Synthesis and phosphorylation of pollen proteins during the pollen-stigma interaction in self-compatible Brassica napus L. and self-incompatible Brassica oleracea L.

A technique is described which permits the in vivo study of protein synthesis and phosphorylation in the pollen of Brassica spp. during the early stages of the pollen-stigma interaction. In Brassica napus and B. oleracea, compatible pollination is followed by a dramatic activation of protein synthesis in the pollen involving the synthesis of approximately 40 proteins. After incompatible pollinations in B. oleracea, virtually no newly synthesised polypeptides were detected in the pollen except for a small group of high molecular weight proteins which were not normally synthesised during compatible pollinations. Both compatible and incompatible pollinations were followed by the appearance of newly phosphorylated proteins in the pollen; these fell into four distinct groups. In B. oleracea, the number of phosphorylated proteins and the degree of phosphorylation of individual proteins within the four groups differed between compatible and incompatible pollinations. One group of phosphorylated proteins appeared to correspond with the small group of high molecular weight polypeptides which were synthesised in pollen after incompatible pollinations. These findings are discussed in the perspective of cell signalling during the pollen-stigma interaction in Brassica and also in terms of their possible implication in sporophytic self-incompatibility.     

Effects of Promoters and Inhibitors of Ethylene and ABA on Flower Senescence of Hibiscus rosa-sinensis L.

Hibiscus rosa-sinensis L. flowers (cv La France) senesce and die over a 12-h period after opening. The aim of this study was to examine the physiological mechanisms regulating the senescence process of ephemeral hibiscus flowers. Different flower stages and floral organs were used to determine whether any interaction existed during flower senescence between endogenous abscisic acid (ABA) and the predisposition of the tissue to ethylene synthesis. This was carried out on whole flowers treated with promoters and inhibitors of ethylene and ABA synthesis or a combination of them. Treatments with 1-aminocyclopropane-1-carboxylic acid (ACC), a precursor of ethylene biosynthesis, enhanced flower senescence, whereas amino-oxyacetic acid (AOA) and fluridone, an ethylene and an ABA inhibitor, respectively, extended flower longevity. These effects were more significant when applied before anthesis. Ethylene evolution was substantially reduced in all organs from open and senescent flowers treated with fluridone and AOA. Similarly, endogenous ABA accumulation was negatively affected by AOA and fluridone treatments. Application of fluridone plus ACC reduced ethylene evolution and increased ABA content in a tissue-specific manner but did not overcome the inhibitor effect on flower longevity. AOA plus fluridone treatment slightly accelerated flower longevity compared to AOA-treated flowers. Application of ABA alone promoted senescence, suppressed ethylene production, and, when applied with fluridone, countered the fluridone-induced increase in flower longevity. Taken together, these results suggest that the senescence of hibiscus flowers is an endogenously regulated ethylene- and ABA-dependent process.     

Changes in ethylene production and 1-aminocyclopropane-1-carboxylic acid content of pollinated carnation flowers

Pollination of flowers of standard carnation (Dianthus caryophyllus L. cv. White Sim) with pollen from flowers of miniature carnations (D. caryophyllus L. cv. Exquisite) caused them to wilt irreversibly within 1 to 2 days. Pollination stimulated a sequential increase in ethylene production by stigmas, ovaries, receptacles, and petals of the flowers. The ACC content of the stigmas increased rapidly in the first few hours after pollination. The possibility that subsequent production of ethylene by other parts of the flower is stimulated by translocated ACC is discussed. Ethylene production and ACC content of other parts of the flower reached their maximum 24 h after pollination. The petal tissues contributed the bulk of the ethylene productionper flower thereafter. There appears to be a qualitative difference between the enzyme in the stigmas converting ACC to ethylene and that in other parts of the flower.     

