Extreme temperature is a widespread abiotic stressor that adversely affects metabolism and inhibits plant growth and productivity [1]. Plants critically depend on ambient temperature, and they survive solely by the cause of internal defense mechanisms [2]. To reduce the effects of temperature damage and improve adaptation to adverse temperature conditions, plants have developed certain physiological and molecular adaptations aimed at regulating metabolism [3]. Plant responses to low and high temperatures are often considered antagonistic. Heat stress causes cellular damage and includes hampering ion homeostasis, ROS production, and subsequent oxidative stress. Cold stress changes the physical state of the membrane bilayer [4]. However, a common response to heat and cold stress activates antioxidant defense systems, which include secondary metabolites: free amino acids (AAs) and total phenolic compounds (TPHs).

Accumulation of free AAs is a nonspecific reaction of plants to abiotic stresses. Free AAs play an important role in the formation of stress resistance as osmoregulators [5]. They are involved in pH regulation, ROS detoxification, synthesis of specific enzymes, and precursors of secondary metabolites and act as energy donors in the tricarboxylic acid cycle [6, 7]. Free AAs act as regulatory and signaling molecules [5] and serve as precursors in the synthesis of phytohormones and low molecular weight nitrogenous substances [6]. TPHs are synthesized mainly in the aboveground organs of plants: leaf and flower tissues, pollen, seeds, and bark. They play an important role in protection from pathogens and abiotic stresses [8]. The activity of a TPHs is determined by the structure of the molecule, specifically the number and position of hydroxyl groups in it. Phenolic compounds are actively involved in the regulation of plant cell growth and differentiation; they stabilize membranes, prevent lipid peroxidation, protein denaturation, and DNA damage, and provide protection against ROS [9]. Membrane stabilization is ensured by the ability of THPs, and especially flavonoids (TFs), to bind to individual integral membrane proteins and phospholipids [10]. The high antioxidant and radical-neutralizing potential of TFs is due to their specific chemical features [11].

The content of photosynthetic pigments (chlorophyll a and b, carotenoids) indicates changes in the photosynthetic apparatus under adverse environmental conditions [12]. The pigment complex actively responds to signals from the environment, and changes in the content and ratio of pigments serve as a marker that allows assessing the impact of a certain factor on the state of the plant. The amount of chlorophyll (a + b) is closely related to the productivity of photosynthesis and is useful as a marker of the effects of stress on plants [13]. The degree of formation and functioning of the photosynthetic apparatus under the action of unfavorable environmental factors characterizes the ratio between chlorophylls a and b. The change in the ratio of chlorophyll occurs mainly due to the lability of chlorophyll a [14]. The ratio of chlorophyll a/b is considered one of the traits of photosynthetic activity and under stress is used as a marker of tolerance [15]. The chlorophyll a + b/carotenoid ratio is another informative indicator of the intensity of damage to the photosynthetic apparatus. Lower ratios correspond to greater damage caused by aging or stressors [16]. Carotenoids are involved in the absorption of low-wavelength spectra of light and in the protection of chlorophyll molecules from oxidative stress [17]. Antioxidant effects of carotenoids are determined by their ability to neutralize the damage induced by the formation of triplet chlorophyll and singlet oxygen [18].

Rye (Secale cereale L.) is a diploid Triticeae species of the Poaceae family that belongs to important annual cereals and ranks first among grains in regions where wheat cultivation is difficult or impossible [19]. Among cereals, winter rye is resistant to adverse environmental factors, including drought and frost, and it also can be grown on poorly cultivated soils [20]. Winter rye is grown as a multi-purpose cereal crop for grain production and forage as a cover crop helping soil erosion, weed control, and building soil organic matter [21]. Rye is characterized by a high level of constitutive frost resistance. Single studies report an increase in antioxidant enzyme activities and proline content in rye seedlings under low temperature stress and freezing [22] as well as cold hardening [23]. The example of two winter rye varieties shows differences in amino acid levels during the cold adaptation and overwintering [24]. However, no studies on the physiological response of rye to high temperatures were found, which is apparently because rye is considered a frost-resistant crop.

Winter rye grows and develops to a fully tillered stage under low positive temperatures. The minimum temperatures for rye germination and growth to the start of tillering fluctuate between 1-5 °C and the temperature optimum for rye development is in the range of 15-22 °C [21]. An increase in temperature during the period of germination and the start of tillering is an extremely negative factor, which leads to wilting, rot, damage by pathogens, and yield loss [25].

