INTRODUCTION

Soil pollution with heavy metal (HM) ions is caused by the use of phosphate fertilizers, mining, fuel combustion, and metallurgical industrial processes [1, 2]. Excessive concentrations of HMs in the environment cause a decrease in plant productivity and create risks for animals and humans due to contamination of food and feed with toxic elements. An increased content of mobile forms of HMs is inherent in acidic soils, which account for more than a third of all arable land [2, 3]. One of the most toxic metals is cadmium, the excess concentration of which limits the growth and development of plants [4, 5]. The most intense sources of environmental pollution with cadmium are metallurgy and electroplating as well as the combustion of solid and liquid fuels. Cadmium is toxic even at low concentrations [6]. It negatively affects plants at the morphological, physiological, biochemical, and molecular levels [7]. Numerous studies have shown that cadmium inhibits seed germination, reduces the length and weight of roots, and reduces the number of plant leaves [8]. Along with lead and copper, cadmium is one of the most toxic metals that enter the food chain and can cause carcinogenic and chronic diseases in humans. Soil acidity increases the availability of HM ions, including cadmium, for plants. However, currently accepted technologies for reducing soil acidity can have a negative impact on the environment [9]. The use of compounds of a hormonal nature to increase plant productivity under technogenic stress is an effective and safe technology [3]. In this light, of particular interest is phytomelatonin, a pleiotropic molecule of indole nature, which a number of researchers classify as phytohormones [10]. It has been shown that exogenous melatonin reduces the toxic effect of copper, cadmium, aluminum, and nickel by enhancing root growth, increasing antioxidant activity, increasing the intensity of photosynthesis, and reducing the accumulation of metal ions in different parts of plants [11–13]. However, in most works devoted to the effect of melatonin on plant resistance under conditions of HM excess, high concentrations of both melatonin itself and the studied metals are used. The first is economically unprofitable, while the second does not correspond to the real characteristics of contaminated soils. In addition, the scientific literature hardly discusses the ability of melatonin to induce the state of priming in plants, i.e., the process by which an organism acquires the ability to increase stress tolerance in response to the action of one or another damaging factor in the future [14]. Melatonin can serve as a priming inducer, leading to a significant increase in plant resistance to a variety of abiotic and biotic damaging effects. It seems expedient to study the short-term, compared with the long-term, effect of melatonin on the resistance of cereal plants to the action of heavy metals under conditions of basal cultivation. Data on the mechanisms of plant protection by melatonin in response to the action of cadmium ions, as well as optimization of plant processing technology in order to obtain the maximum protective effect, are essential both for obtaining new fundamental knowledge and for creating a scientific basis for the development of innovative agricultural technologies to increase plant productivity.

The aim of this work is to elucidate the physiological mechanisms of melatonin priming of barley plants against the background of the delayed action of excess cadmium in the medium compared to the long-term action of melatonin during “cadmium stress.”

MATERIALS AND METHODS

Studies have been carried out on barley plants Hordeum vulgare L. varieties Biom. The plants were grown in the soil with the addition of perlite for 5 days at a temperature of 19 ± 2°C in a phytotron with a 16-h photoperiod. Then, within 7 days, the plants were adapted to the conditions of a liquid nutrient medium according to Blamey (pH 4.5) [15] with a low content of phosphorus. This nutrient medium most closely matches the real composition of the soil solution on acidic soils compared to traditionally used nutrient media, where, due to the high pH values of the solution and high concentrations of phosphate ions, the availability of metal ions for plants and, consequently, their toxic effect is significantly reduced [3, 16].

After adaptation, some plants were subjected to a daily root treatment with melatonin at a concentration of 10 μM. For other plants, the nutrient medium was changed for 1 day. All plants were then divided into six variants (Table 1).

Table 1. Experimental scheme
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The concentration of cadmium chloride (CdCl2) were selected based on typical values in the soil solution of industrially polluted acidic soils [16–18]. Six days later, growth parameters (length of axial organs, total leaf surface area), content of photosynthetic pigments, and parameters of photochemical activity of PSII were assessed. Plant material for biochemical analysis was fixed with liquid nitrogen and stored at –70°С.

