Stigma-surface Esterase Activity and Stigma Receptivity in Some Taxa Characterized by Wet Stigmas

Stigma-surface esterase activity and stigma receptivity througha sequence of developmental stages of the pistil have been studiedin four taxa characterized by having wet stigmas — Petuniahybrida, Nicotiana tabacum, Crinum defixum and Amaryllis vittata.The style is solid in the first two and hollow in the lattertwo taxa. In all the taxa, stigma—surface esterase couldbe detected in a thin surface layer (pellicle) from a very earlystage of pistil development, irrespective of the presence orabsence of the exudate. However, the taxa showed variation instigma receptivity. In Petunia and Nicotiana, stigmas from pistilsof all the stages supported pollen germination and tube growth.In Amaryllis and Crinum, stigmas of only the mature pistils,when the exudate is present on the stigma, supported normalpollen germination and tube growth. It is inferred that in taxacharacterized by a wet stigma and solid style, the factors requiredfor pollen germination are present from an early stage of pistildevelopment and the exudate per se is not involved in pollengermination. In taxa characterized by a wet stigma and hollowstyle, however, the pellicle does not carry the factors requiredfor pollen germination and tube growth; they appear to be presentin the exudate. Petunia hybrida Vilm, Nicotiana tabacum L., Crinum defixum, Ker-Gawl, Amaryllis vittata Ait., tobacco, pollination, pollen germination, stigmatic exudate, stigma receptivity, stigma-surface esterase, esterase activity     

Effective Pollination Period and Nature of Pollen-collecting Apparatus in the Gymnosperm, Larix leptolepis

In controlled environment, the effective pollination periodin Larix leptolepis is characterized by its brevity, lastingfor less than 1 d. It is associated with the opening of thepapillae of the pollen collecting apparatus which possess surfaceesterase activity, a condition that has its parallel in thesurface pellicle of angiosperm stigmas. Larix leptolepis, ovule receptivity, morphometric analysis, pollination, stigma surface, esterase activity     

Serine esterases are required for pollen tube penetration of the stigma in Brassica

. We have investigated the diversity of serine esterases in pollen and stigma tissues of Brassica napus and the role of these enzymes in pollen germination and pollen tube penetration of the stigma. The serine esterase-specific inhibitor diisopropyl fluorophosphate was used as a probe in a tritiated form, [³H]-DIPF, to determine the number and diversity of serine esterases in crude protein extracts from pollen and stigma. Seven serine esterases were identified in pollen and at least seven serine esterases were identified in stigma. The most abundant enzymes had molecular weights of 30-50 kDa. In the pollen extract a serine esterase was detected with the same molecular weight, 22 kDa, as an esterase previously shown to be a cutinase. Only one serine esterase (40 kDa) appeared to be shared between pollen and stigma extracts. Butyrate esterase activity in pollen and stigma extracts was assayed using p-nitrophenyl butyrate (PNB), an ester substrate frequently used in 'cutinase' assays. Total PNBase activity in pollen and stigma extracts was shown to be significantly reduced by the serine esterase inhibitors DIPF and ebelactone B. When DIPF and ebelactone B were applied to stigmas prior to pollination, pollen germination was not significantly affected but, at the highest inhibitor concentrations, up to 70% of germinating pollen tubes failed to penetrate the stigma surface. These data demonstrate that serine esterases, most probably cutinase(s), are required for pollen tube penetration of the dry cuticularised Brassica stigma.     

Reproduction in Seagrasses: Nature of the Pollen and Receptive Surface of the Stigma in the Hydrocharitaceae

This paper describes the characteristics of the pollen and thereceptive surfaces of the stigmas in the three marine angiospermsincluded in the Hydrocharitaceae. The pollen in Enhalus acoroidesand Thalassia hemprichii is spherical and has an ornamentedexine. An exine layer is not found in Halophila stipulacea wherereniform pollen grains are contained within transparent moniliformtubes. Cytochemical tests show that the pollen wall in the threespecies contains acidic and neutral polysaccharides and acidhydrolase acitivity is detected in the intine of H. stipulaceaand T. hemprichii. In Thalassia, one of the intine enzymes,acid phosphatase, is unambiguously associated with cytoplasmicinclusions. Flowering in Thalassia is coincident with the spring tides andthe pollen is released as a mass suspended in a thecal slimewhich contains approximately 5 per cent by weight carbohydrate,the principal mono-saccharide being mannose. Electrophoreticanalysis of the pollen-free slime shows a single glycoproteincomponent. The stigmas of the three seagrasses are papillate and of the‘dry’ type possessing a continuous protein-aceouspellicle subtended by a cuticle. The stigma pellicle exhibitscytochemically detectable esterase activity and binds the lectinconcanavalin A. Acid phosphatase activity is localized beneaththe cuticle at the tips of the stigma papillae. The discoveries show that the characteristics of the pollenand stigmas in the seagrasses are comparable with those foundin terrestrial flowering plants. The similarity in enzymaticproperites of the pollen wall and stigma pellicle suggests that,intriguingly, a similar mechanism of cuticle erosion might wellfollow compatible pollination both on land and in the sea. Enhalus acoroides, Thalassia hemprichii, Halophila stipulacea, Halophila decipiens, seagrasses pollen wall, stigma surface, hydrolytic enzymes     

