Keywords

1 Introduction

The plant kingdom has an enormous variety of chemical compounds. A significant part is the metabolites and products of primary metabolism. Another, no less significant, group of compounds that are different from those of primary metabolism vary greatly depending on the family and plant species. They are known as secondary metabolites (SM). According to some authors, secondary metabolites are compounds produced from plants that are not directly relevant for basic photosynthetic or respiratory metabolism [1]. The specificity, as well as the limited distribution of many such compounds, makes it possible to use them as taxonomic markers [2]. The plant kingdom offers a wide range of compounds that exhibit antioxidant properties. Essential oils and polyphenols such as tannins, flavonoids, and phenolic acids are considered excellent natural antioxidants. They are widespread and can be considered as the richest group of secondary metabolites in plants. As they have a positive effect on human health, the plants or fruits that hold them are of great interest to the food and pharmaceutical industry. Prunus persica (L.) belongs to the family Rosaceae and is grown in a huge area of Europe, India, North Africa, and West Asia. From all 3000 species belonging to the Rosaceae family, nearly 200 species are cultivated for their edible fruits and seeds [3, 4].

Peach is an important fruit and some of the major producers are Spain, Italy, China, and the United States (http://www.fao.org). Peaches and nectarines (both Prunus persica) are characterized by a wide range of different varieties, their healthy characteristics, color, and taste being important factors for consumer choice. The red color of the fruits has been the subject of most breeding programs. In particular, high levels of red coloring are sought in varieties intended for fresh consumption. By contrast, the reduction of pigment content in any part of the fruit is the goal of most canning programs for the canning industry [5].

Plants have developed the ability to synthesize and store secondary metabolites as a means of protecting against herbivores, bacteria, fungi, and viruses, as well as other competing plants. Plants typically produce complex mixtures of SMs that can work in an additive or even synergistic ways. The mechanism of the protective action of secondary metabolites is not fully elucidated. Some protecting compounds are directed to a particular target, e.g., the neurotransmitter receptor or the ion channel of the animal pest; others have a broad spectrum of activity and show pleiotropic activity for several purposes. In addition to protective function, secondary metabolites also serve as signal compounds attracting pollinators and seeds spreading animals [6]. A characteristic feature of secondary metabolites is that their metabolism, especially synthesis and accumulation, strongly depends and is regulated by the conditions of the environment. The use of biostimulants can also have a positive effect on the biosynthesis of secondary metabolites, which increases the resistance of plants to various stress factors.

2 Secondary Metabolites: Classification and Function

Plants are a rich source of thousands of secondary metabolites. They consist of low molecular weight compounds that are considered crucial to the survival of the organism that produces them. These compounds are often accumulated by plants in smaller quantities than the major metabolites [7]. Secondary metabolites are produced by plants and play an important role as protective chemicals and signaling molecules. Alkaloids, flavonoids, essential oils, phenols, terpenes, etc. are included in this class of compounds [3, 8]. Signaling messages that regulate plant behavior are delivered from a wide range of chemical compounds. In some cases, they can facilitate communication between members of a species (e.g., pheromones) or between members of different species (e.g., allopathic substances) [9, 10]. These interactions have a largely negative effect on the germination, growth, development, propagation, and behavior of other organisms [7, 11, 12].

There are different classifications of secondary metabolites based on the content or absence of nitrogen in the molecules as well as their biosynthetic pathway or precursor. The most common classifications divide the secondary metabolites into two main groups: nitrogen-containing and non-nitrogenous compounds, each of which is subdivided into subgroups (Table 1).

Table 1 Classification of secondary metabolites in higher plants based on the content or absence of nitrogen in the molecule (by [13])
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Depending on the biosynthetic pathway, the secondary metabolites are divided into three main groups: (1) Terpenoids; (2) Flavonoids and concomitant phenolic and polyphenolic compounds; (3) Nitrogen-containing alkaloids and sulfur-containing compounds [14] (Fig. 1).

Fig. 1
figure 1

Classification of secondary metabolites in higher plants based on their metabolic pathway of synthesis (by [14])

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Terpenoids are the largest and most diverse family of natural products, ranging from linear to polycyclic molecule structures, and ranging in size from five-carbon (C5) hemiterpenes to natural rubber containing thousands of isoprene units (C5). All terpenoids are synthesized by condensation of isoprene units and are classified according to the number of five carbon atoms present in the basic structure [15]. Many aromatic molecules such as menthol, linalool, geraniol, and caryophyllene are formed from monoterpenes (C10) with two isoprene units and sesquiterpene (C15) with three isoprene units. Other bioactive compounds such as diterpenes (C20), triterpenes (C30), and tetraterpenes (C40) show very special properties [13].

A characteristic feature of phenolic compounds is the presence of at least one aromatic ring with one or more hydroxyl groups attached. There are more than 8000 phenolic compounds in the plant kingdom [16]. Phenols range from simple, low molecular, single aromatic rings to large and complex tannins and polyphenol derivatives. They can be classified based on the number and location of their carbon atoms and usually found conjugated to sugars and organic acids. Phenols can be classified into two groups: flavonoids and nonflavonoids [13].

