at the S locus showed only one major protein on two-dimensional gel electrophoresis, while pistils from plants heterozygous at the S locus showed two. No evidence was obtained for the presence of this putative S protein in pollen extracts.     

Synthesis and phosphorylation of pollen proteins during the pollen-stigma interaction in self-compatible Brassica napus L. and self-incompatible Brassica oleracea L.

A technique is described which permits the in vivo study of protein synthesis and phosphorylation in the pollen of Brassica spp. during the early stages of the pollen-stigma interaction. In Brassica napus and B. oleracea, compatible pollination is followed by a dramatic activation of protein synthesis in the pollen involving the synthesis of approximately 40 proteins. After incompatible pollinations in B. oleracea, virtually no newly synthesised polypeptides were detected in the pollen except for a small group of high molecular weight proteins which were not normally synthesised during compatible pollinations. Both compatible and incompatible pollinations were followed by the appearance of newly phosphorylated proteins in the pollen; these fell into four distinct groups. In B. oleracea, the number of phosphorylated proteins and the degree of phosphorylation of individual proteins within the four groups differed between compatible and incompatible pollinations. One group of phosphorylated proteins appeared to correspond with the small group of high molecular weight polypeptides which were synthesised in pollen after incompatible pollinations. These findings are discussed in the perspective of cell signalling during the pollen-stigma interaction in Brassica and also in terms of their possible implication in sporophytic self-incompatibility.     

Biosynthesis of S-associated proteins following self- and cross-pollinations in Brassica campestris L. var. ‘T. 15’

Summary Homozygote plants for the (S) self-incompatibility gene have been produced in Brassica campestris L. var. T 15. Stigmas from plants designated S ₁ S ₁, S ₂ S ₂ and S ₄ S ₄ were extracted and their protein separated on an isoelectric focusing mini-gel. Differences were observed between proteins from stigmas of the three S-homozygous groups: S-genotype specific proteins were determined for S ₁ S ₁ and S ₂ S ₂ stigmas that were absent in the self-compatible S ₄ S ₄ stigmas. Carbon dioxide (CO₂), which is known to block the self-incompatibility reaction in Brassica, was applied to [³⁵S]-methionine unpollinated, self- and cross-pollinated stigmas to observe the effect of external CO₂ on the synthesis of these S-associated proteins. The results indicate that pollination triggers a dramatic reduction in protein synthesis in general and in the synthesis of S₂-associated protein after self-pollination in particular.     

An ultrastructural,cytochemical and immunofluorescence study of postmeiotic development of plasmodial tapetum inTradescantia virginiana L. and its relevance to the pathway of sporopollenin secretion

Summary Establishment of a tapetal plasmodium in postmeiotic stages in anther locules ofTradescantia virginiana encloses the tetrads in membrane-limited compartments. The perispore membrane (PSM), around each tetrad, is derived from composite tapetal cell plasma membranes. The tapetum acquires an abundance of ER and ribosomes and by the late tetrad stage the PSM and its underlying cytoplasm exhibit specialized features, studied here by ZnIO impregnation, osmium maceration, application of indirect immunofluorescence employing antitubulin, conventional thin sectioning and the Thiéry reaction. These features include: labyrinthine convolutions of the PSM resulting from migration of membranous sacs and their partial fusion to the PSM, an intimate relationship of tubular ER with the convoluted PSM, and microtubules underlying the PSM and among the membranous sacs. At the same time membrane-bound granules, comparable to but smaller and simpler than tapetal orbicules of secretory tapeta, form in the convolutions. It is postulated that the ER supplies precursors of sporopollenincontaining parts of the spore wall, that the PSM-associated microtubules stabilise the whole secretory apparatus at the tapetum-spore interface, and that the precursors are expelled into the lumen bounded by the PSM and then accreted upon the orbicule-like granules or the developing spore wall. With dissolution of the callosic wall, the plasmodium invades the intermicrosporal spaces of late tetrads, the PSM unfolding its elaborations and becoming closely appressed to the exinous surfaces of individual spores. Microtubules, although present during this phase of invasion, do not seem to propel the invasion processes and may have roles in shape maintenance. During pollen mitosis and enlargement the tapetal cytoplasm accumulates lipidic globules. A late phase of Golgi activity precedes accumulation of vesicles or vacuoles near the spores, these being bounded by single or multiple tripartite membranes. With anther desiccation, portions of plasmodium are deposited on the pollen surface in the form of tryphine, the deposits containing stacked membrane-like bilayers.     

