Primary style protein expression in the self-incompatible/compatible apricot by the 2D-DIGE technique

In order to explore the molecular mechanism underlying self-incompatibility (SI) in the apricot (Prunus armeniaca L.) at the proteome level, we examined the style proteomes at different stages of flower development: small bud, big bud, 24h after self-pollination and 24h after cross-pollination with cultivar Badanshui in the SI apricot cultivar Xinshiji and the self-compatible (SC) apricot cultivar Katy by 2D fluorescence difference gel electrophoresis (2D-DIGE) and mass spectrometry (MS). About 1500 style protein spots were detected; 66 were expressed differently in the four stages in Xinshiji. About 1600 style protein spots were detected; 143 were expressed differently in the four stages of flower development in Katy. In Xinshiji, one protein was expressed specifically, four proteins showed up-regulated expression and twenty-nine proteins showed down-regulated expression in the cross-pollinated style compared to the self-pollinated style. Thirteen proteins were identified unambiguously. In Katy, three proteins were expressed specifically, five proteins showed up-regulated expression and thirteen proteins showed down-regulated expression in the cross-pollinated style compared to self-pollinated style. Seven proteins were identified unambiguously. The different reactions of the style at the proteomic level were triggered in Xinshiji and Katy by self pollen and non-self pollen.     

Pollination biology and breeding system of Zeyheria montana (Bignoniaceae)

The reproductive biology of Zeyheria montana was studied through field observations of flower visitors and floral events, controlled manual pollinations, and observations of pollen tube growth and ovule penetration by fluorescence microscopy. Analysis of secretory areas of the nectar chamber was made by flower dissections and histology of serial sections. The flower lasted 6–8 days, but pollen exposure and stigma receptivity occurred only up to the end of the first and second days, respectively. Pollination was effected by several species of hummingbirds, especially Colibri serrirostris. The flowers present a rudimentary, non-functional disc, and secretion of nectar is performed by corolla-borne glandular trichomes. Only hand cross-pollinated and natural-pollinated flowers set fruits. Artificially self-pollinated and non-pollinated flowers dried off after anthesis without presenting any swelling of the ovary. Almost all the ovules in selfed and crossed pistils were penetrated 96 h after pollination. However, a delay in ovule penetration in self-pollinated pistils was verified, which indicates the occurrence of late-acting self-incompatibility.We are grateful to Fundo de Apoio ao Ensino e à Pesquisa (UNICAMP) for financial support, and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for the fellowship granted to the first author (this work is part of a Ph.D. thesis presented to UNICAMP, Campinas, SP, Brazil).     

Detection of Proteins Possibly Involved in Self-Incompatibility Response in Distylous Buckwheat

Buckwheat (Fagopyrum esculentum Moench) is a heterostylous plant displaying heteromorphic sporophytic self-incompatibility (SI). In order to detect proteins involved in SI, pistils from both long and short styles were isolated and then selfed or cross-pollinated. One-dimensional gel electrophoresis revealed that short pistils 2 h after selfing contained an unique 50 kDa protein. In the two-dimensional electrophoresis two distinct groups of proteins possibly involved in SI response were detected in the short, and one in the long pistils. This revised version was published online in July 2006 with corrections to the Cover Date.     

Floral-specific polypeptides of the Japanese morning glory

Polypeptides from stems, leaves, sepals, corollas, stamens and pistils of the Japanese morning glory (Ipomoea nil Roth (Pharbitis nil Chois.)) were separated by one- and two-dimensional gel electrophoresis and visualized by silver staining. The majority of polypeptides were expressed in two or more organs, while those specific to only one organ were comparatively rate. Among the polypeptides of the former class were two which appeared to be floral-specific. A 46-kDa (kilodalton) polypeptide was expressed in corollas, stamens and pistils, whereas a 32-kDa polypeptide was observed only in extracts prepared from reproductive organs. Polypeptide spots from the various organs were compared with those from leaves, and it was found that sepals and stems shared 40–50% of their polypeptides with leaves, whereas corollas, stamens and pistils shared 20% or less. The latter organs shared 120 polypeptides or roughly 15% of those identified in the floral extracts. Floralorgan-specific polypeptides comprised nearly 10% of the total floral polypeptides identified.Abbreviations kDa kilodalton - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecylsulfate     

