Feb 27, 2017

Hormonal Status of the Pollen-Pistil System: Role after Pollination

Results of studies of hormonal regulation of petunia (Petunia hybrida L.) male gametophyte germination and growth in vivo, on surface of receiving stigma as well as in conducting style tissue, and also in vitro, on the cultivating medium are presented. The contents of IAA, ABA and cytokinins as well as the rate of ethylene production in the pistils and their parts were measured following compatible and self-incompatible pollinations. The data obtained provide evidence suggesting the participation of phytohormones in pollen-pistil interactions controlling uninterrupted pollen tube growth after compatible pollination and its inhibition after incompatible pollination. For two experimental systems studied, in vitro germinating male gametophyte and pollen pistil, it was established that ethylene is able to control the regulation of development, germination, and growth of male gametophyte and also to be involved in the mechanism of gametophyte self-incompatibility as one of the main barriers of self-fertilization and to behave as a regulator of the gametophyte-sporophyte interactions. According to our findings germination and growth of petunia male gametophyte in vitro is accompanied by changes in the content of endogenous phytohormones and at the same time exhibits sensitivity to exogenous ones. It was shown that the effects of exogenous phytohormones on petunia male gametophyte germination and growth in vitro are related to some changes in the organization of its cytoskeleton. In the experiments carried out on germinating pollen grains, the first data indicating a possible mechanism underlying the action of the phytohormones in question were obtained. It was found that IAA, ABA and gibberellin A3 are capable of significantly changing the cytoplasmic pH of pollen cells and simultaneously resulting in hyperpolarization of the pollen plasma membrane, thereby indicating the phytohormone-induced stimulation of the activity of H+-ATPase in this membrane. The above results, taken together, indicate a significant impact of phytohormones on various cellular activities of petunia male gametophyte in the course of the gametophyte-sporophyte interactions in the progame phase of higher plant fertilization.

Introduction
Angiosperms have evolved a system of sexual reproduction where water is not required at the point of fertilization, immobile sperm being delivered directly to the egg by a pollen tube. This key innovation, termed ‘siphonogamy’, allowed them to reproduce sexually in most terrestrial environments. Before a pollen tube (male gametophyte) can access an ovule to release its two sperms into the embryo sac (female gametophyte) it must first penetrate the gynoecium and navigate its way through this material sporophytic tissue to find an ovule. The events and interactions that occur during this prezygotic cellular and molecular ‘courtship’ between haploid pollen and diploid gynoecium have been termed the pollen-pistil interaction (Heslop-Harrison 1975) and consist of six key stages: pollen capture and adhesion; 2) pollen hydration; 3) germination of the pollen to produce a pollen tube; 4) penetration of the stigma by the pollen tube; 5) growth of the pollen tube through the stigma and style; 6) entry of the pollen tube into the ovule and discharge of the sperm cells. Understanding the physiological and molecular basis of the cellular interactions that occur during the pollen-pistil interactions involving a continuous exchange of signals between the haploid pollen and the diploid maternal tissue of the pistil (sporophyte) is a major goal for plant biologists, because ultimately we all depend on this fundamental biological process for our food. Despite the recent exponential increase in the number of molecules implicated in pollen-pistil interactions in different model plant species and the significant progress that has been made in elucidating the molecular identity of these signals and the cellular interactions that they regulate, no general consensus has yet emerged of a universal set of pollen-pistil mediation molecules that regulate a common program of cellular interactions (Hiscock and Allen 2008).

The first experiments on the involvement of phytohormones [indolyl-3- acetic acid (IAA), zeatin, gibberellic acid (GA), and abscisic acid (ABA)] in fertilization (Stanley and Linskens 1974) allowed researchers to suggest that hormones play a regulatory role in pollen tube germination and extension through pistil tissue. The question of phytohormones involvement in controlling post-pollination events had been discussed for a long time but so far remains unstudied.

Here, results of studies of hormonal regulation of petunia (Petunia hybrida L.) male gametophyte germination and growth in vivo, on surface of receiving stigma and in conducting style tissue, and also in vitro, on cultivating medium are presented.

Hormonal Status of Petunia Pollen-Pistil System

The hormonal status of the pollen-pistil system was investigated in two clones of Petunia hybrida L. (Kovaleva and Zakharova 2003). The contents of IAA, ABA, and cytokinins, as well as the rate of ethylene production in the pistils and their parts (stigmas, styles and ovaries) were measured over an 8-hr period following compatible and self-incompatible pollination. (Figs. 1–3).