Lithium induces cell plate dispersion during cytokinesis inTradescantia

Summary We have found that 10mM LiCl added toTradescantia stamen hair cells prior to early anaphase appears to prevent the vesicle coalescence phase of cell plate formation. In a fashion similar to caffeine inhibition of cell plate formation [Bonsignore and Hepler 1985, Protoplasma 129, 28–35], cell plate vesicle aggregation occurs at its normal time in LiCl, forming an incipient plate which is similar in morphology to that of an untreated cell during the first 10min, but the structure subsequently disperses and the resultant cells are binucleate. The addition of 10–20M myo-inositol was sufficient to reverse the inhibitory effect of Li⁺ in the majority of our experiments while scyllitol, an isomer of myo-inositol, or buffer without myoinositol were usually insufficient to reverse the inhibition. The timing of the addition of myo-inositol was critical for reversal; if the rescue solution was added more than 4 min after the onset of cell plate vesicle aggregation, the cell was usually irreversibly destined to become binucleate. The addition of 100M CaCl₂ within 2min of cell plate vesicle aggregation also overcame Li⁺-induced plate dispersal, but the kinetics of reversal were substantially slower than with myo-inositol. The results show that cell plate formation and in particular, cell plate vesicle coalescence, is sensitive to exogenously applied LiCl.Abbreviations AO Anaphase Onset - CP Cell Plate (completed) - CPD Cell Plate Dispersion - CPVA Cell Plate Vesicle Aggregation - DAG 1,2-diacylglycerol - InsP₃ 1,4,5-inositol trisphosphate - IP inositol-1-phosphate - polyPI polyphosphoinositide     

Stigma proteins of the two loci self-incompatible grass Phalaris coerulescens

Summary Protein extracts from four self-incompatible genotypes of Phalaris coerulescens were subjected to analysis by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and ultra-thin isoelectric focusing. A comparison between stigma, root and leaf extracts showed that there was no significant difference in electrophoretic or isoelectric focusing patterns between the genotypes for both root and leaf proteins. However, stigma protein patterns did vary between genotypes especially within the molecular weight region of 43 000–97 000 and within the pI range 5–7. The stigma-specific changes strongly suggest a link between the self-incompatible genotype and these stigma proteins. However, because there are two loci involved, it is not yet possible to precisely assign particular proteins to each S- or Z-allele.     

Pollen stigma interactions in Brassica oleracea

Summary Recent studies on the mechanism of self-incompatibility in Brassica indicate the location, nature and mode of action of the molecules involved. Characteristics of the pollen surface and the stigma surface are described in detail, together with new information pertaining to the recognition molecules located therein. A sequence of events is outlined leading from pollination, through adhesion, hydration, germination, and tube growth to acceptance and ultimate compatibility. The characteristics of rejection of incompatible grains are described for each stage of the pollen-stigma interaction. It is proposed that recognition of proteins from the coating of self-pollen by the molecules in the pellicle results in the formation of a biologically-active complex which inhibits water supply to the incompatible grain, and that all other manifestations of incompatibility are a consequence of this initial response.     

