Heterostyly inPrimula. 2. Sites of pollen inhibition,and effects of pistil constituents on compatible and incompatible pollen-tube growth

Summary In incompatible (intramorph) pollinations of the heterostylousPrimula vulgaris, pollen germination or tube growth may be partially inhibited in several sites associated with the stigma or style. Blockage may occur, a) on the stigma surface through the failure of germination or of pollen tube penetration after germination, b) in the stigma head during the passage of the tube through the specialized transmitting tissue of the head, or c) in the transmitting tract of the style. None of the barriers is complete, and the prohibition of selfing or intramorph crossing depends upon the cumulative screening effect of one following upon the other. In both morphs, the germination of incompatible pollen on the stigma is enhanced in high ambient relative humidity, but many tubes still fail to penetrate the stigma. Those that do are retarded or blocked in their growth in the transmitting tissues of the stigma head and style. Crude extracts from the tissues of the stigma head and style show some differential effect on the growth of pollen tubesin vitro, and dialysates of extracts containing high molecular weight fractions show a consistent differential effect, those from thrum tissues retarding thrum tubes while having a lesser effect on pin tubes, and those from pin tissues retarding pin tubes while having lesser effect on thrum. It is suggested that the factors influencing tube growth are present in the intercellular secretions of the transmitting tract.     

The Structure and Cytochemistry of the Pistil of Hypericum calycinum: The Stigma

The pistil of Hypericum calycinum has a pentacarpellary, syncarpousovary with five slender styles, each terminating in a smallstigma. The stigma is dry and papillate with a thin lining ofpellicle. The cuticle is thin and continuous around the papillae.A large vacuole filled with tannins occupies the major partof the papillae and the cytoplasm forms a thin lining aroundthe vacuole. The cell wall of the mature papillae show two distinctlayers - an outer layer of loosely woven fibrils and an innerdenser layer with compact fibrils. A large number of small lipoidalbodies accumulate just below the cuticle. The papillae havefewer organelles than those typical of glandular cells. Dictyosomesobserved occasionally are without associated vesicles. The cytoplasmis rich in ribosomes. The basal portions of the papillae mergeinto the transmitting tissue made up of loosely arranged cells.The intercellular matrix of the transmitting tissue is richin lipids. Pollen grains are deposited between the papillae.Upon pollen germination, pollen tubes enter the stigma throughthe interstices between the papillae Hypericum calycinum, cytochemistry, pistil, pollen-pistil interaction, stigma, ultrastructure     

Fine Structure and Cytochemistry of the Stigma Surface and Incompatibility in some Distylous Linum Species

The receptive surface of the stigma in distylous Linum grandiflorumand L. pubescens was studied by electron microscopy and cytochemicaltechniques. In both floral morphs a proteinaceous-lipoidal coatingis present on the papilla surface. In thrum stigmas the cuticleis highly irregular and pitted at the papilla tip. The cuticleis dislodged and torn at anthesis and an osmiophilic secretionproduct is released within a pectinaceous matrix. The secretionproduct stains for proteins and lipids and contributes to adhesionof pollen. In the larger pin papillae the cuticle is wavy, continuous,thicker than in thrum papillae and adjoins the cell wall. Inboth species the surface of the two types of pollen grains iscoated with lipids and protein. A similar behaviour of the male gametophyte is observed in incompatiblepollinations of L. pubescens, L. mucronatum and L. grandiflorum.In intramorph thrum pollinations pollen tubes are arrested withinthe stigma. In intramorph pin pollinations the majority of pollengrains fail to adhere to the stigma. Low permeability to waterin pin papillae, as determined by the neutral red test, maybe a factor preventing imbibition of the few adhering grains.Tubes of the few germinated grains are inhibited inside thestigma. On the part of the stigma, the difference in the major siteof inhibition in the two intramorph incompatible combinationsmay be accounted for by the dissimilar properties of the papillae,i.e. the occurrence of wet thrum stigmas and dry pin stigmas.Functionally, the unusual association of sporophytic incompatibilitywith wet thrum stigmas is attributed to retention of the secretorymaterial on individual papillae. Stigmatic papillae, cuticle, pollen coat, distyly, incompatibility, Linum grandiflorum, L. pubescens, L. mucronatum     

The dry and wet stigmas ofPrimula obconica: Ultrastructural and cytochemical dimorphisms

