In vitro allelopathic effects of pollen from three Hieracium species (Asteraceae) and pollen transfer to sympatric Fabaceae

Pollen allelopathy occurs when pollen toxins inhibit sexual reproduction in heterospecifics. To quantify pollen allelopathic effects in vitro, pollen of Hieracium aurantiacum, H. floribundum, and H. pratense was extracted with double distilled water into concentrations of 0 (control), 0.1, 1, 3, 5, 10, and 25 pollen grains/μl. A subset of these extracts was partitioned by ion-exchange chromatography into acidic, basic, and neutral fractions. Pollen from six sympatric species of Fabaceae (Lotus corniculatus, Medicago sativa, Trifolium hybridum, T. pratense, T. repens, Vicia cracca) and the species of Hieracium was germinated on agar media containing different extract concentrations. As extract concentrations from intact pollen or from acidic fractions increased, mean percent germination of pollen of all Fabaceae decreased nonlinearly. Extract concentrations of 0.1 grains/μl and above inhibited pollen germination in the Fabaceae. In no case was any Hieracium species affected, i.e., there was no autotoxicity. Similar results were obtained using plants from nine populations of H. floribundum and H. pratense across eastern Canada. Observation of floral visitors indicated that there were relatively few movements between Hieracium and the Fabaceae (about 15% of total visits). Hence there is limited opportunity for heterospecific pollen transfer. This was consistent with censuses of stigma, i.e., few pollen grains of Hieracium were found on stigma of the Fabaceae. Thus, pollen allelopathy in Hieracium is unlikely to affect reproductive success in these six species of Fabaceae.     

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.     

PROTANDRY,INCOMPATIBILITY, AND SECONDARY POLLEN PRESENTATION IN CEPHALANTHUS OCCIDENTALS (RUBIACEAE)

Cephalanthus occidentalis L. is protandrous and presents pollen secondarily on the stigma surface. Because self-pollen is present on the stigma, the degree of selling vs. outcrossing in this species will depend on 1) the phenology of pollen presentation and stigma receptivity; 2) whether the species is self-incompatible; and 3) the rates of self vs. crossed pollen tube growth. This study describes floral morphology and phenology, self-incompatibility, and pollen tube growth rates in self- and crosspollinations of C. occidentalis. Light and scanning electron microscopy were used to study stigma morphology after flower opening, while controlled pollinations tested for incompatibility. Stigmas were unreceptive initially but became receptive by the second day after flower opening. Ninety-two percent of cross-pollinated flowers set fruit, compared to 12% fruit set in self-pollinations. Pollen tubes from selfed and out-crossed pollen initially had similar growth rates. Out-crossed pollen tubes began to grow rapidly ca. 5 hr after pollination of a receptive stigma, whereas selfed pollen tubes ceased growth or grew slowly after this time. Pollen tubes from out-crossed pollen grew the length of the style within 24 hr after pollination, while selfed pollen tubes were inhibited at the stigma-style junction. Our results indicate that C. occidentalis has selfincompatibility, in addition to protandry and secondary pollen presentation. Protandry allows removal of self-pollen from the unreceptive stigma, while self-incompatibility prevents fertilization by unremoved self-pollen.     

In vitro test of self-incompatibility in Malus domestica

Summary An in vitro assay in which self-incompatible pollen of Malus domestica is selectively inhibited is described. This assay involves heat-labile substances diffusing from the stylar tissues — in particular, glycoproteins found in the protein extract of styles. In the presence of the self-style extract, a dramatic decrease in total protein concentration in the culture medium was revealed at 30-min germination. Pretreatment of the self-pollen with 100 mM glucose prevented this drop in protein level; moreover, tube growth was entirely restored. A possible explanation in terms of protein-carbohydrate complementation is suggested.     

Papillate Tepal Hoods and Delayed Pollen Germination inAlbucaL. (Liliaceae)

In the genusAlbuca, pollen germination is delayed until thetepals close around the stigma immediately before the onsetof floral senescence. At this time, papillae on the tepal apicesand the stigma swell and produce an exudate in which pollenrapidly germinates. No pollen germination occurs when the tepalsare artificially prevented from closing around the stigma.Copyright1998 Annals of Botany Company Albuca, Liliaceae, delayed pollen germination, gametophytic self-incompatibility, perianth contributing to pollen germination, pollen tube inhibition in ovary.     

