1 Introduction

Reactive oxygen species (ROS) are multifunctional regulators of cellular processes and intercellular communications over short distances (Inupakutika et al. 2016). The role of ROS molecules in reproduction of flowering plants is described (Zhou et al. 2021; Zhou and Dresselhaus 2023). It has been established that a pollen grain receives ROS signals from the female tissues (Breygina et al. 2022), which regulate pollen germination and tube growth, and later the female gametophyte is involved: pollen tube rupture is also mediated by ROS (Duan et al. 2014). Other studies are also devoted to the significance of ROS produced by pollen in flowering plants (Speranza et al. 2012; Jimenez-Quesada et al. 2019), including the regulation of wall stiffness (Smirnova et al. 2013) and control of intracellular gradients (Scholz et al. 2020), however, the data on ROS production and their possible functions in pollen of conifers are scarce (Breygina et al. 2021a) which hinders our understanding of reproductive interactions in an evolutionary perspective.

It was found that ROS production occurs on stigmas of all flowering plants (Hiscock et al. 2007; Zafra et al. 2016), although the dynamics of such production may be different depending on the plant species. Using stigma staining, transcriptomic analysis, and controlled pollination after exposure to antioxidants, the importance of ROS for pollen germination in Fraxinus mandshurica (Oleaceae) was demonstrated (Wang et al. 2024). It was unclear which ROS is the main one on stigma; it was originally thought to be hydrogen peroxide as H2O2 was found in Nicotiana tabacum stigma exudate, which lost its regulatory activity after catalase treatment (Breygina et al. 2021b). However, lily pollen tubes are more sensitive to the stimulation effect of O⋅2− (Podolyan et al. 2021). Recently, total ROS production and H2O2 concentration in stigma exudate studied in plants from different taxonomic groups (Breygina et al. 2023a) included the predominance of either peroxide or superoxide on the wet stigma, the latter being more characteristic of basal angiosperm Aristolochia and monocot Lilium.

Unlike flowering plants, gymnosperm pollen is mostly carried by the wind and lands on a pollination drop (PD)—an extracellular fluid secreted by the female cone. PD retracts/dries up and thus takes part in capture, adhesion and transport of pollen to the ovule (Gelbart and von Aderkas 2002; von Aderkas et al. 2018). The composition of PDs in different species is largely unknown although they were found to contain some inorganic substances, carbohydrates, fatty acids and proteins (Coulter et al. 2012; Prior et al. 2019). Lu et al. employed gas chromatography–mass spectrometry to identify a total of 101 metabolites in Ginkgo biloba L. PDs: the most abundant metabolites were sugars, followed by organic acids and alcohols involved in carbohydrate metabolism, glycine, serine and threonine metabolism (Lu et al. 2020). Proteomic analysis of PDs of some gymnosperms revealed certain similarities with other sugar-rich extracellular fluids (Hou et al. 2019; Prior et al. 2019). It is believed that feeding insect pollinators could have been one of the functions of pollination drops in ancient times (Peña-Kairath et al. 2023). The production and retraction of PDs is a largely mysterious process. Recently, based on the transcriptome of the Ginkgo ovule, an assumption was made about the hormonal regulation of the process (Che et al. 2021). Even less studied in this regard are conifers, which are widespread in the Northern hemisphere and form huge forest areas. In the available literature, we did not find data on the presence of ROS in PDs of any conifer species.

Our working hypothesis was that ROS are present in pollination drops of coniferous plants and modulate the efficiency of pollen germination, similar to what has previously been shown for stigma exudate of flowering plants. If the hypothesis is confirmed, we could conclude that the mechanisms of ROS-based control of germination in seed plants are ancient and highly conserved.

