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

This study explores the hormonal regulation of Petunia hybrida male gametophyte germination and growth in vivo (on the stigma and in the style) and in vitro (on a cultivating medium). The findings highlight the role of phytohormones in pollen-pistil interactions, influencing pollen tube development and self-incompatibility mechanisms. Additionally, phytohormones such as IAA, ABA, and cytokinins, along with ethylene production, were analyzed in pistil tissues after both compatible and self-incompatible pollinations.

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Introduction

Angiosperms have evolved a unique system of sexual reproduction where immobile sperm is delivered directly to the egg via a pollen tube, eliminating the need for water-based fertilization. This adaptation, known as siphonogamy, enables flowering plants to reproduce efficiently in terrestrial environments.

The process of pollen-pistil interaction consists of six stages:

1. Pollen capture and adhesion
2. Pollen hydration
3. Pollen germination and tube formation
4. Penetration of the stigma
5. Pollen tube growth through the stigma and style
6. Entry of the pollen tube into the ovule and sperm cell discharge

Understanding the molecular and physiological mechanisms underlying these interactions is crucial, as they directly impact crop yield and food production. Despite significant progress in identifying molecules involved in pollen-pistil interactions, no universal set of mediators has been established across all plant species.

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Role of Phytohormones in Pollen Germination and Growth

Phytohormones play a regulatory role in pollen tube germination and growth through pistil tissues. The involvement of hormones such as indole-3-acetic acid (IAA), zeatin, gibberellic acid (GA), and abscisic acid (ABA) was first suggested by Stanley and Linskens (1974). However, their precise function in post-pollination events remains under investigation.

This study presents findings on the hormonal status of the Petunia pollen-pistil system, analyzing hormone levels in stigma, style, and ovary tissues following both compatible and self-incompatible pollinations.

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Findings on Phytohormone Regulation

1. Pollen-Pistil Interaction and Ethylene Production
• Ethylene plays a crucial role in controlling pollen germination and tube growth.
• It also contributes to self-incompatibility, preventing self-fertilization by regulating gametophyte-sporophyte interactions.
2. Impact of Phytohormones on Male Gametophyte Development
• Endogenous phytohormone levels fluctuate during pollen germination and growth.
• Exogenous phytohormones influence cytoskeletal organization and germination efficiency.
3. Membrane Polarization and pH Regulation
• IAA, ABA, and GA₃ alter cytoplasmic pH and induce hyperpolarization of the pollen plasma membrane.
• This effect suggests that phytohormones stimulate H⁺-ATPase activity, influencing pollen tube elongation.

Phytohormonal Regulation in Pollen-Pistil Interactions

Hormonal Distribution in the Pollinated Pistil

During both compatible and self-incompatible pollinations, phytohormones were distributed unevenly across the stigma, style, and ovary:

• Stigma: The primary site of ethylene synthesis, containing 90% of the total ABA.
• Style: Held 80% of the total cytokinins present in the pollinated pistil.
• Ovary: Displayed relatively low hormone levels, which did not significantly impact the hormonal balance of the pollen-pistil system.

Interactions between the male gametophyte and stigmatic tissues (pollen adhesion, hydration, and germination) triggered:

• A 7- to 10-fold increase in ethylene production.
• A 1.5- to 2-fold rise in IAA content within 0–4 hours post-pollination.

In self-incompatible pollination, ABA levels in the stigma and style tripled, whereas during compatible pollination, ABA remained stable. Over the next 4 hours, pollen tube growth altered the hormonal profile of the pollen-pistil system:

• Ethylene production declined in both cases, but the decrease was slower in self-incompatible pollination.
• IAA levels increased in both cases but rose more gradually in self-incompatible pollination.
• ABA remained unchanged, but its concentration remained elevated in self-incompatible interactions.

By the 8-hour mark, pollen tube growth inhibition in self-incompatible pollination was accompanied by a 5-fold increase in cytokinin levels in style tissues, whereas in compatible pollination, cytokinin levels remained constant.

These findings highlight the distinct hormonal dynamics of compatible vs. self-incompatible pollinations, supporting the hypothesis that phytohormones regulate pollen-pistil interactions, either facilitating uninterrupted pollen tube growth or triggering its inhibition in self-incompatible cases.

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Role of Ethylene in Male Gametophyte Germination and Growth

Pollination initiates a hormonal cascade in flowers, regulating processes such as petal wilting, pigmentation changes, ovule development, and style or stamen abscission. Ethylene, a gaseous phytohormone, is a key mediator of these post-pollination events. It is synthesized via a two-step pathway:

1. Conversion of S-adenosyl-L-methionine (SAM) into ACC by ACC synthase.
2. Oxidation of ACC by ACC oxidase, producing ethylene, CO₂, and HCN.

