INTRODUCTION

The surface of plant cells is sensitive to changes in tropospheric ozone concentration, salinity, traumatic factors, etc. As protection, various compounds in liquid form (phenols, terpenes, alkaloids) are released, and sometimes deposited on the surface. The excretion can occur over the entire surface or through special secretory structures [1]. First of all, external signals are perceived by the components of the cell wall, and they must create protection against the penetration of toxic substances to the plasmalemma and deep into the cell. It is generally accepted that phenols of plant excreta and even phenolic components of the cell wall have antioxidant protection of plant surfaces. Recently, attention has been payed to the blue pigments of the surface—azulenes. The presence of azulenes is associated with the blue color of plants, which may belong to these pigments in some herbaceous and woody species [2].

A special place in the composition of liquid secretions secreted by the cell can be occupied by signaling compounds known as animal neurotransmitters – acetylcholine and biogenic amines: catecholamines, serotonin, and histamine; they are found in all organisms and are also called biomediators [3]. In many cases, the appearance of biogenic amines is a reaction to environmental factors. Biogenic amines appear in the secretions of unicellular horsetail spores under the influence of salinity [4] or ozone [5]. At this moment, the exogenous alluvial effects of dopamine on plants that have undergone abiotic damage by factors affecting the physiological state of whole plant organisms are also being studied [6, 7]. There are also the first data related to the release of histamine and dopamine by Chara algae [8]. It should be noted that exogenous neurotransmitters secreted by animals, plants, and microorganisms can appear on the cell wall [8, 9]. Chemical interactions between various living organisms (plants, animals, and microbes) with the help of these substances can play an important role in the regulation of the biocenosis, including the stress of the plant organism, where biogenic amines and acetylcholine are involved [10].

Using spectral methods of analysis on model unicellular and multicellular systems of organisms, this article attempts to consider the role of azulenes on the surface of a plant cell in interaction with signaling compounds such as neurotransmitters.

MATERIALS AND METHODS

Objects. Unicellular and multicellular models of species at different stages of evolution were chosen as objects of research. The first are represented by diatoms Ulnaria ulna (Nitzsch) Compere (Bacillariophyta, line 02-903), cultured in vessels with a volume of 100 mL in a nutrient medium that included in μg/L: KH2PO4 6.63, CaCl2 6.51, NaCl 3.47, MgCl2 5, and silicagel (Merck, Austria) 2 μg/L as a source of silicon [11]. Unicellular vegetative microspores of horsetail Equisetum arvense L. (fam. Equisetaceae) were also analyzed and fern spleenwort Asplenium scolopendrium L. (Newman) (fam. Asteraceae) cultured in the laboratory on slides in nutrient medium within Petri dishes as described earlier [12].

The second group of models includes samples with a blue or silver surface, collected in 2020–2022. These were species of central Russia, both herbaceous (pasture ryegrass Lolium perenne L. and white clover Trifolium repens L.) and woody (sea buckthorn shrubs Hippophae rhamnoides L. and white willow Salix alba L.), collected in May–June on the banks of the Oka river of the botanical reserve “Stepnoi” of Pushchino. Southern species were also used as samples—needles and cones of high juniper Juniperus excelsa Bieb. from the Karadag Nature Reserve, Feodosia, Crimea, and juniper Juniperus pfitzeriana 'Blue Cloud’) from the family Cupressaceae from the National Park Arboretum, Sochi, collected in October and January–February, leaves of eucalyptus ashy Eucalyptus cinerea F. Muell. Ex. Benth (fam. Myrtaceae) with a silvery surface and the leaves of the noble laurel Laurus nobilis L. (fam. Lauraceae) from Adler’s Southern Cultures Park, collected in September.

Spectral methods. Absorption (absorbance) and autofluorescence of cells before and after histochemical treatment (see section Histochemical analysis) was measured directly on slides using the microspectrophotometer/microspectrofluorimeter MSF-15 (LOMO, St. Petersburg, Russia) and laser-scanning confocal microscope Leica TCS SP-5 (Germany–Austria–USA). The fluorescence excitation was excited by ultraviolet light 360–380 nm or laser 458 nm.