Summary The stigmatic papillae of the distylous speciesPrimula obconica are studied by means of cytochemical, light and electron microscopic techniques. The papillae on thrum stigmas are smaller than those on pin stigmas. At the bud stage, secretory vesicles are not a conspicious part of the cytoplasm, although certain signs of secretory activity are present. The young papillae bear the thin, superficial pellicle typical to dry stigmas. Small vesicles are numerous in mature papillae of both morphs, and seem to originate from the ER. A layer of closely packed, osmiophilic globuli is present in the outermost part of mature walls of pin papillae. At sites with cuticle disruption, the globuli seem to migrate outwards to be incorporated with the copious, blistery exudate. Due to this exudate the pin stigma is characterized as wet. Cytochemical tests suggest that the exudate contains mainly lipids, and different carbohydrates and protein are detected. It reacts positively in tests for peroxidase, acid phosphatase, and non-specific esterases. The thrum stigma remains dry at maturity, with a distinct pellicle also reacting positively to the enzyme tests. Only a few, scattered osmiophilic globuli, sized and situated as those in the pin papillae walls, are found in the thrum walls, and they do not form a proper layer.Thus the generally accepted correlation between dry stigmas and the sporophytic kind of self-incompatibility system is not substantiated withinP. obconica, and the possible influence of the dimorphisms to the pollen/stigma interaction is discussed.     

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

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

Stigma morphology in distylous and non-heterostylous species ofVillarsia (Menyanthaceae)

Stigma morphology was examined with the SEM in 14 of the 16 species ofVillarsia. In nine of the ten distylous species studied, stigmas of the floral morphs were strongly dimorphic in length, shape, configuration of the receptive surface, and in the size and density of their papillae. Thrum stigmas ofVillarsia, in contrast to those of most other distylous species, are not simply smaller versions of the conspecific pin stigmas, but generally exhibit an array of morph-specific characters. Thrum stigma lobes may be broader than those of pins, they may have undulate margins, lobes subdivided into secondary lobes, papillae more extensively distributed than in pins, and various combinations of these traits occur. The traits that distinguish thrum from pin stigmas achieve an increase in the receptive area and may enhance more efficient pollen capture by the shorter and less accessible thrum stigmas. The morphogenesis of the stigma shape dimorphism appears to involve processes more complex than inhibition of elongation in thrum styles. InVillarsia, the stigma dimorphisms are species-specific. No correlations were found between morphologies of the stigma and the different breeding systems in distylous species. Stigmas of the four non-heterostylous species examined resemble the thrum stigma type found in most distylousVillarsia species.     

Fluorescence and scanning electron microscopic study on self-incompatibility in distylous Averrhoa carambola L.

Various combinations of intermorph, selfing and intramorph pollinations were carried out in Averrhoa carambola and the pollinated pistils were observed under fluorescence and scanning electron microscopes in time-course experiments. In both compatible and incompatible pollinations, similar behavior of pollen germination and penetration was observed in the first 4 h after pollination. In compatible intermorph pollination, pollen tubes were found at the base of the transmitting tract of the style at 8 h and 24 h after pollination in both the pin and thrum morphs. With thrum flowers, selfing resulted in pollen tubes being uniformly arrested at the junction between the stigmatic and stylar tissues. Penetration of pollen tubes into the upper portion of style was observed in thrum intramorph pollination and, when the second member was treated as the gynoecial tissue under the same pollination, penetration of tubes was further enhanced. Pin flowers, on selfing, resulted in pollen tube penetration farther down the style than was the case with thrum selfing. Intramorph pollination of pin morph behaved in a similar manner to selfing and was not affected by genotypes. Beside the stamen-style dimorphism, the receptive surface of the cob stigma was larger in pin than thrum flowers. While pin pollen was round, thrum pollen was oblong in shape with pin to thrum ratio on the polar axis being 1.2 and on the equatorial axis 0.8. The stigma of pin morph belonged to the dry type, while that of the thrum resembled the wet type.     

Structural and Cytochemical Characteristics of the Stigma and Style in Vitis vinifera L. var. Sangiovese (Vitaceae)

Studies were carried out on structural and cytochemical aspectsof the stigma and style ofVitis vinifera . The stigma is ofthe wet papillate type with a continuous cuticle and pellicle.During the development of the papillae, the cell walls increasein thickness and produce a secretion product constituted oflipids that pass through the wall forming the exudate. The styleis solid with a central core of transmitting tissue which hasconspicuous intercellular spaces that increase remarkably fromthe periphery to the centre where the cuticle is present. Theintercellular spaces, where the pollen tubes grow, contain amatrix that includes polysaccharides, pectic substances andscattered areas of lipidic nature. Cytochemistry; stigma; style; ultrastructure; Vitis vinifera     