Flavonoids are polyphenol compounds containing 15 carbon atoms with 2 aromatic rings attached through a triangle bridge. They are the most abundant phenolic compounds and are present in high concentrations in the epidermis of the leaves and the skin of the fruits. Their main function in plants is to participate in protective processes against high UV radiation, infections, oxidative stress. They also contribute to the pigmentation of plant parts, stimulate nitrogen-fixing microorganisms, and increase the resistance of plants to diseases [17]. The major subclasses of flavonoids are flavones, flavonols, flavan-3-ols, isoflavones, flavanones, anthocyanidins, dihydroflavonols, flavan 3,4-diols, coumarins, chalcones, dihydrochalcones, and aurons. A variety of substitutes may be added to the primary flavonoid skeleton. Hydroxyl groups are typically present at 4, 5, and 7 positions. Sugars are very common in most flavonoids naturally occurring like glycosides. Both sugars and hydroxyl groups increase the water solubility of flavonoids, but other substituents such as methyl groups and isopentyl units make the flavonoids lipophilic. This, in turn, determines the sites for their accumulation [13].

Anthocyanins, flavonols, and flavan-3-ols play a central role in determining fruit quality [18]. Flavan-3-ols are the most complex subclass of flavonoids ranging from simple catechin and epicatechin monomers to oligomeric and polymeric proanthocyanidins, also known as fused tannins [13]. Proanthocyanidins give astringency to fresh fruits, fruit juices, and wine. They can be oxidized by forming brown pigments in the seeds and other tissues and can act as substances that inhibit the feeding of various pests in reproductive tissues and in developing fruits [5].

The major nonflavonoids are gallic acid, which is the precursor of hydrolyzable tannins, hydroxycinnamates, and their conjugated derivatives, and polyphenol stilbenes. Phenolic acids are also known as hydroxybenzoates, the main component being gallic acid. For the first time, it is isolated from the juice of specific bumps (gallae) formed in plants after an attack by parasitic insects. The tissue swelling is due to the accumulation of carbohydrates and other nutrients that support the growth of insect larvae. The phenolic composition of the bumps consists of up to 70% of gallic acid esters [19].

Gallic acid is the major unit of gallotannins whereas gallic acid and hexahydroxydiphenoyl residues are both subunits of ellagitannins. Gallotannins and ellagitannins are called hydrolyzable tannins because they are easily degraded, releasing gallic acid and/or ellagic acid, while condensed tannins are not. Condensed tannins and hydrolysis tannins are capable of binding and precipitating collagen proteins in animal skins [13].

The main precursor to phenylpropanoids is cinnamic acid and its derivatives – hydroxycinnamates. The most common hydroxycinnamates are p-coumaric, caffeic, and ferulic acids, which are often accumulated as corresponding tartrate esters, caftaric, and fetaric acids. Conjugates of caffeic acid are common components of fruits and vegetables [13].

Members of the stilbene family having a C6–C2–C6 structure, such as flavonoids, are polyphenol compounds. Phytoalexins are compounds produced from plants in response to an attack by fungal, bacterial, and viral pathogens. Resveratrol is the most common stilbene [20].

Alkaloids are a large and structurally diverse group of compounds. Many are derived from amino acids, but others are the result of modifying different classes of molecules, including polyphenols, terpenes, or steroids. With some notable exceptions, alkaloids are the most soluble aqueous alcoholic solutions and commonly occur as salts (e.g., chlorides or sulfates) or as N-oxides in plants. Most of these have heterocyclic ring nitrogen or a ring system and a basic (alkaline) nature.

3 Distribution of Secondary Metabolites in Peach Tissues

The tissue distribution of secondary metabolites in peaches varies greatly and depends on varieties and environmental conditions. The main pigment responsible for the red coloration of peaches and nectarines is cyanidin – in particular, cyanidine 3-glucoside, one of the most common anthocyanin pigments in fruit. The hydroxycarboxylic acid derivatives, anthocyanins, flavonols, and flavan 3-ols are the most common phenols in peaches and nectarines. Peaches and nectarines contain cyanidin-3-O-glucoside, cyanidin-3-O-rutinoside, flavonols (quercetin-3-O-glucoside and quercetin-3-O-rutinoside), flavan 3-ols (catechin, epicatechin, and proanthocyanidins, including procyanidin B1), and others [5, 21, 22]. Apricots and peaches contain carotenoids mainly in the form of β-carotene [23]. Chlorogenic acids, caffeic acid, catechin, and procyanidin B3 (catechin- (4β-8) -catechin) are the major phenols in peaches. Chlorogenic and non-chlorogenic acids are the main derivatives of hydroxycinnamic acid, while procyanidin B1 (epicatechin- (4β-8) -catechin), catechin, and epicatechin are the predominant flavan 3-ols and flavonols found in peach skin compared to peach flesh. Anthocyanins are mainly found in peach and nectarine skin. Small amounts of pigments can also be found in the tissues near the stone. Cyanidine 3-glucoside and cyanidin 3-rutinoside are the main pigments in nectarines and peaches. Some varieties may also contain cyanidine 3-acetylglucoside and cyanidin 3-galactoside. Quercetin 3-glucoside and quercetin 3-rutinoside are the major flavonols in nectarines and peaches and are found mainly in the skin [24].