Research on orchid, carnation, tobacco, and petunia confirms that ethylene is essential for pollen tube growth and fertilization (Hoekstra & Weges, 1986; Singh et al., 1992; O’Neill et al., 1993). However, its precise role in gametophyte-sporophyte interactions during fertilization and its impact on self-incompatibility mechanisms remain unresolved.

Ethylene Dynamics in Pollination

Ethylene production following pollination occurs in two distinct peaks:

• First Peak: Rapid ethylene release from the stigma, primarily due to the conversion of pollen-derived ACC into ethylene via ACC oxidase.
• Second Peak: Ethylene synthesis in distal floral organs (style, ovary, petals) driven by endogenous ACC synthase and ACC oxidase activity, correlating with gene expression changes (O’Neill et al., 1993; Tang et al., 1994).

Ethylene and Floral Senescence in Petunia

Studies on self-incompatible Petunia inflata show distinct ethylene production patterns between compatible and incompatible pollinations (Singh et al., 1992):

• Compatible Pollination:
• Initial ethylene peak at 3 hours post-pollination.
• Second ethylene surge at 18 hours, leading to floral senescence at 36 hours.
• Incompatible Pollination:
• First ethylene peak remains similar (3 hours).
• Second ethylene surge delayed until 3 days post-pollination.
• Floral senescence delayed by 12 hours compared to compatible pollination.

Gametophyte Response to Ethylene in Petunia

Ethylene and ACC release vary depending on pollination type (Kovaleva et al., 2011):

• Self-Compatible Pollination:
• Higher ACC content in germinating pollen grains.
• Self-Incompatible Pollination:
• Higher ethylene production compared to compatible pollination.

These findings suggest that ethylene not only regulates pollen tube growth but also plays a crucial role in self-incompatibility responses, reinforcing its function as a key signaling molecule in plant reproduction.

Ethylene's Role in Gametophyte Self-Incompatibility

In both compatible and self-incompatible pollinations, ACC and ethylene production were primarily concentrated in the stigma tissues. Our findings suggest that ethylene may play a key role in the mechanism of gametophytic self-incompatibility, acting as a major barrier to self-fertilization. This hypothesis is supported by evidence showing that exposure to high ethylene concentrations (10 μl/l) reduced pollen tube growth rate by 50% in a cultivation medium (Kovaleva et al., 2013).

One proposed mechanism for the inhibition of pollen tube growth following self-incompatible pollination is programmed cell death (PCD) (Wang et al., 2009), which ethylene is known to induce (Rogers, 2006). Based on this, we suggest that the intense ethylene production following self-incompatible pollination triggers PCD in self-incompatible pollen tubes, thereby slowing their growth within the style’s conducting tissues.

Recent preliminary data (K.L.V., unpublished) further support ethylene’s involvement in self-incompatibility. In experiments with self-incompatible petunia clones, treatment with an ethylene synthesis inhibitor significantly enhanced pollen tube growth. The pollen tubes in treated stigmas grew twice as long as the control group. Notably, no signs of PCD were observed in style tissues where these pollen tubes elongated, whereas in control samples (self-pollination of a self-incompatible clone), DNA degradation associated with PCD was evident 8 hours post-pollination, coinciding with the onset of the self-incompatibility response.

Ethylene as a Regulator of Gametophyte-Sporophyte Interactions

Findings from various experimental systems, including anther-male gametophyte and pollen-pistil interactions (Kovaleva et al., 2007, 2011, 2013), suggest that ethylene plays a regulatory role in pollen germination, development, and growth. Recent data (K.L.V., unpublished) indicate that ethylene interacts with other phytohormones—such as IAA, ABA, and gibberellins—in the biosynthesis of ACC, influencing gametophyte-sporophyte interactions during the progamic phase of fertilization.

Studies on Arabidopsis pistil senescence (Carbonell-Bejerano et al., 2011) reveal that ethylene modulates ovule senescence and plays a role in GA-mediated fruit set, reinforcing its importance in reproductive regulation. Additionally, recent research demonstrates that ethylene serves as a key regulator of autophagy in petunia petal senescence, with pollination itself inducing autophagy (Shibuya et al., 2013).

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Hormonal Changes in Germinating Petunia Pollen

The endogenous phytohormone levels in germinating petunia pollen undergo significant fluctuations when cultivated in a 0.4 M sucrose and 1.6 mM H₃BO₃ medium (Kovaleva et al., 2005) (Fig. 5). These hormonal dynamics further highlight the complex regulatory network involving ethylene and other phytohormones in pollen development and fertilization.

