Keywords

5.1 Introduction

A variety of substances or compounds called secondary metabolites are mainly responsible for the adaptation of plants to environmental changes. Plants synthesize a wide range of secondary metabolites such as alkaloids, flavonoids, phenolic, steroids, anthocyanins between others, which are used as pharmaceuticals, agrochemicals, biopesticides, color, additives, etc. Secondary metabolites are not considered to be part of fundamental life processes of plants, however, they play a significant role in protection from insect, pest, herbivores, phytopathogens, and other harsh environmental variables (Thakur et al. 2019). Therefore, the synthesis of secondary metabolites depends on internal and external factors (stressors and stress factors, respectively) that influence plant metabolism, and can affect plant reproduction and productivity (Kranner et al. 2010). The stress factors, that for their nature can be biotic or abiotic, modify positively or negatively the plant metabolism (Cheynier et al. 2013). Biotic stress is the result of the interaction between plant and viral, bacterial, fungi, pheromones, phytohormones, and nucleic acids among others. Meanwhile, abiotic stress can be physical factors (Light spectra, temperature, water stress, acoustic waves, others) and chemical factors (Nanostructures, gasses, nutriment, others) (Vázquez-Hernández et al. 2019).

According to data provided by Agathokleous et al. (2019), exist 109,821 publications that review the topic of “plant stress” only in the period 2000 to 2018. The results indicated that the dose–response is not always linear, observing a biphasic response when the doses allow it. This balance of response between the plant and stress factors provides information on a positive (eustress) or a negative effect (distress), a phenomenon called “Hormesis” (Agathokleous et al. 2019; Vázquez-Hernández et al. 2019). The concept of hormesis is a term used in medicine for the application of toxins in low doses (Calabrese 2004). Paracelsus (1493–1541) defined it as “All things are poison and nothing is without poison, only the dose permits something not to be poisonous” (cited by (Vázquez-Hernández et al. 2019)). Currently, the term is applied in horticultural and agricultural practices as a biphasic response in which doses of a toxic agent could cause inhibition (distress) or can cause stimulation (eustress) (Vargas-Hernandez et al. 2017).

Plants are sessile organisms susceptible to the interaction between various types of stress, which has resulted in an evolved defense system that increases the synthesis of secondary metabolites (Ghorbanpour et al. 2014). The variety of stress factors together can affect the plant physiology, plant-plant interaction, defense type, reproductivity, among others. For example, salinity and low/high-temperature are conditions which restrict plant growth and productivity (Akula and Ravishankar 2011). In the signaling response to pathogens or herbivorous insects, several response pathways are invoked, some of these are induced by infection and some are performed regardless of the antimicrobial nature (Zaynab et al. 2018). Another example is the interaction between plants and herbivory insects that causes the plant to emit volatile organic compounds which influence the plant-to-plant communication, pollinators, and other insects, and increase fluidity of cell membranes for thermo-tolerance and leaf tissue protection from atmospheric oxidants within and around leaves (Faiola and Taipale 2020).

The foregoing indicates that plants can react in various ways in the presence of one or more stress factors and that, in the same way, the response to the stimulus may be the activation of a synthesis pathway of only one metabolite or a series of secondary metabolites. For this reason, this work will focus on stress factors and the production of secondary metabolites in plants.

5.2 Abiotic Stress

“Any unfavorable condition or substance that affects or blocks a plant's metabolism, growth or development” is the definition of plant stress suggested by Lichtenthaler (1996). Currently, this definition has been modified depending on the stimulus origin, defining as stress factors those stimuli that are external to the plant, biotic (fungi, insects, etc.) or abiotic (temperature, luminosity, nanoparticles, metals and polluting salts, water, etc.) (Kranner et al. 2002; Thakur et al. 2019). Abiotic stress origin is not biological and can be divided into chemical or physical (Vázquez-Hernández et al. 2019). For example, the adaptation to cold environments in some plants is the result of an increase in the synthesis of flavonoids due to acclimatization processes at low temperatures or by the application of UV radiation (Samanta et al. 2011; Nakabayashi et al. 2014). In Vitis vinifera, it has been observed that stimulation with heavy metals such as Cadmium (Cd2+), Cobalt (Co2+) and Silver (Ag+), can increase the synthesis of Resveratrol (Cai et al. 2013), while the application of UV-C irradiation induces the synthesis of stilbene (Wang et al. 2010; Liu et al. 2010). This indicates that the synthesis of secondary metabolites will depend on various factors such as the type of stimulus, the concentration, and the form of application. These same observations are appreciated by Feregrino-Perez et al. (2018), where the effect of nanomaterials on germination, development of plants, and synthesis of secondary metabolites is reviewed, concluding that the stress level will depend on the used nanomaterial, the dose and the time of exposition. Low/high-temperature, relative humidity in air, drought, microelements shortage, and CO2 reduction are classical abiotic factors for plants elicitation. The role of novel abiotic stress factors on the production of secondary metabolites is described below.