The position of the maxima in the absorption spectra of intact cell surfaces recorded using the MSF-15 microspectrophotometer was determined according to the Zolotarev method [13] by the option of the reflection spectra differentiation [13]. We photographed living cells before and after histochemical treatment using a Leica DM 6000 B fluorescence microscope, the above-mentioned confocal microscope and an MSF-15 microspectrophotometer/microspectrofluorometer with a Levenhuk M300 Base (USA) camera. The image of the leaves in transmitted light was also obtained using the Invitro Evos M500 microscope (Thermo Fisher Scientific, USA). The absorption and fluorescence spectra of extracts with 100% acetone or 95% ethanol from cells (1 : 10 w/v for 5 min to 1 h or more) in 1–0.5 cm cuvettes or on paper chromatograms were recorded using the Unicam Helios-epsilon spectrophotometer (USA), spectrophotometer Specord M-40 (Germany) and Perlin Elmer 350 MPF-44 B spectrofluorometer (Great Britain).

Detection of azulenes. Azulenes in sample extracts with 95% ethanol and acetone for 10–30 min of exposure were determined spectrophotometrically at 580 nm, as described earlier [2]. The average error of the experiment of three to four repetitions was calculated for each variant and control, respectively.

Histochemical analysis for biogenic amines. The histochemical fluorescent determination of biogenic amines (dopamine, histamine, and serotonin) in cells was carried out according to the methods originally described for animal cells and also used for plant cells [14, 15]. Microspores were applied to slides and moistened with drops of 0.5–1% solutions to determine dopamine with glyoxylic acid; histamine, with orthophthalic aldehyde, and serotonin, with formaldehyde. Histochemical reactions were repeated up to 3–5 times. Autofluorescence and fluorescence after histochemical staining of biogenic amines (see Spectral methods) were used as test reactions of cells on subject glasses (slides). All experiments were carried out at room temperature 20–22°C. After 10–20 min of staining with the reagent, the samples were dried at 50–80°C for 5–10 min. Fluorescence reactions of forming products were studied under a Leica DM 6000 B fluorescence microscope or a Levenhuk camera (USA) when excited with light of 360–380 nm. The fluorescence spectra were recorded by the MSF-15 microspectrofluorimeter/microspectrophotometer (LOMO, St. Petersburg, Russia). The fluorescence intensity at 460 nm was expressed statistically with standard error.

Electron transport in photosynthesis. To analyze the donor properties of azulene, it was tested for electron transport with NADP+ [16], in the model of isolated chloroplasts Kalanchoe pinnata (Lam.) Pers. (Fam. Crassulaceae), which do not contain natural azulenes [17], in contrast to the chloroplasts of pea and clover, which contain these pigments [18]. In addition, a small amount of chlorophyll in the probes made it possible to observe the blue coloration of azulene in a reaction medium. In classical versions of pea or spinach plastids, non-cyclic electron transfer was better observed at 650–660 nm, while cyclic transport, at 700–719 nm. In our model, chloroplasts from Kalanchoe showed photochemical activity with 550–555 nm filters, although the data obtained with 650 nm, 670 and 706 nm interference filters were similar, but with lower acceptor reduction rates [17]. In the probes we used NADP+ 0.9 μmol/mL, ferredoxin from peas 0.1 mg/mL, diuron or 3-(3,4-dichlorophenyl)-1,1-dimethylurea), 10–6 M, antimycin A, 10–5 M. Actinic light (luminous flux intensity 38 K erg cm–2 s–1) passed through an interference filter 550 nm. The amount of NADPH is expressed statistically with a standard error. The interaction of azulene (2 mg/mL) with the isolated components of the electron transport chain (2 mg/mL) was carried out on cytochrome C553 (f) preparations from Chlorella algal cells and plastocyanin, ferredoxin, and ferredoxin-NADP reductase, from pea chloroplasts [17].

Reagents. Azulene and glyoxylic acid (Fluka, Austria/Germany), ortho-phthalic aldehyde, formaldehyde, dopamine, histamine, serotonin (ICN Biomedical/Pharmaceuticals, USA), and NADP+ (Sigma–Aldrich, USA) were used in the study.

RESULTS AND DISCUSSION

Spectral and histochemical methods were used to consider the role of cell surfaces in model systems of unicellular and multicellular organisms.

Study of the surface of different types of cells by spectral methods. The surface of plant cell is the primary sensor of any external signal and can also be a protective barrier if it includes appropriate components that protect the cell from any damage.

With the help of microscopy in transmitted light and fluorescence microscopy, including microspectrofluorimetry/microspectrophotometry and laser-scanning confocal microscopy, we examined a variety of model plant cells from the lowest organisms on the evolutionary ladder—unicellular (diatom Ulnaria ulna, vegetative unicellular microspores of horsetail Equisetum arvense and fern spleenwort Asplenium scolopendrium) to highly organized multicellular angiosperms, obtaining their images (Fig. 1) and the absorbance spectra (Fig. 2). Figure 1 shows external image of the plant cell surface for species of different steps of the evolution.