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 (adhesion, hydration, and germination of pollen grains) 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 hr. Pollen tube germination 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. During the subsequent 4 hr., pollen tube growth was accompanied by some changes in the hormonal status of the pollen-pistil system. Ethylene production by pistil tissues decreased in both cases; however, in the case of incompatible pollination, ethylene content declined more slowly. The IAA content rose in both cases; however, in the case of incompatible pollination, IAA rose more slowly. In both pollinations, the ABA content remained unchanged, but the ABA concentration in the case of incompatible pollination was maintained at the same high level. During incompatible pollination, inhibition of pollen tube growth by 8 hr. was accompanied by a 5-fold increase in cytokinin content in style tissues, whereas it remained unchanged during a compatible pollination. The data obtained provide two lines of evidence suggesting the participation of phytohormones in pollen-pistil interactions controlling uninterrupted pollen tube growth after compatible pollination or its inhibition after incompatible pollination. Firstly, a marked difference in the hormonal status of the pollen-pistil system in the course of compatible and incompatible pollination argues in favor of such a hypothesis. Secondly, pollen grain germination on the stigma surface and pollen tube growth in style tissues appears to occur in tissues differing in their hormonal status and to be accompanied by complex alterations of the hormonal concentrations in stigma and style tissues.

Role of Ethylene in the Control of Male Gametophyte Germination and Growth after Self-Compatible and Self-Incompatible Pollination
In flowering plants, pollination of the stigma sets off a cascade of responses in the whole flower that contribute to the successful sexual reproduction in higher plants. Such post-pollination symptoms as petal wilting, pigmentation changes, ovary and ovule development or style and stamen abscission are mediated by ethylene (O’Neill 1997). The responses of the distal floral organs to the pollination event at the stigma surface are regulated by inter-organ signaling. Compounds implied in signaling are the gaseous hormone ethylene and its precursor 1-aminocyclopropane-1- carboxylic acid (ACC) (Whitehead et al. 1984). Ethylene is produced via a two-step biosynthetic route that starts with the conversion of the Met derivative S-adenosyl-L-Met to ACC and 5’-methyl-thioadenosine by ACC synthase. Next, ACC is oxidized by ACC-oxidase to form ethylene, CO2 and HCN (Yang and Hoffman 1984).

Studies performed on the orchid, carnation, tobacco, and petunia indicate that ethylene induced by pollination is necessary for the growth of pollen tubes and successful fertilization (Hoekstra and Weges 1986; Singh et al. 1992; O’Neill et al.1993; Tang and Woodson 1996; Bui and O’Neill 1998; Holden et al 2003; Kovaleva and Zakharova 2003; Kovaleva et al. 2007, 2011). However, the issue of the physiological role of ethylene in gametophyte sporophyte interactions in the progamic phase of fertilization both at normal development of the reproductive process and in the presence of genetically determined barriers of self-fertilization is still far from being solved. The ethylene that is produced upon pollination is characterized by two peaks of its evolving. The first ethylene burst evolves from the stigma and can be attributed mainly to direct conversion of pollen-borne ACC (Hill et al. 1987) by ACC-oxidase that is abundantly present in the stigma (O’Neill 1997). The second peak of ethylene evolving is produced by flower organs that are distal to the stigma, like the style, the ovary, and the petals, and can mainly be attributed to endogenous ACC-synthase and—oxidase activities. These activities correlate closely with the transient increase in expression of the corresponding genes (O’Neill et al. 1993; Tang et al. 1994; Lindstrom et al. 1999; Weterings et al. 2002).

Ethylene production and floral senescence following compatible and incompatible pollinations were studied in a self-incompatible species, Petunia inflata (Singh et al. 1992). Both compatible and incompatible pollinations resulted in a burst of ethylene synthesis that peaked 3 hr. after pollination. After compatible pollination, a second increase in ethylene synthesis began at 18 hr., and the first sign of senescence appeared at 36 hr. After incompatible pollination, a second increase in ethylene production did not occur until 3d, and the first sign of senescence occurred 12 hr. later.

Depending on the type of pollination, germination of petunia (Petunia hybrida L.) 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 (Kovaleva et al. 2011). 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 (Fig. 4).