Different phenolic compounds have been found in peach fruits. They are one of the richest in antioxidant substances. However, both the qualitative and quantitative profiles of these compounds vary considerably depending on the variety. In addition to phenolic compounds in peach fruit, a number of vitamins are also present, with significant amounts of ascorbic acid (vitamin C) and carotenoids (provitamin A). Different conditions before and after ripening of fruit can change the synthesis and emission of volatile substances from harvested plant products. This affects taste, ripening, and other factors that affect quality or storage potential. The peach content of volatile substances has been thoroughly studied. Up to now, more than one hundred volatile compounds have been identified. Some of the most common are linalool, benzaldehyde, ester terpenoids, norisoprenoids, ketones, and lactones. Color properties are predominantly determined by lactones and fewer aldehydes, alcohols, terpenoids. The chemical composition of the volatile compounds varies between the different parts of the fruit. In the mesocarp, closer to the skin, for example, the concentration of volatile substances such as norisoprenoids and benzaldehydes is higher than in the inner mesocarp close to the stone. Besides the composition during the ripening process, the chemical composition of the volatile substances is changing: the levels of the six carbon compounds are drastically reduced, while the content of lactones, benzaldehyde, linalool, norisoprenoids, and phenylalanine derivatives is increased. Volatile ingredients are also influenced by the conditions of fruit storage [25] (Fig. 2).

Fig. 2
figure 2

Chemical compounds in peaches – fruits, leaves, and stems by [25] with modifications)

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According to their biosynthetic origin, the secondary metabolites in plants can be divided into three main groups: terpenoids, nitrogen-containing compounds (alkaloids, glucosinolates, and cyanohydrins), and phenylpropanoids, also known as phenolic compounds [26]. One of the most important building blocks associated with the biosynthesis of secondary metabolites is obtained from acetyl coenzyme A, shikimic acid, mevalonic acid, and 1-deoxyxylose-5-phosphate. They participate, respectively, in the acetate, shikimate, mevalonate, and deoxyxylose phosphate pathways of biosynthesis [7, 26, 27].

4 Biosynthetic Pathways of Major Secondary Metabolites: Enzymes and Regulation

All plants have the capacity to produce secondary metabolites (SMs). The widest variety of them is found in the flower plants. The majority of these metabolites originate from five different precursors or metabolic pathways. These are acetyl coenzyme A (polyketides such as anthraquinones, flavonoids), active isoprene (various terpenoids), shikimic acid (aromatic amino acids, cinnamic acids, tannins, indole, and isoquinoline alkaloids), glycolysis (sugars, gallic acid), and TCA (alkaloids). These pathways, both individually and in combination, create enormous structural diversity, with around 200,000 currently identified.

This structural diversity is further enhanced by widespread glycosylation and esterification and also by the less frequent inclusion of other primary metabolites, such as certain nonaromatic amino acids and polysaccharides. Plants typically produce complex mixtures of SMs. The ingredients of these mixtures, which differ between plant organs and stages of development, generally belong to several classes of secondary metabolites; for example, terpenoids are often accompanied by phenols. In principle, a limited number of major secondary metabolites and several minor components are commonly found, which are often biosynthetically related to major constituents [28].

Plant secondary metabolites are synthesized by specific pathways. The sites of their synthesis can vary for both the type metabolite and the different plant species. In addition, some molecules can be synthesized in all plant tissues, while others are produced in a specific tissue or even in a cell-specific species [29]. The place of synthesis for SM is not always the place for their accumulation. Secondary metabolites, which are hydrophilic compounds, are predominantly stored in the vacuole, whereas lipophilic SMs are usually isolated in gum channels, oil cells, trichomes, or in the cuticle [7, 30].

Anthocyanins, flavonols and flavan 3-ols are synthesized through the flavonoid pathway, whose genetics and biochemistry are already well-studied. The process consists of several steps common to the synthesis of different flavonoids. Additionally, there are also branches of specific reactions that are specific to each type of flavonoid (Fig. 3). It is assumed that the flavonoid pathway is mainly regulated at the level of transcription of genes coding for enzymes from the pathway. Several transcription factors (TFs) from various plants that control this transcription have been isolated. In particular, the interacting TFs of the R2R3-MYB and bHLH form complex with WD40 proteins (called MBW complex) to activate the genes responsible for the anthocyanin and proanthocyanidin biosynthesis. The MBW complex usually regulates groups of flavonoid biosynthetic genes that vary between species. This regulation is via specific binding to motifs in the promoters of the pathway genes [5].