The cross-fertilization mechanism and reproductive interference of self-pollen of Medicago varia ‘Xinmu No. 1’

Some control tests and a technique employing the fluorescent properties of pollen and pollen tube stained with aniline blue dye were utilized to study the stigma receptivity, the optimum pollination period, the self-pollen clogging effect, pollen germination and the pollen tube growth state in alfalfa. The results can be summarized as follows: (1) The stigma receptivity and flower longevity barely overlap. On the first day after pollination, the pollination efficiency was the highest, with a pod-seting rate per raceme of 65.17%±2.01% (P<0.01). The period of the highest pollination efficiency was the same as the peak of stigma receptivity. When pollination was on the fourth day after flowering, the pod-setting percentage per raceme was still 34.25%±6.73%. (2) Self-pollination led to a lower pollen germination frequency and a lower pollen tube growth rate than cross-pollination. When artificial cross-pollination was applied after self-pollen grains on the stigma was re-moved, the pod-setting percentage per raceme was 72.31%±6.24%, and the number of seed per pod was 3.46±0.25, In contrast, these two values were 51.76%±5.37% (P<0.01) and 2.11%±0.18 respectively (P<0.01) when the self-pollen grains were not removbed in the artificial cross-pollination experiements. Hence, the clog-ging effect of self-pollen to stigma was significant. (3) In the breeding system of alfalfa, there are two mecha-nisms that help the plants to avoid self-fertilization: dichogamy and self-incompatibility. However, due to the structure of flowers, they were not effective enough to improve cross-fertilization rate, even if the stamen matured earlier than the stigma. The typical self-incompatibility system guaranteed the lower self-fertilization rate, but cannot avoid the reproductive interference of self-pollen.     

Pollen-pistil interaction in Brassica oleracea

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

ROLE OF CALCIUM IN THE CALLOSE RESPONSE OF SELF-POLLINATED BRASSICA STIGMAS

In Brassica oleracea, sporophytic self-incompatibility prevents germination of self pollen, or normal growth of self pollen tubes. After self-pollination, the papillae of stigmas synthesize callose. The role of Ca⁺⁺ in the formation of stigmatic callose was tested by adding compounds that interact with Ca⁺⁺ to suspensions of pollen that were known to induce callose formation in self stigmas. The calcium channel antagonist, lanthanum, and the calcium chelating agent, EGTA, reduced or abolished the callose response to self-pollen suspensions. In the presence of Ca⁺⁺, the calcium ionophore, A23187, induced callose in stigmatic papillae when added to pollen suspensions, or alone. Therefore, callose deposition in response to incompatible pollinations appears to be a calcium-dependent process. Pretreatment of pistils with 100 μm 2-deoxy-D-glucose abolished the callose response to self-pollination, while self pollen remained inhibited and cross pollen grew normally in treated pistils. Thus, callose formation in the stigma is not an essential part of the self-incompatibility mechanism preventing the growth of self pollen in Brassica.     

Germination Inhibition in Stigma Extracts of Tobacco and Identification of MeABA,ABA, and ABA-γ-d-Glucopyranoside

Extracts of various flower tissues of tobacco with 70% methanol inhibited tobacco seed germination differently. Among them, extracts of stigma and anther were very inhibitory. When the extracts were partitioned between ethyl acetate and water, the activity of the ethyl acetate layer was stronger than that of the water layer. Stigmas and anthers had more abscisic acid (ABA) than the other floral tissues, which matched the results of the germination tests well. Guided by a bioassay using the inhibitory effects on tobacco seed germination, MeABA, ABA, and ABA-γ-d-glucopyranoside were isolated and identified from stigmas. All of the MeABA isolated did not seem to be an artefact produced by esterification with the solvent, for MeABA was detected even when stigmas were extracted with other solvents than methanol. MeABA and ABA did not inhibit tobacco pollen germination and elongation in vitro.     