Pollination-induced ethylene promotes the early phase of pollen tube growth in Petunia inflata

In Petunia inflata, a species with gametophytic self-incompatibility, pollination triggers two phases of ethylene production by the pistil, the first of which peaks 3 hours after pollination with compatible or incompatible pollen. To investigate the physiological significance of the first phase of ethylene production, pollinated flowers were treated with 2,5-norbornadiene (NBD), an inhibitor of ethylene action. Treatment with NBD reduced pollen tube growth in a dose-dependent manner during the first six hours after pollination; however, pollen tube growth was insensitive to NBD if the treatment was applied 6 hours or more after pollination. Simultaneous application of exogenous ethylene substantially offset the inhibitory effects of NBD in flowers pollinated for 4 hours. Another inhibitor of ethylene action, 1-methylcyclopropene (1-MCP), also produced a strong inhibition of pollen tube growth during the first six hours of pollination. The experiments with 1-MCP pretreatment indicate that pistil tissues are the primary target of the pollination-induced ethylene.     

Ethylene synthesis in petunia stigma tissues governs the growth of pollen tubes in progamic phase of fertilization

In the pollen-pistil system of petunia (Petunia hybrida L.) self-compatible and self-incompatible clones within 7 h after self-pollination, we determined the content of ACC (1-aminocyclopropane-1-carboxylic acid), the activity of two enzymes (ACC synthase and ACC oxidase), and the rate of ethylene production. Depending on the type of pollination, germination of pollen on the stigma surface and the pollen tube growth in the tissues of style were accompanied by different levels of ACC and ethylene release. The pollen-pistil system of the self-compatible clone contained twice more ACC than in the self-incompatible clone, whereas the pollen-pistil system in the self-incompatible clone produced 4–5 times more ethylene than in the self-compatible clone. For both types of pollination, ACC and ethylene were predominantly produced in the stigma tissues. The rate of ethylene production therein was 50 times greater than in the styles and ovaries, and the content of ACC was 100 times higher than in the styles and ovaries. Germination of male gametophyte after both types of pollination was accompanied by elevated ACC synthase activity (especially in the case of compatible pollination), whereas notable increase in ACC oxidase activity was manifested in growing pollen tubes after self-incompatible pollination     

Roles of ethylene and 1-aminocyclopropane-1-carboxylic acid in pollination and wound-induced senescence of Petunia hybrida flowers

Normal senescence of Petunia hybrida L. (cv. Pink Cascade) was associated with a 10-fold increase in their ethylene production. Soon after pollination wounding of the stigma of detached flowers there was a burst of ethylene production by the gynoecium, which reached a maximum after 3 h. A subsequnt more gradual rise in ethylene production by the flowers was accompanied by blueing, wilting, and senescence of the corolla. Treatment with 1 μl ethylene 1⁻¹ accelerated the onset of senescence as measured first by color change and then by wilting of the corolla. These changes were further accelerated by using older flowers or higher concentrations of ethylene. Senescence was also hastened by supplying 1-aminocyclopropane-1-carboxylic acid (ACC) through the flower pedicel. Petunia pollen contained high concentrations of ACC (300 nmol g⁻¹); treatment of stigmas with ACC (1 m M ) caused a 4-fold increase in their ethylene production. Senescence, whether natural or hastened by pollination or piercing, was delayed by treating the flowers with the anionic silver thiosulfate complex.     

Analysis of stylar self-incompatibility competence by use of heat induced inactivation

Summary Immersion of Lilium longiflorum pistils in 49 °C water for increasing durations of 1,2,3, or 4 minutes immediately prior to incompatible pollination resulted in a correspondingly progressive decrease in the stylar self-incompatibility competence, as determined from the lengths attained by pollen tubes during 48 hours growth in the styles at 24 °C. Neither pistils remaining on the plant nor those detached from the plant which were immersed after anthesis in 49 °C water for 5 minutes regained self-incompatibility competence during a 48 hour incubation at 24 °C prior to incompatible pollination. Heat treatment of detached pistils as early as 39 hours prior to bud anthesis also resulted in an inactivation of stylar self-incompatibility competence when incompatible pollination was made at 24 hours after anthesis. Experiments utilizing heat treatment of partial lengths of detached whole styles revealed that pollen tubes which have grown through as much at 45 millimeters of either a physiologically incompatible or compatible portion of the style are still capable of shifting to either a higher growth rate or lower growth rate upon entry into respectively either a physiologically compatible or incompatible portion of the style.     

