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Article

Detection of Abscisic Acid and Jasmonates in Stigma Exudates and Their Role in Pollen Germination

by
Maria Breygina
*,
Dmitry V. Kochkin
*,
Anna Podobedova
,
Maria Kushunina
,
Danil Afonin
and
Ekaterina Klimenko
Department of Plant Physiology, Biological Faculty, Lomonosov Moscow State University, Leninskiye Gory 1-12, Moscow 119234, Russia
*
Authors to whom correspondence should be addressed.
Horticulturae 2025, 11(9), 1146; https://doi.org/10.3390/horticulturae11091146
Submission received: 31 July 2025 / Revised: 18 September 2025 / Accepted: 19 September 2025 / Published: 21 September 2025
(This article belongs to the Special Issue The Role of Plant Growth Regulators in Horticulture)
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Abstract

Pollen–stigma interactions have been studied extensively because they play an important role in sexual reproduction and crop yield. The vast majority of studies have focused on dry stigmas, which are typical of many model and agricultural plants; however, the data obtained are difficult to apply to plants with wet stigmas, such as tomato and tobacco. Pollen germination in this case occurs in a liquid, an exudate, which has a complex, species-specific composition. UPLC-ESI-MS-based hormone screening was carried out for six plant genera belonging to Solanaceae, Bromeliaceae, and Gesneriaceae families and revealed jasmonic acid (JA), abscisic acid (ABA) and/or jasmonoyl-L-isoleucine (IleJA) in stigma exudates of tobacco, tomato, and Streptocarpus sp. To assess the physiological significance of plant hormones in stigma exudate we tested their effect in vitro, finding that JA, IleJA, and MeJa significantly stimulated germination of tobacco pollen, with JA being most effective in accordance with its predominance in the stigma exudate; furthermore, ABA stimulated pollen germination in all tested species including bromeliads despite the lack of this hormone in their exudates. Both JA and ABA had an anti-oxidant effect on germinating pollen. Possible functions of hormones and ROS in exudate as well as ways of implementing the anti-oxidant effect of phytohormones are discussed.

1. Introduction

The interaction between the stigma and pollen at the initial stage is a relevant and significant subject that has been actively studied recently [1,2,3]. In plants with dry stigmas, pollen hydration depends on water supply directly from the stigma papillae, and this process is selective [4,5]. On the contrary, plants with wet stigmas are characterized by pollen germination in a special fluid produced by the stigma cells before pollination [6]. These plants are a minority among the Angiosperms, but they dominate in some families, including economically important ones, such as Solanaceae and Orchidaceae [7]. It was previously believed that the main substances in the exudate are water, lipids, minerals, and sugars [8,9], and that the exudate is a nutrient substance for pollen germination [10] which can also attract pollinators [6]. Later, enzymes that can perform protective functions [11,12], and regulatory low-molecular substances, such as ROS and NO, were discovered in stigma exudates of different plants and found to affect pollen germination in vitro and in vivo [13,14,15].
Of the plant hormones, only abscisic acid (ABA) has been discovered in tobacco stigma exudate up to date [16]. This finding was remarkably consistent with earlier studies that implicated hormones, including ABA, as key regulators of pollen germination. The effect of plant hormones on pollen physiology in vitro has been reported in a series of articles on Petunia hybrida [17,18,19]: ABA and gibberellic acid 3 (GA3) considerably promoted pollen germination and pollen tube growth; and indole-3-acetic acid (IAA) promoted and cytokinins inhibited the growth of pollen tubes [20]. Similar studies were conducted on a plant with a dry stigma type Torenia fournieri L., and the results were quite different: IAA and GA3 stimulated in vitro pollen tube growth, ABA inhibited pollen tube growth, and zeatin (ZT) had no effect. However, what is important is that no significant changes were found in the efficiency of pollen germination [21]. Since T. fournieri has no stigma exudate, the authors analyzed stigma tissues and showed that 0.5 h after pollination ABA content decreased, whereas the content of IAA, ZT, or GA3 did not change significantly. In maize, another species with a dry stigma, low ABA concentration (0.5 μM) significantly increased pollen germination and tube growth, while 500 μM ABA significantly decreased both parameters, thus indicating that ABA can influence the growth of maize pollen in vitro [22].
Other studies have focused on the effects of jasmonates in vitro on pollen from a variety of plant species, including strawberry, apricot, Camellia oleifera C. Abel, and Pinus nigra J.F. Arnold [23,24,25,26]. The reported effects were contradictory, as in some plants jasmonates stimulated pollen germination at moderate concentrations, while in others they inhibited it. The situation is further complicated by the fact that different jasmonates can act on pollen with different efficiency.
Interactions between jasmonate and/or ABA-mediated signals and ROS generation are common in plant stress responses [27] and certain physiological processes such as stomatal closure [28]. Jasmonates can both promote the accumulation of ROS [29,30] and perform the opposite function by activating anti-oxidant systems to scavenge ROS [27,31]. Pollen germination is highly dependent on ROS production by stigmatic tissues in a wide variety of plants [13,32,33], and pollen can produce ROS itself, although this phenomenon is largely unstudied. For example, ROS production is essential for kiwifruit pollen performance, since in the presence of superoxide dismutase/catalase mimic MnTMPP or NADPH oxidase inhibitor pollen tube emergence was severely or completely inhibited [34]. On the other hand, the same substances in low concentrations as well as ascorbic acid stimulated tobacco pollen germination [35,36]. It can be concluded that pollen is very sensitive to the balance of ROS during interaction with the stigma and it can be assumed that hormones in the exudate can influence this balance.
In the present study, to test the hypothesis about the role of hormones in stigma exudate as stimulators of pollen germination we used two approaches: first, by identifying all possible hormone-like substances in stigma exudates of representatives of several families by UPLC-ESI-MS; second, by testing the sensitivity of pollen of different plant species to phytohormones found in the exudate in vitro. Finally, we investigated the potential relationship between hormones and ROS via pollen H2O2-sensitive staining.

