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.     

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.     

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.     

Exogenous IAA and ABA stimulate germination of petunia male gametophyte by activating Ca<Superscript>2+</Superscript>-dependent K<Superscript>+</Superscript>-channels and by modulating the activity of plasmalemma H<Superscript>+</Superscript>-ATPase and actin cytoskeleton

To date, the molecular mechanisms underlying the osmoregulation of pollen grains (PGs) related to the maintenance of their water status and allowing pollen tubes (PTs) to regulate concentrations in them of osmolytes and transmembrane water transport remain to be not so far characterized. In the present work, the data on the participation of IAA and ABA in the osmoregulation of germinating in vitro petunia male gametophyte were obtained. It has been established that the growth-stimulating effect of these phytohormones is due to their action on intracellular pH (pH_c), the membrane potential of plasmalemma (PM), the activity of PM H⁺-ATPase, K⁺-channels in the same membrane and organization of actin cytoskeleton (AC). Two possible targets of the action of these compounds are revealed. These are represented by (1) PM H⁺-ATPase, electrogenic proton pump responsible for polarization of this membrane, and (2) Ca²⁺-dependent K⁺-channels. The findings of the present work suggest that the hormone-induced pH_c shift is involved in cascade of the events including the functioning of pH-dependent K⁺-channels. It was shown that the hormoneinduced hyperpolarization of the PM is a result of stimulation of electrogenic activity of PM H⁺-ATPase and the hormonal effects are mediated by transient elevation in the level of free Ca²⁺ in the cytosol and generation of reactive oxygen species (ROS). The results on the role of K⁺ ions in the control of water-driving forces for transmembrane water transport allowed us to formulate the hypothesis that IAA and ABA stimulate germination of PGs and growth of PTs by activating K⁺-channels. In addition, the studies performed showed that the AC of male gametophyte is sensitive to the action of exogenous phytohormones, with to more extent to the action of IAA. As judged by the action of latrunculin B (LB) the AC may serve as the determinant of the level of endogenous phytohormones that most likely participate in the regulation of the polar growth of PTs impacting on the pool of F-actin in their apical and subapical zones.     

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.     

The effect of temperature on pollen germination, pollen tube growth, and stigmatic receptivity in peach

Temperature is a major climatic factor that limits geographical distribution of plant species, and the reproductive phase has proven to be one of the most temperature-vulnerable stages. Here, we have used peach to evaluate the effect of temperature on some processes of the progamic phase, from pollination to the arrival of pollen tubes in the ovary. Within the range of temperatures studied, 20 degrees C in the laboratory and, on average, 5.7 degrees C in the field, the results show an accelerating effect of increasing temperature on pollen germination and pollen tube growth kinetics, as well as an increase in the number of pollen tubes that reach the style base. For the last two parameters, although the range of temperature registered in the field was much lower, the results obtained in the laboratory paralleled those obtained in the field. Increasing temperatures drastically reduced stigmatic receptivity. Reduction was sequential, with stigmas first losing the capacity to sustain pollen tube penetration to the transmitting tissue, then their capacity to offer support for pollen germination and, finally, their capacity to support pollen grain adhesion. Within a species-specific range of temperature, this apparent opposite effect of temperature on the male and female side could provide plants with the plasticity to withstand changing environmental effects, ensuring a good level of fertilization.     

Establishment of the male germline and sperm cell movement during pollen germination and tube growth in maize

Two sperm cells are required to achieve double fertilization in flowering plants (angiosperms). In contrast to animals and lower plants such as mosses and ferns, sperm cells of flowering plants (angiosperms) are immobile and are transported to the female gametes (egg and central cell) via the pollen tube. The two sperm cells arise from the generative pollen cell either within the pollen grain or after germination inside the pollen tube. While pollen tube growth and sperm behavior has been intensively investigated in model plant species such as tobacco and lily, little is know about sperm dynamics and behavior during pollen germination, tube growth and sperm release in grasses. In the March issue of Journal of Experimental Botany, we have reported about the sporophytic and gametophytic control of pollen tube germination, growth and guidance in maize.1 Five progamic phases were distinguished involving various prezygotic crossing barriers before sperm cell delivery inside the female gametophyte takes place. Using live cell imaging and a generative cell-specific promoter driving α-tubulin-YFP expression in the male germline, we report here the formation of the male germline inside the pollen grain and the sperm behaviour during pollen germination and their movement dynamics during tube growth in maize.Key words: male gametophyte, generative cell, sperm, pollen tube, tubulin, fertilization, maize     

Free IAA in stigmas and styles during pollen germination and pollen tube growth of Nicotiana tabacum

Although many studies have emphasized the importance of auxin in plant growth and development, the thorough understanding of its effect on pollen–pistil interactions is largely unknown. In this study, we investigated the role of free IAA in pollen–pistil interactions during pollen germination and tube growth in Nicotiana tabacum L. through using histo and subcellular immunolocalization with auxin monoclonal antibodies, quantification by HPLC and ELISA together with GUS staining in DR5::GUS -transformed plants. The results showed that free IAA in unpollinated styles was higher in the apical part and basal part than in the middle part, and it was more abundant in the transmitting tissue (TT). At the stage of pollen germination, IAA reached its highest content in the stigma and was mainly distributed in TT. After the pollen tubes entered the styles, the signal increased in the part where pollen tubes would enter and then rapidly declined in the part where pollen tubes had penetrated. Subcellular localization confirmed the presence of IAA in TT cells of stigmas and styles. Accordingly, a schematic diagram summarizes the changing pattern of free IAA level during flowering, pollination and pollen tube growth. Furthermore, we presented evidence that low concentration of exogenous IAA could, to a certain extent, facilitate in vitro pollen tube growth. These results suggest that IAA may be directly or indirectly involved in the pollen–pistil interactions. Additionally, some improvements of the IAA immunolocalization technique were made.     