Role of ethylene in the control of gametophyte-sporophyte interactions in the course of the progamic phase of fertilization

We investigated dynamics of the content of 1-aminocyclopropane-1-carboxylic acid (ACC) and ethylene production in male gametophyte development and germination in fertile (self-compatible and selfincompatible) and sterile clones of petunia. Fertile male gametophyte development was accompanied by two peaks of ethylene production by anther tissues. The first peak occurred during the microspore development simultaneously with the degeneration of both the tapetal tissues and the middle layers of the anther wall. The second peak coincided with dehydration and maturation of pollen grains. In the anther tissues of the sterile line of petunia, tenfold higher ethylene production was observed at the meiosis stage compared with that in fertile male gametophytes. This fact correlated with the degeneration of both microsporocytes and tapetal tissues. Exogenously applied ethylene (1–100 ppm) induced a degradation of the gametophytic generation at the meiosis stage. According to the obtained data, ethylene synthesis in germinating male gametophyte is provided by a 100-fold ACC accumulation in mature pollen grains. The male gametophyte germination, both in vitro, on the culture medium, and in vivo, on the stigma surface, was accompanied by an increase in ethylene production. Depending on the type of pollination, germination of pollen on the stigma surface and the pollen tube growth in the tissues of style were accompanied by various levels of ACC and ethylene release. The male gametophyte germination after self-compatible pollination was accompanied by higher content of ACC as compared with the self-incompatible clone, whereas, after the self-incompatible pollination, we observed a higher level of ethylene production compared with compatible pollination. For both types of pollination, ACC and ethylene were predominantly produced in the stigma tissues. Inhibitor of ethylene action, 2,5-norbornadiene (NBN), blocked both the development and germination of the male gametophyte. These results suggest that ethylene is an important factor in male gametophyte development, germination, and growth at the progamic phase of fertilization.     

EFFECT OF FLORAL AGING ON THE GROWTH OF COMPATIBLE AND INCOMPATIBLE POLLEN TUBES IN LILIUM LONGIFLORUM

Compatible and incompatible pollen tube growth in detached styles of three Lilium longiflorum cultivare varies with the physiological age of the style. Before anthesis both compatible and incompatible pollen tubes grow, in 48 hr, only a fraction of the distance compatible tubes grow after anthesis. Incompatible pollen tubes are restricted to about half the distance of compatible tubes in the four days postanthesis, but thereafter increase up to or three-fourths or more the length of compatible tubes at the time of floral senescence. About 10 days after anthesis, growth of both types of pollen tubes decreases. The detached style method of pollen-tube cultivation is validated in the cultivar ‘Ace’ by seed set obtained following self-pollination in the 6- to 9-day interval and failure of seed set after either self- or cross-pollination after 9 days following anthesis. Also, in agreement with detached style data, self-pollination fails to produce seeds when attempted in bud stages or the five days following anthesis. Cross-pollinations are successful in this period. This material and technique appear well suited for study of the nature of the self-incompatibility reaction.     

Expression of the ACC synthase and ACC oxidase coding genes after self-pollination and incongruous pollination of tobacco pistils

In tobacco, as in other species, ethylene is produced in response to pollination. Although tobacco is a self-compatible species, it displays unilateral incongruity with other Nicotianaplants. Incongruous pollination also results in ethylene production, but this production differs depending on the pollen used and is related to the extent to which pollen tubes grow in the tobacco style. In the investigation reported here we followed the expression of the ACC synthase- and ACC oxidase-coding genes upon pollination of tobacco pistils and compared self-pollination with incongruous pollination. The pattern of expression of these genes also correlated with pollen-tube growth, although wounding alone cannot explain the results obtained. We also examined the expression of these genes upon pollination of immature tobacco pistils, in which different pollen tubes grew indistinctly inside the tobacco style and reached the ovary at the same rate. In this situation no significant differences in gene expression could be observed between the different pollinations. Ethephon, a substance that produces ethylene, could, in some cases, minimize the arrest of incongruous pollen tubes inside the style.     

