Ethylene is Involved in the Control of Male Gametophyte Development and Germination in Petunia

The time courses of 1-aminocyclopropane-1-carboxylic acid (ACC) content and ethylene production in developing anthers of petunia fertile and sterile lines and the effects of exogenously applied ethylene and an inhibitor of ethylene action, 2,5-norbornadiene (NBD), on male gametophyte development and germination were investigated. Fertile male gametophyte development was accompanied by two peaks of ethylene production by anther tissues. The first peak occurred during microspore development simultaneously with degeneration of both tapetal tissues and middle layers of the anther wall. The second peak coincided with maturation and dispersal of pollen grains. The mature pollen is characterized by a high ACC content (up to 300 nmol/g). Exogenously applied ethylene (1–100 ppm) induced degradation of gametophytic generation at the meiosis stage. NBD completely inhibited anther development at the early stages of its development and delayed anther dehiscence. In anther tissues of the petunia sterile line, tenfold higher ethylene production was observed at the meiosis stage compared to that in fertile male gametophytes and this correlated with degeneration of both microsporocytes and tapetal tissues. In vitro male gametophyte germination was accompanied by an increase of ethylene production, whereas NBD completely blocked male gametophyte germination. These results suggest that ethylene is an important factor in male gametophyte development and germination.     

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     

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.     

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.     

Gametophyte–Sporophyte Interactions in the Pollen–Pistil System: 3. Hormonal Status at the Progamic Phase of Fertilization

The content of hormones, IAA, ABA, and cytokinins, as well as the rate of ethylene production in petunia (Petunia hybrida L.) pistils and their parts (stigma, style, and ovary) were determined over 8 h after compatible pollination. At the progamic phase of fertilization in the pollen–pistil system, the phytohormones were virtually absent from the ovary but were present in various proportions in stigma and style. The stigma was the main site of ethylene synthesis and contained 90% of ABA while the style contained 80% of cytokinins of their contents in the whole pollinated pistil. Stigma and style did not differ in their IAA levels. The interaction of the male gametophyte with the stigmatic tissues was accompanied by a threefold increase in the ethylene production and a 1.5-fold increase in the IAA content in the pollen–pistil system within 0–4 h. Growth of pollen tubes in the stylar tissues (4–8 h) was accompanied by a further increase in IAA content and a decrease in the ethylene production by stigmatic tissues, as well as by a decrease in the cytokinin content in the stylar tissues. The ethylene/auxin status of the stigma may be suggested to control the processes of adhesion, hydration, and germination of pollen grains during pollination, while the auxin/cytokinin status of the style controls the pollen tube growth.     

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.     

Auxin abolishes inhibitory effects of methylcyclopropen and amino oxyacetic acid on pollen grain germination,pollen tube growth,and the synthesis of ACC in petunia

As established by us earlier, ethylene behaves as a regulator of germination, development, and growth of male gametophyte during the progamic phase of fertilization. However, the mechanisms of the regulation of these processes remain so far unstudied. It is believed that the main factor providing variety of the ethylene responses is its interaction with other phytohormones. According to our working hypothesis, ethylene controls germination of pollen grains (PGs) and growth of pollen tubes (PTs) by interacting with auxin, which, as the available data indicate, is likely a key regulator of plant cell polarization and morphogenesis and one of the factors modulating the biosynthesis of ethylene at the level of ACC-synthase gene expression. In the present work, on germinating in vitro male gametophyte and the pollen-stigma system for petunia (Petunia hybrida L.) effects of phytohormones (ethylene and IAA) and known blockers repressing ethylene reception (1-methylcyclopropene, 1-MCP), the synthesis of ACC (amino oxyacetic acid, AOA) and transport IAA (triyodbenzoynaya acid, TYBA) on PGs germination, PTs growth and the synthesis of ACC were investigated. According to the data obtained, exogenous ethylene and IAA stimulated both PGs germination and PTs growth. 1-MCP and TYBA completely inhibited the first process, whereas IAA abolished the inhibitory action of 1-MCP and AOA on both the above processes. Etrel only partially weakened the inhibitory effect of TYBA. Examination of ACC synthesis modulation with AOA showed that IAA does not affect the level of ACC in germinating in vitro male gametophyte and nonpollinated stigmas, while this phytohormone insignificantly raised the level of ACC and abolished the inhibitory effect of AOA on its synthesis in the pollenstigma system. Pollination of stigmas with the pollen preliminarily treated with 1-MCP led to 2.5-fold decline in both the rate of PT growth and the level of ACC. At the same time, IAA abolished the inhibitory action of 1-MCP recovering the synthesis of ACC and growth of PTs to the control values. All these results, taken together, provide evidence for the interaction of the signal transduction pathways of ethylene and auxin at the level of ACC biosynthesis in the course of germination and growth of petunia male gametophyte during the progamic phase of fertilization.     

