Apomixis: seasonal and population differences in a grass.

Changes in incidence of apomictic and sexual embryo sacs were detected in Dichanthium aristatum in an experimental population at six stations covering 27 degrees of south latitude, and during the flowering season in a wild population. Differences were associated with photoperiods prevailing during development of inflorescences. Response to length of day was quantitative, differing in the two strains.

Mapping of the antigenic and allergenic epitopes of Lol p VB using gene fragmentation.

The recombinant proteins of Lol p VA and Lol p VB expressed in E. coli reacted with IgE antibodies from sera of allergic patients and mAbs FMC A7 and PpV1. Cross-absorption analyses using these recombinant proteins showed that Lol p VA and Lol p VB possess both similar and unique IgE binding determinants. Gene fragmentation was utilized to localize the antigenic and allergenic determinants of Lol p VB. When full-length cDNA of Lol p VB was digested into three fragments and expressed as the fusions from the glutathione transferase of pGEX vectors, fragments Met1-Val196 and Asp197-Val339 bound IgE while fragment Met1-Pro96 did not. The data suggest that there are at least two IgE binding determinants in Lol p VB. In addition, only fragment Met1-Val196 reacted with mAb PpV1. The localization of these determinants was further resolved using random fragment expression libraries. The mAb PpV1 determinant was near the N-terminal region of Lol p VB molecule. The IgE binding determinants were distributed in the central region: region I (amino acids 111-195) and II (199-254). These IgE binding determinants are conserved in Lol p VA.

Immunological relationships among group I and group V allergens from grass pollen.

Specific IgE antibodies have been affinity-purified from recombinant grass pollen allergens, and used to identify isoforms of the two major allergens of rye-grass pollen, Lol p I and Lol p V and cross-reactive allergens in other grasses. Lol p I-specific IgE (affinity-purified from the recombinant protein expressed by clone 13R which encodes amino acids 96-240 of Lol p I) identified four isoforms of the allergen. The same probe recognized cross-reactive epitopes in pollen proteins from 14 out of 16 grasses. The allergens identified by Lol p V-specific IgE (affinity-purified from the recombinant protein expressed by clones 12R or 19R which encode the full Lol p V protein) varied more in their physicochemical characteristics than the Group I isoforms. At least eight isoforms of Lol p V were identified by the Lol p V-specific IgE. The same probe recognized cross-reactive epitopes in pollen protein from 13 out of 16 grasses. Group I proteins were identified in grasses from two sub-families of the Poaceae, while the Group V allergens were only identified in pollen of grasses from one sub-family, the Pooideae.

Molecular basis of IgE-recognition of Lol p 5, a major allergen of rye-grass pollen.

Grass pollen, especially of rye-grass (Lolium perenne). represents an important cause of type I allergy. Identification of IgE-binding (allergenic) epitopes of major grass pollen allergens is essential for understanding the molecular basis of interaction between allergens and human IgE antibodies and therefore facilitates the devising of safer and more effective diagnostic and immunotherapy reagents. The aim of this study was to identify the allergenic epitopes of Lol p 5, a major allergen of rye-grass pollen, immunodissect these epitopes further so that the amino acid residues critical for antibody binding can be determined and investigate the conservation and nature of these epitopes within the context of the natural grass pollen allergens. Peptides, 12-13 amino acid residues long and overlapping each other by 4 amino acid residues, based on the entire deduced amino acid sequence of the coding region of Lol p 5, were synthesised and assayed for IgE-binding. Two strong IgE-binding epitopes (Lol p 5 (49-60) and (265-276), referred to as peptides 7 and 34, respectively) were identified. These epitopes were further resolved by truncated peptides and amino acid replacement studies and the amino acid residues critical for IgE-binding determined (Lol p 5 (49-60) residue Lys57 and (265-276) residue Lys275). Sequences of these epitopes were conserved in related allergens and may form the conserved allergenic domains responsible for the cross-reactivity observed between pollen allergens of taxonomically related grasses. Furthermore, due to its strong IgE-reactivity, synthetic peptide Lol p 5 (265-276) was used to affinity-purify specific IgE antibodies which recognised proteins of other clinically important grass pollens. further indicating presence of allergenic cross-reactivity at the level of allergenic epitope. Moreover, Lol p 5 (265 276) demonstrated a strong capacity to inhibit IgE-binding to natural rye-grass pollen proteins highlighting the antibody accessibility to these sequences within the context of the natural allergens. Strong IgE-binding epitopes of Lol p 5 have been identified down to single critical amino acid residues and are shown to occur as linear or continuous domains in the natural conformation of natural Lol p 5 and other group 5 grass pollen allergens. The fact that such an allergenic synthetic epitope has the capacity to strongly inhibit IgE-binding to natural allergens highlight its potential for use as a candidate in future therapeutics to treat pollen-associated allergies.