During the hydration and germination of pollen grains, ABA levels dropped almost to zero, while GA, IAA, and cytokinin levels increased 1.5–2.0-fold. As pollen tubes continued to grow, GA content doubled, whereas IAA and cytokinin levels declined significantly.

Chen and Zhao (2008) observed a similar pattern in Nicotiana tabacum, where IAA levels were initially high in certain style regions before pollen tubes penetrated them. However, as the pollen tubes advanced into the style, IAA levels gradually decreased.

Additionally, the application of phytohormones to the cultivation medium had a significant impact on the germination and growth of petunia male gametophytes (Tables 1 and 2).

Influence of Phytohormones on Pollen Germination and Tube Growth

At concentrations ranging from 10⁻¹² M to 10⁻³ M, ABA and gibberellin A3 significantly stimulated pollen grain germination. IAA, at 10⁻¹² M to 10⁻¹⁰ M, also promoted germination, but at higher concentrations (10⁻⁴–10⁻³ M), it became inhibitory. Conversely, the synthetic cytokinin 6-BAP, across the same concentration range, inhibited pollen germination.

Pollen tube length was measured 1 hour after cultivation in a medium containing 0.4 M sucrose and 1.6 mM H₃BO₃, with data averaged from three independent experiments conducted in two replicates (n = 6).

Among the phytohormones tested (ABA, gibberellin A3, and IAA), GA₃ and ABA had the strongest stimulatory effects on pollen germination at 10⁻¹² M. GA₃ exhibited the greatest impact on pollen tube growth, with tubes reaching 450 μm after 6 hours of cultivation. IAA, at 10⁻¹² M, enhanced pollen germination 1.5-fold and pollen tube growth 2–2.5-fold. However, when paclobutrazol—a known gibberellin synthesis inhibitor—was added, pollen still germinated, but tube length was reduced 2–3-fold compared to controls, depending on inhibitor concentration.

ABA, at 10⁻¹² M, maximally stimulated pollen germination three-fold within 30 minutes and sustained a 2–3-fold increase in both pollen germination and tube growth after 1 hour. Conversely, fluridone, an ABA synthesis inhibitor, suppressed both processes, with the degree of suppression depending on the inhibitor concentration.

IAA, at 10⁻¹² M, promoted pollen germination 1.5-fold and pollen tube growth 2–2.5-fold, but only at low concentrations. Higher concentrations led to inhibition. Furthermore, 2,4-chlorophenoxy-2-methylpropionic acid, an IAA transport inhibitor, completely blocked pollen germination at 10⁻³ M, regardless of the presence of ABA or GA₃ in the medium.

Unlike IAA, 6-BAP consistently inhibited pollen germination and tube growth, regardless of its concentration in the cultivation medium.

Proposed Mechanisms of Phytohormone Action

These findings suggest that IAA's polar transport influences male gametophyte germination and growth, whereas ABA regulates intracellular osmotic pressure during these processes. Given ABA’s role in pollen dehydration and its potential regulation of Rop gene expression (Hsu et al., 2010), further exploration of this pathway is warranted.

Additionally, our results highlight the role of gibberellins in pollen tube growth regulation, aligning with previous studies on Arabidopsis mutants exhibiting altered gibberellin levels (Singh et al., 2002).

Phytohormones and Cellular Activities in Male Gametophyte

Pollen germination and polar tube growth require the coordination of multiple cellular functions, including cytoskeletal dynamics, vesicular transport, and transmembrane ion exchange (Franklin-Tong, 1999; Vidali & Hepler, 2001; Holdaway-Clarke & Hepler, 2003; Certal et al., 2008; Cheung & Wu, 2008).

Effects of Phytohormones on Actin Cytoskeleton

Our studies indicate that phytohormones influence pollen germination and tube growth via modifications in the actin cytoskeleton (Voronkov, 2010). Specifically:

• IAA (10⁻¹² to 10⁻⁶ M) increased actin filament density by 37%, enhancing fluorescence intensity of FITC-phalloidin-stained structures. The effect was most pronounced in the apical and subapical regions, which are critical for maintaining polar growth.
• ABA and GA₃, while stimulating pollen tube growth, only altered the zonal distribution of F-actin without affecting the total polymerized actin content.

Thus, IAA accelerates pollen tube growth by increasing polymeric actin, whereas ABA and GA₃ regulate its spatial organization within the growing pollen tube.

Influence of Kinetin on Actin Polymerization and Pollen Tube Growth

Unlike IAA, ABA, and GA₃, kinetin inhibited actin polymerization in pollen tubes, reducing the density of actin filaments by approximately 40% along the entire tube length (Fig. 6). The most pronounced effect was observed in the basal region of the tube.