Pollen-pistil interaction in Brassica oleracea

Quantitative studies of the adhesion of pollen grains to the stigma in Brassica oleracea revealed that self-pollen is initially less firmly bound than cross-pollen. The pollen grain tryphine, believed to be important in the adhesion process, has been shown to differ in mobility following self- and cross-pollination when observed using fluorescent probes. The hydration of the pollen grains has been investigated in vitro by measuring the changes in shape, volume and fresh weight of the imbibing grains. Whilst little change in volume could be detected there was a considerable increase in fresh weight together with a change of shape. The significance of these events, which occur prior to pollen germination, is discussed in relation to their effect upon subsequent germination and expression of self-incompatibility.Abbreviations RH relative humidity - SI self-incompatibility - ConA concanavalin A - I-ANS I-anilino-napthyl-sulfonic acid     

Kinetochore behavior during generative cell division inTradescantia virginiana

Summary Previous observations indicate that division of the generative cell inTradescantia virginiana is characterized by several unusual features, including persistence of surrounding microtubule (Mt) bundles during karyokinesis, lack of a distinct metaphase plate and direct contribution by mitotic Mts to the cytoskeleton of young sperm. We have further probed karyokinesis in these cells using additional antitubulin and chromosome staining, as well as kinetochore visualizations with CREST serum. The CREST antibodies reveal kinetochores as paired and single fluorescent dots similar to those seen in other species stained with this preparation. Double localizations show that the dots are located at the ends of Mt bundles previously identified as kinetochore fibers (Palevitz and Cresti 1989). Before anaphase, paired kinetochores are distributed along the length of the cell. They also tend to be located at the cell periphery or are directly connected to peripheral Mt bundles by their kinetochore (K)-fibers. Twelve pairs of dots can be counted per cell, equal to the expected number of chromosomes. During anaphase, kinetochore separation starts at various positions along the length of the cell, producing single, relatively uniformly distributed kinetochores in the crotches of forks formed by K-fiber trunks and elongating Mt branches attached to the base of the trunks. Eventually, K-fibers with attached kinetochores aggregate in stepwise fashion on thick Mt bundles at both ends of the cell. This pattern is reflected in the cytoskeleton of young sperm. These results further document the unusual distribution of chromosomes and kinetochores inTradescantia generative cells and the origin of the Mt cytoskeleton in sperm cells.Abbreviations CREST Calcinosis, Raynaud's phenomenon, Esophageal dysmotility, Sclerodactyly, Telangiectasia - K-fiber kinetochore fiber - Mt microtubule Dedicated to the memory of Professor Oswald Kiermayer     

Self-incompatibility inCrocus vernus subsp.vernus (Iridaceae)

Outcross, self- and mixed pollinations were performed inCrocus vernus subsp.vernus, a species with bicellular pollen, dry stigmas and hollow style. No differences were noted among the above pollinations concerning the germination of pollen and the growth of pollen tubes until the top of ovary. Within 45 min after pollinations 62% of pollen grains germinated. Pollen tubes penetrated the papilla cuticle extending along the papilla wall; on entry into stigmatic lobes they continued growth in the stylar secretion to ovarian locules. Here, however, self-pollen tubes failed to reach or to enter the ovule micropyle; while pollen tubes from either outcross- or mixed pollinations grew until fertilizing ovules. These observations gave evidence of a self-incompatibility system inCrocus, which appeared to be neutralized by mentor effect. The ovary as site of incompatibility response is discussed.     

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.     

In vitro propagation of two species of commelinaceae from bud cultures and repeated subcultures on a growth regulator-free medium

Summary In our wide-ranging research on in vitro propagation of some monocotyledonous plants, two Commelinaceae species were studied: Tradescantia fluminensis var. foliis variegatis and Tradescantia pallida. Initial cultures were established successfully using nodal and apical meristems that produced single shoots, many roots, and no callus, by utilizing growth regulator-free MS medium. Addition of growth regulators did not increase the activity of explants that produced single or multiple shoots, atypical roots, and no callus. Consecutive cultures were possible using the apical and nodal meristems of the previous generation. The behavior of the different generations in culture did not change and was similar to the initial cultures. Their growth capacity was maintained over many months, also on a growth regulator-free medium. In both species, the chromosome number in the root tips of the mother plant and all morphologically stable in vitro plantlets confirmed a constant ploidy level, in T. fluminensis 2n=72, and in T. pallida 2n=24.     