Cloning of a cDNA encoding a group-V (group-IX) allergen isoform from rye-grass pollen that demonstrates specific antigenic immunoreactivity.

We have isolated and characterized the cDNA clone, 19R, that encodes an isoform of a major rye-grass pollen allergen, Lol p V [previously referred to as Lol p 1b; Singh et al., Proc. Natl. Acad. Sci. USA 88 (1991) 1384-1388; and Lol p IX; Suphioglu et al., Lancet 339 (1992) 569-572]. Clone 19R was isolated from a rye-grass pollen cDNA expression library using grass pollen-specific immunoglobulin E (IgE) antibodies (Ab) from an allergic serum pool. The nucleotide (nt) sequence of clone 19R potentially encodes a 33.8-kDa protein of 339 amino acids (aa). It possesses a leader peptide essentially identical to the previously characterized isoform of Lol p V (Lol p VA). This indicates a mature processed 31.3-kDa protein of 314 aa, correlating well with the size of the polypeptides revealed by Western analysis of pollen proteins using IgE Ab affinity purified from recombinant fusion protein (reFP) encoded by clone 19R as solid matrix. There is no N-glycosylation motif. The protein encoded by clone 19R, designated Lol p VB, has 66.4% identity and 80.4% similarity with Lol p VA. However, a Lol p VA-specific monoclonal Ab, FMC A7, does not recognize reFP encoded by clone 19R, indicating that Lol p VB does not share this epitope. Cross-reactivity studies using affinity purified IgE Ab showed that both isoforms share similar allergenic epitopes. Immunoblot analysis using sera from a population of 30 patients showed that 80% possess IgE Ab that recognize both Lol p V isoforms. Variation occurred in the signal intensities of IgE binding.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular cloning and immunological characterisation of Cyn d 7, a novel calcium-binding allergen from Bermuda grass pollen.

A cDNA coding for a newly identified Bermuda grass pollen allergen, Cyn d 7, with significant sequence similarity to Ca2+-binding proteins, was isolated from a cDNA expression library using serum IgE from an allergic individual. The deduced amino acid sequence of Cyn d 7 contained two typical Ca2+-binding sites (EF hand domains). Depletion of Ca2+ with EGTA led to a loss of IgE-binding capacity of rCyn d 7. A synthetic peptide based on domain II showed high IgE reactivity. Cyn d 7 therefore represents a grass pollen allergen that belongs to a novel class of Ca2+-binding proteins.

Immunofluorescent screening of monoclonal antibodies to surface antigens of animal and plant cells bound to polycarbonate membranes.

We have developed an immunofluorescent screening method in which animal or plant cells are immobilized without fixation on polycarbonate (Nuclepore) membranes. The technique does not involve centrifugation, and virtually no cells are lost during processing. Direct microscopic analysis of antibody binding provides an extremely sensitive and reproducible screening system for hybridoma supernatants.

Male gametes and fertilization in angiosperms.

