Progamic Phase Of Fertilization Research Articles

Hormonal Signaling in the Progamic Phase of Fertilization in Plants

Pollen–pistil interaction is a basic process in the reproductive biology of flowering plants and has been the subject of intense fundamental research that has a pronounced practical value. The phytohormones ethylene (ET) and cytokinin (CK) together with other hormones such as auxin, gibberellin (GA), jasmonic acid (JA), abscisic acid (ABA), and brassinosteroids (BRs) influence different stages of plant development and growth. Here, we mainly focus on the information about the ET and CK signaling in the progamic phase of fertilization. This signaling occurs during male gametophyte development, including tapetum (TAP) cell death, and pollen tube growth, including synergid programmed cell death (PCD) and self-incompatibility (SI)-induced PCD. ET joins the coordination of successive events in the developing anther, including the TAP development and cell death, anther dehiscence, microspore development, pollen grain maturation, and dehydration. Both ET and CK take part in the regulation of pollen–pistil interaction. ET signaling accompanies adhesion, hydration, and germination of pollen grains in the stigma and growth of pollen tubes in style tissues. Thus, ET production may be implicated in the pollination signaling between organs accumulated in the stigma and transmitted to the style and ovary to ensure successful pollination. Some data suggest that ET and CK signaling are involved in S-RNase-based SI.

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.

Role of auxin and cytokinin in the regulation of the actin cytoskeleton in the in vitro germinating male gametophyte of petunia

Pollen tubes germinating in vitro, which has become in recent years the object of detail and intensive research, is a model for studying the mechanisms of sexual reproduction of higher plants. The potential role of the actin cytoskeleton (F-actin) in the transduction of hormonal signals in the progamic phase of fertilization was studied on in vitro germinating male gametophyte of petunia (Petunia hybrida L.). It was established for the first time that treatments with phytohormones, such as auxin (IAA) and cytokinin (kinetin), affected germination and polar growth of the pollen tubes, e.g., stimulated or inhibited these processes, and also induced the actin cytoskeleton rearrangement. The addition of IAA to nutrient medium increased the content of F-actin in the apical and subapical zones of pollen tubes and, as it is supposed, is responsible for the stimulation of their polar growth due to the intensification of the cytoplasm flow and apically directed vesicle transport. The inhibitor of actin polymerization, latrunculin B, disturbed actin cytoskeleton spatial organization, decreased the content of endogenous IAA, and, as a consequence, retarded germination and/or blocked male gametophyte polar growth. In contrast, kinetin suppressed pollen tube growth, reducing the content of polymer actin along the entire tube length. In pollen tubes germinating in the presence of latrunculin B for 4 h, the content of cytokinins was not significantly changed as compared with control. These results allow a conclusion that auxin and cytokinin are involved in the regulation of pollen tube polar growth via their effects on actin polymerization and spatial organization.

Cell division interference in newly fertilized ovules induces stenospermocarpy in cross-pollinated citrus fruit

Seedlessness is a highly desirable characteristic in fresh fruits. However, post-fertilization seed abortion of cross-pollinated citrus fruit is uncommon. The factors regulating stenospermocarpy in citrus are unknown. In this research, we induced stenospermocarpy interfering in newly fertilized ovule cell division. The research also elucidates the most sensitive stage for ovule/seed abortion in citrus. Experiments were conducted with ‘Afourer’ mandarin that cross-pollinates with several cultivars and species. Cross-pollinated fruitlets were treated with maleic hydrazide (MH), a systemic growth regulator that specifically interferes in cell division. MH reduced ovule growth rate, the number of cell layers in nucella and inhibited embryo sac expansion; moreover, the treatment increased callose accumulation in nucella and surrounding the embryo sac. Fruits developed an early-aborted seed type with an immature, soft and edible seed coat. Seed number (−80%) and seed weight (−46%) were reduced in mature fruits. MH also hampered cell division in ovary walls, mesocarp and endocarp, thus reducing daily fruitlet growth and increasing fruit abscission. Stenospermocarpy could only be induced for a short period of time in the progamic phase of fertilization, specifically, when ovules are ready to be fertilized (7 days after anthesis) to early stages of embryo sac development (14 days after anthesis).

Роль этилена в регуляции гаметофитно-спорофитных взаимодействий в прогамной фазе оплодотворения у петунии (Petunia hybridaL.)

We investigated dynamics of the content of 1-aminocyclopropane-l-carboxylic acid (ACC) and ethylene production in male gametophyte development and germination in fertile (self-compatible and self-incompatible) 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 ofACC 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.

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.

Inheritance and Expression of Gametophyte Mutant Gene Adh-PAffecting the Modification of Alcohol Dehydrogenase in Pollen Grains of the Beet Beta vulgaris L.

