Localisation and expression of arabinogalactan-proteins in the ovaries of Nicotiana alata Link and Otto

The transmitting-tissue cells of the style of flowering plants secrete a complex extracellular matrix through which pollen tubes grow to the ovary to effect fertilisation. This matrix is particularly rich in a class of proteoglycans, the arabinogalactan-proteins (AGPs). AGPs from the ovary of Nicotiana alata were found to be developmentally regulated, as the different charge classes of AGPs altered during floral development. The AGPs from the mature ovary had charge characteristics that were distinct from those previously reported for the stigma and style. However, the concentration of AGP (0.6 g/ml fresh weight) in the ovary did not change during development, or in response to either compatible or incompatible pollination. The AGPs of the ovary are mainly associated with the epidermis of the placenta.     

Arabinogalactan-protein secretion is associated with the acquisition of stigmatic receptivity in the apple flower

Background and Aims

Stigmatic receptivity plays a clear role in pollination dynamics; however, little is known about the factors that confer to a stigma the competence to be receptive for the germination of pollen grains. In this work, a developmental approach is used to evaluate the acquisition of stigmatic receptivity and its relationship with a possible change in arabinogalactan-proteins (AGPs).

Methods

Flowers of the domestic apple, Malus × domestica, were assessed for their capacity to support pollen germination at different developmental stages. Stigmas from these same stages were characterized morphologically and different AGP epitopes detected by immunocytochemistry.

Key Results

Acquisition of stigmatic receptivity and the secretion of classical AGPs from stigmatic cells occurred concurrently and following the same spatial distribution. While in unpollinated stigmas AGPs appeared unaltered, in cross-pollinated stigmas AGPs epitopes vanished as pollen tubes passed by.

Conclusions

The concurrent secretion of AGPs with the acquisition of stigmatic receptivity, together with the differential response in unpollinated and cross-pollinated pistils point out a role of AGPs in supporting pollen tube germination and strongly suggest that secretion of AGPs is associated with the acquisition of stigma receptivity.Key words: AGPs, arabinogalactan proteins, apple, Malus × domestica, pollen, pollen tube, stigma, stigmatic receptivity, flower receptivity     

Characterization and localization of the transmitting tissue-specific PELPIII proteins of Nicotiana tabacum

The class III pistil-specific PELP proteins (PELPIII) of Nicotiana tabacum includes at least two members of highly soluble glycoproteins containing glucan modules that are characteristic for arabinogalactan proteins (AGPs). PELPIII accumulates in the style transmitting tissue (TT) during pistil development and, at flower anthesis, is present in the intercellular matrix (IM) of non-pollinated pistils. After pollination, PELPIII appears to be directly and completely translocated from the IM into the pollen tube callose walls, no significant accumulation was observed in the primary wall in the tip. In the spent parts of the pollen tubes these proteins become detectable against the remnants of the tube cell membrane and in the callose plugs. Different protein extraction procedures of PELPIII from pollinated tobacco pistils showed that these proteins remain in the highly soluble protein fraction and are not modified by the growing pollen tubes. These data concur with a role in IM development and pollen tube growth. In addition, the data show that the PELPIII are able to reach the cell membrane, facilitated by an already present or induced high porosity of the tube wall and an additional, yet unknown, mechanism. The differences in behaviour between the three related classes of style IM glycoproteins of Nicotiana, namely, PELPII, TTS and the 120 kDa glycoprotein, are proposed to connect more to their differences in glycosylation than to major differences in amino acid sequence.     