For both types of pollination, ACC and ethylene were predominantly produced in the stigma tissues. Our data indicate the possible participation of ethylene in the mechanism of gametophyte self-incompatibility as one of the main barriers of self-fertilization. This suggestion is confirmed by our data indicating that ethylene at high concentration (10 μl/l) resulted in decreasing by 50% the rate of pollen tube growth on the cultivation medium (Kovaleva et al. 2013). It is believed that one of the reasons for inhibition of the growth of pollen tubes after the self-incompatible pollination can be the programmed cell death (PCD) (Wang et al. 2009) induced by ethylene (Rogers 2006). In this connection, we suggest that intensive production of ethylene after self-incompatible pollination induces PCD in self-incompatible pollen tubes and thereby leads to retardation of their growth in conducting style tissues.

Very recently, we have obtained preliminary data that may be considered as evidence for ethylene involvement in the mechanism of gametophytic self-incompatibility (K. L.V., unpublished data). In the corresponding experiments it was found that treatment of petunia stigmas of self-incompatible clone with inhibitor of ethylene synthesis resulted in significant stimulation of this incompatible pollen tube growth and the resulting length of pollen tubes appeared to exceed more than two times that of the control pollen tubes. In this connection, it is significant to note that in style tissues where an extension growth of these pollen tubes occurred any signs of programmed cell death (PCD) were not revealed, unlike the control growing pollen tubes (self-pollination of self-incompatible petunia clone) where in 8 h after self-pollination, i.e., during the development of self-incompatibility reaction, clear signs of the PCD involving DNA degradation were observed.

In general, all these results obtained for the two experimental systems studied (anther-male gametophyte and pollen-pistil) (Kovaleva et al. 2007, 2011, 2013) allow us to conclude that ethylene controls the regulation of germination, development and growth of male gametophyte. Our recent results (K.L.V., unpublished data) suggest that ethylene is able to behave as a regulator of the gametophyte-sporophyte interactions in the progame phase of higher plant fertilization by exerting its effect on the base of its interaction with other phytohormones, such as IAA, ABA and gibberellins, in the course of the biosynthesis of ACC. In this connection, recent results published by Carbonell-Bejerano et al. (2011) and concerning pistil senescence in Arabidopsis is of great interest because here it was found that ethylene is involved in pistil fate by modulating the onset of ovule senescence and the GA-mediated fruit set. Another interesting result very recently obtained is that ethylene is capable of serving a key regulator of autophagy in petunia petal senescence and autophagy is induced by pollination (Shibuya et al. 2013).

Hormonal Status of in vitro Germinating Petunia Male Gametophyte
Endogenous level of phytohormones in petunia pollen was shown to undergo pronounced changes in the course of its germination on the cultivation medium (0.4 M sucrose and 1.6 mM H3BO3) (Fig. 5) (Kovaleva et al. 2005).

 On stage of hydration and germination of pollen grains ABA content in them declined practically up to zero, while levels of GA, IAA and cytokinins exhibited 1.5–2.0-fold increase. Subsequent growth of pollen tubes was companied by two-fold increase in GA content and marked decrease in IAA and cytokinin levels. Chen and Zhao (2008) in the experiments carried out on Nicotiana tobacco found that the IAA content was markedly high in part of the styles before pollen tubes penetrated into them and then gradually decreased when the latter reached the style region.

Application of phytohormones to the cultivation medium significantly influenced the germination and growth of petunia male gametophyte (Table 1 and 2).

ABA and gibberellin A3 at concentrations of 10–12 M to 10–3 M markedly stimulated the germination of pollen grains, whereas, IAA at 10–12 M to 10–10 M concentrations were stimulatory but higher concentrations (10–4–10–3 M) were inhibitory. Synthetic cytokinin 6-BAP at concentrations of 10–12 M to 10–3 M inhibited the germination.

Pollen tube length was measured after 1hr of pollen cultivation in the medium containing 0.4 M sucrose + and 1.6mM H3BO3. The data are the means and their standard deviations obtained from three independent experiments carried out in two replicates (n = 6).

The phytohormones that were tested (ABA, gibberellin A3 and IAA), stimulated the petunia pollen grain germination and pollen tube growth to different extent. Gibberellin A3 and ABA appeared to be most stimulatory hormones in their action upon pollen germination at concentration of 10–12 M. GA3 exerted a maximal effect on pollen tube growth: after 6-h cultivation their length was 450 μm. IAA at concentration of 10–12 M stimulated pollen germination and pollen tube growth 1.5- and 2–2.5-fold, respectively. At the same time, after addition of paclobutrazol, a known inhibitor of gibberellin synthesis, to the cultivation medium the pollen still germinated but under these conditions the length of pollen tubes appeared to be 2–3-fold lower as compared to the control pollen tubes and depended on concentration of the inhibitor.