Fig. 3
figure 3

Main pathways and branched reactions for biosynthesis of secondary metabolites in plants (by [24] with modifications)

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Phenolic compounds are one of the major classes of secondary metabolites in plants derived from phenylalanine and, to a lesser extent, in some plants also from tyrosine (Fig. 4). Chemically, the phenols can be defined as having an aromatic ring bearing one or more hydroxyl groups including their functional derivatives. The plants contain a wide variety of phenolic derivatives including simple phenols, phenylpropanoids, benzoic acid derivatives, flavonoids, stilbene, tannins, lignans, and lignins. Together with long-chain carboxylic acids, phenols are also components of suberin and cutin. These quite diverse substances are essential for the growth and reproduction of plants and also act as antinutritional and antipathogenic agents [31]. In addition, the phenols function as antibiotics, natural pesticides, symbiosis signaling agents for nitrogen-fixing bacteria, pollinator attractants, ultraviolet light protection agents, insulating materials that make cell walls impermeable to gas and water, and as structural materials that confer stability of the plants [24].

Fig. 4
figure 4

Phenylpropanoid pathway for biosynthesis of secondary metabolites in plants (by [24] with modifications)

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A key enzyme from the phenolic pathway is phenylalanine ammonium lyase (PAL), which catalyzes the deamination of phenylalanine and leads to the formation of a carbon–carbon double bond, resulting in trans-cinnamic acid. In some plants and grasses, tyrosine is converted to 4-hydroxycinnamic acid by the action of tyrosine ammonium lyase (TAL). The introduction of a hydroxyl group in the para-position of the phenyl ring of cinnamic acid proceeds via catalysis with monooxygenase using cytochrome P450 as the oxygen binding site. The p-coumaric acid formed can be further hydroxylated in the 3 and 5 positions by hydroxylase and eventually methylated by O-methyl transferase with S-adenosylmethionine as a methyl donor; this results in the formation of caffeine, ferulic, and sinapic acids (Fig. 5). These compounds have a phenyl ring (C6) and a three-carbon side chain and are collectively called phenylpropanoids, which serve as precursors for the synthesis of lignins and many other compounds [24].

Fig. 5
figure 5

Specific steps of phenylpropanoid pathway (by [24] with modifications)

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Benzoic acid derivatives are obtained by the loss of a bicarbonate residue from phenylpropanoids. Salicylic acid is a benzoic acid derivative and acts as a signal molecule (Fig. 5) [32]. After infection or ultraviolet radiation, many plants increase the salicylic acid content, which can induce biosynthesis of the protective substances. Similar to the phenylpropanoid series, the hydroxylation and eventual methylation of hydroxybenzoic acid lead to the formation of dihydroxybenzoic acid (protocatechuic acid), vanillic acid, syringic acid, and gallic acid. Hydroxybenzoic acids are usually present in the bound form in plants and are often a component of a complex structure such as lignins and hydrolyzable tannins. They are also in the form of organic acids and as sugar derivatives. However, there are exceptions in which they are mainly present in a free form [24].

Flavonoids, including flavones, isoflavones, and anthocyanidins, are formed by condensation of phenylpropane (C6–C3) and malonyl CoA molecules, resulting in the formation of chalcones which subsequently cyclize under acidic conditions (Fig. 6). Thus, flavonoids have the basic skeleton of diphenylpropanes (C6–C3–C6) with a different oxidation level of the central pyran ring. This also applies to stilbene, but in this case, after the introduction of the second phenyl moiety, a carbon atom of phenylpropane is separated. Stilbenes are powerful fungicides in plants, e.g., viniferine from vineyards. In the case of flavonoids and isoflavonoids, flavones, flavanones, flavonols, and flavanonols, as well as flavan 3-ols and related compounds may be formed depending on the substitution and unsaturation patterns. Flavones and flavonols occur as aglycons in foods. Till now about 200 flavonols and about 100 flavones have been identified in plants. Flavonols are different from flavones because they have a hydroxyl group in the 3-position and can be considered as 3-hydroxyflavones [24].

Fig. 6
figure 6

Biosynthetic pathway of stilbenes and flavonoids (by [24] with modifications)

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5 Change of Secondary Metabolites in Abiotic Stress

Plant stress is a state of tension caused by the changing conditions of the external and internal environment that cause a response from the affected organism. During the growing season, plants are often subjected to a stress of a different nature. Traditional abiotic stress factors for plants are low and high temperatures, drought, excess soil moisture, poor nutrition, etc., which greatly influence the formation of yields and the quality of plant production. Under stress conditions, the normal metabolism of plants can undergo changes that lead to increased synthesis of some compounds and weakening of others (Fig. 7). Often these changes are related to the accumulation of compounds having a protective or regulatory function.