Experimental insights on the function of ancillary pollen and stigma polymorphisms in plants with heteromorphic incompatibility

Most heterostylous plants possess a reciprocal arrangement of stigmas and anthers (reciprocal herkogamy), heteromorphic self‐incompatibility, and ancillary polymorphisms of pollen and stigmas. The topographical complementarity hypothesis proposes that ancillary polymorphisms function in the rejection of incompatible pollen thus promoting disassortative pollination. Here, we test this hypothesis by investigating patterns of pollen transfer and capture in populations of dimorphic Armeria maritima and A. pubigera and distylous Limonium vulgare (Plumbaginaceae), and by studying pollen adherence and germination patterns in A. maritima following controlled hand‐pollinations. Armeria lacks reciprocal herkogamy allowing the evaluation of the extent to which ancillary polymorphisms affect the composition of pollen loads. We compared the amounts of compatible and incompatible pollen on stigmas in natural populations and calculated the proficiencies of pollen transfer for each mating type. We detected disassortative pollination in each species, and mating types did not differ in compatible pollen capture, although cob stigmas captured more incompatible pollen. Controlled hand‐pollinations revealed the failure of incompatible pollen to adhere and germinate on stigmas. Our results provided evidence that, while structural in nature, pollen‐stigma dimorphisms are tightly associated with heteromorphic incompatibility and likely function to promote disassortative pollination, especially in the absence of reciprocal herkogamy.     

Endogenous Levels and Transport of 1-Aminocyclopropane-1-Carboxylic Acid in Stamens of Ipomoea nil (Convolvulaceae)

Filament and corolla growth in flowers of Ipomoea nil are inhibited by ethylene production. Anthers inhibited filament growth in vitro during younger stages of development even in the presence of the growth promoter gibberellic acid (GA₃). To test whether the anthers could be sources of 1-aminocyclopropane-1-carboxylic acid (ACC) endogenous levels of ACC and ethylene production were monitored using gas chromatography. To also test whether the filaments could be transport vectors for ACC the movement of [¹⁴C]ACC was assessed by scintillation counting from donor agarose blocks, through filament sections, and into receiver agarose blocks. While ACC levels fluctuated in anthers 87 to 21 h before anthesis, anthers contained increased levels of ACC from 15 to 6 hours before anthesis. Ethylene production also fluctuated but peak levels were shifted about 6 hours closer to anthesis than ACC levels within the anthers. Both ACC and ethylene levels in filaments showed fluctuations similar to those in the anthers. [¹⁴C]ACC movement became increasingly basipetal during development. Older stages showed greater polar [¹⁴C]ACC efflux rates, while all stages showed constant polar influx rates. Low levels of endogenous ACC were transported basipetally from the anther through the filament into agarose blocks at all stages of development. Corresponding levels of endogenous ethylene production remained constant between the various stages during ACC transport. We have evidence that stamens of I. nil have a role as source tissues and transport vectors for ACC, to stimulate corolla growth, such as corolla unfolding and senescence.     

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.     

Disorganization of F-actin cytoskeleton precedes vacuolar disruption in pollen tubes during the in vivo self-incompatibility response in Nicotiana alata