An additional alcohol dehydrogenase (ADH) activity zone denoted ADH-P (“pollen”) has a slightly lower mobility than the major protein ADH1 (the product of structural locus Adh1). This zone is detected in maturing and mature pollen grains and has not been found in any other tissue. ADH-P is detected by electrophoresis in a neutral medium (at pH 7.0–7.2). In an alkaline medium (pH > 8), protein ADH-P is completely inactivated, whereas protein ADH1 retains its activity. ADH-P is a modified variant of the major protein ADH1. Both alleles of the main structural gene (Adh1-F and Adh1-S) undergo modification. The pollen of an FS heterozygote has two variants of the modified enzyme: ADH-PS and ADH-PF. Analysis of segregation in F2 offsprings and test crosses has confirmed that this character is controlled by the only gene Adh-P with allelic variantsAdh-P+ (the presence of the modified ADH protein in the pollen) and Adh-P– (the normal protein). Allele Adh-P+ is transmitted through female gametes at a normal frequency (about 1) and through male gametes at a decreased frequency (0.2–0.6), the mean frequency being about 0.4. The frequency of the transmission of allele Adh-P+ through male gametes depends on the genotype of the female parent and the conditions of pollination. Cytoembryological study of microsporogenesis in the Adh-P+/Adh-P– heterozygotes demonstrated an absence of any disturbances in the formation of microspores and pollen grains. Some differences in the formation of pollen tubes on an artificial medium have been observed. It is assumed that the differences between theAdh-P+ and Adh-P– microgametophytes manifest themselves at the progamic phase of fertilization. The possible mechanisms of the formation of the modified ADH-P protein are discussed in connection with the differential activity of genes in the microgametophytes of angiosperms.

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.

Cytochalasin B Affects Protein Secretion in Germinating Pollen of Malus domestica Borkh.

Summary Apple pollen sensitivity to cytochalasin B was studied during germination in vitro , with consideration of the effect on tube emergence and growth, on protein release in the culture medium, and on secretion of tube wall proteins. Cytochalasin B from 0.05 to 1.0 μg ml −1 induced strong inhibition of tube elongation, whereas the pure emergence was less affected by the drug. At 0.1μg ml −1 the cytochalasin blocked protein export from 90 min germinated pollen tubes and markedly reduced 3 H-proteins of the ionically-bound fraction of tube walls. The inhibition of protein secretion agrees with numerous ultrastructural observations on the cytochalasin B effect in pollen tubes or other plant systems. In the case of pollen, the ionic protein fraction of the tube wall is important for communication with the stigma or style during the progamic phase of fertilization.

Effect of carbon dioxide and relative humidity on self incompatibility in cauliflower, Brassica oleracea

The events of the progamic phase of fertilization have been monitored by in vitro experiments in self compatible (SC), partial self-incompatible (PSI) and self incompatible (SI) lines. The duration of the progamic phase is about 30 h. Treatment with low concentrations of CO2 (3 to 5%) at high relative humidity (rH, 100%) had the following effects: pollen quality, which declines normally during flower ageing, was prematurely reduced; pollen adhesion and germination, both low in SI matings, were increased; the stigma callose response in SI matings was reduced to the low level of SC matings; and the number of pollen tubes in the style after SI matings significantly increased. CO2 concentrations of 4 to 6% applied for 8, 16 or 24 h at 100% rH proved to be the most effective treatment for blocking the SI response in cauliflower.

PM H+-ATPase is a potential target of hormone signaling during pollen tube growth in petunia

Aim. Pollen tube growth is the best known example of highly polarized plant cell expansion. This process is known to require intensive exocytosis at the tip which is supported by dynamic cytoskeleton, vesicle trafficking, and functioning of ion transporters in pollen tube plasma membrane (PM). We have attempted to test the possibility of plant hormones functioning as signal molecules in progamic phase of fertilization. Methods. The effects of exogenous plant hormones, indole-3-acetic acid (IAA), abscisic acid (ABA), gibberellin A3 (GA3), and cytokinin (kinetin) on the growth, PM polarization of in vitro germinating petunia (Petunia hybrida L.) pollen tubes were investigated. Results. Growth-stimulated effects of IAA, ABA and GA3 were accompanied by orthovanadate-sensitive hyperpolarization of PM suggesting possible involvement of PM H+-ATPase activation in this process. Fluorescent labeling the enzyme with H+-ATPase antibodies exhibited IAA- and ABA-induced lateral membrane redistribution of it into the subapical zone of pollen tube PM. Conclusions. All these results, taken together, indicate the potential target of hormone signaling in pollen tubes, PM H+-ATPase, related to the hormone-induced modulation of the above mentioned characteristics of this proton pump.Keywords: Petunia hybrida, Pollen tube, PM H+-ATPase, phytohormones.