Fig. 7
figure 7

Primary and secondary metabolism network

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The content of phenolic compounds in peach fruit depends on many external and internal factors, including variety, the degree of maturity, environmental conditions, and storage conditions. Among them, light, temperature, oxygen, ethylene, growth regulators, nutrients, and pesticides have been shown to affect phenolic metabolism [33,34,35]. Plant phenols are readily oxidized by polyphenol oxidase (PPO), most commonly after tissue damage, as PPO is believed to act as a protective enzyme [36]. Endocarp lignification in the fruit of the peach is carried out in accordance with the separate induction of competitive flavonoid pathways in the mesocarp and the exocarp tissue layers. The induction of flavonoid biosynthesis is preserved among Rosaceae and possibly also in many other fruits, whereas the induction of lignin is not. The coordination of these two processes is likely to be critical to controlling a number of important agronomic situations in fruit and nuts. Furthermore, the development of peach and Arabidopsis endocarp seems to be controlled by very similar mechanisms, which include the regulatory transcription factors (which stimulate endocarp differentiation), negative regulator and factor that cause secondary wall formation, and lignin deposition [37].

5.1 Temperature and Oxidative Stress

Cold is one of several important environmental stresses affecting plant productivity and distribution. Tolerance to low but not freezing temperatures – the phenomenon is known as cold acclimatization – is a complicated response to stress, which involves a complex cross-link between signal transduction and gene expression. In Arabidopsis thaliana, cold acclimatization involves rapid, cold-induced expression of transcriptional activators, followed by expression of genes that are mobilized in response to cold stress. Most fruit species suffer from a negative low-temperature effect when stored in a refrigerator (0 to 7 °C). Prunus spp., including Prunus persica (L.) Batsch, are highly susceptible to chilling stress (overcooling). The main symptoms of overcooling injuries in peach fruits are dehydration (lack of juice), browning or redness of the mesocarp, sharpness or fleshiness of the flesh. Peach cooling symptoms develop during storage at room temperature after prolonged refrigeration storage [38].

Temperature is also one of the most important factors for maintaining the quality of peaches after harvesting. Some of the metabolic activities such as maturation and degradation of substances decrease by decreasing the temperatures. However, the injuries caused by the low temperatures deteriorates significantly the quality of the fruit during storage and the shelf life is limited. The emergence of cooling damage is often associated with oxidative stress due to increased production of reactive oxygen species such as superoxide anion (O2−), hydrogen peroxide (H2O2), hydroxyl radical, nitric oxide, and peroxynitrite. Oxidative damage is considered an early response to sensitive tissues to cooling. If the production of ROS increases dramatically, as happens under stress in the environment, the hydroxyl radical reacts with membrane lipids, resulting in lipid peroxidation and membrane destruction. Malondialdehyde (MDA) is a product of this lipid peroxidation and is used as a stress indicator in some tissues. To deal with ROS, the plants have developed an effective antioxidant defense system that reacts to oxidative stress and prevents the buildup of ROS and restores the oxidative damage. This system includes both lipid-soluble antioxidants (tocopherol and carotene) and water-soluble reductants, including ascorbic acid (AsA), glutathione (GSH), and enzymes such as catalase (CAT), ascorbate peroxidase (ARX), superoxide dismutase (SOD), and glutathione reductase (GR). The substance melatonin (N-acetyl-5-methoxytyptamine), which is a neurohormone secreted from the pineal gland in mammals, is found in plant tissues too. Melatonin has been reported to be involved in the growth, development, and response to stress in plants [39].

5.2 Water Deficiency and Oxidative Stress

Another important factor for the development of plants and in particular peach is the presence of sufficient water for irrigation. Water stress stimulates stinging and reduces CO2 fixation, which can significantly reduce photosynthetic electron transport [40]. If water stress is prolonged and/or severe, part of the energy supplied by photons can be redirected to processes favoring the formation of reactive oxygen species (ROS) such as hydrogen peroxide (H2O2), which leads to oxidative damage to plant tissues [41]. However, the plants may activate ROS neutralizing enzymes and nonenzymatic systems including secondary metabolites such as phenolic compounds, alkaloids, isoprenoids, phenylpropanoids, and other antioxidants such as glutathione and ascorbic acid (AsA) to reduce oxidative damage [42, 43]. In addition, these systems can also play a very important role in the protection of cell membrane integrity [44, 45].

6 Biotic Stress

Except in abiotic stress, it has also been found that the level of phenolic compounds in plants increases as a response to infection by phytopathogens [46], consistent with the proposed role of these compounds in the protective plant mechanism. It has been found that infected plant tissues and resistant tissues are characterized by a general displacement of the metabolic model, which involves the activation of phenol-oxidizing enzymes and peroxidases. In fact, the degree of resistance is related to the number of phenolic compounds oxidized by phenolases [47].