2 Material and methods

2.1 Plant material

Conifer species used in this study were selected under certain criteria: (a) with a pollination drop that could be collected, (b) pollinated in Moscow, Russia, in open ground. Out of all the plants that met these requirements, four species were elected from three different families. Branches of thuja (Thuja occidentalis L.), yew (Taxus baccata L.), pine (Pinus sylvestris L.) and spruce (Picea pungens Engelm.) with mature unpollinated female cones were cut from plants grown on the territory and in the Botanical Garden of Moscow State University. The drops were collected in April (5-12 for thuja, 13-20 for yew) and May (15–22 for spruce, 23–30 for pine) 2023. For pollination drop collection, we immersed 10 yew, 2 pine or 10 thuja seed cones with droplets in 300 µl of distilled water for 30 min. For electron paramagnetic resonance (EPR) spectroscopy experiments, the water contained a spin trap or probe during incubation: 30 min, 0.5 mM (CAT-1H) or 1 h, 0.1 M (DEPMPO). The cones were not cut from the stems to avoid additional ROS generation. The spruce cones are larger, so the drops were transferred to the same volume of water by pipette or needle. The resulting wash was immediately measured.

Male cones of Picea pungens Engelm. were collected in Moscow State University Botanical Garden and kept at 25 °C until the scales opened to shed pollen, which was then stored at − 20 °C. Defrosted pollen samples were used for cultivation.

2.2 ROS measurement

Total ROS production in pollination drops was estimated EPR spectroscopy—a highly sensitive analytical technique for studying free radicals (Senesi and Senesi 2005; Samuni et al. 2022). The physical phenomenon underlying EPR spectroscopy is the interaction between an external magnetic field and the magnetic moment of an unpaired electron. Spin traps and spin probes are used to react covalently with the radicals and form a more stable adduct that can be measured (Haywood 2013). 1-Hydroxy-2,2,6,6-tetramethyl-4-(trimethylammonio)-piperidinium dichloride (CAT-1H) (Dikalov et al. 2018) was used as a spin probe. To quantitatively characterize the signal intensity, we measured the central line intensity in the EPR spectra. For superoxide detection we used specific spin trap DEPMPO (Zielonka et al. 2021) (Cayman Chemical, USA). The spectra were recorded at room temperature (21–22 °C) with RE-1307 spectrometer (Moscow, Russia) operating at microwave power and time constant of 22 mW and 0.1 s, respectively. Each characteristic spectrum is the result of 5 signal accumulations for CAT1-H and 20 for DEPMPO. Since the baseline signal from CAT1-H is nonzero, in each experiment blank samples were recorded; characteristic spectra in Fig. 3a are shown with blank control spectra subtracted; Values for Fig. 3c were normalized to the corresponding blank samples.

H2O2 in PDs was detected with FOX-1 method (Li 2019; Breygina et al. 2021b). Water containing PDs was mixed with equal volume of freshly prepared assay reagent (500 µM (NH4)2Fe(SO4)2·6H2O, 50 mM H2SO4, 200 µM xylenol orange in 200 mM sorbitol). A560 was detected after 5 min with SmartSpec spectrophotometer (BioRad, Hercules, CA, USA).

2.3 Pollen germination in vitro

Pollen was incubated at 25 °C in standard medium containing: 0.3 M sucrose, 1 mM CaCl2 and 1 mM H3BO3 in 15 mM MES-Tris buffer pH 5.5 (Maksimov et al. 2018) with gentle agitation. Germinated pollen grains (PGs) were fixed with 2% paraformaldehyde in 50 mM PBS, pH 7.4 for 30 min at 4 °C. 500 to 900 PGs were counted in each sample.

O•2− generation system included freshly prepared 50 mM xanthine (X) and xantine oxidase (XO) (1 U/mL) solutions (Sigma-Aldrich) (Zhu 2016). The final concentration in pollen suspension was 0.25 mM X and 0.005 U/mL XO. H2O2 was added to a final concentration 0.1 mM.