Sporophyte-gametophyte interactions between anther and male gametophyte in petunia

Sporophyte-gametophyte interactions between anther and male gametophyte were investigated in two (fertile and sterile) clones of petunia (Petunia hybrida L.) with different reproductive strategies. Structural and functional reorganization of sporophyte tissues in the developing anther of fertile clone is closely coordinated with each of the successive stages of male gametophyte development (from meiosis to the formation of binuclear pollen) and comprises not only destruction of tapetum and three middle layers of the wall but also an activation of gas exchange and a rise in the content of sugars (sucrose, fructose, and glucose). In sterile clone, degradation of tapetum and anomalies in the development of sporogenic tissue were simultaneously observed in the prophase of meiosis. The death of microsporocytes and degeneration of tapetum were accompanied by a decrease in the level of sucrose delivered to the anther tissues and changes in the ratio between sucrose and hexoses in favor of glucose.     

Five Nodulation Mutants of White Sweetclover (Melilotus alba Desr.) Exhibit Distinct Phenotypes Blocked at Root Hair Curling,Infection Thread Development,and Nodule Organogenesis

Flavonol aglycones are required for pollen germination in petunia (Petunia hybrida L.). Mutant plants lacking chalcone synthase (CHS), which catalyzes the first committed step in flavonoid synthesis, do not accumulate flavonols and are self-sterile. The mutant pollen can be induced to germinate by supplementing it with kaempferol, a flavonol aglycone, either at the time of pollination or by addition to an in vitro germination system. Biochemical complementation occurs naturally when the mutant, flavonol-deficient pollen is crossed to wild-type, flavonoid-producing stigmas. We found that successful pollination depends on stigma maturity, indicating that flavonol aglycone accumulation may be developmentally regulated. Quantitative immunoblotting, in vitro and in vivo pollen germination, and high-performance liquid chromatographic analyses of stigma and anther extracts were used to determine the relationship between CHS levels and flavonol aglycone accumulation in developing petunia flowers. Although substantial levels of CHS were measured, we detected no flavonol aglycones in wild-type stigma or anther extracts. Instead, the occurrence of a conjugated form (flavonol glycoside) suggests that a mechanism may operate to convert glycosides to the active aglycone form.     

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。     

ABA and IAA control microsporogenesis in <Emphasis Type="Italic">Petunia hybrida</Emphasis> L.

The formation of fertile male gametophyte is known to require timely degeneration of polyfunctional tapetum tissue. The last process caused by the programmed cell death (PCD) is a part of the anther program maturation which leads to sequential anther tissue destruction coordinated with pollen differentiation. In the present work, distribution of abscisic acid (ABA) and indole-3-acetic acid (IAA) in developing anthers of male-fertile and male-sterile lines of petunia (Petunia hybrida L.) was analyzed by using the immunohistochemical method. It was established that the development of fertile male gametophyte was accompanied by monotonous elevation of ABA and IAA levels in reproductive cells and, in contrast, their monotonous lowering in tapetum cells and the middle layers. Abortion of microsporocytes in the meiosis prophase in the sterile line caused by premature tapetum degeneration along with complete maintenance of the middle layers was accompanied by dramatic, twofold elevation in the levels of both the phytohormones in reproductive cells. The data obtained allowed us to conclude that at the meiosis stage ABA and IAA are involved in the PCD of microsporocytes.     

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.     

Ovary and Gametophyte Development Are Coordinately Regulated by Auxin and Ethylene following Pollination

The differentiation and development of ovules in orchid flowers are pollination dependent. To define the developmental signals and timing of critical events associated with ovule differentiation, we have examined factors that regulate the initial events in megasporogenesis and female gametophyte development and characterized its progression toward maturity and fertilization. Two days after pollination, ovary wall epidermal cells begin to elongate and form hair cells; this is the earliest visible morphological change, and it occurs at least 3 days prior to pollen germination, indicating that signals associated with pollination itself trigger these early events. The effects of inhibitors of ethylene biosynthesis on early morphological changes indicated that ethylene, in the presence of auxin, is required to initiate ovary development and, indirectly, subsequent ovule differentiation. Surprisingly, pollen germination and growth were also strongly inhibited by inhibitors of ethylene biosynthesis, indicating that male gametophyte development is also regulated by ethylene. Detailed characterization of the development of both the female and male gametophyte in pollinated orchid flowers indicated that pollen tubes entered the ovary and grew along the ovary wall for 10 to 35 days, at which time growth was arrested. Approximately 40 days after pollination, coincident with ovule differentiation as indicated by the presence of a single archesporial cell, the direction of pollen tube growth became redirected toward the ovule, suggesting a chemical signaling between the developing ovule and male gametophyte. Taken together, these results indicate that both auxin and ethylene contribute to the regulation of both ovary and ovule development and to the coordination of development of male and female gametophytes.     