Genetic architecture of yield and components of yield in mustard (Brassica juncea (L.) Czern & Coss.)

Summary The relative ability of cross- and self-pollen to achieve fertilisation in Brassica was studied by making double pollinations using cross-pollen carrying a dominant seedling marker gene. With simultaneous self- and crosspollination 12–40% self-seed was set, but when cross-pollen was applied to the stigma four hours before self-pollen, only 2–4% self-seed was obtained. In two plants to which cross-pollen was applied at various time intervals after self-pollen there was a tendency for the percentage of self-seed to increase as the time interval increased. In a third plant this trend was not apparent, probably because of a greater degree of self-incompatibility. The competitive advantage of the first pollen to arrive on the stigma is discussed in relation to the strength of the self-incompatibility and the sib problem in F₁ hybrid brassicas.     

Leaf and root extracts of the invasive shrub, Lonicera maackii, inhibit seed germination of three herbs with no autotoxic effects

In addition to effects mediated by resource competition, some invasive plants may impact surrounding vegetation by secreting compounds that are directly inhibitory to growth. Lonicera maackii, an invasive Asian shrub of forests and open areas in eastern and midwestern North America, has devastating effects on understory vegetation, some of which persist even after this shrub is removed. In this study, we explored the potential of aqueous extracts of the leaves and roots of this plant to inhibit seed germination of Impatiens capensis, Alliaria petiolata, Arabidopsis thaliana, and L. maackii in Petri dish bioassays. Both L. maackii root and leaf extracts significantly decreased germination in the three herb species. This inhibitory effect generally increased with increasing extract concentration and was more pronounced with application of leaf extract than root extract. However, when the same extracts were applied to seeds of L. maackii itself, germination was delayed in some cases, but was not significantly reduced by the end of the experiment. Germination of L. maackii seeds even reached significantly higher levels in some extract treatments than in no-extract controls. This implies that L. maackii can successfully inhibit the germination of other plants with few autotoxic effects and may even promote the germination of its own seeds.     

Sulfinylated azadecalins act as functional mimics of a pollen germination stimulant in Arabidopsis pistils

Polarized cell elongation is triggered by small molecule cues during development of diverse organisms. During plant reproduction, pollen interactions with the stigma result in the polar outgrowth of a pollen tube, which delivers sperm cells to the female gametophyte to effect double fertilization. In many plants, pistils stimulate pollen germination. However, in Arabidopsis, the effect of pistils on pollen germination and the pistil factors that stimulate pollen germination remain poorly characterized. Here, we demonstrate that stigma, style, and ovules in Arabidopsis pistils stimulate pollen germination. We isolated an Arabidopsis pistil extract fraction that stimulates Arabidopsis pollen germination, and employed ultra‐high resolution electrospray ionization (ESI), Fourier‐transform ion cyclotron resonance (FT‐ICR) and MS/MS techniques to accurately determine the mass (202.126 Da) of a compound that is specifically present in this pistil extract fraction. Using the molecular formula (C₁₀H₁₉NOS) and tandem mass spectral fragmentation patterns of the m/z (mass to charge ratio) 202.126 ion, we postulated chemical structures, devised protocols, synthesized N‐methanesulfinyl 1‐ and 2‐azadecalins that are close structural mimics of the m/z 202.126 ion, and showed that they are sufficient to stimulate Arabidopsis pollen germination in vitro (30 μm stimulated approximately 50% germination) and elicit accession‐specific response. Although N‐methanesulfinyl 2‐azadecalin stimulated pollen germination in three species of Lineage I of Brassicaceae, it did not induce a germination response in Sisymbrium irio (Lineage II of Brassicaceae) and tobacco, indicating that activity of the compound is not random. Our results show that Arabidopsis pistils promote germination by producing azadecalin‐like molecules to ensure rapid fertilization by the appropriate pollen.     