Synchronicity of pollination and inoculation with Claviceps africana and its effects on pollen-pistil compatibility and seed production in sorghum

Sorghum ergot (caused by Claviceps africana) is a disease that affects sorghum seed development and yield. The interaction between pollen tube growth and hyphal development determines whether ovaries will be fertilized or colonized. Thus their respective deposition times on the stigma are critical. The effect of the time interval between pollination and inoculation on stigma receptivity and seed production was measured under field conditions in the male-sterile line A9 at Montecillo, State of México (2240m altitude). Pollination and inoculation treatments, from simultaneous application to 2 and 4h difference, were imposed when all stigmas on the panicle had emerged. Control panicles were either only pollinated or only inoculated. Eighteen hours later, pollen grains that adhered to, and germinated within the stigma, pollen tubes in the style and ovary, and fertilized pistils were counted. Pistils showing some disease expression (germinated spores, mycelium growth, or tissue necrosis) at 18, 48, and 72h were recorded. The number of diseased florets was registered at the dough growth stage, while number of seeds, grain yield and 100-seeds weight was measured at the physiological maturity. The pathogen applied in a water suspension of macro and secondary conidia caused a decrease in stigma receptivity; the greatest decrease (40-60%) occurred when the pollen and the inoculum were deposited almost simultaneously, regardless of which was deposited first. The route of the pollen tube was also the route for fungal infection. On average, treatments first inoculated had 60% more diseased florets and 36% less grain yield, 30% fewer seeds and seed size decreased 8%, than those first pollinated.     

Pollen tubes enter neighbouring ovules by way of receptacle tissue,resulting in increased fruit-set in Sagittaria potamogetifolia Merr

Using fluorescence microscopy, deposition of pollen on stigmas and pollen tube growth in the gynoecium of Sagittaria potamogetifolia Merr., a monoecious species with an apocarpous gynoecium, were observed. The maximum rate of pollination averaged 83.9 +/- 4.7 %, and the number of pollen grains per stigma ranged from zero to 30. Pollen tubes grew through one stigma to the base of the ovary at almost the same speed, but generally only one of the pollen tubes then turned towards the ovule and finally entered the nucellus through the micropyle. The other pollen tubes grew through the ovary base and the receptacle tissue into ovules of adjacent carpels whose stigmas were not pollinated or which had been pollinated later. This phenomenon is termed pollen tube 'reallocation' by the authors. To verify the direct effect of the phenomenon on fruit set, artificial pollination experiments were conducted in which two or more pollen grains were placed onto only one stigma in each gynoecium; frequently more than one fruitlet was obtained from each flower treated. The reallocation of pollen tubes among pistils in the gynoecium could effect fertilization of ovules of unpollinated pistils and lead to an increase in sexual reproduction efficiency. It would, to some extent, also increase pollen tube competition among pistils of the whole gynoecium.     

授粉诱导蝴蝶兰雌蕊中乙烯合成和ACC氧化酶基因表达

对蝴蝶兰（Ｐｈａｌａｅｎｏｐｓｉｓ “Ｇｅｎｅｒａｌｋｕ”ｈｏｒ．）在授粉后乙烯的合成和１－氨基环丙烷－１－羧酸（ＡＣＣ）氧化酶基因的表达进行了研究。实验结果显示在授粉后１２、２４ 和４８ ｈ,柱头和花柱中乙烯的产生和ＡＣＣ氧化酶ｍ ＲＮＡ 的积累显著下降,而子房中则明显上升,表明授粉后雌蕊中乙烯的产生与ＡＣＣ氧化酶基因的表达密切相关。此外,授粉后雌蕊的柱头中合成的乙烯相对量最多,花柱次之,子房中则较少     

Pollination-Induced Corolla Wilting in Petunia hybrida Rapid Transfer through the Style of a Wilting-Inducing Substance

Pollination or wounding of the stigma of Petunia hybrida flowers led to the generation of a wilting factor and its transfer to the corolla within 4 hours. This was concluded from the effects of time course removal of whole styles. In this 4-hour period, pollen tubes traversed only a fraction of the total distance to the ovaries. Both pollination and wounding of the stigma immediately resulted in an increase of ethylene evolution. Accelerated wilting, however, occured only when treated styles remained connected with the ovaries, and not when they were detached and left in the flower. A wilting factor was found in eluates collected from the ovarian end of the styles, only in the case of previous pollination or wounding. In such eluates, the level of the ethylene precursor 1-amino-cyclopropane-1-carboxylic acid was below detection.