5.2.1 Electromagnetic Sources

Electromagnetic phenomena can be seen as an abiotic stress elicitor to affect plants. In this context, many reports have demonstrated more advantages than disadvantages when strong or weak electric fields, magnetic fields were applied to plants (Dannehl 2018). Electromagnetic sources have been studied as another possibility to increase plant growth and development due to the alteration in the electrostatic balance of the plant system at the cell membrane level (Radhakrishnan 2019). An electromagnetic field is produced by a distribution of electric current and charge. An electric field (EF) can occur under high-voltage lines and the units in the SI are newtons per coulomb or, equivalently volts per meter (V/m); in the same way, a magnetic field (MF) is usually measured in terms of its magnetic flux density whose unit is expressed as Tesla (T) (Dannehl 2018).

Pulsed electric field PEF technology consists of the application of short, high power electrical pulses to products placed in a treatment chamber, confined between electrodes (Soliva-Fortuny et al. 2017). A high electric field can cause cell membrane disruption, whereby inner secondary metabolites are released from intracellular cell compartments, resulting in a high content of bioactive compounds (Odriozola-Serrano et al. 2009; Janositz and Knorr 2010). In the case of MF, several studies have used small boxes with coils, iron bars, a function generator, and a power amplifier. The application of magnetized water to plants is a novel area of indirect application of MF that the scientific community is currently researching (Dannehl 2018).

Light is an electromagnetic wave within the visible spectrum, however, that definition depends upon the sense of sight involving the response of individuals (Koshel 2004), therefore, the UV and infrared parts of the electromagnetic spectrum are roughly included, but will be considered in this chapter due to their importance in the plant production of secondary metabolites. Plants sense light through specific molecules called photoreceptors that trigger specific signals for photomorphogenesis or other defense systems. Depending on the dose rate and exposure time, either insufficient or excess levels, light can become a type of eustress, producing several effects, from damage to cellular components to triggering of defense systems for secondary metabolite production (Alvarado et al. 2019; Muller-Xing et al. 2014; Akula and Ravishankar 2011). The next sections of this chapter will discuss the effect of electromagnetic sources in the production of natural bioactive compounds.

5.2.1.1 Light

Light is one of the most important and obvious requirements for plant growth and development, where the energy of sunlight and artificial light sources is mainly used for photosynthesis. However, light is not only involved in the photosynthesis process but also in the production of natural bioactive compounds, gene expression, and synchronization of the circadian clock in the light/dark cycle (Larner et al. 2018). Changes in the light intensity, quality, direction, and duration are sensed by specialized photoreceptors which are specially designed proteins that sense light, triggering chain reactions that have been studied in terms of photomorphogenesis and primary and secondary metabolites production (Alvarado et al. 2019). Photoreceptors perceive specific light wavelengths of over a continuous spectral range through a small cofactor or chromophore molecule (Burgie et al. 2014). Five photosensory systems have been identified: phytochromes perceiving red (660–700 nm) and far-red (700–750 nm), cryptochromes, phototropins, and members of the Zeitlupe family perceiving blue (495–400 nm) and UV-A (400–315 nm), and UV Resistance Locus 8 (UVR8) perceiving (315–280 nm) (Bantis et al. 2018; Alvarado et al. 2019).

Recent investigation has focused on the effect of light technology in plant growth, developmental traits, and primary and secondary metabolites by using one or more light wavelengths, intensities, and photoperiods. It has been reported that blue light increases phenolic compounds by promoting the production of malonyl CoA and coumaroyl CoA, participating in the synthesis of phenolic compounds (Qian et al. 2016). In addition, red and far-red wavelengths are perceived by the phytochromes photoreceptors, which regulates biosynthetic pathways involved in the synthesis of anthocyanins, molecules that belong to the phenolic compounds known as flavonoids and have many functions in plants including pigmentation (Alokam et al. 2002). In the same way, plants produce secondary metabolites such as flavonoids and anthocyanins to cope with cell damage produced by UV radiation (Jiang et al. 2017b). Serious damage to DNA, membrane, and proteins can be caused by UV-B radiation, whereas UV-A induces DNA damage less efficiently because of the activation of photoreactions forming reactive oxygen species (ROS) (Hideg and Strid 2017; Häder et al. 2015).

Supplemental lighting has been accepted for improving horticultural crops. Light-emitting diode (LED) technology has been linked to controlled environments in horticulture for achieving crop yield, phytochemical content, nutritional value, flowering control, transplant success, pre-harvest and postharvest product quality, and production of regeneration material (Bantis et al. 2018; Alvarado et al. 2019). LEDs have allowed a sustainable and highly efficient use of energy and reproduce true spectral composition of blue, green, red, and far-red wavelengths that matches with plant-specific photoreceptors (Singh et al. 2015). Other light technologies, as high sodium pressure (HSP) and other high-intensity discharge (HID) lamps are still used in greenhouse and plant experimentation, however, LED technology is replacing these devices due to the various advantages LEDs offer. Table 5.1 summarizes some examples of the application of supplemental light on plants or foods with a commercial interest and presents the effect on the production of natural bioactive compounds.