Plant stress response is a dynamic process that can be divided into several phases. The first is the alarm phase, usually very short, lasting several hours, in which the stress signaling system and gene expression are activated. Then comes an acclimation phase measured by days, during which plants' protein and energy metabolism change. If the impact of extreme factors continues, plants either die or adapt to them, corresponding to the resistance phase. If the factors stop, the recovery phase begins [26]. Our study analyzes and compares specific dynamics and distribution of free AAs and phenolic compounds in shoots and roots and photosynthetic pigment composition in leaves of young rye plants ‘Boguslavka’ that have been subjected to short-term high and positive low temperature stresses (alarm phase of response). The study suggests that as a result of the activation of signaling systems and changes in gene expression in the alarm phase, a repartition of metabolites occurs under both cold and heat stress. The results of this study should contribute to a better understanding of rye's tolerance to temperature in a changing climate and provide background information for future cultivation management, especially in regions where the difference between day and night temperatures is very large.

Our study measured nonspecific and specific changes in the nature of the accumulation of AAs in the organs of 14-day-old winter rye plants after temperature stresses (alarm phase of response). Short-term heat stress induced a rise in the pool of AAs in the shoots and a decrease in the roots. On the other hand, after cold stress, the AAs pool increased significantly in the shoots and slightly – in the roots. The changes recorded in the content of AAs after exposure to high temperatures were more pronounced. At the level of individual AAs, GABA, Pro, Cys, Val, and Asp dominated in the shoots of 14-day-old winter rye plants after high temperature stress, while after positive low temperature, Pro, Asp, Ser, and Thr prevailed. In the roots, the content of these AAs decreased significantly compared with the control under both temperature stresses, except for Asp, Ser, Gly and Ala.

Free AAs perform osmoprotective and regulatory functions under stress conditions [31]. Pro and GABA are key osmolites that form the plant responses to abiotic and biotic stresses [32, 33]. An accumulation of Pro in the shoots of 14-day-old winter rye plants after heat and cold stresses were established, and a significant decrease in its content in the roots, especially after hyperthermia. Aghaee et al. [34] showed that low temperature stress induced the accumulation of Pro in the cold-resistant genotype of Oryza sativa. Significant accumulation of Pro and an increase in antioxidant activity were reported in wild Sorghum bicolor genotypes under heat stress [35]. It was previously shown that after cold stress, the pool of free AAs and the content of Pro in shoots and roots in 14-day-old plants of frost-resistance winter wheat ‘Podolyanka’ increased [36]. It was identified that the level of GABA almost doubled in the shoots of 14-day-old rye plants after heat stress, while it did not change after cold stress. There is a relationship between the perception of the stress signal, accumulation of GABA, and physiological responses of plants. GABA is considered involved in signal transduction as a signaling molecule or as a trigger for ROS generation, activation of antioxidant enzymes, interaction with plant hormones, and protection against biotic stressors [37]. An increase in intracellular GABA content induces the expression of signaling and metabolic genes involved in the regulation of growth and stress resistance. Such changes in genome activity positively affect the quality and quantity of the crop [38].

The content of Asp and Gly increased together with GABA in the roots of 14-day-old rye plants under temperature stress. On the contrary, the level of Ser and Ala became elevated under the cold stress, while after heat stress − it decreased. Asp is a precursor in the biosynthesis of asparagine, Thr, Lys, Met, isoleucine (Ill) in higher plants, and such metabolic transformations are activated by abiotic stress [39]. Gly and Ser are two interconverting amino acids that accumulate during photorespiration, which are the precursors for chlorophyll, glutathione, ethanolamine, tryptophan, phosphatidylcholine, and related phospholipids [40]. The synthesis of these AAs in non-photosynthetic cells is considered activated under stress factors [41, 42]. Ala is an activator of the aspartate-derived amino acid pathway in the plant and takes part in carbon and nitrogen metabolisms of the cell [43]. Alanine forms in large quantities under hypoxia in the roots of Arabidopsis thaliana, but after returning the plant to normal conditions, its concentration decreases [44].

The accumulation of AAs under temperature stresses has been reported in a number of studies. For example, an increase in the levels of GABA, Gly, Val, and Ala in seedlings of Arabidopsis was found after the heat and cold stresses [45]. The content of Asp, glutamine (Gln), Val, Arg, Pro, and Gly increased in maturing grains of barley (Hordeum vulgare) after three-hour temperature stress. According to the authors, the elevation in AAs levels was associated with the expression of genes involved in the metabolism of proteins damaged by high temperatures [46].

The results of our study and literature show that the growth of the pool of free AAs and of the content of individual AAS, including Pro, GABA, Asp, and Glu, is a nonspecific response to temperature stresses. Simultaneously, certain specific features depend on the species, tolerance and phase of plant development, plant organs, and the intensity and duration of the stressor.