REGULATION OF THE TIMING OF POLLEN GERMINATION BY THE PISTIL IN TALINUM MENGESII (PORTULACACEAE)

In angiosperms selection among male gametophytes may take place between the time a flower is pollinated and the time its ovules are fertilized. With insect pollination, a surplus of male gametophytes in the form of pollen grains may be deposited on the stigma of a pistil and this provides the potential for gametophytic competition and selection. In all plants examined from two Talinum mengesii populations, pollen germination was delayed for up to two hours after pollination. In plants of two other populations pollen germination was not delayed or the time of germination varied. Reciprocal pollinations between plants with and without the delay trait and pollinations at different times during anthesis revealed that the timing of pollen germination was regulated by the maternal parent. Theoretically, the effect of the delay trait would be to promote the accumulation of pollen on the stigma and induce simultaneous germination. This would intensify intermale competition and provide a mechanism for the maternal parent to influence the paternal parentage of its offspring.     

COSTS OF SELF-POLLINATION IN A SELF-INCOMPATIBLE PLANT,POLEMONIUM VISCOSUM

Plants of Polemonium viscosum are strongly self-incompatible, yet have floral features that enforce spatial and temporal separation of pollen and stigma presentation. In this study, we address the widely held view that such barriers to selfing are maintained because they reduce interference by self pollen in performance of outcross grains. In the first year of the study we found that 24 hr precedence of self pollen on the stigma (24S), reduced outcross pollen germination by 32%, and seed set by 40% in flowers of P. viscosum. In the second year significant negative effects of self pollen on compatible pollen germination were seen when self pollen was applied four hours before (4S) or immediately prior (S×) to outcross pollen. Surprisingly, however, in the second year of study, seed set was not reduced with as much as 50% inhibition of compatible pollen germination. Results suggest that by interfering with normal functioning of outcross grains, incompatible self pollen can increase pollen limitation in P. viscosum. Reduction in seed production as a result of this phenomenon should select for displacement of pollen presentation from stigma receptivity.     

Status of self-pollen in bee pollination efficiency of white clover (Trifolium repens L.)

Flowers of white clover (Trifolium repens L.) are hermaphrodite and self-incompatible; their cross-pollination depends entirely on insect visitors, mainly bees (Apoidea). Because self-pollination of white clover occurs before flower anthesis, we determined whether selfing affected the pollination efficiency of a honeybee visit. We compared pollen deposition in emasculated and intact flowers following (1) a single honeybee visit, (2) open-pollination for a day and (3) enclosure in a cloth bag to prevent insect visits. In emasculated flowers, open-pollination resulted in more pollen deposited than after one visit (+30%) which is consistent with flowers being visited more than once by pollinators during the course of a day. On intact flowers, saturation of the stigma was achieved after the first visit of a honeybee (near 280 grains) because of self-pollination. Additional visits did not increase pollen deposits, but they improved pollen efficiency in terms of numbers of pollen tubes reaching the ovules. In such a context of easily saturated stigmas, self-pollen does not inhibit cross-pollen activity, but represents a constraint for pollination which demands multiple bee visits to each flower to achieve maximum fertilization. Received: 20 May 1997 / Accepted: 25 October 1997     

Self-interference is reduced in a secondary pollen presentation species, Duperrea pavettifolia (Rubiaceae)

Occurrence of secondary pollen presentation (SPP) is evidenced for Duperrea pavettifolia. For this, self-pollen was presented on the stigma and reduced fertilization was observed in this case. The receptive area located in the furrow of the stigma becomes receptive from the third day of elongation growth. Fruit set upon hand pollination with pollen from other individuals was significantly higher than the results of selfed, bagged, emasculated and control treatments. However there was no difference in pollen tube growth rate between selfed and crossed pollen on a receptive stigma. Hawkmoths and butterflies were effective pollinators for D. pavettifolia, but the visiting frequencies were very low. Stingless bees removed pollen from the unreceptive stigma which had no contribution to reproductive success. High level of outcrossing in D. pavettifolia was demonstrated by molecular analyses using the simple sequence repeats (SSR) method. Although the wild populations of D. pavettifolia are small and with fragmented distribution, the genetic diversity of seedlings was high, with fluctuations among years. Our results indicated that protandry and the visitation of stingless bees reduced the amount of self-pollen on the still unreceptive stigma and self-incompatibility prevented fertilization by un-removed self-pollen.     