2. Materials and Methods

2.1. Plant Material and Stigma Exudate Collection

Plants of Nicotiana tabacum L. cv. Petit Havana, cv. Samsun, and Lycopersicon esculentum Mill. cv. Pugovka plants were grown in a climatic chamber in controlled conditions (25 °C, 16 h light) in vermiculite. The plants were watered with a nutrient solution [37]. Streptocarpus cv. Salmon Sunset plants were grown in the same climatic chambers but in soil and watered with tap water.
Bromeliaceae plants were grown in greenhouse conditions as a part of the main collections of the Botanical Garden of Lomonosov Moscow State University and Tsitsin Main Botanical Garden. The following species were used for exudate and/or pollen collection (Figure 1): Aechmea caudata Lindm., Vriesea carinata Wawra, Vriesea erythrodactylon (É.Morren) É.Morren ex Mez, Vriesea rubyae E.Pereira, Tillandsia ionantha Planch., Tillandsia capitata Griseb., Tillandsia xerographica Rohweder, and Puya mirabilis (Mez) L.B.Sm. For Tillandsia and Vriesea the results obtained on several species of the respective genera that bloomed simultaneously were combined into one sample.
Stigma exudate was collected by the “cap method”, which was developed by us earlier [13]. Briefly, a certain amount of dH2O was put on the stigmatic surface without damaging any parts of the flower, incubated for 30 min, carefully removed, and frozen at −80 °C. Since the stigmatic surfaces of different plants differ in size and position, we selected the volume of liquid and the method of collection individually. The minimum volume for measurements was 500 µL, so one sample was the sum of washes from a certain number of flowers.

2.2. Chromato-Mass-Spectrometric Detection of Phytohormones

In general, a previously developed method was used with some modifications [16].
Sample preparation. Aqueous solutions of pistil exudates of the studied plants were diluted with methanol 1:1 (v/v) and evaporated under vacuum on a VVMicro rotary evaporator (Heidolph Instruments GmbH, Schwabach, Germany). The dried sample was dissolved in 1 mL of 70% (v/v) aqueous methanol solution and centrifuged at 15,294× g for 10 min (MiniSpin, Eppendorf, Germany). The supernatant was used for further analysis.
Qualitative and quantitative liquid chromatography-mass spectrometry (UPLC-ESI-MS, negative ion registration) was performed on an Agilent 1260 Infinity instrument (Agilent Technologies, USA) equipped with a mass-selective detector (6100, Agilent Technologies, USA). Column–ACQUITY UPLC BEH C18 1.7 μm, 2.1×75 mm (Waters, USA). Column temperature—61 °C. Mobile-phase flow rate—0.39 mL/min. Injection volume—5–10 μL.
A 0.1% (v/v) solution of formic acid (Fluka, USA) in water (solvent A) and a 1% (v/v) solution of formic acid in acetonitrile (solvent B) were used as the mobile phase. During the analysis, the composition of the mobile phase was changed as follows (B, vol. %): 0–1 min—15%, 1–3 min—15→55%, 3–4 min—55→65%, 4–13 min—65%, 13–13.5 min—65→95%, and 13.5–16 min—95%. The analysis was carried out in the negative ion detection mode (m/z range 100–500, fragmentor—70). Ionization source parameters: quadrupole temperature—100 °C, carrier gas temperature (N2)—350 °C, nitrogen feed rate (nebulizing gas)—10 L/min, nitrogen pressure—1035 Torr, and capillary voltage—4 kV. The chromatograms were recorded in the total ion current mode. Abscisic acid, jasmonic acid, and jasmonoyl-L-isoleucine were identified in the samples based on a comparison of the chromatographic and mass spectrometric characteristics of the detected compounds with the characteristics of standard samples of the corresponding phytohormones. The following standard samples were used to check the presence of other phytohormones in the samples: salicylic acid (Laverna, Russia), trans-zeatin (FlukaChemie AG), kinetin (Sigma-Aldrich, St. Louis, MO, USA), 6-benzylaminopurine (ICNBiochemicals Inc., Aurora, OH, USA), 3-indoleacetic acid (Sigma, USA), indole-3-butyric acid (ICNBiochemicals Inc., USA), and gibberellin GA3 (Honeywell Riedel-deHaën AG, Seelze, Germany). However, these compounds were not detected in any of the samples.
Quantitative analysis of abscisic acid (ABA), jasmonic acid (JA), and jasmonoyl-L-isoleucine (IleJA) in aqueous solutions of stigma exudates was carried out by external calibration against standard samples. Standard samples of JA and ABA were purchased from Sigma-Aldrich (USA). The standard sample of jasmonyl isoleucine was obtained from JA (Sigma-Aldrich, USA) and L-isoleucine (Sigma-Aldrich, USA). According to the standard method [38], the product was purified by column chromatography on silica gel and identified by NMR spectroscopy [38]. In the working concentration ranges, the calibration curves were approximated by straight lines with R2 above 0.99. The relative standard deviation of the retention times of chromatographic peaks for standard samples of phytohormones did not exceed 3%, and for the areas of the corresponding chromatographic peaks it did not exceed 5%. The results obtained were processed using the OpenLAB program (Agilent Technologies, USA).

