Self-incompatibility and the pollen-stigma interaction inTradescantia ohiensis rafin

Summary Self-incompatibility (SI) is reported for an accession ofTradescantia ohiensis. Pollen tube inhibition is stigmatic. The stigma is wet and papillate, the papillr surface bearing conspicuous blebs particularly in the mid- and basal regions of the cell. A proteinaceous pellicle is present on the surface of the papillar cuticle. The penetration of the stains calcofluor white and alcian blue into the cell wall of fresh stigma papillae strongly indicates that the permeability of the papillae is greatest at the mid-region of the cell and not at the tip. When single pollen grains are attached to the tip of a papilla there is either no response at all or the pollen adheres to the papilla. When attached in the mid-region pollen adheres, and often germinates. It is concluded that the sites of pollen receptivity are the mid- and basal regions of the papular cell.     

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

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

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.     

Ultrastructure of the dimorphic pollen and stigmas of the cleistogamous species,Collomia grandiflora (Polemoniaceae)

Summary An ultrastructural study of the pollen and stigma of the dimorphic flowers in the cleistogamous speciesCollomia grandiflora reveals significant differences in cytoplasmic features in the pollen and wall features in the papillae. Both pollen types contain lipid and starch reserves, but the smaller CL (cleistogamous) pollen shows a much greater abundance of starch compared to the CH (chasmogamous) pollen. In addition to the papular size and shape differences between the two stigma types, there is a more extensive cuticular stretching and wall microfibrillar loosening over the CH papillar tip. There is no apparent pellicle on the cuticle surface of either type of papilla, only scattered lipidic deposits. It is proposed that these structural differences may contribute to the cross incompatibility between the two floral morphs.     

THE STIGMATIC PAPILLAE OF AMYEMA (LORANTHACEAE): DEVELOPMENTAL RESPONSES TO PROTANDRY AND SURFACE ADAPTATIONS FOR BIRD POLLINATION

The flowers of Amyema miquelii and A. miraculosum are protandrous and pollinated by birds. Their dry-type stigmatic surface is composed of unicellular papillae. At the male phase, these papillae are constricted with rugulose surfaces. During the transition to the female (pollen receptive) phase these cells expand, almost doubling in width while their surface becomes much smoother. Beneath the thin proteinaceous pellicle, the papillar wall consists of an extraordinarily thick bi-layered cuticle overlying the primary wall. The two layers of the cuticle are stained by lipid dyes, but are distinguished by their different responses to other cytochemical tests. The reaction product for the enzyme esterase is present within crenulations on the papillar surface in small amounts, and in dense deposits in the cuticular clefts at the base between papillae. Not surprisingly, pollen tubes are unable to penetrate the thick papillar cap and enter the style through these clefts. The unusual thickness of the cuticle is interpreted as an adaptive response to pollination by perching birds (passerines) probing for nectar.     

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.     

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

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

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.     

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 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.     

Bee-pollination inHibbertia fasciculata (Dilleniaceae)

In direct contrast to mostHibbertia spp., the flowers ofH. fasciculata R. Br. ex D. C. bear only a single whorl of stamens and these stamens are arranged separately (not in typical bundles). The short filaments are appressed to the three carpels so that the inflated, porose and introrsive anthers form a centralized cluster obscuring the three ovaries. The three slender styles emerge at right angles from between the filaments. These styles curve upward and the stigmas form the three points of a triangle; each stigma is approximately one millimeter outside the centralized cluster of anthers. The flowers are nectarless and bear a bright yellow corolla. A pungent and unpleasant fragrance appears to be concentrated within the pollenkitt. When native bees attempt to forage for the pollen, within the cluster of anthers, the ventrally deposited loads of pollen, on the bees' abdomens, contact the outer triangle of stigmas. The major pollinators ofH. fasciculata are female bees in the polylectic genera,Lasioglossum (subgenusChilalictus, Halictidae) andLeioproctus (Colletidae). These bees carry an average of more than two pollen taxa when they are caught foraging onH. fasciculata. 78% of the 47 bees, captured onH. fasciculata carried the pollen from at least one sympatric taxon bearing nectariferous flowers (e.g., genera in theMyrtaceae, Compositae, andEpacridaceae). The pollination biology ofH. fasciculata is assessed in relation to the known radiation of bee-pollinated flowers in the genusHibbertia, and within theDilleniaceae s. l.     

The pollination biology ofHibbertia stricta (Dilleniaceae)

The exines of pollen grains ofHibbertia stricta (DC.)R. Br. exF. Muell. (Sect.Pleurandra) wear an oily, yellow pollen coat that stains positively for lipids. The pollen is collected by asocial bees, exclusively. The most common floral foragers are members of the genusLasioglossum (subgenusChilalictus;Halictidae) and they harvest pollen via thoracic vibration. As these bees cling to the inflated anthers their pollen smeared bodies come in contact with either of the two wet, nonpapillate stigmas. The stigmas respond positively to cytochemical tests for the presence of esterase immediately following expansion of the corolla, indicating the effective pollination period. The foraging patterns of the bees are narrowly to broadly polylectic. AsH. stricta flowers are nectarless, it is not surprising that bees bearing mixed pollen loads always carry the pollen of at least one nectariferous, coblooming plant. The pollination biology ofH. stricta is compared with otherHibbertia spp. and with pollen flowers in general.     