Dissecting Pistil Responses to Incompatible and Compatible Pollen in Self-Incompatibility <Emphasis Type="Italic">Brassica oleracea</Emphasis> Using Comparative Proteomics

Angiosperms have developed self-incompatibility (SI) systems to reject self-pollen, thereby promoting outcrossing. The Brassicaceae belongs to typical sporophytic system, having a single S-locus controlled SI response, and was chosen as a model system to study SI-related intercellular signal transduction. In this regard, the downstream factor of EXO70A1 was unknown. Here, protein two-dimensional electrophoresis (2-DE) method and coupled with matrix-assisted laser desorption ionization/time of flight of flight mass spectrometry (MALDI-TOF -MS) and peptide mass fingerprinting (PMF) was used to further explore the mechanism of SI responses in Brassica oleracea L. var. capitata L. at protein level. To further confirm the time point of protein profile change, total proteins were collected from B. oleracea pistils at 0 min, 1 h, and 2 h after self-pollination. In total 902, 1088 and 1023 protein spots were separated in 0 min, 1 h and 2 h 2-DE maps, respectively. Our analyses of self-pollination profiles indicated that proteins mainly changed at 1 h post-pollination in B. oleracea. Moreover, 1077 protein spots were separated in cross-pollinated 1 h (CP) pistil 2-DE map. MALDI-TOF-MS and PMF successfully identified 34 differentially-expressed proteins (DEPs) in SP and CP 1 h 2-DE maps. Gene ontology and KEGG analysis revealed an array of proteins grouped in the following categories: stress and defense response (35%), protein metabolism (18%), carbohydrate and energy metabolism (12%), regulation of translation (9%), pollen tube development (12%), transport (9%) and cytoskeletal (6%). Sets of DEPs identified specifically in SP or only up-regulated expressed in CP pistils were chosen for funther investigating in floral organs and during the process of self- and cross-pollination. The function of these DEPs in terms of their potential involvement in SI in B. oleracea is discussed.     

沙田柚配子体自交不亲和花柱消减文库的构建及分析

分别以沙田柚自交花柱cDNA为tester,异交花柱cDNA为driver,利用抑制性消减杂交技术构建了消减文库,文库的重组率高于95%,插入片段集中在100～500 bp之间,对文库部分克隆进行测序并与GenBank中的同源序列进行比较,发现了一些类似于SI、S9-RNase、激酶类等与自交不亲和相关的基因.     

Partial self‐incompatibility in the polyploid endemic species Scalesia affinis (Asteraceae) from the Galápagos: remnants of a self‐incompatibility system?

Different pollination treatments of capitula were used to examine the breeding system of individuals of the tetraploid endemic species Scalesia affinis from the Galápagos Islands. All types of crossings resulted in approximately 35 achenes per capitulum, but in actively and passively self-pollinated capitula these were mostly without embryos. Among self-pollinated individuals a large variation was found in the production of embryos. Some individuals failed completely to set filled achenes while others produced few or many. Additionally, we found a clear difference in female phenology of florets in self-pollinated capitula compared with florets in cross-pollinated capitula. Female florets in self-pollinated capitula remained receptive for longer time. These results suggest that Scalesia affinis is partly self-incompatible. Outcrossing is assured by the endemic carpenter bee, Xylocopa darwini , which proved to be an important pollinator of Scalesia affinis . Nevertheless, geitonogamy seemed to be considerable in the focal population, for which reason open pollinated capitula produced fewer achenes than controlled outcrossed capitula.  © 2003 The Linnean Society of London, Botanical Journal of the Linnean Society , 2003, 142 , 93–101.     

Characterization of a gene family abundantly expressed in Oenothera organensis pollen that shows sequence similarity to polygalacturonase.