Table 5.1 Effect on natural bioactive compounds of experiments where supplemental light was the stress factor on plants or food with commercial interest
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5.2.1.2 Electric and Magnetic Fields

Magnetic fields (MFs) are considered an abiotic factor that can induce eustress with significant effects on the growth and development of plants. The effect of light, gravity, mechanical damage, and electrical signaling on plants has been studied and documented over the past years concluding strong facts relating to phototropism, gravitropism, and thigmotropism (Maffei 2014). The geomagnetic field (GMF) is a natural component of our environment, however, its impact on plant growth and development is not well-understood, moreover, the effects of artificial magnetic fields on plants have been poorly studied (Maffei 2014). Several experiments with lower and higher values than the GMF has been conducted with predominantly positive effects depending on the plant, time of exposure and intensity. For example, an increase in germination or subsequent seedling growth barley, corn, beans, wheat, hornwort, mung bean, pea, chickpea, tomato, and okra, but it was reduced in seeds of rice. In a similar way, the effect on roots, shoots, gravitropism, photosynthesis, and lipid composition present a similar pattern (Maffei 2014).

Several theories and studies about the biological effect on MF have been proposed. A polar structure in various chemical bonds in the organic material may be linked to the polar water molecules and dissociated ions of mineral salts conferring magnetic properties (Chepets et al. 1985). A MF can decrease the disease index of plants due to the modulation of calcium signaling, and proline and polyamines pathways (Radhakrishnan 2019). The plant cells contain about 4500 iron atoms in the ferritin molecules involved in growth and metabolism. The magnetic rotator moment of ultimate iron atoms creates an external MF which collectively generates an atom re-positioning in the direction of MF that leads to an increase of the plant temperature (Vaezzadeh et al. 2006). Photoreceptors have been also proposed to be potential magnetoreceptors since cryptochromes and phytochromes produce radical pairs after the exposure to their corresponding light wavelength triggers (Maffei 2014; Dhiman and Galland 2018). Cryptochrome-dependent responses such as blue-light-dependent anthocyanin accumulation and blue-light-dependent degradation of CRY2 protein were enhanced at higher magnetic intensities in Arabidopsis mutants lacking cryptochromes (Ahmad and Jones 1979). Limited information is available on the molecular basis and the function of the MF receptors and their activation by physiological signals, therefore, their involvement in directing the overall response in different plant organs is yet to be determined (Radhakrishnan 2019).

Static magnetic field (SMF) exposition in plants has been found to be an effective and emerging tool to control diseases and increase tolerance against the adverse environment (Radhakrishnan 2019). However, a small number of studies have been attempted to determine the role of MF on plant tolerance against various stress conditions (Radhakrishnan 2019). The effect on secondary metabolites production has been scarcely studied; presenting an opportunity area in this field.

Electrostatic fields (EF) has also been used in the horticultural industry. Most of the applications of this technology are for germination and seedlings improvement due to its importance in the supply chain. For example, drought resistance and removal of free radicals in maize seedlings with electric field intensity 200 kV/m, pulse width 80 ms and frequency 1 Hz were analyzed by He et al. (2017), where the growth of root, the ability of self-organization, and the respiration metabolism of root cells was improved. Another example with potato tubers subjected to pulsed electric field (PEF) through a treatment voltage across the potatoes of 5 kV and a discharge capacitance of 450 pF prior to planting had an increase in the yield of 22–29% (Gachovska et al. 2015). In the same way, winter wheat seeds increased the germination energy up to 32.41% and weight up to 23.8% under PEF (Starodubtseva et al. 2018). In the experiment of Yan et al. (2017), PEF through a high-voltage pulse power supply and arc electrode was studied on cotton seeds vigor with different frequencies of 1, 5, 10, 20, and 50 Hz at the voltage of 16 and 20 kV, and the treatment time was 40 s, finding that when the frequency of the electric field increased, the effects increased and reached the maximum at 10 Hz, and after 10 Hz, as the electric field frequency increased, the effects began to decrease. This last experiment exemplifies the hormetic curve in a vigor treatment where there is a maximal dose at which a maximum response value is reached and then vigor starts decreasing.

In several studies, PEF pretreatments in plants, whole fruits, or other food sources result in an increase in the natural bioactive compounds. When biological cells are exposed to an EF, the charge accumulates along the plasma membrane causing electroporation, a transmembrane potential difference which causes porosity, and thus the diffusion of intracellular components in cellular juice increasing the extractability of natural bioactive compounds by the release of solutes into the solvent (El Kantar et al. 2018; Vicaş et al. 2017; Barba et al. 2015; Hendrawan et al. 2019). The time exposure and intensity of the EF are critical since a lower EF may form smaller pores allowing the ions to pass through, but large molecules may not get out of the cell, however, higher EF are suspected to damage antioxidant compounds due to long exposure to high-voltage electric current (Hendrawan et al. 2019). Table 5.2 summarizes some examples of the application of magnetic or electric fields on plants or foods with a commercial interest and presents the effect on the production of natural bioactive compounds.