The use of pesticides and fertilizers has been found to modulate the biosynthesis of phenols in plants [48,49,50]. Consequently, the increase in the polyphenols content observed in organically grown peaches and pears may support the hypothesis [48,49,50] that protective mechanisms against infects are related to an increase of endogenous polyphenols when there are no external pesticides that are widespread in conventional agriculture. Many plants show that the regulation of phenolic metabolism depends on several factors. Changes in the level of phenols and in the amount and activity of oxidizing enzymes, especially phenol oxidase, are part of the mechanism of disease resistance that would be realized by inhibiting the polygalacturonase of the pathogen by oxidized phenols [47]. It is also possible that biochemical protections are present all the time in healthy plants, although observed variations in sensitivity to age seem to indicate that they can develop at certain stages [34, 47].

PAL is a rate-determining enzyme in the activation of the phenylpropanoid pathway, and the increase in PAL activity is associated with the biosynthesis of active metabolites such as phytoalexins, phenols, lignins, and salicylic acid in plant protection pathways [51]. POD participates in cell wall building processes, such as phenol oxidation, suberisation, and lignification, during the protective response against pathogenic agents [52]. PPO participates in the oxidation of polyphenols in quinones (antimicrobial compounds) and lignification of plant cells during the microbial invasion [53]. In addition, the accumulation of phenolic compounds is associated with disease resistance in a number of interactions between plants and a pathogen. The high level of phenolic compounds at the site of pathogen invasion may limit or slow down its growth [54, 55].

In a number of infectious diseases, the metabolism of the affected parts varies considerably under the influence of the pathogen. In leaf curl disease caused by Taphrina deformans (Berk.) Tul., it induces serious changes in the biochemical status of the infected plants, which are detectable not only in the tissues with observable symptoms but also in distally situated ones. These changes include the elevation of the activity of antioxidant enzymes (peroxidases), reduced polyphenols content and plastid pigments, alterations of antiradical activity, anthocyanin, and free proline concentrations [56]. The metabolism of the peach leaves affected by the pathogen resembles strongly the characteristic of the still immature leaves. A reduction in photosynthetic function is observed, and the import of sugars into the leaves is dominated by their exports. In addition, the content of both soluble carbohydrates and the enzymes involved in their metabolism is similar to that of young leaves, not mature (Fig. 8). Many of the effects of the disease on the metabolism of peach leaves are similar to those caused by other plant diseases on the metabolism of photosynthetic organs [57].

Fig. 8
figure 8

The changes of metabolism caused by infection with PLC disease. The following enzymes are shown: glucokinase (EC 2.7.1.1), fructokinase (EC 2.7.1.4), and sucrose synthase (SUSY, E.C. 2.4.1.13); the invertases [both soluble and particulate acid invertase E.C.3.2.1.26, and neutral invertase (also known as alkaline invertase) E.C.3.2.1.27]; sucrose phosphate synthase (SPS, E.C. 2.4.1.14); NADPdependent aldose-6-phosphate reductase (A6PR, E.C. 1.1.1.200); sorbitol dehydrogenase (SDH,E.C. 1.1.1.14); ADP-glucose phosphorylase (AGP, EC 2.7.7.27); and phosphoenolpyruvate carboxylase (PEPC, EC: 4.1.1.31) (by [57])

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Like other crops, peach also is attacked by many plant pathogens such as fungi, bacteria, and viruses. Such pathogen-associated infections in plant tissues, particularly local and resistant (hypersensitive) infections, show a general metabolic change that involves the accumulation of amounts of secondary metabolites (phenols, flavonoids, coumarins, terpenoids, steroids, etc.). This change in the spectrum of secondary metabolites is mainly in response to the infectious agent or physiological stimuli and stress.

Besides playing a vital role in the normal development of healthy plants, the temperature is also a key factor in determining the nature of the interactions between plants and pathogens. Any major change in environmental conditions, especially temperature, will affect not only plants but also pathogens and therefore plant diseases [58]. Different temperature regimes are expected to have a direct impact on biochemical compounds in both healthy and infected plants and the most pronounced effect can be visualized in the total phenolic content (TPC). Polyphenols have antioxidant and antimicrobial action [59]. The accumulation of polyphenol compounds in and around the local lesions in the plant is a reliable evidence of a hypersensitivity reaction. Naturally occurring antibiotic compounds that are found endogenously in healthy plants are embedded with chemical barriers to protect plants against attack by a wide range of fungal and bacterial pathogens. There are also viruses that cause peach diseases such as Plum pox virus, Prunus necrotic ringspot virus, and others. The viral infection causes necrosis of the cells at and near the site of the infection where the viral movement is often restricted. Otherwise, in the absence of necrosis, a systemic infection occurs and the reason for this is a significant change in the concentration of polyphenols due to viral infection. The extent of changes in the metabolism of virus-infected plants (respiration and photosynthesis) is often associated with the severity of symptoms and is greatest when tissues become necrotic [60].