The wet and dry biomass of plant material was estimated gravimetrically using a Sartorius CP 622 analytical balance (Germany). The dry weight was determined after fixing the material and drying it to constant weight. The water content (% of wet weight) was calculated from the ratio of the difference between wet and dry biomass, referred to wet weight. The leaf area was calculated using the Anikeev and Kutuzov formula [19].

The photochemical activity parameters of PSII were measured using a PAM fluorimeter (MINI-PAM-II, Heinz-Walz, Germany). The parameters of fluorescence coefficients and relative electron transport rates were calculated using the MINI-PAM-II software.

Analysis of the content of metals in the root system and aboveground parts of plants was carried out by inductively coupled plasma mass spectrometry (ICP-MS) (Agilent 7900 mass spectrometer, United States). In order to desorb metal ions from the apoplast space, washing with calcium chloride solutions was used.

The LPO value was measured spectrophotometrically by the concentration of compounds active in the reaction with thiobarbituric acid upon heating (TBARS) according to Buege and Aust [20].

The concentration of photosynthetic pigments in solution was determined according to Lichtenthaler [21]. Leaves (70 mg) were added to a test tube with 96% ethanol (1.5 mL) and calcium carbonate, vortexed, centrifuged for 10 min (10 000 g), and the supernatant was carefully transferred into test tubes. This extraction was performed three times, and the tubes were kept in the dark. The amount of alcohol in all tubes was adjusted to the same volume (up to 5 mL), mixed, and the optical density was measured.

The content of endogenous proline was determined according to Bates et al. [22].

To assess the activity of superoxide dismutase (SOD) and peroxidase (PO), plant samples were triturated in liquid nitrogen with insoluble polyvinylpyrrolidone, extracted with potassium phosphate buffer (pH 7.4), then centrifuged for 20 min (8000 g, t = 4°C) (Eppendorf 5430R, Germany). Aliquots were taken from the resulting supernatant to determine the activity of SOD and PO. Total SOD activity was determined according to the method described by Beauchamp and Fridovich [23]. PO activity was determined according to Shevyakova et al. [24]. The protein content in preparations was measured by the Esen method [25].

The results obtained are presented in the tables and figures as an arithmetic mean with a standard error. To compare independent samples obeying the normal distribution law, we used Student’s parametric test. Values of t-criteria were found for 95% significance level (R ≤ 0.05).

RESULTS

Effect of Cadmium Chloride and Melatonin on the Morphometric Parameters of Barley Plants

Under optimal growing conditions, melatonin did not have a significant effect on the growth of barley. The addition of 2 μM cadmium to the solution caused the suppression of stem and root growth by 11 and 24%, respectively (Figs. 1a, 2). Melatonin, regardless of the duration of exposure, partially reduced the negative effect of “cadmium stress” on stem and root growth.

Fig. 1.
figure 1

Influence of cadmium chloride and melatonin on the growth of barley plants: (a) stem and root length; (b) total area of the leaf surface. * P ≤ 0.05 when compared with the control value; # P ≤ 0.05 compared to CdCl2. (1) Control; (2) melatonin (1 day); (3) melatonin (6 days); (4) CdCl2 (6 days); (5) melatonin (1 day) + CdCl2 (6 days); (6) melatonin (6 days) + CdCl2 (6 days).

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Fig. 2.
figure 2

Appearance of barley plants grown in a medium with the addition of cadmium chloride and melatonin: (1) control; (2) melatonin (1 day); (3) melatonin (6 days); (4) CdCl2 (6 days); (5) melatonin (1 day) + CdCl2 (6 days); (6) melatonin (6 days) + CdCl2 (6 days).

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The negative effect of “cadmium stress” affected not only the size of the axial organs but also the leaf surface area, which decreased by 12% (Figs. 1b, 2). Priming with melatonin followed by “cadmium stress,” as well as prolonged exposure to melatonin, partially maintained the assimilating surface area compared to the action of cadmium in the absence of melatonin.