Fig. 1.
figure 1

Images of the surface of plant objects. Fluorescence of the cells of diatom Ulnaria ulna (a) and vegetative spore of horsetail Equisetum arvense (b), when excited by light of 430 nm. (c) Spore-bearer (1) and spores (2) of fern Aslenium scolopendrium in transmitted light (1) and when fluorescence excited by the light at 430 nm (2). (d) Leaves of Eucalyptus cinerea in transmitted light under a conventional microscope (1): greenish-yellow oil glands are visible on a dark green surface and an Invitro Evos M5000 microscope (2): where blue wax plates cover the entire surface of the leaf, including yellow secretory cells of the glands. (e) Sea buckthorn Hippophae rhamnoides under a laser-scanning confocal microscope Leica TCS SP5, autofluorescence excited by laser 458 nm: (1) in blue (488–500 nm), (2) green (505–540 nm), and (3) red (580–700 nm) channels; (4) superposition of images (1)–(3) of the laser-scanning confocal microscope of the surface glands of. Scale bars for images in (a) and (b), 75 and 25 μm; in (c), 100 and 20 μm; (d) and (e), 100 and 75 μm, respectively.

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Fig. 2.
figure 2

The absorbance spectra from the surface of various plants (the first derivative of curves for identifying maxima). (a) The initial sample for the Salix alba leaf; (b) the upper parts of the graphs for the species studied. The estimated maxima of azulenes are marked with circles, and asterisks indicate the maximum of chlorophyll.

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According to microscopy in transmitted light and when excited by light that causes fluorescence of the object, in the model system of unicellular almost colorless diatoms Ulnaria ulna, we were able to assess the state of the surface only by exciting the fluorescence of chlorophyll with light of 430 nm (Fig. 1a). If red emission is not observed, it means this cell is dead, and solid shells (frustules) impregnated with silicon compounds fluoresce with green light [11]. In the non-germinated vegetative microspore of the horsetail Equisetum arvense in transmitted light looks as green. When excited by light of 430 nm, the outer solid shell with external exine lightened in the blue or blue-green region, and only the developing cell has red emission, and the shell is missed, when the cell begins to develop (Fig. 1b). At a higher stage of evolution in ferns, unlike horsetail, spores no longer have such rigid shell, but they are enclosed in spore-bearers with solid shell, which fluoresces in green-yellow, when excited by light of 430 nm, and spores inside are green (Fig. 1c). The germinated spore emitted in green for a long time—up to a week after wetting the spore-bearer—with a maximum of 530 nm, until maximum of 680 nm appears in the fluorescence spectrum, referring to the already synthesized chlorophyll.

The surface of the cell of diatoms and spores of horsetails is a dense cellulose shell, including silicon compounds. In the fern, all the spores are enclosed in a spore-bearer, the surface of which is also rigid shell, but there is no such shell as in diatom cells and horsetail spores. In all three models of undeveloped diatom, horsetail and fern cells, the surface of the cells emitted in green with a maximum of 520–530 nm. Chlorophyll is synthesized and usually shines through with red light from inside the cell after the onset of development. Unlike the leaves of higher plants eucalyptus Eucalyptus cinerea, sea buckthorn Hippophae rhamnoides and laurel Laurus nobilis, dry non-germinated spores of horsetail and fern do not have chlorophyll, which can be seen in their absence of red fluorescence [12].

Multicellular models of angiosperms (Figs. 1d, 1e) do not have such dense cell membranes, but there are special secretory structures (Figs. 1d, 1e). In ashy eucalyptus, yellow-green secretory glands are clearly visible in transmitted light (Fig. 1d, 1), and they fluoresce in the green region of the spectrum [2]. Moreover, with the help of a microscope, bluish wax plates are visible in transmitted light, covering both these glands and the entire surface of the leaf outside the secretory cells. Apparently, these are plates of azulene, previously found in this species in ethanol or acetone 10-min leaf washes (leachates) [2]. Laser-scanning confocal microscopy showed that fluorescence in blue-green light is also characteristic of multicellular glands on the leaf surface of sea buckthorn Hippophae rhamnoides (Fig. 1e). Fluorescent methods made it possible to show the structure of the cell surface marked by autofluorescence, as we can see from the emission in the blue and blue-green regions of the spectrum, and to estimate the viability of cells, mainly by the presence of chlorophyll inside the cells. What kind of compounds are on the surface of cells is still unknown. It is not understood, how this may be associated with the release of secretory products. In previous work on ozone resistance indicators, the attention was to be attracted to woody plants with a blue or silver surface color, where azulenes were found [2]. Usually, a lot of material was required for similar analysis. An image of the surface of a eucalyptus leaf in transmitted light (Fig. 1d) using an Invitro Evos M5000 microscope (Thermo Fisher Scientific, USA) made it possible to see azulene-containing wax plates. We supposed that their absorbance maxima can also be seen in the spectra of intact leaves. In this work, a microspectrophotometer was used for this purpose to obtain data on the absorbance spectra from the surface of objects, which are presented in Fig. 2.