3 Results and discussion

PDs of coniferous plants from three families: Cupressaceae (Thuja occidentalis), Taxaceae (Taxus baccata) and Pinaceae (Pinus sylvestris, Picea pungens) were analyzed (Fig. 1). Spruce and pine represent a relatively basal clade, yew and thuja belong to more divergent conifers; Thuja can be considered the most advanced, in accordance with current phylogeny (Lu et al. 2014; Ran et al. 2018; Yang et al. 2022).

Fig. 1
figure 1

Systematic position of the studied species. Scheme is adopted from the recent phylogeny of gymnosperms (Yang et al. 2022)

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The mechanism of pollination is quite well studied in these genera (Owens et al. 1998; Breygina et al. 2021a), as is the biochemical composition of ovular secretion in species with relatively large drops (for example, Taxus baccata). The major components are carbohydrates and amino acids with addition of peptides, inorganic phosphate, calcium, malic and citric acids, and proteins (Gelbart and von Aderkas 2002; Prior et al. 2013; von Aderkas et al. 2018).

PD collection is a seasonal operation, droplets are present for a short period of time (about a week for each species), and their appearance depends on weather: on cold or windy days, drops did not appear, which is consistent with the dependence on water potential of a tree (ψ) reported previously for Picea engelmannii (Owens et al. 1987). In warm calm days in yew and thuja drops appeared massively throughout the pollination period (Fig. 2a, c). In thuja, PDs can be seen on several ovules in a cone (Fig. 2b) which is typical for this genera (Owens and Molder 1980a; Jin et al. 2020). In yew, each cone is carrying one PD (Fig. 2d), which is consistent with the literature data (Stützel and Röwekamp 1999).

Fig. 2
figure 2

Female cones of the studied species: Branches with mature female cones (a, c) and pollination drops on female cones (b, d–f) during the receptive period: Thuja occidentalis a, b; Taxus baccata c, d; Picea pungens e, Pinus sylvestris – f. Female cones are marked with arrows (a, c). PDs are marked with arrowheads (e, f). Scale bar 1 mm (b), 5 mm (a, c–f)

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For spruce and pine, branches were kept in a humid atmosphere to collect PDs. The difficulty of observing droplets in natural conditions is consistent with data from other spruce species: in Picea sitchensis, ovules within a female cone exude drops asynchronously, in an acropetal manner during 1 week (Owens and Molder 1980b). Up to 6 PDs on one spruce cone could be observed at a time, an example of a cone with several drops can be seen in Fig. 2e. In pine PDs were small and hardly noticeable on a cone (Fig. 2f).

EPR spectroscopy with a non-specific, highly sensitive CAT-1H spin probe was used to assess total ROS. The signal was detected in all the studied samples (Fig. 3a) albeit in varying amounts. We do not undertake to compare species quantitatively because the number of drops on one cone, as well as the volume of one drop in the studied species are different, but we found that PDs from different cones of the same species, collected on different days, give constant EPR signal, that is, the concentration of ROS is a characteristic property of PDs of each species (Fig. 3c). A similar situation is observed in flowering plants, where the concentration of ROS in the stigma exudate can be assessed for each species but can be hardly compared between species due to the different pistil size (Breygina et al. 2023a). In this case, pistil staining provides an opportunity to compare the dynamics of ROS (but not amount of ROS) between species (Zafra et al. 2016).

Fig. 3
figure 3

ROS in conifer pollination drops, including total ROS (a, c), superoxide radical (b), and hydrogen peroxide (d); a, c characteristic spectra (a) and results of calculation of EPR data (c) for CAT-1H, reflecting the total production of ROS; b characteristic spectra for DEPMPO reflecting O⋅2− generation; d H2O2 concentration in the PDs (diluted in H2O). Mean values and standard errors are presented in c and d. We do not compare different objects with each other; control – blank sample containing CAT-1H (a) or DEPMPO (b) incubated in the same conditions as PDs

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EPR spectroscopy was also used to assess the generation of superoxide radical in PDs, with a specific spin trap DEPMPO (Zhu 2016; Breygina et al. 2022). O⋅2− was found in PDs of three species (Fig. 3b). In thuja no DEPMPO signal was detected.