Effect of Cadmium Chloride and Melatonin on Photosynthetic Parameters of Barley Plants

Chlorophyll fluorescence analysis is a widely used method for detecting changes under stress not only in PSII but also in the photosynthetic apparatus as a whole. One of the main criteria for the efficiency of photochemical processes in PSII is the maximum quantum efficiency (Fv/Fm). The data obtained indicate that Fv/Fm plant leaves decreased by 6% under the action of cadmium ions relative to the control variant (Table 2).

Table 2. Effect of cadmium chloride and melatonin on the parameters of photochemical activity of PSII in barley leaves
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Under the action of 2 μM CdCl2, the photochemical efficiency of PSII somewhat decreased, which can be judged from the trend toward a decrease in the effective quantum yield of PSII (Y(II)) and an increase in uncontrolled energy dissipation in PSII (Y(NO)). Photodamage of PSII is the result of excess absorption of light energy by the photosystem over the ability to use energy during photosynthesis [26].

PSII in plants pretreated with melatonin retained a higher photochemical activity under conditions of contamination with cadmium chloride. Thus, the value of the parameter of the effective quantum yield of PSII (Y(II)) in the variant with priming was almost equal to the control one (Table 2), as in long-term exposure to melatonin. In addition, long-term treatment with melatonin against the background of the action of cadmium ions reduced the controlled thermal dissipation of excitation energy (Y (NPQ)) by 19% and short-term treatment by 23% compared with the stress variant.

Another negative effect of HM is a decrease in the level of photosynthetic pigments, including due to the suppression of their biosynthesis [27]. Under control conditions, the leaves of barley plants contained 0.77 mg/g chlorophyll a, 0.22 mg/g chlorophyll b, and 0.19 mg/g carotenoids, on average. In response to the action of melatonin, the content of chlorophylls (a, b) and carotenoids increased, with the degree of increase determined by the duration of exposure. Thus, during short-term treatment with melatonin, the content of pigments increased by 14–26%, while it increased only by 4–7% during long-term treatment (Fig. 3). The impact of cadmium chloride had a significant impact on the content of photosynthetic pigments in the leaves. Thus, the addition of 2 μM CdCl2 to the medium reduced the amount of chlorophyll a, chlorophyll b, and carotenoids by 31, 18, and 24%, respectively (Fig. 3).

Fig. 3.
figure 3

Effect of cadmium chloride and melatonin on the content of pigments in barley leaves. * P ≤ 0.05 when compared with the control value; # P ≤ 0.05 compared to CdCl2. (1) Control; (2) melatonin (1 day); (3) melatonin (6 days); (4) CdCl2 (6 days); (5) melatonin (1 day) + CdCl2 (6 days); (6) melatonin (6 days) + CdCl2 (6 days).

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In the presence of melatonin in a medium containing cadmium chloride, the amount of chlorophylls (a, b) and carotenoids by 9–14 and 11%, respectively, exceeded the content of these pigments in the leaves of plants that were under similar conditions but in the absence of melatonin. However, melatonin priming was more effective in reducing the toxic effect of cadmium on chlorophyll content: the amount of chlorophyll a increased by 18% (0.67 mg/g) relative to the variant with cadmium chloride (0.53 mg/g), while the amount of chlorophyll b (0.21 mg/g) was almost equal to the control value.

Effect of Cadmium Chloride and Melatonin on Lipid Peroxidation and Antioxidant Status of Barley Plants

The LPO value in barley plants was estimated by the content of products active in the reaction with thiobarbituric acid (TBARS) when heated. Under control conditions, the leaves, stems, and roots of barley plants contained an average of 39 nM/g, 97 nM/g, and 69 nM/g, respectively, fresh weight of TBARS (Fig. 4). In response to the action of melatonin, no significant increase or decrease in the level of TBARS was observed either for short-term treatment or for long-term treatment. As can be seen from the data presented in Fig. 4, the addition of 2 μM CdCl2 in the nutrient solution increased the content of TBARS by 49% in leaves and by 32% in stems and roots, which indicates the development of oxidative stress in barley plants.

