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.     

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.     

与授粉有关的因子调节的乙烯生物合成基因在兰花花器官中的表达

分析了与授粉有关的因子调节的ＡＣＣ合酶和ＡＣＣ氧化酶基因在朵丽蝶兰（ＤｏｒｉｔａｅｎｏｐｓｉｓｈｙｂｒｉｄａＨｏｒｔ．）花中的表达。生长素和乙烯均可诱导ＡＣＣ合酶和ＡＣＣ氧化酶的ｍＲＮＡ在花器官中积累。然而,去雄却不能诱导这两个基因在花器官中表达。生长素和乙烯所诱导的ＡＣＣ合酶和ＡＣＣ氧化酶的ｍＲＮＡ在花器官中的积累模式相似。原位杂交结果表明,生长素和乙烯处理后ＡＣＣ氧化酶的ｍＲＮＡ在柱头的表皮和薄壁细胞中积累。根据ＡＣＣ合酶和ＡＣＣ氧化酶基因表达的结果,对生长素、乙烯和去雄在兰花授粉后乙烯生物合成过程中的作用进行了分析。     

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。     

Auxin and flavonoids in the progame phase of fertilization in petunia

The contents of IAA and flavonoids (Fls) were monitored in developing anthers, in vitro growing pollen tubes, and in the in vivo pollen-pistil system of two petunia (Petunia hybrida L.) clones, self-compatible and self-incompatible. In both clones, the development of male gametophytes was accompanied by the increase in the IAA (from 10 to 60–70 ng/g fr wt) and Fls (from 2 to 20 mg/g fr wt) contents. In both clones, pollen grain germination was accompanied by a substantial (by 10–30%) increase in the IAA content during the first two hours and Fl content during the first hour. Treatments with IAA and Fls stimulated both in vitro pollen grain germination and pollen tube growth by 25–30%. Male gametophyte germination in vivo, on the pistil surface, was accompanied by the increase in the IAA content from 90 to 200 ng/g fr wt during 8 h, whereas the content of Fl increased from 2 to 3 mg/g fr wt during the first hour and was maintained later at this level. In the pollen-pistil system, IAA and Fls were distributed evenly in the tissues of stigma, style, and ovary. On the basis of data obtained, we concluded that Fls might be endogenous mediators of IAA transport, which is one of the principal regulators of male gametophyte growth and development in the progame phase of fertilization, but are not involved in the mechanism of gametophyte incompatibility.     

Ethylene synthesis and auxin augmentation in pistil tissues are important for egg cell differentiation after pollination in maize

The role of ethylene and auxin in stigma-to-ovule signalling was investigated in maize (Zea mays L.). Maturation of the egg cells in an ear was stimulated before actual fertilization by the application of fresh pollen grains or quartz sand to fully receptive stigmas. Ethylene emission by maize ears increased in response to those treatments. Silks and ovaries were involved in ethylene synthesis after pollen or sand was shed over the silks. The content of ethylene precursor [1-aminocyclopropane-1-carboxylic acid (ACC)] increased in both pistil parts soon after pollination. ACC rise was delayed by 4 h in the ovaries, and by 8 h in the silks after mock-pollination with sand. The auxin level increased rapidly in the silks and ovaries after pollination, and it was very high in the pollinated silks due to the high indole-3-acetic acid (IAA) content of pollen grains. IAA rise also appeared in the silks and ovaries after treatment with sand but it was delayed by 8 h. Application of ACC (10 microM) or IAA (6 microM) solutions to non-pollinated silks stimulated maturation of the egg cells. Moreover, the response of the egg cells to pollination was cancelled by l-alpha-(2-aminoethoxyvinyl)-glycine, alpha-aminoisobutyric acid or 2,3,5-triiodobenzoic acid applied to the silks before pollination. Thus ethylene synthesis and polar auxin transport in the silks pollinated with fresh pollen were necessary to evoke accelerated differentiation of the egg cells in maize ovules. Differences in pistil responses found between true- and mock-pollination suggest that signalling pathways are at least partially different for the reception of pollen grains and sand crystals on maize stigma.     

Temporal and spatial regulation of 1-aminocyclopropane-1-carboxylate oxidase in the pollination-induced senescence of orchid flowers.

Pollination of many flowers initiates a sequence of precisely regulated developmental events that include senescence of the perianth and development of the ovary. The plant hormone ethylene is known to play a key role in regulating the biochemical and anatomical changes that constitute the postpollination syndrome. For this reason, we have studied the pollination syndrome in Phalaenopsis orchids by examining the spatial and temporal location of ethylene biosynthesis within the orchid flower, and how this biosynthesis is regulated by factors that influence expression of genes that encode key enzymes in the ethylene biosynthetic pathway. In particular, we examined the role in the postpollination syndrome of the expression of the gene for 1-aminocyclopropane-1-carboxylate (ACC) oxidase, which catalyzes the conversion of ACC to ethylene. In vivo incubation of tissues with the ethylene precursor ACC demonstrated that ACC oxidase activity increases after pollination in the stigma, contrary to the observation that activity is constitutive in petunia and carnation gynoecia. RNA blot hybridization of floral tissues indicates that the increase in ACC oxidase activity is due to de novo synthesis of mRNA and presumably protein, which is induced after pollination. Furthermore, the pattern of induction is consistent with a model of coordinate regulation of gene expression in which the pollination signal travels to other organs of the flower to induce their ethylene production. We have also used in situ hybridization to define further the temporal and spatial expression of ACC oxidase within the floral organs, showing that expression, and,by inference, the capability to oxidize ACC to ethylene, is induced in all living cells of the tissues examined after pollination. These findings contrast with work in petunia that suggests that ACC oxidase is localized to the stigmatic surface.     