Background and Aims The integrity of actin filaments (F-actin) is essential for pollen-tube growth. In S-RNase-based self-incompatibility (SI), incompatible pollen tubes are inhibited in the style. Consequently, research efforts have focused on the alterations of pollen F-actin cytoskeleton during the SI response. However, so far, these studies were carried out in in vitro-grown pollen tubes. This study aimed to assess the timing of in vivo changes of pollen F-actin cytoskeleton taking place after compatible and incompatible pollinations in Nicotiana alata. To our knowledge, this is the first report of the in vivo F-actin alterations occurring during pollen rejection in the S-RNase-based SI system. Methods The F-actin cytoskeleton and the vacuolar endomembrane system were fluorescently labelled in compatibly and incompatibly pollinated pistils at different times after pollination. The alterations induced by the SI reaction in pollen tubes were visualized by confocal laser scanning microscopy. Key Results Early after pollination, about 70 % of both compatible and incompatible pollen tubes showed an organized pattern of F-actin cables along the main axis of the cell. While in compatible pollinations this percentage was unchanged until pollen tubes reached the ovary, pollen tubes of incompatible pollinations underwent gradual and progressive F-actin disorganization. Colocalization of the F-actin cytoskeleton and the vacuolar endomembrane system, where S-RNases are compartmentalized, revealed that by day 6 after incompatible pollination, when the pollen-tube growth was already arrested, about 80 % of pollen tubes showed disrupted F-actin but a similar percentage had intact vacuolar compartments. Conclusions The results indicate that during the SI response in Nicotiana, disruption of the F-actin cytoskeleton precedes vacuolar membrane breakdown. Thus, incompatible pollen tubes undergo a sequential disorganization process of major subcellular structures. Results also suggest that the large pool of S-RNases released from vacuoles acts late in pollen rejection, after significant subcellular changes in incompatible pollen tubes.     

生长素与乙烯对兰花授粉后花发育的调节作用(英文)

以朵丽蝶兰为材料,对乙烯和生长素调节的授粉后花的发育进行了研究。实验结果显示,切花和植株上的花授粉后,乙烯的产生和花的发育无明显差异;花瓣的衰老、子房发育、花粉萌发和花粉管的伸长受乙烯调节;与切花相比,植株上花的子房内无ＡＣＣ合酶和ＡＣＣ 氧化酶ｍＲＮＡ 的积累。用生长素运输抑制剂２ ［（１ｎａｐｈｔｈａｌｅｎｙｌａｍｉｎｏ）ｃａｒｂｏｎｙｌ］ ｂｅｎｚｏｉｃａｃｉｄ（ＮＰＡ） 处理柱头,授粉诱导的子房发育在很大程度上受到抑制, 表明授粉后子房的发育需要转运来的生长素。     

Cytokinin-induced ethylene biosynthesis in nonsenescing cotton leaves

The influence of cytokinins on ethylene production was examined using cotton leaf tissues. Treatment of intact cotton (Gossypium hirsutum L. cv LG 102) seedlings with both natural and synthetic cytokinins resulted in an increase in ethylene production by excised leaves. The effectiveness of the cytokinins tested was as follows: thidiazuron BA isopentyladenine ≥ zeatin kinetin. Using 100 micromolar thidiazuron (TDZ), an initial increase in ethylene production was observed 7 to 8 hours post-treatment, reached a maximum by 24 hours and then declined. Inhibitors of 1-aminocyclopropane-1-carboxylic acid (ACC) synthesis and its oxidation to ethylene reduced ethylene production 24 hours post-treatment; however, by 48 hours only inhibitors of ACC oxidation were effective. The increase in ethylene production was accompanied by a massive accumulation of ACC and its acid-labile conjugate. TDZ treatment resulted in a significant increase in the capacity of tissues to oxidize ACC to ethylene. Endogenous levels of methionine remained constant following TDZ treatment. It was concluded that the stimulation of ethylene production in cotton leaves following cytokinin treatment was the result of an increase in both the formation and oxidation of ACC.     

Fluorescence and scanning electron microscopic study on self-incompatibility in distylous Averrhoa carambola L.

Various combinations of intermorph, selfing and intramorph pollinations were carried out in Averrhoa carambola and the pollinated pistils were observed under fluorescence and scanning electron microscopes in time-course experiments. In both compatible and incompatible pollinations, similar behavior of pollen germination and penetration was observed in the first 4 h after pollination. In compatible intermorph pollination, pollen tubes were found at the base of the transmitting tract of the style at 8 h and 24 h after pollination in both the pin and thrum morphs. With thrum flowers, selfing resulted in pollen tubes being uniformly arrested at the junction between the stigmatic and stylar tissues. Penetration of pollen tubes into the upper portion of style was observed in thrum intramorph pollination and, when the second member was treated as the gynoecial tissue under the same pollination, penetration of tubes was further enhanced. Pin flowers, on selfing, resulted in pollen tube penetration farther down the style than was the case with thrum selfing. Intramorph pollination of pin morph behaved in a similar manner to selfing and was not affected by genotypes. Beside the stamen-style dimorphism, the receptive surface of the cob stigma was larger in pin than thrum flowers. While pin pollen was round, thrum pollen was oblong in shape with pin to thrum ratio on the polar axis being 1.2 and on the equatorial axis 0.8. The stigma of pin morph belonged to the dry type, while that of the thrum resembled the wet type.     