Double fertilization appears to have evolved as a product of change directly related to an accelerated rate and timing of reproduction. In this review, the focus is on the angiosperm male gametophyte, where changes include a reduction in the number of mitoses, establishment of the male germ unit and involvement of both members of the pair of sperm cells in reproduction. The organization of the generative cell during mitosis indicates that there may be basic similarities between this process in plant and animal cells. The microtubular organization of generative cells alters after isoiation. However, mitosis in Allamanda, proceeds as usual during in vitro culture. The presence of actin microfilaments within generative cells has previously been shown in Rhododendron and here we provide further evidence that actin microfilaments are indeed present in generative cells. Two different kinds of intermediate-filament-like systems (IFS) are present in the generative cells of Allamanda: one in the cytoplasm and the other closely associated with the surface domain of chromosomes, both identified by the use of monoclonal antibodies. This is the first report of an IFS existing in the vegetative nucleus of pollen. Two alternate views have been proposed for the involvement of sperm cells in double fertilization of angiosperms. First, the chance hypothesis assumes that sperm fusions with the egg and central cell are random interactions. Second, the specific receptor hypothesis proposes that one of the pair of sperm (the true male gamete) is destined to fuse specifically with the egg. Support for this latter view has come from demonstrations of sperm cell dimorphism, both in size and content of mitochondria and plastids. The production of monoclonal antibodies which bind to surface domains on the reproductive cells of higher and lower plants, and specifically to the cytoplasm of generative and sperm cells also suggest that directed fertilization occurs. Recently, the existence of translatable mRNA pools within the generative and sperm cells indicates that, with the use of recent technological advances such as the polymerase chain reaction, the potential exists to identify male gamete-specific genes. Contents Summary 679 I. Introduction 680 III. A cell biological perspective 681 IV. Two hypotheses for double fertilization 687 V. Isolation of living sperm from flowering plants 687 VI. Sperm surface antigens of plants 688 VII. Molecular characterization 690 VIII. Conclusions 691 Acknowledgements 691 References 692.

PHYLOGENETIC ANALYSIS OF THE MALE ONTOGENETIC PROGRAM IN AQUATIC AND TERRESTRIAL MONOCOTYLEDONS.

The male program of ontogeny in flowering plants encompasses the events from meiosis of microsporocytes to fertilization. Three main sequences are discussed; the deposition of cell walls, changes in cytoplasmic organelles, and the program of nuclear divisions leading to the formation of two sperm cells and a vegetative cell in each pollen grain. Variations in these ontogenetic sequences are particularly apparent in the monocotyledons, which exhibit diversity in pollen morphology, wall structure, and mode of pollination. The male program of development has been compared in selected terrestrial monocotyledons belonging to the Liliaceae and Gramineae and aquatic members of the Cymodoceaceae, Najadaceae, and Zannichelliaceae. A total of 26 characters from the male program are discussed and then used to construct a cladogram derived only from developmental data for the five species. The polarity of only a few of the character transformations has been determined directly by observation of developmental sequences; most have been interpreted by outgroup analysis.

Recombinant expression and epitope mapping of grass pollen allergens.

We have studied the expression of recombinant forms of Group 1 allergens from rye-grass and Bermuda grass pollens. Recombinant Lol p 1 expressed in bacteria bound serum IgE from allergic patients. Based on analysis of fragments of the Lol p 1 cDNA clone, the major IgE-reactive epitope has been mapped to the C-terminus. However, although SDS-denatured natural Cyn d 1 (from Bermuda grass) bound IgE, the full or partial recombinant proteins expressed in bacteria did not bind IgE. We have since expressed Cyn d 1 in the yeast Pichia pastoris and restored IgE binding. cDNA clones encoding two isoforms of Lol p 5, Lol p 5A and Lol p 5B, have been expressed in bacteria and resulting polypeptides show IgE-binding. Random fragments of these clones have been generated and when expressed as partial recombinant proteins in bacteria, allowed us to identify the major IgE-binding epitopes. The allergenic epitopes were localised towards the C-terminal half of the molecule. Although both isoforms shared similar IgE-reactive epitopes, Lol p 5B did not recognise the Lol p 5A-specific monoclonal antibody A7. At sequence level, there appear to be several amino acid differences between the antigenic epitopes of these two isoallergens. These results aid in the design of diagnostics and in grass pollen immunotherapy.

Callose and determination of pistil viability and incompatibility.