Pollen and pistil in the progamic phase

The progamic phase, the period of pollen tube growth through the pistil, is a period of specific interactions between the male gametophyte and the pistil. Understanding of pollen germination and pollen tube growth are relevant for the study of pollen-pistil interactions and for understanding the function of components specifically accumulated in the transmitting tissue cell walls and intercellular matrix that may interact with pollen tubes. Received: 18 January 2001 / Accepted: 19 June 2001     

Ethylene Synthesis and Floral Senescence following Compatible and Incompatible Pollinations in Petunia inflata

Ethylene production and floral senescence following compatible and incompatible pollinations were studied in a self-incompatible species, Petunia inflata. Both compatible and incompatible pollinations resulted in a burst of ethylene synthesis that peaked 3 hours after pollination. P. inflata pollen was found to carry large amounts of the ethylene precursor, 1-aminocyclopropane-1-carboxylic acid (ACC). The amount of pollen-held ACC varied in different genetic backgrounds, and the magnitude of the peak correlated with the amount of ACC borne by the pollen. Aminooxyacetic acid (AOA), an inhibitor of ACC synthesis, had no inhibitory effect on this ethylene response, indicating that pollen-borne ACC was largely responsible for the early synthesis of ethylene. After compatible pollination, a second increase in ethylene synthesis began at 18 hours, and the first sign of senescence appeared at 36 hours. Upon treatment with AOA, the second phase of ethylene production was reduced by 95%, indicating that endogenous ACC synthesis was required for this phase of ethylene synthesis. AOA treatment also delayed senescence to 6 days after anthesis. After incompatible pollination, a second increase in ethylene production did not occur until 3 days, and the first sign of senescence occurred 12 hours later. Unpollinated flowers showed an increase in ethylene production 3 to 4 days after anthesis and displayed signs of senescence 1 day later. The significance of the early and late phases of pollination-induced ethylene synthesis is discussed.     

Germination and <Emphasis Type="Italic">In Vitro</Emphasis> Growth of Petunia Male Gametophyte Are Affected by Exogenous Hormones and Involve the Changes in the Endogenous Hormone Level

The data obtained characterize the changes in the contents of endogenous phytohormones (IAA, cytokinins, GA, and ABA) in germinating pollen grains and growing pollen tubes of a self-compatible clone of petunia (sPetunia hybrida L.) within an 8-h period under in vitro conditions. The hydration and initiation of germination of pollen grains brought the ABA content down to a zero level, while the levels of GA, IAA, and cytokinins increased 1.5–2-fold. Later, in the growing pollen tubes, the GA content increased twofold, while the levels of IAA and cytokinins decreased. The exogenous ABA and GA₃ considerably promoted pollen germination and pollen tube growth; however, only the treatment with GA₃ produced the maximum length of pollen tubes. The exogenous IAA promoted and the exogenous cytokinins hindered the growth of pollen tubes. The membrane potential, as assessed with a potential-sensitive dye diS-C₃-(5), considerably increased in the pollen grains treated with ABA and benzyladenine, whereas IAA and GA₃ did not practically affect it. The authors conclude that the mature pollen grains contain the complete set of hormones essential for pollen germination and pollen tube growth. ABA, GA, and IAA together with cytokinins control the processes of pollen grain hydration, germination, and pollen tube growth, respectively.__________Translated from Fiziologiya Rastenii, Vol. 52, No. 4, 2005, pp. 584–590.Original Russian Text Copyright © 2005 by Kovaleva, Zakharova, Minkina, Timofeeva, Andreev.     

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.     

The involvement of ethylene in in vitro maturation of mid-binucleate pollen of Nicotiana tabacum

The role of ethylene during in vitro maturation of Nicotianatabacum pollen from the mld-binucleate (MB) stage was analysedby the addition of aminooxyacetic acid (AOA), aminoethoxyvinylglycine(AVG), CoCl₂ and AgNO₃ to the maturation medium (AMGLu). Anincrease in ethylene production was obtained in both isolatedpollen and pollen surrounded by sporophytic tissue during insitu maturation. in vitro maturation of pollen was inhibitedby AOA and AVG; ACC and ethrel were able to overcome this inhibitoryeffect. Cyclohexylamine (CHA) reverted the inhibition provokedby both Ag⁺ and Co²⁺ The results reported in this paper indicatethat ethylene is one of the factors implicated in in vitro maturationof MB pollen of Nicotiana tabacum. Key words: Nicotiana tabacum, maturation, germination, pollen, ethylene     

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.