The role of phytohormones in alleviating the adverse effects of both abiotic and biotic stress factors is well known. Among plant herbs, salicylic acid (SA) acts as a signaling and regulatory molecule in plant environmental stress responses by SA-mediated control of metabolic and molecular processes [61, 62].

Horsakova et al. [63] found that in the Plum pox virus infection in two peach varieties (“Symphony” and “Royal Glory”), the antioxidant activity (expressed by DPPH, ABTS, FRAP, DMPD, and Free Radicals) of all polyphenol compounds increases significantly. The increased antioxidant activity in the fruit of PPV-infected peach trees is probably due to the function of protective systems that regulate the production of reactive oxygen species and thus protect cells from oxidative damage. Peach fruits contain a whole range of natural substances that have a positive effect on human health. Carotenoids, vitamin C, and polyphenol compounds [64,65,66,67] are considered to be major antioxidants.

Free radicals are reactive oxygen species (ROS), namely atoms or molecules that, due to the absence of an electron, show high reactivity. Under normal circumstances, the production of ROS in the cell is low; however, the oxidative stress caused by PPV infection may lead to an increase in ROS [68], which in turn leads to a distortion of the balance between production and the elimination of ROS [69]. However, plants have developed very good protective mechanisms to neutralize ROS, thus protecting cells from oxidative damage. Protective antioxidant systems prevent the initiation of chain oxidation by removing partially reduced oxygen species such as superoxide and hydrogen peroxide [70]. Superoxide dismutase (SOD) catalyzes the conversion of the superoxide radical into hydrogen peroxide, which is subsequently converted by catalase (CAT) or ascorbate peroxidase (APX) into water [68]. Other processes occur in the so-called ascorbate-glutathione cycle when, during the ascorbate peroxidase catalysis, the hydrogen peroxide reacts with the ascorbate to form two molecules of water. At the same time, MDHA is formed which either is disproportionated to dehydroascorbate (DHA) and ascorbate or is reduced from NAD (P) H to ascorbate by dependent MDND. Dehydroascorbate is transformed into ascorbate during reduction with glutathione in a dehydroascorbate reductase (DHAR) catalyzed reaction. Oxidation of glutathione leads to the formation of disulfide (GSSG) between the cysteine residues of two glutathione molecules [71]. Oxidized glutathione is reduced by glutathione reductase using NADPH [72].

7 Influence of Biostimulants on the Content of Secondary Metabolites and Increase of Plant Tolerance in Stress Factors

Based on the biochemical mechanisms of plant cell protection and the importance of a number of metabolites for the detoxification of active oxygen species and radicals, a study has been developed to study the impact of biostimulants (substances without nutritional effect but affecting different processes) to increase plant tolerance to stress factors. Ascorbic acid and the ascorbate-glutathione cycle play an important role in the detoxification of ROS and the modulation of other fundamental functions in plants under stress conditions [42, 73, 74]. Ascorbic acid is also the major nonenzyme antioxidant in the apoplast [75], where it also plays a key role in the perception of stressful environmental stimuli and stress signaling [76, 77]. Plant tolerance to environmental stressors can be enhanced by the exogenous use of useful molecules such as proline, amino acids, humic acid, and other antioxidants [78]. The physiological responses of herbaceous plants to the exogenous AsA have been extensively studied [79,80,81,82]. However, the effects of exogenous AsA applications on fruit tree species subject to water stress have been poorly studied, and there are currently no studies on the impact of exogenous AsA on water-stressed deciduous fruit trees and their responses after wetting. Water stress can inhibit the growth of young fruit trees and reduce the growth, yield, and quality of fruits of mature trees [83].

Ascorbic acid is the richest plant antioxidant [84] and is important for the photoprotection and regulation of photosynthesis by stomatal or nonstomatal factors [85, 86]. Foliar application of AsA in young peach trees can be a useful practice to overcome short periods of water scarcity. With regard to gas exchange, exogenous uses of AsA to young water-stressed peach trees significantly increased the assimilation of CO2 in both varieties (Scarletprince and CaroTiger) to the control levels in a restorative watering step. Biosynthesis of AsA occurs on the internal mitochondrial membrane by the oxidation of L-galacto-1,4-lactone (L-GalL). The exogenous application of L-Gal, which is a precursor to ascorbate synthesis, increases CO2 assimilation, photosynthetic electron transport velocity, and ultraviolet conduction [87]. Also, AsA plays a role in photosynthesis and donates electrons to photosystems I and II when the primary electron donor system is damaged [88]. Application of ascorbate results in increased photosynthesis, growth rate, and chlorophyll concentration in wheat plants under water stress compared to untreated plants [81]. This is of the utmost importance to alleviate the negative effects of water stress on the reduction of photosynthesis in young trees in commercial orchards experiencing a period of water stress (especially in areas where the current practice is to start irrigation after the second year); in young container-grown and field-grown trees in nurseries not only in drought periods but also when field trees are excavated and very fine roots are destroyed causing temporary water stress on trees while the roots are not recovering. Accumulation of osmolytes such as proline in water-stress plants can contribute to lower osmotic potential after wetting and allow water to move into cells [89].