Interorgan translocation of 1-aminocyclopropane-1-carboxylic Acid and ethylene coordinates senescence in emasculated cymbidium flowers

In Cymbidium flowers, emasculation by removal of the pollinia and the anther cap leads within 24 hours to red coloration of the labellum (lip). Lip coloration, being the first sign of senescence in these flowers, has been ascribed to the action of ethylene in the lip. When a small incision in the base of the lip is made prior to emasculation, or when the lip is excised and placed in water within 10 to 15 hours after emasculation, coloration is considerably delayed. This indicates that a coloration-associated factor is moving in or out of the lip. Measurements of ethylene production of excised flower parts, isolated at different times after emasculation, showed an increase only in the central column; the other flower parts, including the lip, did not show a measurable change. In contrast, in situ measurements of the ethylene production of the central column and the remaining portion of the flower revealed a simultaneous increase in all the flower parts following emasculation. Similarly, application of radiolabeled 1-aminocyclopropane-1-carboxylic acid (ACC) to the top of the central column in situ leads to the production of radiolabeled ethylene by all the flower parts. In addition, the ethylene production of isolated lips, measured immediately after excision, was initially high but ceased within 10 to 15 minutes. Treatment of the central column in situ with ethylene or ethephon did not stimulate ACC production but did stimulate lip coloration and this was accompanied by an increased internal ethylene concentration in the lip. The data indicate that endogenously produced as well as applied ACC is rapidly translocated from the site of production or application to all the other flower parts where it is immediately converted into ethylene. By excision of a flower organ, the influx of ACC is prevented, causing a rapid decrease in ethylene production. In addition, it was found that ethylene may also be translocated in physiologically significant amounts within the flower. The roles of ACC and ethylene as mobile senescence or wilting factors in emasculation- and pollination-induced senescence is discussed.     

2-Aminooxyisobutyric acid inhibits the in vitro activities of both 1-Aminocyclopropane-1-carboxylate (ACC) synthase and ACC oxidase in ethylene biosynthetic pathway and prolongs vase life of cut carnation flowers

2-Aminooxyisobutyric acid (AOIB) has a partial structure of aminooxyacetic acid (AOA) in its whole structure, and resembles 2-aminoisobutyric acid (AIB) in their tetrahedral structures. Both AOA and AIB are inhibitors of ethylene biosynthesis; AOA inhibits the action of 1-aminocyclopropane-1-carboxylate (ACC) synthase and AIB inhibits that of ACC oxidase. The present study showed that AOIB inhibited the in vitro activities of both ACC synthase and ACC oxidase, which were synthesized heterologously in E. coli cells from corresponding carnation cDNAs, and the magnitudes of inhibition were similar to those caused by AOA and AIB; AOIB and AOA at 0.1 mM inhibited ACC synthase action by 75%, and AOIB and AIB at 10 mM inhibited ACC oxidase action by 16.3 and 22.5%, respectively. AOIB at 1 mM caused 91.5% reduction of maximum ethylene production rate as compared to the control in cut ‘Excerea’ carnation flowers undergoing senescence, thereby lengthening their vase life to 7 d from 3 d of the control flowers. The inhibition by AOIB was probably caused by its action resembling AOA, but not AIB. AOIB also extended significantly the vase life of cut flowers of ‘Pax’ carnation, and tended to do so in ‘Primero Mango’ carnation. The present findings suggest the potential of AOIB as a new preservative for carnations and other ornamentals in which ethylene plays a key role in the induction of senescence.