Pollen-pistil interaction in Linum grandiflorum

A detailed investigation of the receptive surface of the stigma of a dimorphic taxon, Linum grandiflorum, was carried out using scanning electron microscopy, cytochemistry and polyacrylamide gel electrophoresis. The stigma surfaces of the pin and thrum morphs showed distinct differences. The stigma of the pin morph was of the dry type and the papillae were covered with a uniform cuticle-pellicle layer. The stigma of the thrum morph, on the other hand, resembled the wet type; the cuticle-pellicle layer was disrupted at places and a secretion product was released onto the surface of the stigma. Coomassie blue staining material was present on the surface of only the thrum stigma. Although esterases and acid phosphatases were present on the stigma of both the morphs, their activity was invariably higher on the thrum stigma. Polyacrylamide gel electrophoresis of stigma leachates also showed distinct differences in the protein profiles of the two morphs.     

Heterostyly inPrimula. 3. Pollen water economy: a factor in the intramorph-incompatibility response

Summary The pollens from pin (long-styled) and thrum (short-styled) flowers ofPrimula differ markedly in water economy. While pin pollen is little affected by atmospheric humidity, the germinability of thrum pollen in liquid medium is sensitive to the conditions experienced during the immediately preceding period, being depressed when the grains are partly desiccated, although capable of being restored with subsequent controlled rehydration. The tubes produced by incompletely rehydrated thrum pollen show abnormal growth forms, suggesting that the effect is on the membranes of the vegetative cell, a conclusion supported by observations on membrane state using the fluorochromatic reaction. Thrum pollen stripped of part of the material carried in the cavities of the exine by rinsing with a lipid solvent loses much of its capacity for the uptake of atmospheric water, indicating that, in this morph, some factor facilitating hydration is transferred from the sporophytic parent during pollen maturation.Implications of these differences in the physiology of the pin and thrum pollens for the functioning of the intramorph-incompatibility system are discussed.     

Heterostyly and Pollen-tube Interactions in Luculia gratissima (Rubiaceae)

Observations on the floral biology of Luculia gratissima (Rubiaceae)showed that this species is distylous with complementary positioningof anthers and stigmas in the two floral forms. Unusual featuresof distyly in this species include the larger size of the corolla,the stigma surface and the stigmatic papillae in the thrum flowerscompared to the pin ones. Stigmatic surfaces have similar secretionsbut they appear more copious in thrum than pin. The floral dimorphismwas accompanied by a very effective self-incompatibility systemand no seed was set on selfing. Seed number per capsule on crossingwas significantly greater in thrum flowers compared to pin.Incompatible pollen tubes were inhibited within 24 h at thebase of the stigma/top of the style in both morphs. Amputationof this region of the gynoecium removed the self-incompatibilityreaction in thrum but not pin flowers. Pollination with a mixtureof compatible and incompatible pollen and sequential pollinationwith self followed by cross pollen showed that there were interactionsbetween the two types of pollen tube. The presence of compatibletubes was found to cause the excessive swelling of the pollen-tubetip of the incompatible ones. The incompatible tubes did notappear to have any effect on the growth of compatible ones. Luculia gratissima, distyly, floral biology, self-incompatibility, pollen-tube interactions     

GYNOECIAL DEVELOPMENT,POLLINATION, AND THE PATH OF POLLEN TUBE GROWTH IN THE TEPARY BEAN,PHASEOLUS ACUTIFOLIUS

The gynoecium of Phaseolus acutifolius var. latifolius, a self-compatible legume, is characterized by a wet non-papillate stigma, an intermeditae hollow/solid style type, and secretory cells on the ventral surface of the ovary which direct pollen tube growth. The stigma is initially receptive 5–6 days prior to anthesis. Production of stigmatic secretions, composed primarily of carbohydrates and lipids, fragment the cuticle covering epidermal cells of the stigma early in ontogeny; the lipidic aspect of the copious secretions apparently serves to inhibit desiccation after the cuticle is ruptured. Stylar canal development occurs as a combination of elongation of a basal canal present early in development, and dissolution of part of a solid transmitting tract tissue just below the stigma. Anthers dehisce and the tricolporate pollen is released onto the receptive stigma one day before anthesis. Following initial growth in intercellular spaces in the transmitting tract of the stigma, pollen tubes adhere to epidermal secretory cells along the ventral side of the stylar canal and upper ovary; here the transmitting tract is apparently limited in the number of tubes it can accommodate, providing a possible site of selection of male gametes.     