ABA at concentration of 10–12 M exerted a maximal, three-fold stimulation of pollen germination in the first 30 min and 2–3 fold stimulation of pollen germination and pollen tube growth 1.0 h after cultivation. Fluridone, a known inhibitor of ABA synthesis, was found to suppress both pollen germination and pollen tube growth, and intensity of this effect depended on concentration of this inhibitor.

IAA at concentration of 10–12 M stimulated pollen germination and pollen tube growth 1.5- and 2–2.5-fold, respectively, with the stimulation of the latter process observed only at low enough concentrations of this hormone, whereas its high concentrations resulted in inhibition of the given process. The 2, 4-chlorphenoxy-2-methylpropionic acid, a known inhibitor of IAA transport, at concentration of 10–3 M completely blocked the pollen germination in both absence and presence of phytohormones, such as ABA and gibberellin A3, in the cultivation medium.

In the presence of 6-BAP, in contrast, only suppression of the processes in questions was observed, and such a character of action of this hormone did not depend on its concentration in the cultivation medium.

Based on these results, we put forward the hypothesis that polar transport of IAA impacts the germination and growth of male gametophyte, while ABA is involved in the regulation of their intra-cellular osmotic pressure during these processes. In this connection, an experimental approach for studying the role of ABA in the processes under study is highly intriguing if we take into account the putative signal transduction pathway for this hormone associated with pollen dehydration and leading to corresponding regulation of Rop gene expression (Hsu et al. 2010). Our experiments also showed the involvement of gibberellins in the regulation of pollen tube growth. Earlier, requirement of gibberellins for pollen tube growth was established in the experiments carried out on Arabidopsis mutants characterized by both decreased and increased level of gibberellins (Singh et al. 2002).

Evidence Indicating Impact of Phytohormones on Some Cellular
Activities of Petunia Male Gametophyte
It is well known that both pollen germination and maintenance of polar growth of pollen tube require temporal and spatial coordination of many cell functions including dynamic organization of cytoskeleton elements, intracellular vesicular transport delivering the material for cell wall production during exocytosis and endocytosis, transmembrane transport of basic physiologically important ions (H+, Ca2+, K+), and, in addition, transient changes of some parameters of intracellular ionic homeostasis, such as pH and pCa (Franklin-Tong 1999; Vidali and Hepler 2001; Holdaway-Clarke and Hepler 2003; Certal et al. 2008; Cheung and Wu 2008).

Effects of exogenous phytohormones on the actin cytoskeleton of petunia male gametophyte
In the course of our studies it has been established that the effects of exogenous phytohormones on petunia male gametophyte germination and growth in vitro are related to some changes in organization of its actin cytoskeleton (Voronkov 2010). In particular, it was found that IAA at concentrations of 10–12 and 10-6 M caused the increase by 37% of total amount of actin fi laments of pollen tube, and this was expressed in enhancement of fluorescence of these structures stained with FITS-falloidine (Fig. 6), with the largest effect was observed in both apical and subapical regions of pollen tube.

Thus IAA added to the cultivation medium resulted in acceleration of growth of pollen tube through increasing the amount of polymeric actin in the regions in question having the most important significance for maintenance of the polar growth process.

Unlike the above IAA action, stimulating influence of ABA and GA3 on pollen tube growth was accompanied only by tube zonal redistribution of F-actin, although a total amount of polymer actin in pollen tube remained unchanged (Fig. 6).

Kinetin, unlike IAA, ABA and GA3, led to suppression of actin polymerization in pollen tubes decreasing a density of actin fi laments by about 40% along all the length of pollen tube (Fig. 6), with the strongest effect observed in its basal part.

The above data provide evidence for sensitivity of actin cytoskeleton of germinating petunia male gametophyte to the action of exogenous phytohormones. Here, IAA and kinetin exhibited the most pronounced effects. IAA resulted in accelerating growth of pollen tubes by enhancing the content of polymeric actin in their apical and subapical zones, whereas kinetin, in contrast, inhibited their growth by decreasing the content of polymeric actin in all zones of the tube.

Evidence for possible role of phytohormones in petunia male gametophyte in the energization of pollen plasma membrane due to the activity of H+-ATPase during pollen germination and pollen tube growth Taking into account the above findings concerning the phytohormones-induced stimulation of germination and growth of petunia male gametophyte in subsequent experiments we attempted to elucidate the mechanism underlying such an action of the phytohormones in question. First of all, it was found that the latter, namely IAA and ABA, are capable of significantly changing cytoplasmic pH (pHc) of pollen cells, namely resulting in alkalization of intracellular medium (Fig. 7) (Andreev et al. 2007).