Fig. 4.
figure 4

Effect of cadmium chloride and melatonin on the magnitude of lipid peroxidation in different parts of barley plants. * P ≤ 0.05 when compared with the control value; #P ≤ 0.05 compared to CdCl2. (1) Control; (2) melatonin (1 day); (3) melatonin (6 days); (4) CdCl2 (6 days); (5) melatonin (1 day) + CdCl2 (6 days); (6) melatonin (6 days) + CdCl2 (6 days).

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Priming with melatonin followed by the action of cadmium partially reduced LPO and short-term pretreatment was somewhat more effective for plant leaves than adding melatonin to the nutrient solution against the background of a stressor. To reduce the negative impact of oxidative stress in plants, antioxidant defense systems are activated, the action of which is aimed at extinguishing reactive oxygen species. Important enzymes of the antioxidant system are SOD and PO. Short-term and long-term exposure to melatonin increased SOD activity by 30 and 44%, respectively, compared with the control. In response to the action of cadmium chloride in barley plants, SOD activity increased by 62% relative to control values (Fig. 5a).

Fig. 5.
figure 5

Effect of cadmium chloride and melatonin on the activity of antioxidant enzymes in barley plants. (a) SOD activity, arbitrary units/mg of protein per minute; (b) PO activity, arbitrary units/mg of protein per minute; * P ≤ 0.05 when compared with the control value; # P ≤ 0.05 compared to CdCl2. (1) Control; (2) melatonin (1 day); (3) melatonin (6 days); (4) CdCl2 (6 days); (5) melatonin (1 day) + CdCl2 (6 days); (6) melatonin (6 days) + CdCl2 (6 days).

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Plant priming with melatonin followed by the action of cadmium chloride did not cause a significant increase in SOD activity relative to the action of the stressor alone. However, prolonged exposure to melatonin led to an increase in SOD activity by 96% relative to control values and by 34% relative to the variant with cadmium (Fig. 5a). The use of melatonin under optimal barley growing conditions did not lead to a significant change in the PO activity, while cadmium chloride increased the PO activity by 39% (Fig. 5b). Priming with melatonin followed by the action of a stressor increased the studied parameter by 60%. Long-term exposure to melatonin significantly increased the activity of PO not only in relation to the control but also in comparison with the action of CdCl2. Thus, long-term use of melatonin was more effective than priming in increasing the activity of antioxidant enzymes (SOD and PO) in barley plants exposed to cadmium.

A typical plant response to stress conditions is the accumulation of proline [12], an amino acid that performs various functions, including osmoprotectant, protein stabilizer, metal chelator, and antioxidant [28]. Under optimal growing conditions, melatonin, regardless of the part of the plant and the duration of treatment, did not significantly change the content of proline in barley. Cadmium chloride increased the accumulation of proline in the stems and roots of barley plants by 21 and 24%, respectively (Figs. 6b, 6c). Priming with melatonin followed by environmental pollution contributed to an increase in the content of proline in the leaves and roots of barley, not only relative to control values but also in comparison with the action of a stress factor. Long-term exposure to melatonin also increased the accumulation of proline in the organs of barley plants against the background of cadmium chloride and demonstrated greater efficiency compared to priming (Fig. 6).

Fig. 6.
figure 6

Effect of cadmium chloride and melatonin on the accumulation of proline in different parts of barley plants. * P ≤ 0.05 when compared with the control value; # P ≤ 0.05 compared to CdCl2. (1) Сontrol; (2) melatonin (1 day); (3) melatonin (6 days); (4) CdCl2 (6 days); (5) melatonin (1 day) + CdCl2 (6 days); (6) melatonin (6 days) + CdCl2 (6 days).