In the absorbance spectrum of the surface, it is possible to detect maxima characteristic for various compounds (400–460 nm, terpenoids; 500–530 nm, phenols; 570–620 nm, azulenes, and 660–666 nm, chlorophyll) [18]. To determine the maxima in reflection spectroscopy by the microspectrophotometer/microspectrofluorimeter, there is a differentiation option to identify maxima in the absorbance spectra according to [13]. An example of such a method is shown in Fig. 2a for the sample of white willow Salix alba. To identify maxima in the region of 550–670 nm, it was enough to provide information with positive values (top of the graphic), which is done in Fig. 2b for all samples.

The first derivative of the external reflection spectrum for low-intensity bands made it possible to directly determine the presence of the maxima of 580–620 nm and 660–666 nm characteristic of azulenes and chlorophyll, respectively, in the spectra after differentiation (Fig. 2). In the absorption spectra of colorless diatoms of Ulnaria ulna (Fig. 2), along with the chlorophyll maximum, there is a maximum of 600–610 nm. Azulene has also been found in some brown algae [19]. In bluish vegetative microspores of horsetail Equisetum arvense contained azulenes [18] maxima 610–612 nm, peculiar to the compounds, are clearly seen. Peaks, characteristic for azulenes, have been found in all plants studied, except fern Asplenium scolopendrium and laurel Laurus nobilis. In bluish vegetative horsetail microspores containing azulenes [18], the maxima of 610 and 612 nm characteristic of these compounds are clearly visible. The affiliation of the absorption maxima of 580–630 nm was further confirmed by experiments with the extraction of azulenes in ethanol and acetone.

Azulenes in extracts from surfaces. Table 1 shows data on the azulenes of the studied plants, calculated by the intensity of the absorption bands at 580 nm in 10-min extracts – washes off from the whole surface [2]. First of all, the presence of azulenes in extracts confirmed experiments on the detection of maxima characteristic of blue pigments in the absorbance spectra of cell surfaces in the region of 580–610 nm (Fig. 2). The spores of the fern Asplenium and the leaves of the laurel Laurus did not have such maxima in the spectra, and there were no azulenes in their extracts. The benthic freshwater diatom Ulnaria ulna had similar maxima, but it is difficult to isolate them in extracts with organic solvents. It is fundamentally important that sesquiterpene lactones of the azulene series, which have the ability to affect other organisms, were found in algae, in particular the brown Dictyota dichotoma [19] and the benthic gorgonian inhabitants of the Arctic [20]. They either disrupted insect food chains or hindered the development of marine life organisms that causes fouling of the bottom of ships.

Table 1.   The content of azulenes on the surface of plant leaves (10 min extraction with ethanol)
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When compared in Table 1, it turned out that horsetail, ryegrass, juniper have a high blue pigment value per mg of wet weight, while sea buckthorn and juniper are significantly less azulene extracted in 10 minutes. Probably, in the latter species, the pigment was present in the deeper layers of the cuticle or even the cell wall. All these studied species are characterized by blue or blue leaf coloration. One of the azulenes was previously isolated from the bluish spores of horsetail, as well as from the chloroplasts of clover Trifolium repens [18]. It is known that in some junipers, azulenes were found in oil distillation products [21]. Previously, the presence of azulenes was shown in 10 minute alcohol or acetone leaf surface extracts from eucalyptus Eucalyptus cinerea and a number of conifers with blue needle surfaces that were resistant to ozone [2]. Blue pigments, we believe, serve as a protective optical filter against ultraviolet radiation and ozone formed with its participation [22]. The pigments can be primary targets for ozone in these species, and their antioxidant properties determine low sensitivity to ozone and reactive oxygen species.

The presence of azulenes is associated with the blue color of the surfaces, which may belong to these pigments in some herbaceous and woody plants.