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     

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.     

Pollination-Induced Ethylene Synthesis and 1-Aminocyclopropane-1-Carboxylate Oxidase Gene Expression in the Gynoecium of Phalaenopsis Orchid Flower

Pollination-induced ethylene production plays an important role in regulating flower development. As gynoecium is one of the more important parts in Phalaenopsis orchid flower, the authors have investigated ethylene production and 1-aminocyclopropane-l-carboxylate (ACC) oxidase gene expression in gynoecium following pollination. Experimental studies revealed that ethylene production and ACC oxidase mRNA accumulation decreased steadily in stigma and style, in contrast, exhibited a crescendo increase in the ovary at 12.24 and 48 h after pollination, indicating a close relationship between ethylene production and ACC oxidase gene expression in the gynoecium. In addition ethylene production was found most in stigma and least in ovary among the three parts after pollination.     

Pollination and stigma wounding: same response, different signal?

In Petunia hybrida flowers, both pollination and stigma woundinginduced a transient Increase in ethylene production and hastenedcorolla senescence. Ethylene production by different flowerparts was measured in situ using laser photoacoustic (LPA) spectroscopy.In pollinated flowers, ethylene was exclusively produced bythe stigma/style region whereas wounding of the stigma Inducedethylene production both by the stigma/style region and by theremaining flower parts. In aminoethoxyvinylglycine (AVG)-treatedflowers, subsequent treatment of the unwounded stigma with 1-aminocyclopropane-1-carboxylicacid (ACC) induced ethylene production exclusively by the stigma/styleregion whereas treatment of a previously wounded stigma withACC induced a simultaneous increase in ethylene production bythe stigma/style region and the remaining flower parts. Theseresults suggest that following stigma wounding, either ACC orethylene is involved in inter-organ communication. Followingpollination, the signal is apparently not directly related toethylene. In vivo ACC oxidase activity of most flower parts, includingthe gynoecium, was higher in light than in dark. Light or darkdid not influence the relative contributions of stigma/styleand remaining flower parts to the total pollination, woundingor ACC-induced ethylene production, indicating that ACC is nottranslocated. Both in excised styles and intact flowers, radiolabelledACC and its analogue -aminoisobutyric acid (AIB), applied eitherto an intact or wounded stigma, were largely immobile confirmingthat ACC is not likely to play a role in inter-organ signalling. The results collectively suggest that following stigma wounding,translocation of ethylene may be the signal responsible forinitiation of corolla senescence; following pollination thesignal is not directly related to ethylene. Key words: 1-Aminocyclopropane-1-carboxylic acid (ACC), ethylene, flower senescence, Petunia hybrida, pollination, stigma wounding     

Does cholinesterase participate in the intercellular interactions in pollen-pistil system?

Hydrolysis of acetylthiocholine and butyrylthiocholine has been observed in aqueous extracts from petunia pollen and pistils. The reproductive organs of self-compatible clone showed a higher rate of choline ester hydrolysis than those of self-incompatible clone. The highest rate of acetylthiocholine hydrolysis blocked by the cholinesterase inhibitors (physostigmine and neostigmine) was characteristic for the pollen of self-compatible clone. The incomplete (25 - 40 %) inhibition of hydrolysis in pistil extracts of self-compatible clone suggests the presence of unspecific esterases. The eight-fold lower hydrolysis was observed in the pistils of self-incompatible clone as compared to the pistils of compatible clone; neostigmine completely blocked this low hydrolytic activity. The treatment of flower buds with physostigmine and neostigmine (10-5 - 10-3 M) decreased the seed production by 10 - 20 % in compatible clone. When the surfaces of pistil stigmae were treated with physostigmine and neostigmine (10-5 - 10-3 M) before pollination, the seed formation was inhibited by 95 % after both self- and cross-pollination. This revised version was published online in July 2006 with corrections to the Cover Date.     

Peroxynitrite mediates programmed cell death both in papillar cells and in self-incompatible pollen in the olive (Olea europaea L.)