2.3. Pollen Collection and Germination In Vitro

For pollen collection, anthers of tobacco cv. Petit Havana were removed from the flowers on the eve of opening and dried in a thermostat for 2 days, after which the pollen was collected with a specially equipped vacuum cleaner. Dry pollen was stored at −20 °C. According to our long-term observations, pollen does not lose its germination capacity for at least a year. Streptocarpus pollen was collected from mature fresh flowers. Pollen of the Bromeliaceae genera was collected from the same plants that were used for exudate washout (see Section 2.1) and germinated the same day. Pollen of these plants loses its germination capacity even after short-term storage.
Tobacco pollen germination efficiency was assessed after 1 h of incubation at 25 °C in standard medium containing 1.6 mM H3BO3, 3 mM Ca(NO3)2, 0.8 mM MgSO4, 1 mM KNO3, and 0.3 M sucrose in a 25 mM MES-Tris buffer, pH 5.8 at 2 mg pollen/mL [39].
Streptocarpus pollen germination efficiency was assessed after 4 h of incubation at 25 °C in standard medium containing 0.81 mM H3BO3, 0.31 mM Ca(NO3)2·4H2O, and 0.3 M sucrose, with gentle agitation [40].
Bromeliaceae pollen germination efficiency was assessed after 2 h of incubation at 25 °C in standard medium containing 1.6 mM H3BO3, 0.5 mM Ca(NO3)2·4H2O, 0.8 mM MgSO4·7H2O, 1 mM KNO3, and 0.15 mM sucrose, with gentle agitation [41].
Before cultivation, all pollen was pre-hydrated in a humid atmosphere for 2 h.
Germinated pollen was fixed in 2% paraformaldehyde in 50 mM Na-phosphate buffer, pH 7.4 for a minimum of 30 min at 4 °C. Between 500 and 1500 pollen grains from each suspension were counted.
Plant hormones were added at the beginning of incubation from stock solutions (1 mM), bringing them to the desired concentration with the pollen germination medium. To prepare the stock solutions of JA, methyl jasmonate (Sigma-Aldrich, St. Louis, MO, USA), and IleJA, dry hormones were dissolved in DMSO. To prepare ABA stock solution, dry hormone was dissolved in a small volume of 70% alcohol, after which it was adjusted with water to the required volume. Stock solutions were stored at −20 °C.

2.4. ROS and Ca2+ Level Measurements

To assess the level of H2O2, tobacco pollen grains were cultured in vitro in the same medium as in the germination experiment. Plant hormones were added either before germination (resulting in 1 h incubation) or after germination simultaneously with staining (resulting in 15 min incubation). The cells were stained with 10 μM pentafluorobenzenesulfonyl fluorescein (PFBSF) (Santa Cruz Biotechnology, Santa Cruz, CA, USA). PFBSF is a fluorescein derivative. When exposed to hydrogen peroxide (but not other ROS) the sulfonyl bond cleaves, releasing fluorescein [42]. The dye concentration was 10 μM and the incubation time was 15 min., without washing. The staining was performed at 25 °C. To visualize cytoplasmic Ca2+ pollen grains were incubated with 10 μM Fluo-4 AM (Sigma, USA) and 0.1% Pluronic F127 (Sigma, USA) at 29 °C for 60 min. Fluorescence of both dyes was excited with a mercury lamp with a 485 ± 20 nm filter and recorded at 515–656 nm.

2.5. Measuring the Stigma Surface Area

To accurately measure the surface area of a complex shape stigmas we used the “replica method” that had been developed earlier [13]. Briefly, an imprint was made using solidifying liquid glue. The glue was applied in a thin layer to the stigma using a micro-brush. After the glue had hardened, the resulting replica was removed with tweezers and carefully transferred to a measuring grid for photographing. The area of the replica was determined using the Image J V1.54k open software.