Table 5.2 Effect on natural bioactive compounds of experiments where magnetic or electric fields was the stress factor on plants or food with a commercial interest
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5.2.2 Acoustic Emissions

Acoustic emissions (AE) stimulus is one of the recent physical abiotic factors whose beneficial effects on plant growth, development, and health have been discussed. AE from ecological conditions or artificially applied can initiate diverse signals that trigger transduction cascades, similar to other abiotic stress factors (Alvarado et al. 2019). From a bioacoustics perspective, chewing serves as an alarm signal to plants and has been demonstrated that applying recorded insect chewing sounds caused an increase of phytochemical production (Appel and Cocroft 2014). In the same way, Jeong et al. (2014), reported an improvement of natural protection responses in rice plants caused by amplification at 100 decibels of a wide range of frequencies between 0 and 1.5 kHz. Moreover, Hassanien et al. (2014), found a higher disease resistance in pepper, cucumber, and tomato after AE treatments.

The biological mechanism of how sound affects plants is still under discussion. A mechano-stimuli perception of waves has been proposed, but a reliable explanation of sound-specific structure for recognition by plants has not been completely elucidated (Alvarado et al. 2019). This mechanism consists of the second messenger of calcium ion (Ca2+) signals. The channels that mediate Ca2+ flux are possibly located in the plasmatic membrane where Ca2+ is sensed possibly through various Ca2+ sensors and/or CDPKs (Calcium-dependent protein kinase), which pass the message through phosphorylation/dephosphorylation to different signaling proteins or to transcription factors (Mishra et al. 2016). In that way, it is strongly suggested that AE can influence the synthesis of secondary metabolites. The most common acoustic emission utilized for the stimulation of bioactive compounds is ultrasound (US). Several mechanisms of how the US interacts within the cell have been proposed. When the US is applied, cavitation bubbles creates a pressure zone change that occurs and increases up to 400 km h−1, causing higher porosity, rupture or removal of cell membranes, facilitating the mass transfer from the cells’ interior when imploding (Toma et al. 2001; Vinatoru 2001). In that way, the increase of natural bioactive compounds may be caused by better extractability due to rupture of membranes of cell organelles, however, when a decrease is presented, it could be explained by the creation of reactive forms of oxygen (ROS) during cavitation, and that the collapsing bubbles release high doses of energy, raising the temperature (>5000 K) enough to decompose polyphenols (Witrowa-Rajchert et al. 2014; Kentish and Ashokkumar 2011). The second possible explanation is an enhancement of enzymes activity when the US is applied by contact, leading to phenolic compounds’ reduction, more significantly after longer treatment time (Wiktor et al. 2016). Ampofo and Ngadi (2020) established that elicitation of common beans with the US, increased the accumulation of stress markers from the onset until the process was arrested, signifying a demand for sprout protection, resulting in an elevated stimulation of defense phenolic triggering enzymes (PAL and TAL), and final biosynthesis of phenolic compounds. In that way, application in food or plants of US treatment should be studied to find the optimal time, frequency, and intensity in order to optimize the production of secondary metabolites. Table 5.3 summarizes some examples of the application of acoustic emissions on plants or foods with a commercial interest and presents the effect on the production of natural bioactive compounds.

Table 5.3 Effect on natural bioactive compounds of experiments where the magnetic or electric field was the stress factor on plants or food with a commercial interest
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5.2.3 Nanoparticles

Nanoparticles (NPs) vary in size from 1 to 100 nm and have physicochemical properties, due to their dimensions, which generate a high added value for the nanotechnology industry (Yokel and MacPhail 2011). Nanoscale materials can be found on medical imaging, drug delivery, personal care products, cosmetics, clothing, electronics, agrochemicals, motor vehicles, among other products and applications (Vance et al. 2015; Yokel and MacPhail 2011). The metal-based NPs most commonly studied and found in industrial products are Cd (cadmium) in various complexes, GaAs (gallium arsenide), Au (gold), Ni (nickel), Pt (platinum), Ag (silver), Al2O3 (aluminum oxide or alumina), CeO2 (cerium dioxide or ceria), SiO2 (Silicon dioxide or silica), TiO2 (titanium dioxide or titania), ZnO (zinc oxide), CuO (copper oxide), and Fe3O4/Fe2O3 (iron oxides) (Khot et al. 2012; Yokel and MacPhail 2011). Among the carbon-based nanomaterials often studied are fullerene, single-walled carbon nanotubes (SWCNTs), and multiwalled carbon nanotubes (MWCNTs) (Balbus et al. 2007).