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Effect of Cadmium Chloride and Melatonin on the Accumulation of Inorganic Ions in Barley Plants

The content of sodium, magnesium, potassium, calcium, phosphorus, and cadmium in the shoots and roots of barley plants was analyzed in the norm and under conditions of “cadmium stress.” Cadmium chloride reduced the amount of sodium ions by 52 and 13%, magnesium by 15 and 19%, and phosphorus by 19 and 25% for overground (Table 3) and underground (Table 4) plant parts, respectively. It is expected that the accumulation of cadmium ions increased many times both in the root and shoot of barley, on average, by 72 times relative to the control values (Table 3, 4). Melatonin, regardless of the duration of use, against the background of the action of 2 μM CdCl2 restored the calcium content in the shoots of barley plants. In addition, priming with melatonin followed by “cadmium stress” led to a decrease in the amount of cadmium ions in the aerial parts of plants relative to the effect of cadmium in the absence of melatonin (Table 3).

Table 3. Influence of cadmium chloride and melatonin on the content of inorganic ions in shoots of barley plants
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Table 4. Effect of cadmium chloride and melatonin on the content of inorganic ions in the roots of barley plants
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DISCUSSION

The study of barley responses to the action of cadmium chloride makes it possible to evaluate the direct toxic effects of excessive concentrations of heavy metals on cereal crops. The action of cadmium chloride led to a significant decrease in the content of photosynthetic pigments. One of the main reasons for this is the suppression of chlorophyll a and b biosynthesis [27], which occurs mainly due to the inhibition of protochlorophyllide reductase, which is the key enzyme of this process [29].

A decrease in the level of pigments, together with a decrease in the area of the assimilating surface and a decrease in the activity of PSII photosynthetic reactions, indicates the inhibition of photosynthesis processes. In this regard, the ability of melatonin to prevent the degradation of chlorophyll a and b and maintain the photochemical activity of PSII, thereby delaying the senescence of barley plant leaves, as we have shown earlier [30], is one of the protective mechanisms under the action of heavy metals.

The stress caused by the action of cadmium ions is evidenced by an increase in the degree of lipid peroxidation in the organs of barley plants. The antioxidant properties of melatonin were manifested in an increase in the activity of antioxidant enzymes, namely superoxide dismutase and guaiacol-dependent peroxidase, as well as in maintaining the level of carotenoids, which are nonenzymatic antioxidants. The positive effect of melatonin on photosynthetic pigments is possible due to an increase in the activity of antioxidant enzymes that remove reactive oxygen species. Numerous studies demonstrate that melatonin enhances the activity of various antioxidant enzymes in response to other stress factors. For example, exogenous melatonin increased the activity and concentration of antioxidant enzymes, increasing the tolerance of cucumber plants to salinity (at a concentration of 50–150 µM) [31], melon plants to low temperatures (at a concentration of 50–400 µM) [32], and watermelon plants to vanadium pollution (at a concentration of 0.1 μM) [13], and hickory to drought (at a concentration of 25–200 μM) [33].

Evidence of a decrease in oxidative stress by exogenous melatonin is a decrease in the degree of LPO in the leaves of barley plants against the background of the action of a stress factor. In addition, for aboveground organs, short-term pretreatment was more effective than long-term melatonin exposure to reduce LPO levels.

In the study, an excess of cadmium in the medium led to its multiple growth in roots and shoots. According to the obtained results, melatonin had no significant effect on the accumulation of cadmium ions in barley plants, except for a decrease in the accumulation of cadmium in plant shoots for the variant with pretreatment with melatonin and subsequent “cadmium stress.” Exogenous application of melatonin inhibited Cd accumulation both in roots and leaves of radish [35], rice [36], safflower [37], and tomato [38]. It is possible that the positive effect of melatonin on barley plants was not realized through the influence on the accumulation of the metal.

We have shown that, in relation to some protective mechanisms, for example, maintaining the level of photosynthetic pigments and reducing lipid peroxidation under the action of cadmium chloride, short-term root pretreatment with melatonin was more effective than its long-term exposure. Data on the mechanisms of plant protection by melatonin in response to the action of cadmium ions, as well as optimization of plant treatment in order to obtain the maximum protective effect, are important not only for obtaining new fundamental knowledge but can also become the basis for the development of innovative agricultural technologies to increase plant productivity.