These observations suggest a material nature of the wilting factor in Petunia flowers, which rapidly passes through the style to the corolla, but which is different from 1-aminocyclopropane-1-carboxylic acid.

     

Pollen tube reallocation in two preanthesis cleistogamous species, Ranalisma rostratum and Sagittaria guyanensis ssp. lappula (Alismataceae)

Pollen deposition on stigmas and pollen tube growth in two apocarpous species, Ranalisma rostratum and Sagittaria guyanensis ssp. lappula (Alismataceae), were examined with fluorescence microscopy. The reallocation of pollen tubes among pistils was observed in both species. The percentage of pollinated stigmas per flower was only 22.0% in R. rostratum and 51.0% in S. guyanensis, though the seed/ovule ratios are higher than 65% in both species. The number of pollen grains on each single stigma ranged from 0 to 96 in R. rostratum, and from 0 to 125 in S. guyanensis. When more than one pollen grain deposited on a stigma, all pollen tubes grew to the ovary, but only one of them turned towards the ovule and finally entered the nucleus. The other tubes grew through the receptacle tissue into ovules of adjacent carpels whose stigmas were unpollinated or pollinated later. The intercarpellary growth of pollen tubes could be a mechanism to increase the efficiency of sexual reproduction in an apocarpous gynoecium with low pollination on the pistils.     

Gametophyte–Sporophyte Interactions in the Pollen–Pistil System: 4. The Hormonal Status and the Mechanism of Self-Incompatibility

Following 4 and 8 h after self-incompatible pollination of Petunia hybrida plants, ethylene evolution and the contents of IAA, ABA, and cytokinins were measured in pistils and their parts (stigma, style, and ovary). The germination and initial growth of pollen tubes within the initial 4 h of the experiment were accompanied with an almost tenfold increase of the rate of ethylene production by the stigma and a twofold increase of the ABA content in the stigma and style. The inhibition of pollen tube growth in the style tissues during next 4 h coincided with a fivefold increase in the cytokinin content in the style, while high ABA content was maintained in the stigma and style. The authors conclude that phytohormones participate in the mechanism of gametophyte self-incompatibility.     

Chasmogamous and Cleistogamous Pollination in Salpiglossis sinuata

Pollen from chasmogamous flowers of Salpiglossis sinuata L. could not be induced to germinate in vitro unless stigmatal extract was applied to the culture medium. The substance that induces pollen germination in the stigmatal extract is water-soluble and heat-stable. Crosses could not be achieved between chasmogamous and cleistogamous flowers because of structural incompatibility. Pollinated pistils of chasmogamous flowers release a large amount of ethylene. The burst of ethylene release is due to an interaction between pollen tubes and stylar tissue and is directly proportional to the quantity of pollen placed on the stigma. Cleistogamous flower buds also produce a burst of ethylene at the time of pollination within the closed flower. The ethylene release may be a cause of reduced corolla development associated with cleistogamous flowers.     

Polyamines and transglutaminase activity are involved in compatible and self-incompatible pollination of Citrus grandis