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     

Localization of adenylate cyclase activity in Populus: its relation to pollen-pistil recognition and incompatibility

Summary Adenylate cyclase has been localized cytochemically in female and male parents as well as during the pollen-stigma interaction with an original technique employing strontium as the capture ion and adenyl imidodiphosphate as the specific substrate. The specificity of the reaction was checked by using several controls. No final specific reaction product was detected in unpollinated P. deltoides stigmas or in the P. deltoides or P. alba pollen grains used for compatible and incompatible pollinations. In the compatible cross between P. deltoides × P. deltoides, fine dense precipitates were observed in the dictyosomes and the plasma membrane and exterior to the exine of hydrated pollen grains adhering to the stigma surface. Labeling of the stigmatic pellicle was also observed after pollen adhesion and hydration. This was accompanied by a strong reactivity of the cell wall and plasmalemma of the stigma papillae at the sites of pollen tube germination on the stigma surface and at the sites of penetration of pollen tubes between adjacent papillae. In the incompatible cross between P. deltoides x P. alba, adenylate cyclase activity was still present but reduced at the stigma surface following adhesion, hydration, and germination of P. alba pollen. This activity was completely abolished after the penetration of pollen tubes between stigma papillae. These findings suggest that in Populus, adenylate cyclase activity is correlated to pollen adhesion, hydration, and germination at the stigma surface, and that the abolition of this enzyme activity could be one of the cellular events governing the gametophytic phenotype of incompatibility in the cross between P. deltoides and P. alba.     

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.     

Profiling and functional classification of esterases in olive (Olea europaea) pollen during germination

Background and Aims

A pollen grain contains a number of esterases, many of which are released upon contact with the stigma surface. However, the identity and function of most of these esterases remain unknown. In this work, esterases from olive pollen during its germination were identifided and functionally characterized.

Methods

The esterolytic capacity of olive (Olea europaea) pollen was examined using in vitro and in-gel enzymatic assays with different enzyme substrates. The functional analysis of pollen esterases was achieved by inhibition assays by using specific inhibitors. The cellular localization of esterase activities was performed using histochemical methods.

Key Results

Olive pollen showed high levels of non-specific esterase activity, which remained steady after hydration and germination. Up to 20 esterolytic bands were identified on polyacrylamide gels. All the inhibitors decreased pollen germinability, but only diisopropyl fluorophosphate (DIFP) hampered pollen tube growth. Non-specific esterase activity is localized on the surface of oil bodies (OBs) and small vesicles, in the pollen intine and in the callose layer of the pollen tube wall. Acetylcholinesterase (AChE) activity was mostly observed in the apertures, exine and pollen coat, and attached to the pollen tube wall surface and to small cytoplasmic vesicles.

Conclusions

In this work, for the first time a systematic functional characterization of esterase enzymes in pollen from a plant species with wet stigma has been carried out. Olive pollen esterases belong to four different functional groups: carboxylesterases, acetylesterases, AChEs and lipases. The cellular localization of esterase activity indicates that the intine is a putative storage site for esterolytic enzymes in olive pollen. Based on inhibition assays and cellular localization of enzymatic activities, it can be concluded that these enzymes are likely to be involved in pollen germination, and pollen tube growth and penetration of the stigma.     

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 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.     