Pistil-Specific and Ethylene-Regulated Expression of 1-Aminocyclopropane-1-Carboxylate Oxidase Genes in Petunia Flowers

The differential expression of the petunia 1-aminocyclopropane-1-carboxylate (ACC) oxidase gene family during flower development and senescence was investigated. ACC oxidase catalyzes the conversion of ACC to ethylene. The increase in ethylene production by petunia corollas during senescence was preceded by increased ACC oxidase mRNA and enzyme activity. Treatment of flowers with ethylene led to an increase in ethylene production, ACC oxidase mRNA, and ACC oxidase activity in corollas. In contrast, leaves did not exhibit increased ethylene production or ACC oxidase expression in response to ethylene. Gene-specific probes revealed that the ACO1 gene was expressed specifically in senescing corollas and in other floral organs following exposure to ethylene. The ACO3 and ACO4 genes were specifically expressed in developing pistil tissue. In situ hybridization experiments revealed that ACC oxidase mRNAs were specifically localized to the secretory cells of the stigma and the connective tissue of the receptacle, including the nectaries. Treatment of flower buds with ethylene led to patterns of ACC oxidase gene expression spatially distinct from the patterns observed during development. The timing and tissue specificity of ACC oxidase expression during pistil development were paralleled by physiological processes associated with reproduction, including nectar secretion, accumulation of stigmatic exudate, and development of the self-incompatible response.     

长花柱型滇丁香小孢子发生及雄配子体发育

利用常规石蜡切片法和细胞学压片法,对异型花柱植物滇丁香的长花柱型植株的小孢子发生、雄配子体发育及花粉萌发进行观察。结果表明:(1)长花柱型滇丁香具5枚花药,花药4室。(2)花药壁由1层表皮、1层花药内壁、2层中层和1~3层绒毡层组成;花药壁发育方式为基本型,绒毡层类型为腺质绒毡层。(3)小孢子母细胞减数分裂的胞质分裂为同时型,四分体排列方式为四面体型,偶有左右对称型;不同药室间小孢子母细胞减数分裂不同步。(4)成熟花粉粒为二细胞型。(5)小孢子发生和雄配子体发育过程正常,表明长花柱型滇丁香属于发育正常的两性花。(6)授粉4h后,长花柱型滇丁香的花粉在长、短两种花柱柱头上的萌发率分别达(91.8±1.6)%和(93.2±1.1)%,且两者间无显著性差异(t=1.585,df=8,p=0.152),表明长花柱型滇丁香的成熟花粉粒在长、短两种花柱柱头上的萌发均正常。     

蓝猪耳小孢子发生和雄配子体发育

利用常规石蜡切片和超薄切片技术研究蓝猪耳(Torenia fournien)小孢子发生和雄配子体发育过程.蓝猪耳雄蕊4枚,花药具4个花粉囊.小孢子母细胞经减数分裂成四分体,其排列方式为四面体形或左右对称形.成熟花粉属2细胞型,具3个萌发孔.花药壁发育为双子叶型,腺质绒毡层.小孢子母细胞在四分体时期频繁出现细胞质降解的异常现象,其它发育阶段均正常;小孢子母细胞不正常的减数分裂可能导致花粉败育,这可能是蓝猪耳结实率低的原因之一.     

Role of abscisic acid and ethylene in the control of water transport-driving forces in germinating petunia male gametophyte

Data on ABA involvement in osmoregulation of in vitro germinating petunia (Petunia hybrida L.) male gametophyte were obtained. Two potential targets of ABA action in a pollen tube (PT) are identified. These are represented by (1) plasma membrane (PM) H⁺-ATPase, electrogenic proton pump participating in PM polarization, and (2) Ca-dependent K⁺-channels localized in the same membrane. It was established that a stimulatory effect of ABA on electrogenic activity of H⁺-ATPase is mediated by the increase in free Ca²⁺ level in the cytosol of a PT and reactive oxygen species (ROS) generation. Based on the results obtained on the role of K⁺ ions in the hormonal control of water transport-driving forces in a PT, the hypothesis suggesting that ABA stimulated pollen grains (PGs) germinating and PT growth by activating K⁺-channels was put forward. The revealed ABA-induced shift in cytoplasmic pH (pH_c) is suggested to be involved in a cascade of the events of the progamic phase of fertilization, including pH-dependent K⁺-channels functioning. It was established that ABA abolishes the inhibitory effects of ethylene receptors blocker, 1-methylcyclopropene (1-MCP), and blockers of ACC and ABA synthesis (aminooxyacetic acid, AOA, and fluridone, respectively) on PT germination and growth, whereas ethrel blocks the inhibitory effect of fluridone on PT growth. In stigmas pretreated with ABA and AOA before pollination, this phytohormone was found to suppress inhibitory effect of AOA on ACC synthesis in the pollen-pistil system. All these findings, taken together, led us to the conclusion that ABA is involved in petunia male gametophyte osmoregulation interacting with ethylene at the level of ACC synthesis in the progamic phase of fertilization.