The extracellular lipase EXL4 is required for efficient hydration of Arabidopsis pollen

Pollination in species with dry stigmas begins with the hydration of desiccated pollen grains on the stigma, a highly regulated process involving the proteins and lipids of the pollen coat and stigma cuticle. Self-incompatible species of the Brassicaceae block pollen hydration, and while the early signaling steps of the self-incompatibility response are well studied, the precise mechanisms controlling pollen hydration are poorly understood. Both lipids and proteins are important for hydration; loss of pollen coat lipids and proteins results in defective or delayed hydration on the stigma surface. Here, we examine the role of the pollen coat protein extracellular lipase 4 (EXL4), in the initial steps of pollination, namely hydration on the stigma. We identify a mutant allele, exl4-1, that shows a reduced rate of pollen hydration. exl4-1 pollen is normal with respect to pollen morphology and the downstream steps in pollination, including pollen tube germination, growth, and fertilization of ovules. However, owing to the delay in hydration, exl4-1 pollen is at a disadvantage when competed with wild-type pollen. EXL4 also functions in combination with GRP17 to promote the initiation of hydration. EXL4 is similar to GDSL lipases, and we show that it functions in hydrolyzing ester bonds. We report a previously unknown function for EXL4, an abundant pollen coat protein, in promoting pollen hydration on the stigma. Our results indicate that changes in lipid composition at the pollen–stigma interface, possibly mediated by EXLs, are required for efficient pollination in species with dry stigmas.     

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.     

In vitro chemotropism of pearl millet pollen tubes to stigma tissue: a response to glucose produced in the medium by tissue-bound invertase

Summary Water-homogenized stigma pellets of pearl millet and precipitates resulting from dialysis of their salt extracts were observed to: (1) chemotropically attract pearl millet pollen tubes on a sucrose-containing pollen germination and growth medium, (2) have acid invertase activity as assayed by the arsenomolybdate method, (3) hydrolyze sucrose in the pollen germination and growth medium to glucose as assayed by coupled glucose oxidation with Nitro Blue Tetrazolium, and (4) lose chemotropic and invertase activities upon heat treatment. The results indicate that the in vitro chemotropic attraction of pearl millet pollen tubes to water-homogenized stigma pellets is a response to glucose produced by homogenate-pellet-bound invertase hydrolyzing the sucrose present in the pollen germination and growth medium. Yeast and tomato invertases used as controls verified this conclusion. Water extracts of whole stigmas contained water-soluble acid invertase. The results are discussed in relation to the identification of possible in vivo chemotropic factors of pearl millet and other plants by in vitro assays.Abbreviations dH₂O Deionized, house-distilled water - NBT Nitro Blue Tetrazolium, NBT-medium - PGG medium, pollen germination and growth medium (10% sucrose, 1 mM H₃BO₃, and 1% agarose); - WHS pellet, water-homogenized stigma pellet On Specific Cooperative Agreement 58-6612-8-002 with the Department of Biochemistry, University of Georgia, Athens, GA 30602, USA     

Mixed Mating System Are Regulated by Fecundity in Shorea curtisii (Dipterocarpaceae) as Revealed by Comparison under Different Pollen Limited Conditions

The maintenance of mixed mating was studied in Shorea curtisii, a dominant and widely distributed dipterocarp species in Southeast Asia. Paternity and hierarchical Bayesian analyses were used to estimate the parameters of pollen dispersal kernel, male fecundity and self-pollen affinity. We hypothesized that partial self incompatibility and/or inbreeding depression reduce the number of selfed seeds if the mother trees receive sufficient pollen, whereas reproductive assurance increases the numbers of selfed seeds under low amounts of pollen. Comparison of estimated parameters of self-pollen affinity between high density undisturbed and low density selectively logged forests indicated that self-pollen was selectively excluded from mating in the former, probably due to partial self incompatibility or inbreeding depression until seed maturation. By estimating the self-pollen affinity of each mother tree in both forests, mother trees with higher amount of self-pollen indicated significance of self-pollen affinity with negative estimated value. The exclusion of self-fertilization and/or inbreeding depression during seed maturation occurred in the mother trees with large female fecundity, whereas reproductive assurance increased self-fertilization in the mother trees with lower female fecundity.