In contrast to its benefits, the release of nanomaterial-containing wastes has become a threat, since they cause pollution to air, water, and soil (Oberdörster et al. 2005). Their size, equivalent to that of cellular components, allows them to easily permeate cells, causing adverse biological effects (plants and animals) (Shang et al. 2014; Vecchio et al. 2012). In plant cells, NPs can enter from the apoplast and cross the plasma membrane towards cytosol or other organelles via endocytosis, specific membrane-bound transporter proteins or through induction of new pores by using ion carrier substances; subsequently, they can be transported between cells through symplastic flow (Anjum et al. 2019; Marslin et al. 2017). Despite its potential of toxicity, studies have reported positive effects on plant development and physiology, which are dependent on the nature of the nanomaterial, dose and time of exposure, the plant species, and growth conditions (Cox et al. 2016).

Positive physiological effects using carbon-based NPs include increased water uptake, and enhanced assimilation of CO2 in broccoli (Martínez-Ballesta et al. 2016), promotion of seed germination and root growth in rice (Jiang et al. 2014), enhanced germination and seedling growth in sweet corn, barley, rice soybean, switchgrass, and tomato (Lahiani et al. 2015; Tiwari et al. 2014), increase fruit yield in tomato and bitter melon (Khodakovskaya et al. 2013; Kole et al. 2013), among many others. Studies using metallic nanoparticles have reported similar effects. In wheat, CeO2 particles improved plant growth, shoot biomass, and grain yield (Rico et al. 2014). Au nanoparticles in chinese mustard (Brassica juncea) had positive effects on growth parameters and seed yield (Arora et al. 2012). The response of maize exposed to ZnO nanoparticles showed enhanced germination, seedling vigor, and zinc biofortification of grains (Subbaiah et al. 2016).

The effect of NPs on secondary plant metabolism is still largely unknown compared to physiological and phenotypic responses. However, studies have shown that a constant response between species is the induction of reactive oxygen species (ROS) (Marslin et al. 2017). Studies reporting NPs-elicitation of specialized metabolites often report a reduction in the photosynthetic rate and inhibition of growth. These phytotoxic effects have been linked to the inhibition of Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activity and decreased photo-protective capacity of PSII (Jiang et al. 2017a; Wang et al. 2016). When NPs permeate the cells, damage on the photosynthetic apparatus is done because of its accumulation in chloroplasts, at the same time, when they cross the plasma membrane they probably dissociate into ions (rather than stay as intact particles) and bound to NADPH oxidases, causing the production of ROS at the apoplast (Jiang et al. 2017a; Sosan et al. 2016). Besides oxidative burst, it has been reported that NPs also induce reactive nitrogen species (*NO, nitric oxide) (Marslin et al. 2017).

Initial responses also include calcium ion (Ca2+) spikes, Ca2+ flux movements, and upregulation/phosphorylation of mitogen-activated protein kinase (MAPK) cascades that together with ROS production, ultimately lead to the activation of the pathways of specialized metabolites biosynthesis (Anjum et al. 2019; Marslin et al. 2017). As expected, plants exposed to stressful concentrations of NPs have shown to cope with the oxidative stress and lipid peroxidation through the upregulation of enzymatic antioxidants such as superoxide dismutase (SOD), ascorbate peroxidase (APX), glutathione-S-transferase (GST), and catalase (CAT) (Dimkpa et al. 2012; Fu et al. 2014; Mirzajani et al. 2014; Zhao et al. 2012).

Through these findings, the concept of “nano-elicitors” have recently emerged as a novel alternative to stimulate the production of valuable bioactive compounds that might be used as additives in food, cosmetics, and pharmaceutical products. The most widely used nano-elicitors with this purpose are carbon nanotubes, silver, gold, copper, zinc oxide, and titanium dioxide (Anjum et al. 2019). The way to supply NPs to plants can be carried out through foliar spray, directly in the soil or through the nutrient solution applied. Table 5.4 summarizes some published studies in recent years reporting the enhanced production of commercially important specialized metabolites using metallic-, metal oxide-, and carbon related-NPs.

Table 5.4 Summary of the effects of different types of nanoparticles used as elicitors of secondary metabolites in different plant species
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5.2.4 Metals and Salt Metals

Metals, at high concentrations, act as stress agents to plants, therefore, they can induce changes in the secondary metabolism causing an elicitation effect. Exposure of plants to metals, such as Ni, Ag, Fe, and Co, has shown increased production of secondary metabolites in a variety of plants (Zhao et al. 2001). For instance, cadmium (Cd2+) and copper (Cu2+) are known for their toxicity and for not having any value for plants (Das et al. 1997). However, Cd and Cu treatments resulted in enhanced phenolic accumulation on the medicinal plant Gynura procumbens (Ibrahim et al. 2017). Several factors influence the response of plants to metal exposure, mainly depending on the chemical metal species and concentration, the plant species, climate conditions, growth stage, among others (Lajayer et al. 2017).

The use of nonfood crops with the capacity of absorbing and accumulating heavy metals is an alternative for remediation of contaminated environments. It has been shown in certain medicinal and aromatic plants that this practice can lead to the accumulation of secondary metabolites, which can be phytoextracted to obtain high-value compounds (Lajayer et al. 2017). Metabolic changes by the action of heavy metals can lead to inhibition of enzymes involved in the production of photosynthetic pigments, sugars, proteins, and nonprotein thiols (Naik and Al-Khayri 2016; Nasim and Dhir 2010). To date, many studies have shown increases in medicinal plant performance following exposure to heavy metal stress. For example, in a study where garden mint (Mentha crispa L., Lamiaceae) was used for phytoaccumulation of lead (Pb), the chemical composition of the essential oil of the plant was affected by improving the production of carvone, a major component of essential oils (Sá et al. 2015).