2.6. Data and Statistical Analyses

All experiments were performed in at least three independent executions. The data are provided as the means ± SEM. Statistical analysis was performed using Origin Lab software 9.7 (Northampton, MS, USA) according to Mann–Whitney test or Student’s t-test in case of large samples tested for normality (* p < 0.05, ** p < 0.01).

3. Results

3.1. Phytohormones Such as ABA and JA Are Present in Stigma Exudates of Some Dicot Plants

The method for hormone screening of stigma exudates by UPLC-ESI-MS was previously developed and applied to tobacco and lily [16]. Here we analyzed stigma exudates collected from the representatives of different taxonomic groups: Bromeliaceae (Tillandsia, Vriesea, and Aechmea), Solanaceae (Nicotiana (two cultivars), Lycopersicon), and Gesneriaceae (Streptocarpus) (Figure 1).
To check the presence of phytohormones in the samples, the following standard samples were used: salicylic acid, abscisic acid, jasmonic acid, jasmonoyl-L-isoleucine, trans-zeatin, kinetin, 6-benzylaminopurine, 3-indoleacetic acid, indole-3-butyric acid, and gibberellin GA3. None of the hormone-like substances were found in the exudates of bromeliads. Three substances were found in tomato and tobacco exudates: jasmonic acid (JA), jasmonoyl-L-isoleucine (IleJA), and abscisic acid (ABA) (Table 1, see chromatograms in Supplementary Materials, Figures S1–S3). Jasmonates were also present in stigma exudate of another dicotyledonous plant, Streptocarpus, a member of the Gesneriaceae family (Table 1, Figure S4). Examples of original chromatograms that contain no hormones (Bromeliaceae species) and standards of ABA and jasmonates can be found in Supplementary Materials (Figures S5–S7).
Next, a quantitative analysis of jasmonates and ABA in stigma exudates was carried out. Recalculation of the amount of hormones per stigma surface area allows for a semi-quantitative comparison of the hormone production in plants with different stigmas, although we did not set the task of statistically identifying differences between species. Comparison of two tobacco cultivars allows us to estimate the possibility of variation within one species and, thus, makes the data on tobacco more reliable (Table 1). A 2-fold difference between cultivars was observed: Petit Havana had more jasmonates in the exudate while Samsun had more ABA. The highest level of JA was observed in tomato exudate, and ABA in Samsun tobacco cultivar. Streptocarpus had high levels of jasmonates, while ABA was absent in the exudate. Species from the same family may apparently exhibit differences in phytohormone levels up to two orders of magnitude. This prompted us to test how significant such differences were for pollen germination in vitro.

3.2. Jasmonic Acid and Its Conjugate Stimulate Pollen Germination

Since all three hormones were detected in the exudate of Petit Havana tobacco (Table 1), we compared the effects of JA and two of its derivatives, MeJa, which has been commonly used in earlier studies, and IleJA, on tobacco pollen germination.
Both jasmonic acid derivatives affected pollen germination (Figure 2). However, there was no clear concentration dependence: low concentrations had no significant effect, medium (25–100 μM) concentrations had a moderate stimulating effect, and high concentrations strongly inhibited germination. In contrast to both derivatives, unconjugated jasmonic acid had a much stronger effect under the same conditions (Figure 3). In this case, clear concentration dependence was observed with a peak at 5 μM JA and strong inhibition after 100 μM (Figure 3A).
Based on the results obtained for tobacco pollen, we assumed that JA was the most active stimulator of germination. We tested the effect of this hormone on Streptocarpus pollen germination. This plant belongs to the Gesneriaceae family, and jasmonate and its derivative are present in its stigma exudate in relatively large quantities (Table 1). We have found that jasmonate significantly stimulates germination of Streptocarpus pollen in low and moderate concentrations (Figure 3B).

3.3. ABA Is an Effective Stimulator of Germination in All Studied Species

While jasmonates are considered as a potential pollen germination stimulant in many species [43], there is much less data on ABA. We have found that ABA strongly stimulates tobacco pollen germination after 1 h incubation, consistent with our previous data [16]. We also tested the effect of 10 μM ABA on pollen of Bromeliaceae (Aechmea, Puya, Tillandsia, Vriesea) and Streptocarpus. The plants studied had different germination percentages in control: from quite high in Puya to almost zero in Vriesea. Nonetheless, ABA reliably stimulated germination in all the plants studied (Figure 4). In some cases, more than two-fold stimulation was observed.