Structural analysis of stigma development in relation with pollen–stigma interaction in sunflower

Structural analysis of stigma development in sunflower highlights the secretory role of papillae due to its semi-dry nature. Production of lipid-rich secretions is initiated at the staminate stage of the flowers in stigma development and increases at the receptive stage, coinciding with an extensive development of elaioplasts and endoplasmic reticulum network in the basal region of the papillae. Transfer cells, earlier identified only in the wet type of stigma, are also present in the transmitting tissue of the sunflower stigma. Attainment of physiological maturity by the stigmatic tissue, accompanying development from bud to pistillate stage, appears to affect the initial steps of pollen–stigma interaction. The nature of self-incompatibility in Helianthus has also been investigated in relation with pollen adhesion, hydration and germination. Pollen adhesion to the stigma is a rapid process in sunflower and stigma papillae exhibit greater affinity for pollen during cross pollination as compared to self-pollination. Components of the pollen coat and the pellicle on the surface of stigmatic papillae are critical for the initial phase of pollen–stigma interaction (adhesion and hydration). The lipidic components of pollen coat and the proteinaceous and lipidic components from the surface of the papillae coalesce during adhesion, leading to the movement of water from stigma to the pollen, thereby causing pollen hydration and its subsequent germination. Pollen germination (both in self-and cross-pollen) on the stigma surface and the growth of the pollen tube characterize the flexibility of self-incompatibility in sunflower. Compatible pollen grains germinate and the pollen tube penetrates the stigma surface to enter the nutrient-rich transmitting tissue. The pollen tube from incompatible pollen germination, however, fails to penetrate the stigmatic tissue and it grows parallel to the papillae. Present findings provide new insights into structural and functional relationships during stigma development and pollen–stigma interaction.     

Modification of the Pollen-Stigma Interaction in Brassica oleracea by Water

The presence of a film of distilled water on the stigma surfaceof freshly opened flowers results in complete inhibition ofpollen following both incompatible and compatible pollinationsin self-incompatible (SI) genotypes of Brassica oleracea, SIgenotypes of B. campestris and one self-compatible (SC) genotypeof B. campestris. The application of water to the stigmas afterpollination also resulted in a marked reduction in pollen germinationand tube penetration. An increase in the time intervals betweenthe application of pollen onto the stigma and the water treatmentprogressively reduced this inhibition. Pollen germination wasalso completely inhibited when stigmas from freshly-opened flowersof SI B. campestris and B. oleracea genotypes were washed inwater, dried and pollinated with pollen grains of either compatibility.The ability of stigmas to induce pollen germination and tubegrowth was restored over a period, the length of which was dependenton the incompatibility (S) genotype. Stigmas of B. napus (SC)and SC mutants of SI B. campestris were found to be affectedby washing, but stigmas of a SC variety of B. campestris andthe immature stigmas from buds of B. oleracea were found tobe considerably less affected. Microscopic examination of pollenplaced on washed stigmas reveals that grains, irrespective oftheir compatibility, fail to hydrate normally. When inducedto hydrate by raising atmospheric humidity, pollen grains onwashed stigmas did germinate, but most of the tubes failed topenetrate the papillar wall and very few entered the style.It is proposed that the water treatment mobilises componentsof the pellicle which reorganize to block the activity of molecules,present in both SC and SI individuals, responsible for establishingfull contact between the pellicle and pollen grain coating. Brassica, pellicle, pollen, recognition, self-incompatibility     

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,STYLE, AND OBTURATOR OF SOYBEAN,GLYCINE MAX (L.) MERR. (LEGUMINOSAE) AND THEIR FUNCTION IN THE REPRODUCTIVE PROCESS

Soybeans have a wet stigma overtopped by a pellicle that originates from the cuticle. There are numerous exudate-filled, axially oriented channels between cells of the transmitting tissue in the stigma and style. Pollen tubes grow in these channels and receive nutrition and mechanical guidance. Transmitting-tissue cells of the obturator are secretory also, but the obturator in soybean does not appear to control direction of pollen tube growth mechanically. The significant function of transmitting tissue in soybeans is to provide nutrition and to control direction of pollen tube growth.     

Stigma behavior in Mimulus aurantiacus (Scrophulariaceae)

The bilobed stigma of many species in the order Scrophulariales closes in response to touch by an animal pollinator. In hummingbird-pollinated bush monkey flower, Mimulus aurantiacus (Scrophulariaceae), closure is rapid, occurring within seconds of tactile stimulus. We investigated the proximate causes of stigma closure and subsequent reopening in M. aurantiacus, as well as potential costs and benefits of stigma closure for female fitness. Stigma closure is elicited by both touch and pollen, but closure in response to pollen is much slower, requiring 0.5–1.5 h. Stigmata reopen within 2.5–4.5 h if touch, but no pollen, is applied. Upon receipt of pollen, most stigmata remain closed for the remaining lifetime of the flower, even if less pollen is received than is needed for full seed set. Those stigmata that do reopen after pollination generally require between 20 and 28 h to do so, much longer than for unpollinated stigmata. Reopening after pollination appears to be a response to low seed set rather than to low pollen load. Natural pollination of stigmata manipulated to prevent closure shows that closure does not increase capture of pollen or seed set. In fact, closure reduces the average pollen load received by flowers. Despite this, there is no evidence that stigma closure has any negative effect on female fitness in terms of seed set or germinability. Hypotheses for the adaptive significance of stigma closure are discussed. Understanding proximate causes of stigma closure and reopening is essential in the evaluation of these hypotheses.     