Heavy metals have also shown to have a role in stress amelioration through changes in antioxidant balance which often comes hand in hand with increased secondary metabolites. A study subjecting Camellia sinensis (L) plants to drought stress was performed to understand the role of Zn in modulating stress conditions. Results showed decreases in hydrogen peroxide (H2O2) and lipid peroxidation, and at the same time increases in phenolics content and differential expression of antioxidant enzymes, such as superoxide dismutase (SOD), catalase (CAT), peroxidase (POX), polyphenol peroxidase (PPO), glutathione reductase (GR), and ascorbate peroxidase (APX) (Upadhyaya et al. 2013). Similar results were obtained in Brassica napus exposed to cadmium (Cd) stress, exogenous application of low concentrations of selenium (Se) increased the tolerance of plants meanwhile concentrations of ascorbic acid and reduced glutathione were increased (Hasanuzzaman et al. 2012).

Metallic salts have also shown enhanced production of secondary metabolites during in vitro root cultures treatments such as two tropane alkaloids, scopolamine and hyoscyamine, by eliciting with silver nitrate (AgNO3) and cadmium chloride (CdCl2) in Brugmansia candida (Angelova et al. 2006), increases in tanshinone contents using AgNO3 in Perovskia abrotanoides (Zaker et al. 2015) and sesquiterpenoid–defensive compounds using cadmium salts in Datura stramonium (Furze et al. 1991).

5.2.5 Volatile Organic Compounds

Volatile organic compounds (VOCs) are low-molecular weight compounds that are emitted in the atmosphere in vapors or gaseous form. VOCs are produced as secondary metabolites by micro- (bacteria and fungi) and macro-organisms (animals and plants) and play vital roles, such as regulation of physiological processes and inter-organismal communication (Fincheira and Quiroz 2018; Rakshit et al. 2020). Plants can emit VOCs constitutively to attract pollinators and seed dispersers, or in response to a stimulus as a defense against insects or predators, plant-to-plant communication, thermo-tolerance, and environmental stress adaptation (Vivaldo et al. 2017). Plant VOCs can be classified into terpenoids, fatty acid derivatives, phenylpropanoids/benzenoids, and amino acid derivatives (Dudareva et al. 2013). Under-ground VOC’s are emitted by plants through their roots (Rakshit et al. 2020), where they also interact with bacteria and fungi in the rhizosphere zone giving rise to a deep symbiotic plant-microorganisms relation (Dessaux et al. 2016). Microorganisms benefit from root’s exudates while they produce nonvolatile metabolites that affect the plant’s nutrient assimilation and benefits plant growth (Dotaniya and Meena 2015).

A new plant–microbe interaction involving microbial volatile organic compounds (mVOCs) was discovered by (Ryu et al. 2003). In their study, they identified that volatile compounds from Bacillus subtilis act as strong promoters of growth in Arabidopsis thaliana. Since then, studies focusing on mVOCs as potential compounds with practical applications on regulating characteristics of agronomic importance have emerged. Bacterial and fungal volatile compounds may activate defense responses against biotic and abiotic stress, induce systemic resistance, promote growth, and enhance health processes in plants (Kanchiswamy et al. 2015; Piechulla and Degenhardt 2014). There are approximately 1000 mVOCs produced by bacteria and fungi reported in the literature, a few examples include 3-hydroxy-2-butanone (acetoin), 2,3-butanediol, 2-pentylfuran, or dimethylhexadecylmine (Fincheira and Quiroz 2018; Piechulla and Degenhardt 2014).

The idea of using VOCs to elicit secondary metabolites in plants is novel and still very little studied. There are cases of success in the literature to this purpose, which are summarized in Table 5.5, most of them have shown that different volatile compounds from bacterial can increase commercially valued components of essential oils, such as monoterpenes, pulegone, menthone, menthol, limonene, menthyl acetate, terpineol, and eugenol (Banchio et al. 2009; Santoro et al. 2011, 2016; Zhou et al. 2016). These studies are often performed in sterile plastic boxes or petri dishes with divided into two compartments by a physical barrier so that microbial and plant cultures interact without physical contact.