3.4. ABA and JA Affect H2O2 Production in Pollen Grains and Tubes

To test whether stimulating hormones influence the H2O2 concentration in tobacco pollen tubes, we used the standard method of specific staining for hydrogen peroxide with PFBSF. Two approaches were used: (1) pollen germination in a medium with hormones—“1 h incubation”; (2) addition of hormones to already germinated grains at the stage of polar growth—“15 min incubation” (Figure 5). First, we tested the standard stimulating concentration of hormones. During 1 h incubation, both ABA and JA (10 μM) significantly reduced peroxide levels in pollen tubes; during 15 min incubation, only ABA had significant effect (Figure 5A,B). This shows that the effect of ABA is realized quickly and it acts not only on germinating pollen grains, but also on growing tubes. However, since incubation for 1 h yielded more reliable changes, we used this protocol in the subsequent experiment. Surprisingly, at lower concentrations during an hour-long incubation, the hormones had the opposite effect; JA at a concentration of 1 μM and ABA at concentrations of 1 and 5 μM significantly increased the peroxide content in pollen grains, although the values increased by no more than 25% and, thus, did not indicate oxidative stress (Figure 5C). Since 1 and 5 μM JA (Figure 3) and 2 μM ABA stimulate pollen germination with the same efficiency as at 10 μM, these levels of peroxide are not toxic for germinating pollen grains.
Thus, our results demonstrate a complex interplay between hormonal and ROS signaling during tobacco pollen germination. To test the involvement of intracellular calcium in this process, we stained pollen grains with the widely used calcium-sensitive probe Fluo-4 and treated them with hormones during and after staining. We found no significant effects of hormones on intracellular calcium levels (Table 2).