Tissue Development in the Stigma of Prunus avium L.

The tissue inside the stigma of Prunus avium L, through whichpollen tubes grow, undergoes a specific pattern of developmentwhich is different from that of the papillae on the stigmaticsurface or the transmitting tissue of the style in several importantaspects An elaborate system of intercellular spaces developsconsisting of small lacunae and aerenchymatous tissue The majorityof spaces contain no intercellular substances and appear tobe air canals, although the small lacunae may participate insecretion Aerenchymatous tissue on the peripheral regions ofthe stigma is characterized by several cytological featureswhich change during ontogeny, such as nuclear inclusions, amyloplastinclusions, dumbbell-shaped mitochondria, cytoplasmic sequestrationand isolated segments of endoplasmic reticulum Occasional clustersof amyloplasts were also observed Prunus avium L, sweet cherry, stigma, cytology, ultrastructure     

The Structure and Cytochemistry of the Pistil of Sternbergia lutea (Amaryllidaceae)

Studies were carried out on structural and cytochemical aspectsof the pistil of Sternbergia lutea (L.) KerGawl. The stigmais of the wet papillate type; the papillae are unicellular andare arranged densely around the rim of a funnel-shaped stigma.The stigma exudate is limited and is confined to the bases ofthe papillae and the inner lining of the stigma. The papillaeare smooth in the distal part and are covered with intact cuticle-pelliclelining. The cuticle is disrupted at places towards the baseof the papillae releasing the exudate. The exudate is rich inpectins and other polysaccharides but poor in proteins and lipids.The papillae show dense cytoplasmic profiles with extensiveendoplasmic reticulum (ER), abundant mitochondria, polyribosomesand active dictyosomes. The style is hollow. The stylar cavityis surrounded by two to four layers of glandular cells. In theyoung pistil the canal is lined with a continuous cuticle, butin the mature pistil the cuticle becomes disrupted and the canalis filled with the secretion produced by the cells of the surroundingglandular tissue. Ultrastructurally, the cells of the glandulartissue are very similar to the stigmatic papillae. The innertangential wall of the cells bordering the canal is uniformlythicker than other walls. The secretion in the stylar canal,as well as the intercellular spaces of the glandular tissue,stain intensely for pectins and polysaccharides but poorly forproteins and lipids. Pollen tubes grow through the stylar canal.Structural and cytochemical details of the pistil of Sternbergiaare compared with other hollow-styled systems. Pistil, Sternbergia lutea (L.) Ker-Gawl., stigma and style, structure and cytochemistry     

Tissue organisation,pollen receptivity and pollen tube guidance in normal and mutant stigmas of the grass Pennisetum typhoides (Burm.) Stapf et Hubb.

Summary The normal stigma of Pennisetum typhoides is twin-branched, each branch bearing unbranched trichomes. As is general among the grasses, the papillate cells of the trichomes possess a discontinuous cuticle with overlying protein and polysaccharide secretions. These adaptations for pollen capture and hydration are absent from the stigma axes. Pollen tubes emerging from grains received on the trichomes are guided into the axes with the tips directed towards the ovary by the architecture of the basal cell complex. There are no defined transmitting tracts in the stigma axes, and further passage is through intercellular spaces of a tissue of elongated cells between the epidermis and the central vascular strands. In the mutant tr, tr, the stigmas are twin-branched, but lack trichomes. However, the principal adaptations of the trichome cells for the capture and hydration of pollen are expressed in the epidermal cells of the branches, which have permeable cuticles and the characteristic surface secretions. Pollen tubes emerging from grains germinating on the branches enter between the files of epidermal cells, or at their ends. In the absence of the guidance provided by trichome structure in the normal stigma, they pass indifferently either towards or away from the ovary. The implications of the comparison between the normal and mutant genotypes for understanding the requirements for pollen capture, germination and pollen-tube guidance in the grasses are discussed.     

Pollen flow inPrimula veris (Primulaceae)

Observations on two natural and one artificial distylous populations ofPrimula veris in Britain revealed very high proportions of intramorph pollen on pin stigmas and moderate proportions of intermorph pollen on thrum stigmas. The average ratio of pin: thrum pollen production per flower was 2.4:1. Large amounts of pollen remained in dehisced anthers, perhaps reflecting low levels of pollinator activity. Population size and density had no clear effect on pollen flow patterns, which are asymmetrical for the two morphs.     