We have isolated and characterized cDNA clones of a gene family (P2) expressed in Oenothera organensis pollen. This family contains approximately six to eight family members and is expressed at high levels only in pollen. The predicted protein sequence from a near full-length cDNA clone shows that the protein products of these genes are at least 38,000 daltons. We identified the protein encoded by one of the cDNAs in this family by using antibodies to beta-galactosidase/pollen cDNA fusion proteins. Immunoblot analysis using these antibodies identifies a family of proteins of approximately 40 kilodaltons that is present in mature pollen, indicating that these mRNAs are not stored solely for translation after pollen germination. These proteins accumulate late in pollen development and are not detectable in other parts of the plant. Although not present in unpollinated or self-pollinated styles, the 40-kilodalton to 45-kilodalton antigens are detectable in extracts from cross-pollinated styles, suggesting that the proteins are present in pollen tubes growing through the style during pollination. The proteins are also present in pollen tubes growing in vitro. Both nucleotide and amino acid sequences are similar to the published sequences for cDNAs encoding the enzyme polygalacturonase, which suggests that the P2 gene family may function in depolymerizing pectin during pollen development, germination, and tube growth. Cross-hybridizing RNAs and immunoreactive proteins were detected in pollen from a wide variety of plant species, which indicates that the P2 family of polygalacturonase-like genes are conserved and may be expressed in the pollen from many angiosperms.     

Comparative studies on the reproductive systems ofCardamine flexuosa,C. impatients,C. scutata,andC. lyrata,cruciferae

The reproductive systems of fourCardamine species were compared.C. flexuosa ssp.flexuosa, a self-pollinated winter annual in fallow paddy fields, produces numerous small seeds.C. flexuosa ssp.fallax, a self-pollinated year-long annual in gardens and roadsides, also produces many small seeds.C. impatiens, a self-pollinated annual in forest margins, similarly produces numerous small seeds.C. scutata is a self-pollinated perennial in ill-drained paddy fields and creeks; its vegetative reproduction is not predominant, and it produces relatively many seeds.C. lyrata, a cross-pollinated perennial with self-incompatibility in ill-drained paddy fields and creeks, produces fewer and heavier seeds, but it does produce many ramets. Moreover, in seed-dispersal mechanisms and germination behavior of fourCardamine species several adaptive characteristics to their habitats are found. These observations on the reproductive systems of fourCardamine species apparently present different characteristics of life-history strategy for each of them.     

Effects of Protein Phosphatase Inhibitors and Calcium Antagonists on Self-Incompatible Reaction in Buckwheat

Isolated pistils of dimorphic buckwheat (Fagopyrum esculentum Moench.) flowers were treated with phosphatase inhibitors (ocadaic acid and cantharidin) and with calcium antagonists (verapamil, La³⁺, and A23187). They were subsequently cross- or self-pollinated, and the growth of pollen tubes was observed under the fluorescence microscope. All treatments suppressed inhibition of pollen tubes growth suggesting that protein phosphatases and calcium signaling may be involved in self-incompatibility signal transduction in buckwheat. This revised version was published online in July 2006 with corrections to the Cover Date.     

Pollen tube growth and early ovule development following self- and cross-pollination in<Emphasis Type="Italic"> Eucalyptus nitens</Emphasis>

Controlled self- and cross-pollinations were conducted on flowers of five mature Eucalyptus nitens trees. Levels of self-sterility of the trees ranged from 25.8 to 93.6%. Pollen tube numbers in styles and ovule penetration by pollen tubes was investigated 2 weeks after pollination by fluorescence microscopy. There were no significant differences between treatments in the number of pollen tubes present in styles or in the percentage of ovules penetrated by pollen tubes. Embryology of material harvested 2 and 4 weeks after pollination was investigated by bright-field microscopy. Fertilisation had taken place by 2 weeks after pollination with nearly every ovule showing evidence of fertilisation. Cross-pollination resulted in a greater proportion of healthy, developing ovules, at both 2 and 4 weeks after pollination, compared with self-pollination. The proportion of degenerating ovules increased from 2 to 4 weeks after pollination. The reduced ability of E. nitens to set self-pollinated seed compared with cross-pollinated seed appears to be controlled by a post-zygotic mechanism. Differences in ovule size may potentially assist in the identification of trees incapable of setting self-pollinated seed.     