Pollination of pummelo (Citrus grandis L. Osbeck) pistils has been studied in planta by adding compatible and self-incompatible (SI) pollen to the stigma surface. The pollen germination has been monitored inside the pistil by fluorescent microscopy showing SI altered morphologies with irregular depositions of callose in the tube walls, and heavy callose depositions in enlarged tips. The polyamine (PA) content as free, perchloric acid (PCA)-soluble and -insoluble fractions and transglutaminase (TGase) activity have been analyzed in order to deepen their possible involvement in the progamic phase of plant reproduction. The conjugated PAs in PCA-soluble fraction were definitely higher than the free and the PCA-insoluble forms, in both compatible and SI pollinated pistils. In pistils, pollination caused an early decrease of free PAs and increase of the bound forms. The SI pollination, showed highest values of PCA-soluble and -insoluble PAs with a maximum in concomitance with the pollen tube arrest. As TGase mediates some of the effects of PAs by covalently binding them to proteins, its activity, never checked before in Citrus, was examined with two different assays. In addition, the presence of glutamyl-PAs confirmed the enzyme assay data and excluded the possibility of a misinterpretation. The SI pollination caused an increase in TGase activity, whereas the compatible pollination caused its decrease. Similarly to bound PAs, the glutamyl-PAs and the enzyme activity peaked in the SI pollinated pistils in concomitance with the observed block of the pollen tube growth, suggesting an involvement of TGase in SI response.     

Sunny-side up: flower heliotropism as a source of parental environmental effects on pollen quality and performance in the snow buttercup, Ranunculus adoneus (Ranunculaceae)

Floral traits affect mating success via their influence on the microenvironment in which sexual reproduction occurs as well as their impact on pollinator attraction. Here we investigate the importance of flower heliotropism as a source of parental environmental effects on pollen quality and performance. Flowers of the snow buttercup, Ranunculus adoneus, closely track the sun's rays. We experimentally restrained flowers to test for effects of heliotropism on pollen quality and performance after pollination. When equivalent amounts of pollen were transferred to recipient pistils, pollen from solar-tracking donor flowers exhibited a 32% advantage in germination compared to pollen from stationary (tethered) donor flowers. By the end of anthesis, pistils of tracking flowers contained 40% more germinating pollen grains and 44% more pollen tubes midway down the style than pistils of stationary ones. Solar tracking had no direct effect on pollen tube growth. The greater amount of germinating pollen in tracking flowers accounted for the treatment effect on pollen tube density. A survey of pollen receipt and pollen germination in naturally tracking flowers indicated that solar tracking primarily affects pollen tube density by promoting pollen germination rather than pollen deposition. We conclude that flower heliotropism, by enhancing the paternal environment for pollen development and the maternal environment for pollen germination, represents a source of positive parental environmental effects on pollen performance in snow buttercups.     

Studies on flower longevity in Digitalis

Flower lifespan was terminated by corolla abscission 5–6 days after stigma opening in the unpollinated flower. Increased pollen loads produced increased seed set and reduced flower longevity progressively to a minimum of one day after pollination with pure pollen. Weakening of the abscission zone was detectable 8 h after pollination, whilst the pollen tubes were still within the stigmatic zone, suggesting that a stimulus, moving at 4 mm h^–1 minimum, was transmitted through the style and ovary. Soon after pollination removal of the stigma prevented the pollination-induced corolla abscission. Later it was necessary to remove the stigma and upper style, and later still the whole style to delay abscission. The progressive induction of the stigma and style took place at a rate of 1.5 mm h^–1, in advance of the pollen tubes which grew at 0.75 mm h^–1. It was not possible to reproduce the pollination effects by application of indoleacetic acid (IAA) to the stigma or the style.Abbreviation IAA Indoleacetic acid     

烟草柱头和花柱中阿拉伯半乳糖蛋白的定位

通过Western印迹法、免疫组织化学和超微细胞化学等技术,研究了烟草柱头和花柱中阿拉伯半乳糖蛋白（arabinogalactan-proteins,AGPs）的分布。结果表明烟草柱头和花柱组织中含有大量的AGPs,主要分布于柱头表皮细胞的细胞质和分泌层细胞的胞外基质中,且授粉前后AGPs的分布情况差异不明显;而花柱中的AGPs主要分布于表皮细胞的外层细胞壁、维管组织周围细胞的细胞质及引导组织的胞外基质中;花粉管通过后,引导组织胞外基质中AGPs减少,而花粉管细胞质和花粉管壁中检测到大量AGPs。     