Phenols and flavonoids, together with free AAs, participate in the protection of plants from adverse impacts [8, 47]. Activity of phenylalanine ammonia lyase and other enzymes involved in the biosynthesis of phenolic compounds has been reported to increase in response to high [48] and low temperatures [49]. It was detected that the content of TPHs decreased in the shoots and roots of 14-day-old rye plants after heat stress, whereas the amount of TFs did not change appreciably. The TPHs level was higher after cold stress as compared with heat stress, while the concentration of TFs in the shoots declined significantly, and in the roots, it remained at the control level. Information on the effect of temperature on the content of TPHs in different plant species is contradictory. For example, an accumulation of TPHs was established after heat stress (+ 35 °C) in plants of Lycopersicon esculentum, for which the optimal temperature is in the range of + 22-26 °C. Similar results were obtained following cold stress (+ 15 °C) in plants of Citrullus lanatus with an optimal growth temperature of + 33-35 °C [50]. It was reported that the leaves of a cold-sensitive grape variety (Vitis vinifera L.) were characterized by a lower content of TPHs than the leaves of a resistant variety [9]. A decrease in the content of TPHs in the leaf tissues of the grape after cold stress (+7 °C and + 10 °C) and its gradual increase during recovery (+18 °C and + 25 °C) was detected [51]. At the same time, enhanced synthesis of TPHs in soybean seedlings was shown under cold stress (+1^o and + 10 °C) [52]. However, following recovery (+ 24 °C), [53]. The content of TPHs diminished in the roots of Pisum sativum L. under short-term temperature + 8 °C, but it gradually rose following prolonged exposure to cold [54]. During adaptation to low temperature of the two Triticum aestivum L. varieties, which differ in the levels of frost resistance, a significant increase in the content of TFs, especially in the cold-resistant variety, was noted [55]. Simultaneously, a decrease in the content of TFs was detected at low temperatures in plants of spring and winter wheat [56] and Solanum tuberosum L. [57]. During the hardening of Secale cereale L. seedlings, a gradual increase in the total amount of TPHs was recorded [58]. Our previous studies revealed a slight increase in the content of TPHs and TFs after short-term heat stress (+ 40 °C, 2 h) and no significant changes after short-term cold stress (+ 4 °C, 2 h) in 14-day-old Triticum spelta plants [59].

The absolute content of photosynthetic pigments and their ratio in plants can vary widely depending on environmental conditions, particularly concerning the intensity and spectral composition of light, temperature, nutrition, etc [14]. Photosynthetic pigments are among the first targets of stressors, hence the absolute content of pigments and their ratio after stressor impact is a specific trait of heat tolerance and could further serve as markers during the screening and the creation of tolerant varieties [60]. The content of photosynthetic pigments and the ratio of chlorophyll a/b decreased in wheat flag leaves in the grain filling phase under high temperature. More pronounced changes occurred in the sensitive genotype, which was interpreted as evidence for slower degradation of pigments and better capture of solar energy by the heat-tolerant genotype [61]. In earlier investigations, the effects of short-term temperature stressors on the accumulation of photosynthetic pigments in the leaves of two kinds of 14-day-old winter wheat and wheat spelt were analysed. Heat stress decreased the amount of photosynthetic pigments and increased the ratio of chlorophyll a/b in the Triticum spelta 'Frankenkorn' variety [59] and wheat 'Volodarka' variety [12] studied plants, both of which are known for their cold and frost resistance. This indicates that the pigment complex in these genotypes is sensitive to high temperatures. Simultaneously, an increase in the content of pigments and a decrease in the ratio between chlorophylls a and b were shown in plants of heat-tolerant wheat 'Yatran 60' variety [62]. In terms of total chlorophyll content, the studied rye plants significantly exceeded the previously analyzed winter wheat and spelt plants. Changes in the content and ratio of photosynthetic pigments recorded in our study indicate that in the early stages of the vegetation, the photosynthetic apparatus of winter rye plants of the ‘Boguslavka’ variety responded more actively to low temperatures.

It was shown that under simulated short-term temperature stresses (alarm phase of response) at the initial stages of the vegetation, the nature of accumulation and distribution of secondary metabolites in the organs of rye plants is changed, indicative of a primary response to stress. Following heat stress, free AAs accumulated in the shoots. Following cold stress-free AAs pools increase both in the roots and in the shoots. GABA, Pro, Asp, Cys, and Val were dominant following temperature changes. Both temperature stresses inhibited the accumulation of TPHs, while the accumulation of TFs in the shoots under heat stress rose and fell under cold stress, both in the roots and shoots of plants. Quantitative and qualitative changes in the accumulation and distribution of low molecular weight protectors AAs and TPHs in rye plants provide evidence of the involvement of these compounds in the formation of the response to temperature changes. The decrease in the content of photosynthetic pigments indicates the sensitivity of the photosynthetic apparatus of rye plants to temperature regime changes. The results obtained in this study should contribute to a better understanding of the resistance of winter rye plants of the ‘Boguslavka’ variety to short-term temperature changes.