4. Discussion

The method we used here for hormone screening was previously developed for tobacco stigma exudate, and the exudate of Lilium longiflorum was also analyzed [16]. In tobacco, ABA was found in the first analysis and the concentration of this hormone increased as the stigma matured, while in the lily exudate, phytohormones were absent. Continuing this line, in the present study we collected exudate from other plants with wet stigmas: bromeliads, tomato, and Streptocarpus. The idea was to collect exudate and pollen from the same species, and to include representatives of different families, both monocots and dicots, in the study. Of course, we have too few analyzed families and their representatives to draw conclusions (a total of seven representatives of four families in two studies), but so far the observed trend has persisted: representatives of dicotyledonous plants have hormones in the exudate while representatives of monocotyledons do not (Table 1, Figure 1). Comparing the amounts of hormones per surface area of the stigma, we found that tomato and streptocarpus have a higher production of JA and tobacco has a higher production of ABA, the latter being consistent with previous data. The synthesis and interconversions of jasmonates and their functions on the stigma have yet to be studied, but the data obtained suggest that the amount of this hormone in stigma exudates can vary by orders of magnitude between representatives of the same family. We did not find any other studies on the hormonal composition of stigma exudate, but in a recent article Xiong et al. analyzed hormone levels in tobacco tissues, comparing mature and immature stigmas [44]. Compared to the latter, ABA and salicylic acid were significantly upregulated in the former, while JA and IleJA contents showed the opposite trend [44]. A separate issue worthy of study is the distribution of various hormones between the tissues of the stigma and its exudate, which comes into contact with pollen earlier during pollination.
What could be the functions of hormones in stigma exudate? The main hypothesis is the regulation of pollen germination. In the current study, we tested jasmonic acid and two of its derivatives, one of which (MeJA) has been extensively studied as a pollen germination regulator while the other (IleJA) has never been tested in this aspect. All three jasmonates can be considered more or less effective germination activators, but they block germination at high concentrations (Figure 2). The literature data on previously studied jasmonates are quite contradictory. Thus, pollen germination in strawberry was decreased by 0.5 mM JA from 42.5 to 5.8% [23]; JA and MeJA significantly inhibited Camellia oleifera pollen germination and pollen tube elongation in vitro already at 50 μM, and at 1 mM it was completely blocked [45]. In the same species, however, treatment of stigmas with MeJA increased the number of pollen tubes during self-pollination in vivo [26]. A total of 0.1 and 0.25 mM MeJA promoted while 0.5 and 1 mM MeJA inhibited pollen germination in apricot [24]. Pollen germination rate increased after MeJA treatments in Pinus nigra, the most efficient concentration being 50 μM MeJA: no germination was observed at 2.5 mM MeJA [25]. MeJA also affected actin cytoskeleton organization, changed callose distribution, and altered pollen tube growth of Pinus nigra [25]. MeJA activated Arabidopsis pollen germination with the highest percent of germination on the medium supplemented with 500 μM MeJA [46]. It can be concluded that there are both species-specific and concentration-dependent features in the pollen response to jasmonates. We observed the most significant stimulation of tobacco and Streptocarpus germination by low concentrations of JA (Figure 3), which suggests that in studies where all concentrations were considered inhibitory, lower concentrations may not have been tested.
Previous studies have demonstrated that ABA stimulates pollen germination in maize [22] and two Solanaceae species: tobacco [16] and Petunia [17]. To check whether sensitivity to ABA is associated with the presence of this hormone in the exudate, we treated pollen from five plant genera that lacked this hormone in their exudate with 10 μM ABA. Interestingly, reliable, sometimes very strong, stimulation was observed in all cases (Figure 4). Thus, ABA is a strong stimulator of pollen germination for a number of species from three different families, and sensitivity to this hormone does not correlate with the presence of ABA in the exudate. But if hormones are not present in the exudate, how can they influence pollen in vivo? One potential source is pollen itself. Plant hormones are present in the pollen grains; both ABA and JA have been found in pollen from a variety of species including tobacco [46,47] and may be linked to their eventual germination, along with hormones produced by the stigma. JA and its active conjugate, IleJA, represented the most conspicuous derivatives in developing male gametophytes of four tobacco species [48]. In N. tabacum, the concentration of JA significantly exceeded that of IleJA at the earlier developmental stages while a progressively increasing accumulation of IleJA was observed in pollen at the late stages. For N. tabacum, pollen maturation was also accompanied by ABA accumulation [49]. Later, the same was shown for Lilium [50]. Two jasmonates, including IleJA, were identified in pollen grains of Pinus mugo [51]. In pollen of another gymnosperm, Picea pungens, the dynamics of ABA were similar to that in Petunia: the concentration of this hormone was the highest in mature pollen and decreased during germination [17,47]. Thus, the source of hormones can be either the exudate of the stigma or its tissue or pollen grains, and they can influence each other, forming a complex system of interactions.
According to classical concepts, ABA is an inhibitory plant hormone [52]. This idea is mainly based on the well-known activity of ABA as an inhibitor of embryo development and seed germination, as well as spore germination in Fusarium species [53]. It is now generally accepted that the physiological effects of plant hormones, as well as the pathways of their biosynthesis, are tissue-, organ- and species-specific [54,55]. For example, ABA can have diverse effects on different parts of the root system depending on the concentration and external conditions [56,57]; low ABA can stimulate root growth, while high concentrations, on the contrary, inhibit root growth. In addition, ABA can have variable effects on the growth of the primary and lateral roots [57,58]. The effects of ABA on root hair growth and development are also highly complex and variable [59]. The results obtained in the present work also suggest that, despite the proven stimulating effect of ABA on pollen grain germination for a wide variety of plants, its biosynthesis and accumulation in pistil exudates may strongly depend on the systematic position of the plant. Clarification of the physiological, biochemical, and evolutionary significance of these differences for plants is an interesting topic for future research.
In order to investigate the relationship between low-molecular regulators produced both by stigma and pollen during germination, the most important of which are ROS and hormones, we treated pollen germinating in vitro with different concentrations of ABA and JA. Since pollen generates H2O2 during germination, it was expected that hormones would either decrease or increase peroxide production. A reliable anti-oxidant effect of both hormones at 10 μM was found, while lower stimulating concentrations had the opposite effect (Figure 5). This is consistent with data on the stimulating effect of both low concentrations of anti-oxidant enzymes/ascorbate in vitro and H2O2 treatment on tobacco pollen germination [35,36] and the relationship between ROS/H2O2 level in the exudate/on stigma and pollen germination success in vivo [33,60,61]. The results confirm that the relationship between the ROS levels outside and inside the pollen grain and its germination success is very complicated.
How exudate hormones influence peroxide has not yet been established, but there are some assumptions based on data from other model systems [62]. In plant stress and disease responses, JA functions as antagonist of salicylic acid (SA) and can perform anti-oxidant function by blocking SA-induced ROS wave [27,31,63]. Thus, in Arabidopsis leaves, application of JA suppressed the ROS wave in response to high light stress or local wounding [27]. In guard cells, ABA and jasmonates are synergists and perform a signal crosstalk leading to limited ROS generation and stomata closure in response to drought [28]. At a certain stage of signal transduction, the ABA receptor RCAR/PYR1/PYL interacts with downstream type 2C protein phosphatases (PP2Cs) to activate the SNF1 (Sucrose-Non-Fermenting Kinase 1)-related protein kinase OPEN STOMATA1 (OST1)/SnRK2. OST1 targets to bind RBOHF on the plasma membrane, and then generates H2O2 through the SOD outside the plasma membrane [62]. However, this generation in guard cells leads to a strong increase in intracellular calcium, and we did not observe such an effect in pollen grains (Table 2). The possibility of subtle regulation of the ROS level, depending on the strength of the ABA signal, was shown in rice, where exogenous ABA can activate the NOX activity of genes encoding OsNox2, OsNox5, OsNox6, OsNox7, etc., and then promote the generation of ROS in guard cells [64]. Consequently, the expression of OsNox5 and OsNox are dependent on low and high concentrations of ABA, respectively, indicating that OsNox5 and OsNox7 have a significant correlation with the level of ABA in plant tissues [65]. In Arabidopsis seedlings, ABA induces the transcription of genes related to the ascorbate–glutathione cycle in the ROS scavenging system [66]. In addition, ABA may stabilize DELLA proteins which promote ROS detoxification, thus in some cases ABA may indirectly reduce ROS levels [62,63]. The intriguing mechanism of ABA and JA on ROS generation/quenching and their possible interaction with each other in stigma exudate may become a topic for further research.

