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).
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.
No comments:
Post a Comment