The measurement of H2O2 concentration showed that this substance is absent in detectable amounts in all PDs, except for thuja (Fig. 3d). For each measurement, a calibration curve was made; the detection limit according to it was approximately 1 µM peroxide.

Thus, depending on the species, the main ROS in a PD can be either peroxide or superoxide. To check if pollen grains are sensitive to these ROS, Picea pungens pollen suspension was used, being a convenient model system to test the sensitivity of pollen to various substances (Maksimov et al. 2018; Breygina et al. 2023b). Two time points with low germination efficiency in control were taken in order to identify a possible stimulating effect. We found that total germination efficiency of spruce pollen is sensitive to both H2O2 and O⋅2−, but the stimulating effect of the latter was more pronounced though appeared later (after 14 h) (Fig. 4a). When calculating germination, we noted that there are more grains with two pollen tubes in O•2−-treated suspensions, so the ratio of bipolar germination to all germinated grains was also assessed (Fig. 4b, c). No changes were found in the case of peroxide (Fig. 4b), while in the presence of O⋅2− the proportion of bipolar germination increased more than twofold (Fig. 4b–d). Previously, bipolar germination was found to be sensitive to incubation conditions: it was the most abundant in optimal conditions (Breygina et al. 2019). Our data indicate that pollen germination medium with superoxide radical is “more optimal” compared to the ROS-free medium. This is consistent with the high production of O⋅2− in PDs (Fig. 3b). The detection of ROS in all studied PDs and previously, on the stigmas of all flowering plants (Zafra et al. 2016; Breygina et al. 2023a; Wang et al. 2024) demonstrates the conservative nature of ROS-based mechanisms in the reproduction of seed plants. Apparently, this mechanism is more ancient than previously thought.

Fig. 4
figure 4

Stimulation of Picea pungens pollen germination in vitro by H2O2 (100 µM) and O•2.− generated in X/XO system: a, b total germination efficiency (a) and proportion of bipolar pollen germination (b) after 9 and 14 h of incubation (each result normalized to control germination efficiency). Mean values and standard errors are given, significance is indicated by Student’s t-test: * P < 0.05, ** P < 0.01; c, d ratio of unipolar and bipolar germination after 14 h of incubation in control (c) and in the medium + X/XO system (d)

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The difference in ROS balance between thuja and other species indicates the diversity of ROS-based regulatory mechanisms in the reproduction of conifers. Based on the obtained data, we made an assumption that O⋅2− is the main regulatory ROS in PDs of yew, spruce and pine while in thuja it is converted into H2O2. Other possible source of ROS in the latter species could be the oxidation of polyamines: this reaction directly produces hydrogen peroxide (Wu et al. 2010; Do et al. 2019). In addition to regulating pollen germination, ROS in the pollination drop may perform related functions, such as protection against fungal and bacterial infection, similar to what has been suggested for stigmatic ROS in flowering plants (Sharma et al. 2023).

Comparing these differences with the systematic position, we can make a preliminary conclusion about the greater value of superoxide in the reproduction of Pinaceae and Taxaceae, which may to some extent reflect an evolutionary arrengement, since they represent more basal clades compared to Cupressaceae (Yang et al. 2022). This is consistent with previously proposal for angiosperms (Breygina et al. 2023a). Undoubtedly, for convincing conclusions on the whole families, more objects will be included in the study.

4 Conclusions

ROS are present in pollination drops of all studied coniferous plants, including thuja, yew, pine and spruce, and influence pollen germination efficiency in vitro. We can conclude that ROS-mediated interaction between the male gametophyte and sporophyte is characteristic not only of flowering plants, but also of conifers. ROS can play an important role in the sexual reproduction of conifers as a signal from the female tissues stimulating pollen germination in vivo.