In this connection, it is important to note that the observed increase of intracellular pH appeared to be abolished by orthovanadate, a known inhibitor of P-type ATPases in cell membranes of eukaryotes including H+-ATPase in the plasma membrane of plant cells. It is known that this enzyme acting as proton pump plays very important role in plant cell physiology thanks to its capability to energize the plasma membrane by means of generation of transmembrane proton gradient on it and thereby fueling transport of various ions and metabolites through this membrane. Based on these observations we suggested that the phytohormone-induced shift in pHc is due to activation of the given proton pump. In order to test further a validity of this hypothesis other series of our experiments had a purpose to elucidate whether the phytohormones used are able to exert a hyperpolarization of plasmalemma of the same pollen cells. In these experiments transmembrane potential (ΔΨ) changes induced by the phytohormones were followed with voltage-sensitive dyes, the cationic probes Dis-C3-(5) (not shown) and safranin O, often applied to monitor the membrane potential (negative inside) and ion permeability in a variety of cells of different origin. As follows from Fig. 8, addition of IAA to pollen grain suspension in the K+-free assay medium immediately initiated their plasma membrane hyperpolarization, as judged by increase in differential absorption of safranin O related to inward flow of the dye across the membrane, with the effect achieving a saturation level in approximately 10–15 min. Reversal of the hormone-induced fluorescence quenching due to leakage of the dye was observed after addition of KCl (60 mM) to pollen grain suspension as a result of the depolarization of the pollen cells, while subsequent addition of the K+-ionophore valinomycin had practically no effect on the K+-induced dissipation of the membrane potential. In the given case, a sensitivity of the hormone-induced effect to the external potassium ions can be considered as one of the suitable approaches to specifically select a related dye response to the plasma membrane energization from the total dye signal. To test the contribution of H+-ATPase pump to the plasma membrane hyperpolarization due to IAA, orthovanadate, the above specific inhibitor of plasmalemma ATP-driven H+-pump in plant cells, was used. This, as follows from Fig. 8, completely abolished effect caused by IAA indicating that the proton pump was active, exhibited an electrogenic activity and most likely was involved in the IAA-induced hyperpolarization of the plasma membrane. According to our results in fact, similar effects were observed by us in the presence of ABA and GA3 as well but we did not practically observe any effect in the case of kinetin (Fig. 8).



The fact that the membrane hyperpolarization driven by the phytohormones is indeed affected through the stimulation of ATPdependent plasmalemma proton pump is additionally confirmed by the data obtained with fusicoccin, a known stimulator of plant plasma membrane H+-ATPases. This fungus toxin induced pronounced effect greatly resembling the action of IAA or ABA, and in its presence the observed effect of these phytohormones was completely abolished. Recently, we have evidence presented for involvement of both Ca2+ ions entering into petunia pollen grain cells from the external medium and active oxygen species in transduction of the hormonal signal triggered by IAA (Voronkov et al. 2010). In addition, it is important to note that all the above effects of phytohormones were observed by us only in the case of viable, germinating pollen grains. To our knowledge, the above data, taken together, indicated, for the first time, one of possible functions of phytohormones in cell of male gametophyte during its germination related to activation of the plasmalemma H+-ATPase, whose fundamental role in germination and growth of male gametophyte becomes increasingly evident (Certal et al. 2010).

In conclusion, it is important to note that despite the fact that the mechanisms by which IAA and other phytohormones regulate pollen tube growth are still poorly understood. To date it is known that the IAA-induced changes underlying this process involve not only stimulation of the activity of H+-ATPase but indeed some other effects as well, such as the increase in secretory vesicles, mitochondria and the modification of pectin and cellulose microfibrils in the tube wall (Wu et al. 2008). In this connection, it cannot be excluded the possibility that the above impact of IAA on pollen plasma membrane H+-ATPase resulting in corresponding shifts in the membrane potential and the cytosolic pH of pollen cells may serve as a certain stimulus triggering signal transduction pathways, leading to changes in expression of specific genes encoding the proteins required for initiation of the processes involved in the polar growth of pollen tubes. Although it is known that, at present, modeling pollen tube growth largely neglects the roles of phytohormones (Michard et al. 2009; Liu and Hussey 2011), our results may provide a certain base for taking them into account as well as upon developing its future models. It is clear, however, that such modeling will require much more information about the action of phytohormones on different key functions underlying pollen tube growth regulation compared to that accumulated to date. 

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