Programmed cell death (PCD) has been found to be induced after pollination both in papillar cells and in self-incompatible pollen in the olive (Olea europaea L.). Reactive oxygen species (ROS) and nitric oxide (NO) are known to be produced in the pistil and pollen during pollination but their contribution to PCD has so far remained elusive. The possible role of ROS and NO was investigated in olive pollen-pistil interaction during free and controlled pollination and it was found that bidirectional interaction appears to exist between the pollen and the stigma, which seems to regulate ROS and NO production. Biochemical evidence strongly suggesting that both O(2)(-) and NO are essential for triggering PCD in self-incompatibility processes was also obtained. It was observed for the first time that peroxynitrite, a powerful oxidizing and nitrating agent generated during a rapid reaction between O(2)(-) and NO, is produced during pollination and that this is related to an increase in protein nitration which, in turn, is strongly associated with PCD. It may be concluded that peroxynitrite mediates PCD during pollen-pistil interaction in Olea europaea L. both in self-incompatible pollen and papillar cells.     

Unilateral incompatibility within the brassicaceae: further evidence for the involvement of the self-incompatibility (S)-locus

Unilateral pollen-pistil incompatibility within the Brassicaceae has been re-examined in a series of interspecific and intergeneric crosses using 13 self-compatible (SC, Sc) species and 12 self-incompatible (SI) species from ten tribes. SC x SC crosses were usually compatible, SI x SC crosses showed unilateral incompatibility, while SI x SI crosses were often incompatible or unilaterally incompatible. Unilateral incompatibility (UI) is shown to be overcome by bud pollination or treating stigmas with cycloheximide — features in common with self-incompatibility. Treating stigmas with pronase prevents pollen tubes from penetrating the stigma in normally compatible intra-and interspecific pollinations. The results presented show that the presence of an incompatibility system is important in predicting the outcome of interspecific and intergeneric crosses and, combined with the physiological similarities between UI and SI, would suggest an involvement of the S-locus in UI.     

Lack of Control by Early Pistillate Ethylene of the Accelerated Wilting of Petunia hybrida Flowers

Well before pollen tube penetration, ethylene has begun to disseminate from pollinated styles of Petunia hybrida flowers. Previous stigmatic application of aminoethoxyvinylglycine (AVG) completely prevented this ethylene synthesis, indicating that the endogenous 1-aminocyclopropane-1-carboxylic acid (ACC) in pollen is not readily converted on the stigma. Compared to other flower parts, the capacity of the ethylene forming enzyme was largest in the stigma. When applied to the stigma, ACC caused ethylene synthesis, but did not accelerate wilting, unless high concentrations (20 nanomols) were used. Upon pollination or stigma wounding, the early ethylene evolved exclusively from the gynoecium, much later followed by the synthesis of corolla ethylene. Employing wideneck Erlenmeyer flasks, the competitive inhibitor of ethylene action, norbornadiene, was applied to entire flowers in situ, with delaying effects on wound-induced wilting. In contrast, norbornadiene treatment of styles alone, using capillaries, could not postpone wilting. Pollination with foreign pollen species did not lead to accelerated corolla wilting, notwithstanding considerable synthesis of ethylene during the first 5 hours. In situ treatment of the stigma with AVG considerably delayed wound- and pollination-induced wilting. Removal of the entire AVG-treated style 6 hours after stigma wounding still allowed for the postponement of the accelerated wilting, even at very low concentrations of AVG. It is concluded that early stylar ethylene does not play a role in the acceleration of wilting but that, much later, corolla ethylene does, induced by a mobile wilting factor from the stigma, which is ACC.     

Three 1-Aminocyclopropane-1-Carboxylate Synthase Genes Regulated by Primary and Secondary Pollination Signals in Orchid Flowers

The temporal and spatial expression patterns of three 1-aminocyclopropane-1-carboxylate (ACC) synthase genes were investigated in pollinated orchid (Phalaenopsis spp.) flowers. Pollination signals initiate a cascade of development events in multiple floral organs, including the induction of ethylene biosynthesis, which coordinates several postpollination developmental responses. The initiation and propagation of ethylene biosynthesis is regulated by the coordinated expression of three distinct ACC synthase genes in orchid flowers. One ACC synthase gene (Phal-ACS1) is regulated by ethylene and participates in amplification and interorgan transmission of the pollination signal, as we have previously described in a related orchid genus. Two additional ACC synthase genes (Phal-ACS2 and Phal-ACS3) are expressed primarily in the stigma and ovary of pollinated orchid flowers. Phal-ACS2 mRNA accumulated in the stigma within 1 h after pollination, whereas Phal-ACS1 mRNA was not detected until 6 h after pollination. Similar to the expression of Phal-ACS2, the Phal-ACS3 gene was expressed within 2 h after pollination in the ovary. Exogenous application of auxin, but not ACC, mimicked pollination by stimulating a rapid increase in ACC synthase activity in the stigma and ovary and inducing Phal-ACS2 and Phal-ACS3 mRNA accumulation in the stigma and ovary, respectively. These results provide the basis for an expanded model of interorgan regulation of three ACC synthase genes that respond to both primary (Phal-ACS2 and Phal-ACS3) and secondary (Phal-ACS1) pollination signals.     

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