5. Conclusions

Hormone screening of stigma exudates of Lycopersicon esculentum (Solanaceae), Streptocarpus hybr. (Gesneriaceae), Aechmea caudata, Tillandsia spp., and Vriesea spp. (Bromeliaceae) was carried out for the first time. UPLC-ESI-MS revealed ABA, JA, and IleJA in tobacco stigma exudate, and JA and IleJA in Solanum lycopersicum and Streptocarpus exudate. In vitro experiments showed that the principal function of these hormones may be the regulation of pollen germination and ROS balance in the germinating pollen: JA, ABA, IleJA, and MeJa significantly stimulated pollen germination. The lower and higher concentrations of JA and ABA had multidirectional effects on H2O2 in germinating tobacco pollen: at 10 μM these hormones acted as anti-oxidants while in the concentration of 1–5 μM both of them served as moderate pro-oxidants. It can be assumed that hormones in stigma exudate function as pollen germination stimulators and their effect is associated with ROS balance in pollen.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/horticulturae11091146/s1, Figures S1–S6: A representative UPLC-ESI-MS chromatogram of stigma exudate of Nicotiana tabacum cv. Petit Havana (S1), Nicotiana tabacum cv. Samsun (S2), Lycopersicon esculentum (S3), Streptocarpus hybr. (S4), of a standard sample of ABA (S5), of model mixture of standard samples of jasmonates (S6). On chromatograms S1–S6: The first (upper) panel is the total ion current (TIC mode, negative ions); the second panel is the results of filtering the TIC signal by the m/z 209 value for the [M-H]- jasmonic acid ion; the third panel is the results of filtering of the TIC signal by the m/z 263 value for the [M-H]- abscisic acid ion; the fourth (lower) panel is the results of filtering the TIC signal by the m/z 322 value for the [M-H]- jasmonoyl-L-isoleucine ion; Abscissa axis—time, minutes; ordinate axis—detector signal, cps. Figure S7: A representative UPLC-ESI-MS chromatogram ((TIC mode, negative ions) of stigma exudate of Vriesea spp. (upper panel), Aechmea racinae spp. (second panel); the third and fourth (lower) panels are the results of filtering of the TIC signals by the m/z 209 value for the [M-H]- jasmonic acid ion. Abscissa axis—time, minutes; ordinate axis—detector signal, cps.

Author Contributions

Conceptualization, M.B.; experimental design M.B. and D.V.K.; UPLC-ESI-MS, D.V.K. and D.A.; pollen germination, A.P.; plant care and exudate collection, M.K. and E.K.; writing—original draft preparation, D.V.K. and M.B.; writing—review and editing, M.B., D.V.K. and M.K. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Data Availability Statement

Original data are contained within this article and Supplementary Materials.

Acknowledgments

The authors express their deep gratitude to the Botanical Garden of Lomonosov Moscow State University, and especially to the curator of the bromeliad collection, Vitaly Alyonkin. We also thank Nikita Zdravchev and Viktoriya Drokina (Tsitsin Main Botanical Garden of Russian Academy of Sciences) for providing access to the bromeliad collection, Oksana Luneva for stigma surface measurements and our ex-lab member Ksenia Babushkina for her important contribution to the study at its initial stage. The study was conducted under the state assignment of Lomonosov Moscow State University.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
ZTZeatin
ABAAbscisic Acid
GA3Giberellic Acid 3
IAAIndole-3-acetic Acid
JAJasmonic Acid
MeJAMethyl Jasmonic Acid
Ile-JaIsoleucine-Jasmonic Acid
SASalicylic Acid