Table 5.5 Summary of the effects of different sources of VOCs used as elicitors of secondary metabolites in different plant species
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5.2.6 Nutrient Deficiency

The soil provides water and nutrients to plants. Fourteen mineral nutrients are required for plant correct growth and development which are divided into macronutrients (N, P, K, Ca, Mg, and S) and micronutrients (Cl, Fe, B, Mn, Zn Cu, Ni, and Mo). Macronutrients form structural and energy compounds in plants. On the other hand, microelements are related to enzymatic responses. For example, Zn, Fe, Mn, and Cu are components of enzymatic antioxidants, which regulate oxidation processes in the plant (Hajiboland 2012; Nath and Tuteja 2016). Due to the indispensable role of nutrients, plant roots have developed an efficient sensing and signaling system to maintain nutrient homeostasis. Low availability of nutrients in the soil is detected by roots, and in response, chemical signaling and chain reactions are produced. Plants employ signaling players as phytohormones, reactive oxygen species (ROS), sugars, and transcription factors to maintain nutrients homeostasis within the plant (Nath and Tuteja 2016; Isah 2019).

Nutrient deficiency can produce metabolic responses that cause an increased accumulation of secondary metabolites. Natural bioactive compounds are sought in bioproduction processes and improvement of nutritional quality of vegetables and fruits, therefore a nutrient deficiency of specific macro or micronutrients may be an alternative. However, this stress can cause a decrease in crop yields (Hawkesford et al. 2012). To produce secondary metabolites of interest without a considerable loss of growth and biomass, it is necessary to generate eustress in the plant (El-Nakhel et al. 2019). Currently, nutritional eustress is a strategy used in protected production systems, where soilless crops allow greater control of nutrient supply through nutrient solutions.

Nitrogen is a macronutrient constituent of primary metabolites (e.g., protein, peptides, amino acids, and nucleic acids), phytohormones and secondary metabolites. Plants can uptake nitrogen as nitrate and ammonium (mineral form) (Isah 2019). Research has shown an inverse relationship between low nitrogen availability and the synthesis of phenolic compounds. According to this hypothesis, low nitrogen availability increases synthesis of metabolites that contain C, H, and O in their structure. Therefore, terpenes and phenolic compounds synthesis will be favored. On the contrary, metabolites that contain N in its structure such as, alkaloids, nonprotein amino acids, and cyanogenic compounds, will decrease its synthesis (Nath and Tuteja 2016). For example, growing lettuce (Lactuca sativa) increases its content of phenolic compounds and antioxidant capacity in the presence of nitrogen deficiency and drought (Galieni et al. 2015). Table 5.6 shows more examples of the effect on phenolic compounds of experiments where nitrogen deficiency was the stress factor on plants or food with commercial interest.

Table 5.6 Effect on phenolic compounds of experiments where nitrogen deficiency was the stress factor on plants or food with commercial interest
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Phosphorus (P) is a component of molecules such as nucleic acids, lipids and nucleotides with an energy function (ATP and ADP). Plants uptake P in the form of inorganic orthophosphate (Pi, HPO42−, and H2PO4−) which deficiency promotes anthocyanin synthesis (Jezek et al. 2016). Peng et al. (2019) proposed a model of com-modulation (miR399d) and epigenetic modification as a regulatory mechanism of anthocyanin synthesis that depends on the P availability. Sulfur is a structural component of amino acid precursors of secondary metabolites. Therefore, its deficiency negatively affects the biosynthesis of lycopenes and carotenoids (Mohammed et al. 2015). Micronutrient deficiencies are shown to have a negative impact on the synthesis of phenolic compounds and terpenes. Micronutrients such as Cu, Fe, Mo, and Mn, act as factors for the synthesis of secondary metabolites.

5.3 Biotic Stress

Biological stressors are those considered within living organisms (plants or pathogens) including bacteria, insect or herbivores, fungi, phytohormones, and miRNA, among others, that results in biotic stress (Patel and Krishnamurthy 2013). The action mechanism of this factor includes activation or inactivation of enzymes, interaction with receptors, ion channels, stimulation of bioactive compounds, and so forth (Joshi et al. 2019). Some biotic stress factors and their role in the synthesis of secondary metabolites in plants are described below.

5.3.1 Bacteria and Viruses

Plants are exposed to interactions with other living things. The interaction between microorganisms and plants can have positive effects. Microorganism colonization (pathogens and non-pathogens) triggers the resistance mechanism of the plant, conferring resistance against other stressors (Nejat and Mantri 2017; Choudhary et al. 2016). Nonpathogenic microorganisms act as plant biostimulants. A plant biostimulant is defined as any substance or microorganism applied to plants in order to improve nutritional efficiency, tolerance to biotic and abiotic stress, and quality (Van Oosten et al. 2017; Du Jardin 2015). Arbuscular mycorrhizal fungi, Trichoderma, and plant growth-promoting rhizobacteria are biostimulant microorganisms used in crops.

Microorganisms can confer a certain degree of tolerance against abiotic stress conditions. Colonized plants produce a wide range of enzymes and metabolites that allow them to generate tolerance to stress (Miliute et al. 2015). Some genera of bacteria like Rhizobium, Bacillus, Pseudomonas, Pantoea, Paenibacillus, Burkholderia, Achromobacter, Azospirillum, Microbacterium, Methylobacterium, variovorax, Enterobacter, have been shown to induce tolerance to abiotic stress (Choudhary et al. 2016; Naveed et al. 2014; Gururani et al. 2013). Tolerance generated by pathogen attack can induce resistance to abiotic stress factors. The biochemical response generated by the attack of the pathogen is similar to the response generated by abiotic factors. Plants attacked by Verticillium dahliae (pathogenic fungus) develop tolerance to drought due to the formation of xylem but reducing the growth rate (Tani et al. 2018).