Histochemical studies on the distribution of non-specific esterase in the germinating pollen grains ofPortulaca grandiflora

The study deals with the distribution of non-specific esterase in germinating pollen grains ofPortulaca grandiflora. Intense activity of the enzyme is seen in small granules distributed homogeneously in pollen grains stigma hairs and throughout the length of pollen tubes. Further the walls of pollen grains also demonstrate intense activity. The functional significance of the enzyme in these locales has been discussed.     

SD-RLK28 positively regulates pollen hydration on stigmas as a PCP-Bβ receptor in Arabidopsis thaliana

Pollen hydration on dry stigmas is strictly regulated by pollen–stigma interactions in Brassicaceae. Although several related molecular events have been described, the molecular mechanism underlying pollen hydration remains elusive. Multiple B-class pollen coat proteins(PCP-Bs) are involved in pollen hydration. Here, by analyzing the interactions of two PCP-Bs with three Arabidopsis thaliana stigmas strongly expressing S-domain receptor kinase(SD-RLK), we determined that SD-RLK28 directly intera...     

Pollen and stigma size changes during the transition from tristyly to distyly in Oxalis alpina (Oxalidaceae)

• Pollen and stigma size have the potential to influence male fitness of hermaphroditic plants, particularly in species presenting floral polymorphisms characterised by marked differences in these traits among floral morphs. In this study, we take advantage of the evolutionary transition from tristyly to distyly experienced by Oxalis alpina (Oxalidaceae), and examined whether modifications in the ancillary traits (pollen and stigma size) respond to allometric changes in other floral traits. Also, we tested whether these modifications are in accordance with what would be expected under the hypothesis that novel competitive scenarios (as in distylous‐derived reproductive system) exert morph‐ and whorl‐specific selective pressures to match the available stigmas.
• We measure pollen and stigma size in five populations of O. alpina representing the tristyly–distyly transition.
• A general reduction in pollen and stigma size occurred along the tristyly–distyly transition, and pollen size from the two anther levels within each morph converged to a similar size that was characterised by whorl‐specific changes (increases or decreases) in pollen size of different anthers in each floral type.
• Overall, results from this study show that the evolution of distyly in this species is characterised not only by changes in sexual organ position and flower size, but also by morph‐specific changes in pollen and stigma size. This evidence supports the importance of selection on pollen and stigma size, which increase fitness of remaining morphs following the evolution of distyly, and raises questions to explore on the functional value of pollen size in heterostylous systems under pollen competition.

     

Pollen tube growth in association with a dry-type stigmatic transmitting tissue and extragynoecial compitum in the basal angiosperm Kadsura longipedunculata (Schisandraceae)

Spatial features of pollen tube growth and the composition of the extracellular matrix (ECM) of transmitting tissue in carpels of Kadsura longipedunculata, a member of the basal angiosperm taxon Schisandraceae, were characterized to identify features of transmitting tissue that might have been important for pollen-carpel interactions during the early history of angiosperms. In addition to growing extracellularly along epidermal cells that make up stigmatic crests of individual carpels, pollen tubes grow on abaxial carpel epidermal cells between unfused carpels along an extragynoecial compitum to subsequently enter an adjacent carpel, a feature important for enhancing seed set in apocarpous species. Histo- and immunochemical data indicated that transmitting tissue ECM is not freely flowing as previously hypothesized. Rather, the ECM is similar to that of a dry-type stigma whereby a cuticular boundary with associated esterase activity confines a matrix containing methyl-esterified homogalacturonans. The Schisandraceae joins an increasing number of basal angiosperm taxa that have a transmitting tissue ECM similar to a dry-type stigma, thereby challenging traditional views that the ancestral pollen tube pathway was similar to a wet-type stigma covered with a freely flowing exudate. Dry-type stigmas are posited to provide tighter control over pollen capture, retention, and germination than wet-type stigmas.