Callose provides a useful phenotypic bioassay in plant breeding to determine: incompatibility system; gametophytic competition; and stigma and ovule viability. Callose appearance in ovules may be associated with senescence, and used to determine the effective pollination period. In incompatible matings, callose formation is specific and related to rejection phenomena. The stigma callose response is induced by informational molecules carried by the germinal line, i.e. self or interspecific pollen, but not by the somatic line. Several methods of visualizing callose are reviewed. The role of callose in pollen-stigma interactions has many analogies with host-parasite interactions, and a model is proposed based on relationships between callose, boron and inhibitor (phytoalexin-like) synthesis. The callose response provides a useful tool for the biotechnology of seed production.

Pollen-wall proteins: quantitative cytochemistry of the origins of intine and exine enzymes in Brassica oleracea.

Simultaneous coupling methods for detection of acid phosphatase and non-specific esterase produce a coloured reaction product that is quantitatively related to enzyme content in freeze-sectioned Brassica pollen and tapetal cells. The intine-located acid phosphatase has 2 periods of synthesis: the first in late vacuolate period, associated with the completion of deposition of the intine polysaccharides; the second during pollen maturation, apparently reflecting cytoplasmic synthesis, Esterase activity accumulates in the tapetal cells until dissolution at early maturation period, when there is a dramatic rise in pollen-wall esterase activity, reflecting the transfer from tapetum to exine cavities. These quantitative studies confirm the gametophytic and sporophytic origins of the intine and exine proteins.

Characteristics of pollen diffusates and pollen wall cytochemistry in poplars.

Pollen diffusates, known to be important in pollen-stigma interactions controlling interspecific incompatibility between Populus deltoides and Populus alba, have been partly characterized and shown to contain more than 20 protein bands by polyacrylamide gel electrophoresis, at least 4 of these being glycoproteins. Seven fractions had antigenic activity in rabbits and several enzyme activities were also present. Peroxidase and leucine aminopeptidase isoenzymes were detected in the diffusates, demonstrating the extracellular location of these 2 enzymes. Isoenzyme patterns of peroxidase, esterase and acid phosphatase were complex, with some bands common to both species. Localization of acid phosphatase in the intine and esterase in the exine was demonstrated after brief aldehyde fixation and low-temperature embedding in glycol methacrylate. The intine and exine sites were distinguished by their chemical and structural features. Calcofluor white M2R new proved to be an excellent stain for differentiating the intine. Aniline blue-positive material, probably beta-1,3-glucan, is present associated with the intine of many ungerminated as well as germinating grains: production of this material may be a response to damage.

Rapid batch fractionation of ryegrass pollen allergens.

A rapid batch fractionation procedure employing ion-exchange matrices has been used to fractionate ryegrass pollen antigens and allergens. After sequential elution through CM-Sephadex and DEAE-Sephadex, a group of four electrophoretically distinct allergens with a molecular weight of approximately 32,000 was fractionated. These allergens correspond to the group I allergens previously isolated by Johnson and Marsh. This material may be suitable for clinical use where a characterised group I allergen preparation is required.

A cytochemical study of the leaf-gland enzymes of insectivorous plants of the genus Pinguicula.

Cytochemical methods have been used to study the distribution of acid phosphatase, esterase, ribonuclease, amylase and protease activity in the stimulated and unstimulated leaf glands of Pinguicula grandiflora, P. vulgaris, P. lusitanica, and P. caudata. Two gland types are present, stalked and sessile. The stalked glands bear a muco-polysaccharide secretion droplet, and are concerned with capture of the prey; the sessile glands are specialised for digestion. In unstimulated glands of both classes, acid phosphatase, esterase and ribonuclease activity is associated with the anticlinal walls of the head cells, which have a characteristic spongy inner surface, comparable with that of transfer cells. Acid phosphatase and esterase activity was also detected in the vacuoles of the head cells of the sessile glands. Substrate film tests showed that amylase is readily released from the stalked glands but not from the sessile ones, while in contrast proteolytic activity is mainly associated with the sessile glands.On stimulation by suitable nitrogenous materials, the glands begin to sectete fluid onto the leaf surface within 1 hr. During the process the enzymes held in the spongy walls are discharged, and activity is also lost from the intracellular sites in the sessile glands.Digestion on the leaf surface and resorption of the products has been followed autoradiographically after feeding of (14)C-labelled protein. Within 2 hr, digestion products enter the leaf, and move towards the margin in the vascular system. Movement out of the leaf begins within 12 hr. Microautoradiographs showed a concentration of products around the bases of the sessile glands and in the cells of the gland head, showing that these glands are involved in resorption as well as secretion.A possible mechanism of gland function is discussed.