Cytoskeletal changes during generative cell division and sperm formation inTradescantia virginiana

Summary Cytoskeletal organization and chromosome behavior were studied inTradescantia generative cells prior to and during sperm formation using in vitro grown pollen tubes and fluorescence staining methods. Before pollen germination, the crescent-shaped generative cell contains a reticulate microtubule (Mt) system. The cell elongates dramatically after germination, and its Mts assume a helical to longitudinal arrangement. Chromosome condensation is evident approximately 3hr after germination. Kinetochores appear as dark interruptions in the Mt array, and thus seem to attach directly to interphase fibers. No metaphase plate typical of other cells is observed with either DAPI or anti-tubulin staining. Instead, the chromosomes adopt a twisted or braided arrangement, with kinetochores distributed along the length of the cell and kinetochore fibers linked to each other and to surrounding fibers. Anaphase is characterized by a staggered, overlapping separation of chromosomes and by elongation of Mt branches connecting opposing kinetochore fibers. Cytokinesis appears to utilize a furrowing process; a phragmoplast or cell plate was never seen. As a result of these events, the sperm directly inherit their cytoskeleton from generative cell Mts involved in division. No actin fibers are observed at any stage using rhodamine-phalloidin staining. The results are discussed in terms of other reports on sperm formation, possible mitotic and cytokinetic mechanisms, and past distinctions between Mt arrays in higher plant somatic cells.Abbreviations CD cytochalasin D - DAPI 46-diamidino-2-phenyl-indole - DMSO dimethylsulfoxide - K-fiber kinetochore fiber - Mf microfilament - Mt microtubule - PPB preprophase Mt band - RP rhodamine phalloidin     

Ultrastructural and physiological differences between buds and mature flowers of Petunia hybrida prior to and following pollination

The structural events accompanying the maturation of the pistil of Petunia hybrida have been studied in detail, together with the changes in the protein spectrum of the transmitting tissue that occur over this period. These events have been considered in terms of the acquisition of the self-incompatibility response, which occurs while the pistil is enclosed in the bud. Apart from several minor differences, the young pistils differ only from the mature in that their transmitting tissue cells fail to respond to pollination by undergoing characteristic ultrastructural changes. Data from electrofocusing indicates that several proteins, mobilised in the mature transmitting tissue some three hours after pollination, are absent from bud pistils. It is proposed that the pollination-stimulated release of certain polypeptides, believed to be involved in the self-incompatibility response, does not take place in young pistils. These observations are considered with reference to currently-accepted models of the operation of the self-incompatibility mechanism in Petunia.     

Sequence variability and gene structure at the self-incompatibility locus of Solanum tuberosum

Summary Allelic complexity is a key feature of self-incompatibility (S) loci in gametophytic plants. We describe in this report the allelic diversity and gene structure of the S locus in Solanum tuberosum revealed by the isolation and characterization of genomic and cDNA clones encoding S-associated major pistil proteins from three alleles (S ₁, S _r1, S ₂). Genomic clones encoding the S₁ and S₂ proteins provide evidence for a simple gene structure: Two exons are separated by a small intron of 113 (S ₁) and 117 by (S ₂). Protein sequences deduced from cDNA clones encoding S₁ and S_r1 proteins show 95% homology. 15 of the 25 residues that differ between these S ₁and S _r1alleles are clustered in a short hypervariable protein segment (amino acid positions 44–68), which corresponds in the genomic clones to DNA sequences flanking the single intron. In contrast, these alleles are only 66% homologous to the S ₂allele, with the residues that differ between the alleles being scattered throughout the sequence. DNA crosshybridization experiments identify a minimum of three classes of potato S alleles: one class contains the alleles S ₁, S _r1and S ₃, the second class S ₂and an allele of the cultivar Roxy, and the third class contains at present only S ₄. It is proposed that these classes reflect the origin of the S alleles from a few ancestral S sequence types.