Characteristics of self-incompatibility in Schlumbergera truncata and S. x buckleyi (Cactaceae)

The influence of self-incompatibility (SI) on fruit set, seed set, and pollen tube growth was investigated in Schlumbergera truncata (Haworth) Moran and S.xbuckleyi (T. Moore) Tjaden. Four Schlumbergera clones were crossed in a complete diallel to verify the presence of SI. Fruit did not set when the clones were selfed or when two of the clones were crossed reciprocally, but all other outcrosses yielded fruit which contained 100–200 seeds each. Compatible outcrosses were characterized by large numbers of pollen tubes in the style and ovary cavity at 72 h after pollination. When pistils were selfed or incompatibly crossed, pollen tubes were inhibited in the upper third of the style and few pollen tubes reached the base of the style by 72 h after pollination. Schlumbergera exhibits several characteristics often associated with sporophytic SI systems (tricellular pollen and dry stigmas with elongate papillae), together with those commonly observed in gametophytic SI systems (stylar inhibition of incompatible pollen tubes and absence of reciprocal differences in outcrosses).Publication no. 3174 of the Massachusetts Agricultural Experiment Station     

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     

Pollen movement in the micropylar canal ofLarix and its simulation

InLarix pollen captured by the ovule and rested at the distal end of the micropylar canal is transferred upward to the nucellus before it develops a pollen tube. This upward movement occurs after the canal is filled with secreted fluid, despite the fact that the pollen sinks in the fluid. We examined the mechanism of the movement based on the morphology of the canal and its simulation using pipettes. When a water column moves upward in a waxed pipette, suspended particles also move upward carried by the meniscus. InL. x eurolepis the inner surface of the integument lining the micropylar canal is coated by a cuticle layer. This layer is further coated by an integumentary membrane before the fluid is secreted. This membrane, however, becomes distorted or disappears during fluid secretion. The exposed cuticle and the degenerated hydrophilic nucellar apex may facilitate the movement of the meniscus toward the nucellus as in the simulated pipette. Pollen is interpreted to move by being carried by the meniscus when the fluid recedes.     

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.     

Wide hybridization: pollination of Zea mays L. by Sorghum bicolor (L.) Moench

Summary Foreign pollen tubes in the stigma of Zea mays can be prevented from reaching the ovary cavity by the unusual length of the pollen tube pathway. A simple and rapid procedure is described for overcoming this difficulty by pollinating the basal parts of the stigmas without removing the ensheathing bracts (husks). The method maintains high humidity in the vicinity of the ovaries, and by conserving photosynthetic tissues probably also ensures a more normal O₂ /CO₂ balance in the neighbourhood of the stigmas than do bagging procedures. It is shown that Sorghum pollen tubes readily reach the ovary after pollination by the method. Their presence induces some of the characteristic post-pollination effects caused by Zea pollen tubes, but they frequently also stimulate premature enlargement of the nucellus and lysis of nucellar cells. Although Sorghum tubes have been traced across the inner ovary wall, they have not been seen to enter the micropyle, and hybrid embryos have not yet been obtained.     

Structure,ultrastructure and evolution of floral nectaries in the twinflower tribe Linnaeeae and related taxa (Caprifoliaceae)

Linnaeeae is a small tribe of Caprifoliaceae consisting of six genera and c. 20 species. In Linnaeeae, floral nectaries are located on the corolla‐filament‐tube and nectar is produced from unicellular glandular hairs. We studied 23 taxa using scanning electron microscopy (SEM), light microscopy (LM) and transmission electron microscopy (TEM) and found two distinct nectary morphologies, zonate and gibbous types, and two distinct types of glandular hair, clavate and smooth base types. Plesiomorphic characters associated with the nectary and identified in the tribe include hypocrateriform corollas, dichogamous flowers, zonate nectaries, wet papillate stigmas, vestigial nectary disc and smooth pollen grains. Apomorphic characters include bilabiate corollas, homogamous flowers, bulging nectaries, dry papillate stigmas and echinulate pollen grains. The nectary structure is similar in Vesalea and Linnaea and differs from the rest of the tribe, in accordance with recent phylogenetic results. Nectar secretion is typically granulocrine with subcuticular accumulation of nectar, which we compared with the secretion in multicellular hairs of Adoxa moschatellina. The cuticle on the hair becomes detached from the cell wall and large subcuticular spaces filled with nectar are formed. Nectar is probably released in areas with a thin cuticle. In Zabelia, the smooth basal part of the hair could help to build up the hydrostatic pressure.     

Esterases in pollen and stigma of Brassica

The dry type stigma of Brassica is covered with a continuous layer of cuticle. Cutinase and non-specific esterases may be involved in breakdown of this cuticle barrier during pollen-stigma interaction, but only a little is known about their nature and characteristics. We report here the presence of two distinct esterases from stigma and pollen of Brassica. A 33 kD esterase assayed using MU-butyrate substrate shows high activity in stigma papillae. A similar esterase from Tropaeolum pollen has been shown to possess active cutinase activity. The esterase activity in anther tissue is due to a 24 kD enzyme with substrate specificity toward acetate esters. Both enzymes require sulfhydryl groups for their catalytic activity. Immunogold labelling of antibodies raised against these esterases localised the proteins at the subcellular level. Antibodies for MU-butyrate hydrolase gave a positive signal in the cell walls of mature stigma papillae and in the tapetum and microspores during early stages of anther development. In the mature anther, a positive signal in the cytoplasm of pollen grains with some detectable localisation in the exine layer of the pollen wall was obtained. Similar results were obtained with acetate hydrolase antibodies. These esterases are thus spatially and temporally regulated in stigma and anther tissues.Abbreviations MU methyl umbelliferyl - pAbC anti-butyrate hydrolase polyclonal antibodies - pAbE anti-acetate hydrolase polyclonal antibodies