濒危植物安徽羽叶报春两种花型的繁育特性及其适应进化

二型花柱的维持机制和自然选择压力多年来一直是生态学和进化学研究领域的热点之一。通过实验室栽培和野外观察统计相结合的方法,对安徽羽叶报春两种花型(长柱花和短柱花)的形态特征、花粉活力、柱头可授性、花粉/胚珠比、自然授粉及结籽能力、自交亲和性等繁育特性进行了比较研究。结果表明:长、短柱花的花冠直径和裂片宽无明显差异,而花冠筒、雌蕊和雄蕊高、花粉数目及大小、P/O比均有显著差异。在自然条件下,长柱花所接受的总花粉数要明显高于短柱花的总花粉数,但所接受的异型花花粉数和平均每果结籽数两者无显著差异。长柱花和短柱花的花粉和柱头活力相似,均能在较长时间内维持较高活力,仅在开花末期显著下降。两种花型的花在自花授粉、同型异花授粉、异型花授粉条件下均能结籽,但异型花授粉的结籽数均明显高于自花授粉和同型异花授粉结籽数。在长柱花各种授粉方式中,花粉萌发率无明显差异,但异型花花粉管的生长速度明显比同型异花花粉和自花花粉的快,而在短柱花柱头上表现为异型花授粉的萌发率最高,但只要萌发后在花柱中的生长速度无显明差异。此外,综合上述结果,对二种花型花部综合征的维持机制及自然选择压力进行了探讨。     

濒危植物肉苁蓉（Cistanche deserticola）繁育系统研究

在自然居群中通过野外定位观测,运用杂交指数（OCI）;花粉—胚珠比（P/O）;去雄、套袋、人工授粉等方法对濒危植物肉苁蓉（Cistanche deserticola Ma）的花序发育、开花特性、花粉活力、柱头可授性、传粉方式、繁育系统进行了研究,结果表明：肉苁蓉穗状无限花序的生长发育期可明显分为拱土期、出土期、开花期、结实与种子成熟期四个阶段。两性花,单花开放时间2~3d,花序有花期持续20~25d。种群开花历时36~38d。开花时柱头略高出花药,柱头较大,下垂。重力玻片法检测,风媒导致的异株异花传粉完全可以忽略。主要访花昆虫为蜂类。扫描电镜观察虫体,蜂类携带大量花粉,但传粉飞行距离大部分为0。杂交指数为3;花粉—胚珠比为35.45±8.56;结合去雄、套袋和人工授粉试验结果,认为肉苁蓉属于以自交为主的繁育系统。     

Hormonal status of the pollen-pistil system at the progamic phase of fertilization after compatible and incompatible pollination in Petunia hybrida L.

The hormonal status of the pollen-pistil system in Petunia hybrida L. during the progamic phase of fertilization was investigated. The contents of indolyl-3-acetic acid (IAA), abscisic acid (ABA), and cytokinins, as well as the rate of ethylene production in the pistils and their parts (stigma, style, and ovary) were measured over an 8-h period following compatible and self-incompatible pollination. In both pollinations, the phytohormones were present in various proportions in the stigma, style and ovary: the stigma was the main site of ethylene synthesis and contained 90% of the ABA, while the style contained 80% of the total cytokinin content in the pollinated pistil. Relatively low levels of hormones in the ovary did not influence the hormonal status of the pollen-pistil system. The interaction of the male gametophyte with the stigmatic tissues was accompanied by a 7- to 10-fold increase in ethylene production and a 1.5- to 2.0-fold increase in IAA content in the pollen-pistil system over 0–4 h. Pollen tube growth after self-incompatible pollination, in contrast to compatible pollination, was accompanied by a 3-fold increase in the ABA content in the stigma and style and by a 5-fold higher cytokinin content in the stylar tissues. Thus, the ethylene/ABA status of the stigma may play a role in controlling the processes of adhesion, hydration, and germination of pollen grains during pollination while the auxin/cytokinin status of the style may be involved in controlling pollen tube growth.     

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).