The Site of the Self-incompatibility Mechanism in Tradescantia pallida

Cytochemical and ultrastructural investigations were carriedout on unpollinated, self-pollinated and cross-pollinated pistilsof Tradescantia pallida. A protein secretion was found to occuron the unicellular papillac of the stigma in increasing amountstowards the base of the cells. The active cytoplasm of the cellsseemed to be responsible for this secretion. Compatible cross-pollinationresulted in pollen tube growth through the secretion and downthe stylar canal, whereas self-pollination resulted in pollentube growth being halted at the base of the stigma. It is suggestedthat the secretion may play an important role in the self-incompatibilityresponse. Tradescantia pallida, self-incompatibility, self-pollination, cross-pollination     

Effects of self-pollination and outcrossing with cultivated plants in small natural populations of American ginseng, Panax quinquefolius (Araliaceae)

For rare plants, self-pollination and inbreeding can increase in small populations, while unusual levels of outcrossing can occur through restoration efforts. To study both inbreeding and outcrossing, we performed experimental pollinations using Panax quinquefolius (American ginseng), a wild-harvested plant with a mixed mating system. For inbreeding, plants were either cross-pollinated within the population or self-pollinated, which resulted in a higher proportion of seeds from self-pollinated flowers. For outcrossing, wild plants were either cross-pollinated within the population or with cultivated plants from West Virginia or Wisconsin. Offspring of all crosses were followed for 4 yr. Two-yr-old seedlings from self-pollination had 45% smaller leaf areas and 33% smaller heights relative to those from cross-pollination. Leaf area is a positive predictor of longer-term survival in wild populations. Our results suggest inbreeding depression, which is unexpected in this self-fertile species. Seedlings from crosses with cultivated plants had 127% greater leaf area and 165% greater root biomass relative to outcrosses within the population. The accelerated growth suggests genetic differences between wild and cultivated populations, but outbreeding depression may not appear until later generations. Assessment of the ultimate fitness consequences of introducing cultivated genotypes requires monitoring over longer time periods.     

Histological study of post-pollination events in Spathodea campanulata beauv. (Bignoniaceae), a species with late-acting self-incompatibility

The reproductive biology of Spathodea campanulata was investigated by means of hand-pollination experiments, observations of pollen tube growth using fluorescence microscopy, and serial sections of ovules in selfed and crossed pistils. Only cross-pollinated flowers developed fruits, and all selfed flowers abscised within 3-4 d. However, self pollen tubes grew successfully to the ovary, penetrating and fertilizing the majority of ovules by 48 h, indicating that S. campanulata is a species with late-acting self-incompatibility. The incidences of ovule penetration, fertilization and endosperm initiation were all significantly slower in selfed vs. crossed pistils, although no other signs of malfunctioning were detected. The possible role of such slow self pollen tube effectiveness as a recognition event is discussed within the context of the slow but not entirely suppressed self pollen tube growth reported for some species with conventional homomorphic self-incompatibility.     

Relationships between five species ofLolium (Poaceae)

Morphological analysis and electrophoresis of seed proteins of fiveLolium species disclosed that they form two distinct groups corresponding to those recognized from compatibility data.Lolium temulentum andL. remotum of the self-pollinated group were shown to be distinct but closely related species. Morphological intergradation and high similarities between protein profiles ofL. perenne, L. multiflorum andL. rigidum (cross-pollinated species) suggest little genetic differentiation between these taxa. This implies that treatment at the infraspecific level might better accomodate the data.     