In addition to ascorbate, other biologically active molecules also have a positive effect on a number of plants. Recently, melatonin has been shown to have a regulating effect on ripening and preventing disease. For example, pre-melatonin-treated grape berries exhibit a higher endogenous accumulation of melatonin, which not only increases grain size and weight but also enhances the synchronized grain maturation. The application of melatonin after harvesting effectively delays aging and maintains the quality of the peaches stored at ambient temperature. Exogenous melatonin pretreatment improves anthocyanin accumulation by regulating gene expression and increases antiradical activity in cabbage sprouts. Melatonin reduces injuries caused by low temperatures in peach fruits by increasing the protective power in the fruit. However, the main physiological and molecular mechanism of inducing tolerance to low-temperature stress caused by melatonin remains unclear. As a positive regulator of the anti-ROS process, the data show that melatonin can not only directly purify some ROS but also modulates antioxidant enzymes and improves cellular antioxidant protection. Melatonin increases peach tolerance to cooling after harvest. Compared to control peaches, melatonin treatment slows down and reduces cooling injuries in fruit during storage in refrigeration chambers. Melatonin increases the expression of the genes involved in the antioxidant protective system, and also causes an increase in ascorbate and regulating genes involved in the ascorbate-glutathione cycle. AsA and GSH can directly detoxify ROS and thus contribute to the nonenzymatic ROS removal [39].

The role of phytohormones, alleviating the adverse effects of abiotic and biotic stress in plants, is widely described in the literature. Among the plant hormones, salicylic acid (SA) acts as a signaling and regulatory molecule in plant responses to environmental stresses by SA-mediated control of metabolic and molecular processes [61, 62].

There are different pathways for salicylic acid biosynthesis. One of them is found in peaches and its precursor is mandelonitrile (MD) [90]. In this pathway, MD acts as an intermediate molecule between the cyanogenic glycosidic cycle and SA biosynthesis [91]. The contribution of the different pathways to the total amount of SA varies according to plant species, their physiological status, and their rate of development [92,93,94,95,96]. For example, although it is generally accepted that the contribution of phenylalanine (Phe) ammonium lyase (PAL) pathway to the total amount of SA is small, this pathway becomes important during the interactions between the plant organism and the pathogen [62]. Furthermore, it has been found that treatment with MD increases the SA content and provides partial protection against the Plum pox virus (PPV) infection in peach plants [91].

The cyanoglucoside pathway (CNglcs) is involved, at least in part, in the biosynthesis of SA in peach plants, and MD acts as an intermediate molecule between SA biosynthesis and the CNglcs cycle [91]. It is known that SA is a signaling molecule in the plant protection response that can cause tolerance to various abiotic and biotic loads [61, 97]. Various authors have shown that SA can alleviate NaCl-induced injuries. This response, however, is somewhat controversial, and the results depend on plant species and their developmental phase in addition to the concentration of SA and the mode of administration [61, 98, 99]. In terms of biotic stress, peach plants GF305 are commonly used for plant–pathogen interaction studies with PPV, and it has been reported that PPV infection can cause oxidative stress at the subcellular level in these plants [92]. At least 10% of the total SA content in micropropagated peach trees was found to be due to the cycles of CNglcs by MD [91]. Under salt stress conditions, the increase observed in the concentration of SA in untreated (control) and Phe- treated micropropagated peaches correlated with elevated levels of SA precursor MD, whereas in PPV-infested shoots this correlation was observed only in control plants. Taken together, these results suggest that under stress conditions the major part of SA should come from isochorismate (IC) and PAL pathways [93, 94].

It is believed that the pathway of PAL is the main pathway for SA biosynthesis in saline stress [100] Nicotiana tabacum infected with tobacco mosaic virus [101]. In addition, CNglcs is believed to play a possible role in unfavorable environmental conditions [102], which is why MD can potentially play a role in the plant’s responses.

SA content increased in both control and Phe-treated plants. Salt stress also increases the levels of ABA and JA in control and Phe-treated plants, but not in MD plants. In control plants, an SA/JA ratio increased as a result of the salinity stress, whereas in the MD treated, the SA/JA ratio was slightly decreased. This response correlates with the fact that sodium stress does not affect the development of MD-treated plants. ABA is a key modulator of the response to abiotic stress because of its important role in regulating the closure of the stomata. Furthermore, JA appears to act as a regulator of ABA biosynthesis [103]. Under physiological conditions, there was an increase in ABA levels in control plants and Phe-treated plants, which correlate with a significant increase in JA.

8 Conclusions

Knowledge of the properties and functions of the secondary metabolites as well as their biosynthetic pathways allows the use of different biostimulants in order to increase their biosynthesis and hence the resistance of the plants to various stresses factors. This can be successfully used in modern sustainable agriculture to reduce pesticide use.