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Horticulturae 11 01146 g001
Figure 1. Characteristic flower images of the plant genera used for screening of hormone-like substances in stigma exudate using UPLC-ESI-MS (Agilent Technologies, Santa Clara, CA, USA): (A)—Aechmea racinae var. tubiformis (Bromeliaceae); (B)—Tillandsia ionantha (Bromeliaceae); (C)—Vriesea incurvata (Bromeliaceae); (D)—Nicotiana tabacum cv. Petit Havana (Solanaceae); (E)—Lycopersicon esculentum (Solanaceae); and (F)—Streptocarpus cv. Salmon Sunset (Gesneriaceae).
Figure 1. Characteristic flower images of the plant genera used for screening of hormone-like substances in stigma exudate using UPLC-ESI-MS (Agilent Technologies, Santa Clara, CA, USA): (A)—Aechmea racinae var. tubiformis (Bromeliaceae); (B)—Tillandsia ionantha (Bromeliaceae); (C)—Vriesea incurvata (Bromeliaceae); (D)—Nicotiana tabacum cv. Petit Havana (Solanaceae); (E)—Lycopersicon esculentum (Solanaceae); and (F)—Streptocarpus cv. Salmon Sunset (Gesneriaceae).
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Figure 2. Effect of jasmonic acid derivatives methyl jasmonate (MeJA) (A) and jasmonoyl-L-isoleucine (IleJA) (B) on pollen germination in Nicotiana tabacum cv. Petit Havana; * p < 0.05, ** p < 0.01.
Figure 2. Effect of jasmonic acid derivatives methyl jasmonate (MeJA) (A) and jasmonoyl-L-isoleucine (IleJA) (B) on pollen germination in Nicotiana tabacum cv. Petit Havana; * p < 0.05, ** p < 0.01.
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Figure 3. Effect of jasmonic acid on pollen germination in Nicotiana tabacum cv. Petit Havana (A) and Streptocarpus cv. Salmon Sunset (B); ** p < 0.01.
Figure 3. Effect of jasmonic acid on pollen germination in Nicotiana tabacum cv. Petit Havana (A) and Streptocarpus cv. Salmon Sunset (B); ** p < 0.01.
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Figure 4. Effect of abscisic acid (10 μM) on pollen germination in Nicotiana tabacum and different genera of Bromeliaceae and Gesneriaceae families; ** p < 0.01.
Figure 4. Effect of abscisic acid (10 μM) on pollen germination in Nicotiana tabacum and different genera of Bromeliaceae and Gesneriaceae families; ** p < 0.01.
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Figure 5. Effect of jasmonic (JA) and abscisic (ABA) acids on H2O2 level in tobacco pollen tubes assessed by fluorescent PFBSF staining during short-term (15 min) and long-term incubation (1 h): (A) typical fluorescent images (10 μM); (B) mean fluorescence intensity (10 μM); and (C) the effect of different concentrations of hormones on fluorescence intensity during long-term treatment; * p < 0.05, ** p < 0.01 to control meanings.
Figure 5. Effect of jasmonic (JA) and abscisic (ABA) acids on H2O2 level in tobacco pollen tubes assessed by fluorescent PFBSF staining during short-term (15 min) and long-term incubation (1 h): (A) typical fluorescent images (10 μM); (B) mean fluorescence intensity (10 μM); and (C) the effect of different concentrations of hormones on fluorescence intensity during long-term treatment; * p < 0.05, ** p < 0.01 to control meanings.
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Table 1. Plant hormones in stigma exudates collected during the stage of full fertility, nmol/mm2 stigma surface.
Table 1. Plant hormones in stigma exudates collected during the stage of full fertility, nmol/mm2 stigma surface.
Species/VarietyPhytohormone Content, nmol/mm2 Stigma Surface
Jasmonic AcidJasmonoyl-L-IsoleucineAbscisic Acid
Nicotiana tabacum cv. Petit Havana2.9 ± 0.71.0 ± 0.13.7 ± 0.4
Nicotiana tabacum cv. Samsun1.3 ± 0.3traces *6.7 ± 1.0
Lycopersicon esculentum cv. Pugovka211.5 ± 9.9tracestraces
Streptocarpus cv. Salmon Sunset73 ± 4.611.2 ± 1.9not found
*—concentrations less than 0.0025 nmol/mm2 were considered as traces.
Table 2. Calcium-sensitive probe Fluo-4 fluorescence in hormone-treated germinating tobacco pollen grains.
Table 2. Calcium-sensitive probe Fluo-4 fluorescence in hormone-treated germinating tobacco pollen grains.
TreatmentFluorescence Intensity, rel.un.
Control74.4 ± 3.9
ABA, 10 μM78.4 ± 4.1
JA, 10 μM74.7 ± 3.9
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Breygina, M.; Kochkin, D.V.; Podobedova, A.; Kushunina, M.; Afonin, D.; Klimenko, E. Detection of Abscisic Acid and Jasmonates in Stigma Exudates and Their Role in Pollen Germination. Horticulturae 2025, 11, 1146. https://doi.org/10.3390/horticulturae11091146

AMA Style

Breygina M, Kochkin DV, Podobedova A, Kushunina M, Afonin D, Klimenko E. Detection of Abscisic Acid and Jasmonates in Stigma Exudates and Their Role in Pollen Germination. Horticulturae. 2025; 11(9):1146. https://doi.org/10.3390/horticulturae11091146

Chicago/Turabian Style

Breygina, Maria, Dmitry V. Kochkin, Anna Podobedova, Maria Kushunina, Danil Afonin, and Ekaterina Klimenko. 2025. "Detection of Abscisic Acid and Jasmonates in Stigma Exudates and Their Role in Pollen Germination" Horticulturae 11, no. 9: 1146. https://doi.org/10.3390/horticulturae11091146

APA Style

Breygina, M., Kochkin, D. V., Podobedova, A., Kushunina, M., Afonin, D., & Klimenko, E. (2025). Detection of Abscisic Acid and Jasmonates in Stigma Exudates and Their Role in Pollen Germination. Horticulturae, 11(9), 1146. https://doi.org/10.3390/horticulturae11091146

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