The proteins released by isolated barley aleurone layers before and after gibberellic-acid treatment.

Isolated barley (Hordeum vulgare L.) aleurone layers released a number of proteins into an aqueous medium in the absence of gibberellic acid (GA3). Evidence from molecular weight determinations and a number of immunological tests indicated that these proteins were water-soluble endosperm proteins which apparently arose from endosperm cells which adhered to the layers during isolation. They were not aleurone-cell proteins. By means of immunofluorescence, the water-soluble endosperm proteins were found to be concentrated around starch grains in the starchy endosperm. These proteins were resistant to hydrolysis by GA3-induced hydrolases released from aleurone tissue.Isolated aleurone layers could be washed free of soluble endosperm proteins. After treatment with GA3, such layers released another group of proteins which were shown by immunological and electrophoretic methods to be uncontaminated by soluble endosperm proteins. The pure GA3-induced proteins were separated, using SDS-acrylamide gel disc electrophoresis, into 12 components which had molecular weights (monomer) from 15500 to 81000. Ten of these protein bands became radioactive if GA3-treatment of layers was carried out in the presence of radioactive amino acids, and therefore probably contained de novo synthesized proteins. The two protein bands which were not labelled contributed about 40% of the protein released by washed aleurone layers after GA3 treatment, and their production appeared to be dependent on proteolysis.

Quantitative cytochemistry of beta-galactosidase in normal and enzyme deficient (gal) pollen of Brassica campestris: application of the indigogenic method.

The available cytochemical methods for localization of beta-galactosidase have been evaluated using pollen grains of Brassica campestris. beta-Galactosidase-deficient pollen (gal), served as a control. Azo dye methods involving naphthyl substrates showed high and nonspecific background staining to the exine. The indigogenic method, employing 5-bromo-4-chloro-3-indoxyl beta-D-galactoside (X-gal) as the enzyme substrate, gave specific opaque-blue final reaction product, while mutant pollen grains remained colourless. Final reaction product formation was blocked by D-galactono-1,4-lactone, thus demonstrating the specificity of the enzyme reaction. Using microspectrophotometry, the absorbance of the final reaction product was found to be a linear function of incubation time and section thickness in cryostat sections up to 8 micron thick and was only slightly reduced by glutaraldehyde prefixation. The validity of the indigogenic method for quantitative analysis was confirmed by using an enzyme-containing polyacrylamide gel model system and enzyme-coupled Sepharose 4B beads. Cellular sites of enzymic activity have been determined using plastic sections: final reaction product occurred in the intine wall layer and peripheral cytoplasm.

Invertases of Lilium Pollen : Characterization and Activity during In Vitro Germination.

Two different forms of invertase are found in pollen of lily (Lilium auratum). One form is cytoplasmic (Invertase 1) and the other is bound to the pollen wall (Invertase 2). Invertase 1 has been partially purified and is a glycoprotein (apparent molecular weight, 450 kilodaltons) with a K(m) of 0.65 millimolar for sucrose. The two invertases differ in pH optimum and thermal stability. Invertases of lily pollen are beta-fructofuranosidases which can hydrolyze sucrose but not melizitose. The mature pollen grains have enzyme activity in both cytoplasmic and wall fractions, and no increase in the activity of either occurs during germination. The wall-bound enzyme could not be released by treatments with detergents or high salt concentrations.

Tissue origins, cell lineages and patterns of cell division in the developing dermal system of the frut of Vitis vinifera L.

Cell division during development of the dermal system of fruit of the grape cv. Gordo is confined to the first growth period. The epidermis is conserved with anticlinal proliferative cell divisions providing for increase in cell number. The hypodermis is the layer of origin of the collenchymatous dermal system. Six or seven layers are differentiated by periclinal cell divisions early in the first growth period, and later increase in size is obtained by proliferative anticlinal cell divisions. These observations are related to developmental and genetic control of fruit shape and volume.