Ultrastructure and histochemistry of Citrus limon (L.) stigma

Citrus limon has a wet stigma which can be divided in two zones: a glandular superficial one formed by papillae, and a non-glandular one formed by parenchymatic cells. The stigmatic exudate is produced by the papillae after the latter have reached their ultimate size. The papillae of the mature pistil are of varying size and composition. Both the unicellular and multicellular ones are present. The cells at the base of the papillae are rich in cytoplasm, whereas the tip cells are vacuolated. Histochemical analysis has shown that the exudate of Citrus is composed of lipids, polysaccharides, and proteins. Our results indicate that the lipidic component is produced and secreted first, followed by production and secretion of the polysaccharidic component. The lipidic component of the exudate is produced in the basal papillae cells and accumulates as droplets in dilated parts of the smooth endoplasmic reticulum (SER). Subsequently the lipid droplets are transported to the plasma membrane, and transferred by the latter into the cell walls. Then the exudate component is accumulated in the intercellular spaces and in the middle lamellar regions of the walls. Subsequently, the polysaccharidic component of the exudate is produced and secreted by the tip cells of the papillae.Abbreviations RER rough endoplasmic reticulum - SER smooth endoplasmic reticulum     

Structure of the stigma and style of <Emphasis Type="Italic">Callaeum psilophyllum</Emphasis> (Malpighiaceae) and its relation with potential pollinators

The family Malpighiaceae, particularly in the Neotropic, shows a similar floral morphology. Although floral attraction and rewards to pollinators are alike, stigmas and styles show more diversity. The stigmas were described covered with a thin and impermeable cuticle that needs to be ruptured by the mechanical action of the pollinators. However, this characteristic was only mentioned for a few species and the anatomy and ultrastructure of the stigmas were not explored. In this work, we analyze the morphology, anatomy, and ultrastructure of the stigma and style of Callaeum psilophyllum. Moreover, we identify the potential pollinators in order to evaluate how the disposition of the stigmas is related with their size and its role in the exposure of the receptive stigmatic surface. Our observations indicate that Centris flavifrons, C. fuscata, C. tarsata, and C. trigonoides are probably efficient pollinators of C. psilophyllum. The three stigmas are covered by a cuticle that remained intact in bagged flowers. The flowers exposed to visitors show the cuticle broken, more secretion in the intercellular spaces between sub-stigmatic cells and abundant electron-dense components inside vacuoles in stigmatic papillae. This indicates that the stigmas prepares in similar ways to receive pollen grains, but the pollinator action is required to break the cuticle, and once pollen tubes start growing, stigmatic and sub-stigmatic cells release more secretion by a granulocrine process.     

The control of incompatibility in distylous Pulmonaria affinis Jordan (Boraginaceae)

In P. affinis, pin pollen is shorter on average than thrum pollen. Pins have more coronal hooks on stigmatic papillae than thrums, but gaps between papillae are relatively smaller for thrums. Pin stigmas receive more pollen than thrum stigmas. Thrum stigmas receive more (dissortive) pin pollen, but pin stigmas are assortively pollinated. Pollen only germinates when trapped below papilla coronas. On thrum stigmas, most trapped pollen is pin. Pollen germination is better on thrum stigmas than pin stigmas, and thrum stigmas show a close relationship between numbers of legitimate pollen grains, numbers of germinating grains, and numbers of pollen tubes in the style. There is no inhibition of illegitimate pollen germination. Illegitimate pollen tubes are inhibited in the style. Incompatibility operates by a combination of dissortive pollination, dissortive pollen trapping, and stylar pollen tube inhibition. All heteromorphic features differing between pins and thrums are implicated in the inhibition of within-morph fertilization in thrums.     

The transmitting tract in Trimezia fosteriana (Iridaceae). III. Pollen tube growth in the stigma, style and ovary

Trimezia fosteriana is a self-incompatible plant with an open style. The stigma was found to be receptive for approx. three hours. Pollen tube growth in the entire transmitting tract was followed with LM, SEM and TEM. The cuticle that covers the mature papillae is continuous but in the rest of the transmitting tissue it is thin and ruptured. The pollen tubes grow in a mucilage mixed with cuticle remnants. In the style, however, larger parts of a cuticle film remains which gives the impression that pollen tube growth occurs under a cuticle. The secretion contains proteins and carbohydrates including pectic substances. The pollen tube growth rates were estimated to 2 mm/hour in the stigma, 1–2 mm/hour in the style and 0.5 mm/hour in the ovary.