Azulenes have significant antioxidant and anti-allergenic activity in animal cells [23, 24]. In model experiments on vegetative horsetail microspores, the antihistamine properties of exogenous azulene and proazulene sesquiterpenes were demonstrated [25].

Neurotransmitters on the cell surface. Endogenous neurotransmitters can be released to the surface of the cell (can be considered as a physiological response of the cell to external influences or intracellular signaling in the direction out the cell). Compounds such as dopamine and histamine may be normal in some species, but often appear under stress. They accumulate under the influence of various stress factors, both abiotic (ultraviolet radiation, ozone, salinity, drought, nutrient deficiencies, etc.) and biotic (insect damage, for example) in the biocenosis [26, 27].

Biogenic amines were detected by histochemical methods by characteristic fluorescence at 460 nm in model systems of organisms that directly isolate them in nature – in unicellular diatoms Ulnaria ulna (Nitzsch) [11] and unicellular vegetative microspores of horsetail Equisetum arvense L. (fam. Equisetaceae) [14]. Table 2 compares the levels of three biogenic amines in this diatom and the single-cell spore of the terrestrial fern plant Asplenium scolopendrium. In the first object, dopamine predominates in the cells, and the least of all serotonin, while the second object turned out to have more histamine, judging by fluorescence, while similar data were noted for dopamine and serotonin in spores. In the secretion released from the spore, serotonin is significantly less than other amines studied.

Table 2.   Biogenic amines in diatoms Ulnaria ulna L. and vegetative spores of fern spleenwort Asplenium scolopendrium L.
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Specialized secretory surface cells (glands, hairs, etc.) can also secrete biogenic amines, since they contain enzymes that regulate their content, as well as antineurotransmitters, substances that reduce the toxic effect of neurotransmitters or block receptors. In angiosperms, as shown for the ashy eucalyptus glands in Eucalyptus cinerea, biogenic amines can be excreted both by the entire leaf surface (dopamine) and by specialized secretory structures (histamine) of the surface [28]. Figure 3 shows a bright yellow emission, characteristic for high concentrations of dopamine [29] in the secretory glands of the medicinal plant sea buckthorn Hippophae rhamnoides after histochemical treatment with glyoxylic acid. Moreover, dopamine is present in all parts of the scaly structure. In contrast, in response to histamine, blue emission was observed only in the middle of the multicellular gland. The appearance of biogenic amines is often associated with stress, and it is possible that this is not only a marker of stress, but also to some extent a protective reaction.

Fig. 3.
figure 3

Histochemical reactions to dopamine and histamine in the secretory scale-gland of sea buckthorn leaves Hippophae rhamnoides L. Above is a view of the gland under a fluorescent microscope when excited by light of 360–380 nm. Below are the fluorescence spectra.

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Effects of exogenous azulene on the release of biogenic amines from cells and possible mechanisms. To know that the leaves of the studied species contain azulenes in the surface layer, as we have shown above, an interaction between azulenes and released biogenic amines is capable. To assess this possibility, model experiments were carried out on unicellular vegetative horsetail microspores (Fig. 4), which emit significant amounts of biogenic amines under salt stress with 1% sodium sulfate, as in natural conditions [4]. In model experiments on vegetative horsetail microspores, the antihistamine properties of exogenous azulene and proazulene sesquiterpenes were previously demonstrated [25]. Figure 4 shows that under the same conditions, azulene and proazulene grosshemine influenced the content of dopamine in cells and the released secretions.

Fig. 4.
figure 4

Effects of azulene and proazulene grosshemine on dopamine content, estimated by fluorescence of 460 nm after histochemical treatment of cells of vegetative microspores of horsetail Equisetum arvense under conditions of salinization with 1% sodium sulfate.

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The most effect of the reduction by dopamine was observed for proazulene grosshemine and azulene from the five studied sesquiterpenes, both in cells and secretion (Fig. 4) under the conditions of salinization of the medium with 1% sodium sulfate, which stimulates the release of biogenic amines to the outside. Biogenic amines in large quantities can be toxic to the cell.