These findings demonstrate that the actin cytoskeleton of germinating petunia male gametophytes is highly sensitive to exogenous phytohormones. Among them, IAA and kinetin exhibited the most significant effects:

• IAA stimulated pollen tube growth by increasing the amount of polymeric actin in the apical and subapical regions.
• Kinetin, on the other hand, inhibited pollen tube growth by reducing polymeric actin content across all regions.

Role of Phytohormones in Plasma Membrane Energization During Pollen Germination and Tube Growth

Given the observed stimulatory effects of phytohormones on pollen germination and tube growth, further experiments were conducted to explore the underlying mechanisms. It was found that IAA and ABA significantly influenced the cytoplasmic pH (pHc) of pollen cells by inducing intracellular alkalization (Fig. 7) (Andreev et al., 2007).

A key observation was that this increase in intracellular pH was abolished by orthovanadate, a well-known P-type ATPase inhibitor, which includes the H⁺-ATPase found in plant plasma membranes. This enzyme acts as a proton pump, playing a crucial role in plasma membrane energization by establishing a transmembrane proton gradient. This gradient, in turn, drives the transport of ions and metabolites across the membrane. These findings suggest that phytohormones activate this proton pump, leading to intracellular alkalization.

To further test this hypothesis, additional experiments were conducted to determine whether phytohormones could induce hyperpolarization of the plasma membrane. Changes in transmembrane potential (ΔΨ) were measured using voltage-sensitive dyes, including Dis-C3-(5) and Safranin O, which are commonly used to monitor membrane potential and ion permeability.

As shown in Fig. 8, the addition of IAA to a K⁺-free medium containing pollen grains immediately triggered plasma membrane hyperpolarization. This was indicated by an increase in differential absorption of Safranin O, corresponding to the inward flow of the dye across the membrane. The effect reached saturation within approximately 10–15 minutes.

Further supporting this observation, adding KCl (60 mM) to the pollen grain suspension caused membrane depolarization, leading to dye leakage. However, subsequent addition of valinomycin, a K⁺-ionophore, had no effect on K⁺-induced dissipation of the membrane potential. This confirmed that the observed changes in membrane potential were directly related to plasma membrane energization rather than non-specific ionic interactions.

To confirm the involvement of H⁺-ATPase activity in IAA-induced hyperpolarization, orthovanadate was applied. As demonstrated in Fig. 8, orthovanadate completely abolished the hyperpolarization effect, indicating that the proton pump was active, electrogenic, and directly involved in the process.

Similar effects were observed with ABA and GA₃, reinforcing their role in plasma membrane energization. However, kinetin did not produce any significant effect (Fig. 8), suggesting it does not contribute to H⁺-ATPase activation or membrane potential regulation in pollen cells.

Phytohormone-Induced Membrane Hyperpolarization and the Role of H⁺-ATPase

The membrane hyperpolarization induced by phytohormones is directly linked to the stimulation of ATP-dependent plasmalemma H⁺-ATPase activity. This conclusion is further supported by fusicoccin, a well-known activator of plant plasma membrane H⁺-ATPases. The application of this fungal toxin produced a pronounced effect similar to that of IAA and ABA, and in its presence, the effects of these phytohormones were completely abolished.

Furthermore, recent findings indicate that Ca²⁺ influx into pollen cells and the involvement of reactive oxygen species (ROS) play key roles in the hormonal signal transduction pathway triggered by IAA (Voronkov et al., 2010). Notably, all these phytohormone effects were observed only in viable, germinating pollen grains, underscoring their role in male gametophyte development.

Taken together, these findings provide the first direct evidence of a possible function of phytohormones in male gametophyte cells during germination—the activation of plasmalemma H⁺-ATPase, an enzyme whose critical role in pollen germination and tube growth is becoming increasingly clear (Certal et al., 2010).

Conclusion and Future Perspectives

Despite advances in understanding IAA and other phytohormones' regulation of pollen tube growth, the underlying molecular mechanisms remain poorly understood. Current knowledge suggests that IAA-induced changes in pollen cells extend beyond H⁺-ATPase activation to include:

• Increased secretory vesicles
• Mitochondrial activity enhancement
• Modification of pectin and cellulose microfibrils in the pollen tube wall (Wu et al., 2008).

It is also plausible that IAA’s impact on plasma membrane H⁺-ATPase, leading to shifts in membrane potential and cytosolic pH, acts as a trigger for signal transduction pathways. This, in turn, could regulate gene expression necessary for pollen tube elongation and polar growth.

Although current pollen tube growth models largely neglect the role of phytohormones (Michard et al., 2009; Liu & Hussey, 2011), our findings suggest that incorporating phytohormonal regulation could enhance future theoretical models of pollen tube growth. However, such modeling efforts will require significantly more information on how phytohormones influence key cellular functions involved in pollen tube development.