Regulations of the C2H4-forming system and the H2O2-scavenging system by heat treatment associated with self incompatibility in lily

The mechanism of self incompatibility in pistils of Lilium longiflorum Thunb. cv. Hinomoto, which is overcome by heat treatment, was analyzed. Immersing detached pistils in a distilled water bath held at 45°C for 5 min suppressed levels of ethylene and activities of 1-aminocyclopropane-1-carboxylate (ACC) oxidase at 6 h after self- and cross-incompatible pollination. However, the levels and activities showed no significant difference 48 h after pollination. Levels of ACC and activities of ACC synthase at 6 h after self-incompatible pollination were lower in pistils with heat treatment. Moreover, the heat treatment suppressed the activity of superoxide dismutase and enhanced the activity of catalase, ascorbate peroxidase, dehydroascorbate reductase, and glutathione reductase. In addition, the amount of hydrogen peroxide (H₂O₂) was reduced by heat treatment. In summary, heat treatment suppressed the ethylene-forming system and also enhanced the hydrogen peroxide-scavenging system in self-pollinated pistils associated with self incompatibility. A possible correlation between self incompatibility and stress in pistils after self-incompatible pollination is discussed based on the results obtained using heated pistils. Received: 12 April 2000 / Revision accepted: 19 September 2000     

The role of autonomous self-pollination in floral longevity in varieties of Impatiens hypophylla (Balsaminaceae)

The floral longevity of unpollinated, hand self-, and hand cross-pollinated flowers was compared in two varieties of Impatiens hypophylla, which contrasts with their mating systems. When flowers were emasculated and hand-pollinated every day after anthesis, their longevity was reduced. In the absence of emasculation and hand pollination, the staminate phase of the flowers of both varieties was 1 d longer. After the staminate phase, flowers of the outcrossing variety dropped their androecium, exposing the stigma and initiating the pistillate phase, which lasted for ～2 d. In contrast, flowers of the mixed-mating variety self-pollinated autonomously and then terminated their flowering. Under great seasonal variation in the pollinator visitation rate, which was observed in their natural populations, the outcrossing variety should maximize expected outcross success through the phenology of floral sex phases, whereas the mixed-mating variety self-pollinated ovules that were not outcrossed within the staminate phase. Based on these results, I suggest that the autonomous self-pollination in I. hypophylla induced differences both in the selfing coefficient and in floral longevity between the varieties.     

AMONG- AND WITHIN-FLOWER COMPARISONS OF POLLEN TUBE GROWTH FOLLOWING SELF- AND CROSS-POLLINATIONS IN DIANTHUS CHINENSIS (CARYOPHYLLACEAE)

We compared the rate of pollen tube growth following self- and cross-pollinations both among and within flowers of two clones of Dianthus chinensis L. For among-flower comparisons, both styles of a flower were pollinated with either self- or cross-pollen. Within-flower comparisons were made between the two styles of the same flower, one of which was self-pollinated and the other cross-pollinated. Comparisons between flowers indicated that self-pollen grew slower than cross-pollen in both clones. However, differences in the growth rate of pollen tubes from self- and cross-pollinations were greater when comparisons were made between the two styles of the same flower than when pollinations were made in different flowers. These results suggest the existence of interstyle interactions in pollen tube growth.     

Influence of phloem transport on flower abscission in Hibiscus rosa-sinensis

Summary Flowering cultivars of Hibiscus rosa-sinensis L. were either cross-pollinated or self-pollinated. Fruit set was observed on 52% of the cross-fertilized flowers, while only 4.6% of the self-fertilized flowers were not abscised. Once during fruit and seed growth, the subtending leaf was exposed to ¹⁴CO₂, and translocation of labelled photoassimilate was recorded by macro- and microautoradiography. Phloem transport into the raphe occurred in both fruits with fertilized and fruits with non-fertilized ovules. Since empty ovules showed some sink strength, it is assumed that growth of vegetative seed-tissue signalizes the retardation of completion of the abscission process. During fruit growth a considerable amount of starch is deposited in the distal layer of the abscission zone. Part of this starch is consumed during growth of cross-fertilized fruits.