Table 3.   Change in the absorbance intensity in the initial ethanol solutions of azulene and dopamine 5 min and 24 h after mixing the solutions
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Taking into account that azulene and dopamine can enter into redox reactions due to double bonds, we have done experiments with their ethanol solutions (Table 4), simulating the possibility of their interaction on the surface of cells. Dopamine in high concentrations in the presence of oxygen through a chain of reactions superoxide-anion radical → peroxide can be converted into toxic red dopamine chrome with the absorption maximum of 480 nm. In the absorbance spectra, it was observed whether changes occur at 480 nm (possible increase and appearance of red color, characteristic of dopaminechrome) and at 580 nm (maximum of azulene absorption). The sequence of addition of reagents was as follows: Dopamine + Azulene or Azulene + Dopamine in a ratio of 3 : 1 by weight of the starting substance. In the first case, only an increase in absorption at 580 nm was noted, and in the second case, an increase in optical density at 480 nm (possible formation of a small amount of dopaminechrome) and the decrease at 580 nm were observed. However, there was no apparent change in the overall blue color of the mixture in a short observation time. Only after 24 h there was a change in color in the Dopamine + Azulene variant. In comparison with the 5-min probe the absorbance almost doubled at 480 nm and decreased sharply at 580 nm, so that the blue color became hardly noticeable, and a faint pink color of dopaminechrome was already visible. In the Azulene + Dopamine variant, when the concentration of azulene is 3 times greater, dopaminechrome did not form at all after 24 h (there is no absorption at 480 nm), but the blue color of azulene, related to absorbance at 580 nm, disappeared. In the histamine and serotonin variants, the changes were only at 580 nm – a slight increase the in uptake for the first neurotransmitter and a slight decrease for the second. The change in color of the azulene was not noticeable.

Table 4.   Effects of azulene (final concentration of 0.01 mg in 0.5 mL sample) on photosynthetic electron transport in isolated Kalanchoe pinnata chloroplasts. Actinic light, 550 nm
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Electrochemical possibilities of interaction should also be considered. According to the work of Plemenkov et al. [30], the redox potential of azulene in acetonitrile is +0.36 V. The peak current corresponds to the transfer of two electrons per molecule. In the voltammogram, two reduction peaks are recorded at potentials of +0.17 and = +0.04 V. On isolated pea chloroplasts, the dopamine/ascorbate pair (dopamine at concentrations greater than 10–5 M) is able to reduce oxidized cytochrome f (redox potential +0.365 V) and plastocyanin (redox potential + 0.37 V) [31] and be an electron donor between cytochrome f and plastocyanin in the electron transport chain. Blue azulene, if added to a solution of cytochrome f (C 553) isolated from Chlorella cells (1 : 1 ratio of reactants by weight), became colorless after the loss of an electron. The same but weaker reaction was noted when interacting with plastocyanin isolated from pea plants. In mixtures of azulene solutions with individual ferredoxin, ferredoxin-NADP reductase or NADP+ due to their more negative redox potentials than that of blue pigment, no such reaction occurred.

In model experiments on isolated chloroplasts Kalanchoe pinnata (Lam.) Pers. (fam. Crassulaceae), which are devoid of natural azulene, we have shown (Table 4) that azulene can be the electron donor in the electron transport chain of photosynthesis between cytochrome f (+0.36 V) and plastocyanin (+0.37 V), participating in non-cyclic (inhibited by diuron) and cyclic (inhibited by antimycin A) electron transport. Table 4 shows that even in the case of blockade with an inhibitor of diuron or antimycin A, azulene is able to restore electron transport and is apparently an electron donor.

The presence of azulenes in the chloroplasts of peas and clover [18] seems to be no coincidence, and is also a protective mechanism in case of the damage, where azulene donates electrons to the electron transport chain. Given that this material requires special consideration in another publication, we note the main thing in the possible mechanism of interaction between azulene and dopamine on the surface of plant cells: azulenes (and probably proazulenes) as antioxidants can prevent or slow down the formation of toxic dopaminechrome. For histamine and serotonin, there is probably another mechanism of interaction with azulene.

CONCLUSIONS

The use of fluorescence microscopy with modifications of laser-scanning confocal microscopy and microspectrofluorimetry/microspectrophotometry mades it possible to obtain information about the surface of various species on the evolutionary ladder, from diatom frustules (shells) and hard (rigid) covers of vegetative horsetail microspores to leaves of angiosperms. In a significant number of plants with a blue or silver surface, 580–620 nm maxima were found in the absorbance spectra, characteristic for blue azulene pigments, which is confirmed by their appearance in 10-min extracts with ethanol or acetone. In most of the species studied, biogenic amines – dopamine, histamine and serotonin, known as animal neurotransmitters, can be released from the cells outside. Isolation occurs both by the entire surface and by specialized secretory structures. It is likely that the azulene layer (or layers), due to its antioxidant properties, serves as a certain protective filter against endogenous and exogenous biogenic amines.