Viruses are considered symbiotes. They can behave as pathogens or mutualists depending on the environmental conditions where the host is (Roossinck 2015). Research suggests that the mutualistic behavior of a virus occurs when the titer virus is low and the environmental disturbance is low (Bao and Roossinck 2013). Plant viruses can have a positive effect like other pathogens. The presence of the virus in the plant can increase its ability to cope with biotic and abiotic stress factors because of the activation of the plant defense system. Metabolomic studies in infected plants have shown a significant increase in the quantity and diversity of secondary metabolites. This metabolic effect allows the plant to cope with the stress caused by the infection, as well as other stressors present in the environment. For example, Sade et al. (2015), reported a significant impact on the metabolome in tomato plants infected with Tomato yellow leaf curl virus (TYLCV) where resistant and susceptible cultivars showed a major expression of the phenylpropanoid pathway which is related to the production of antioxidant compounds, among others. In the same research, the expression in resistant cultivars was more significant in terms of the production of flavonoids and other antioxidants. On the other hand, rice plants infected with Brome mosaic virus (BMV) and beet plants (Beta vulgaris) infected with Cucumber mosaic virus (CMV) increased the accumulation of osmoprotectants and antioxidant compounds, conferring drought tolerance to both crops (Xu et al. 2008).

5.3.2 Fungi

Plants have a strong symbiosis relationship with some fungi and bacteria present in the substrate where they are grown. These microorganisms, endophytes or exogenous, can induce eustress to the crop, increasing the production of specialized metabolites, e.g.,Aspergillus sp. applied as an elicitor in Artemisia annua L. callus culture, enhanced the production of artemisinin, an endoperoxide sesquiterpene lactone and an effective antimalarial agent (Yuliani et al. 2018). Soil-borne beneficial microbes have shown a protecting potential against pathogens and herbivores via the elicitation of plant responses e.g. plant growth-promoting fungi (PGPF) and arbuscular mycorrhizal fungi (AMF) (Pappas et al. 2018). Fungal elicitation (including yeas extract) is one of the most used to enhance the production of secondary metabolites (Singh et al. 2018).

Fungi with a beneficial effect on plant development that associate to plant roots are called PGPF and are considered the first prevention mechanism in the pathogen infection. Plants need to detect PGPFs and take advantage of the presence of microbe-associated molecular patterns (MAMPs) that can be recognized by pattern recognition receptors (PRRs). PGPFs can stimulate the defense system of plants involving the modification of cell walls by the accumulation of lignin, callose, phenols, etc., preventing the growth and proliferation of pathogens. In addition, elicitors such as chitin, chitosan, and β-glucan that are part of the fungal cell wall have been researched (Naziya et al. 2020; Fesel and Zuccaro 2016; Li et al. 2016).

Recent studies have been performed in different plants, e.g., fungal elicitation in Leguminosae increased the accumulation of isoflavonoids and stilbenoids (Araya-Cloutier et al. 2017). Moola and Diana (2019) used Aspergillus niger, Penicillium notatum, and Rhizopus oligosporus as elicitors on hairy root culture of Beta vulgaris to enhance betalain synthesis. Trichoderma spp. is one of the most widely used microorganisms as a pathogen biocontrol agent and elicitor. In addition, this fungus has been shown to colonize roots and can also induce systemic resistance (ISR), which favors plant growth, increases nutrient availability and enhances disease resistance. The mechanisms employed by Trichoderma spp. are the modulation of plant hormonal mechanisms and the production of secondary metabolites (Nandini et al. 2020; Guzmán-Guzmán et al. 2019; Silva et al. 2019; Martínez‐Medina et al. 2017). PGPFs can be used as bio-fertilizers, improving the quality and quantity of products, and reducing the contamination of the agricultural environment by lowering the use of chemical fertilizers (Pereira et al. 2019; Zhou et al. 2018). Table 5.7 shows some of the fungi used to induce the production of secondary metabolites in plants.

Table 5.7 Plants elicited by fungi application to increase the production of secondary metabolites
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5.3.3 Phytohormones

Phytohormones are molecules synthesized by defined organs that regulate plant growth and have a prominent impact on plant metabolism. Additionally, they play a vital role in the stimulation of response mechanisms of plant defense against stress. Auxins, gibberellins, cytokinins (CK), abscisic acid (ABA), jasmonates (jasmonic acid (JA), methyl jasmonate (MeJA)), salicylic acid (SA), brassinosteroids, strigolactones, cinnamic acid (CA), among others, are examples of phytohormones. Auxins and the auxin índole-3-acetic acid (IAA) act as promoters of growth and developmental events in plants (cell división, elongation, and differentiation). CKs are involved in the maintaining of cellular proliferation, differentiation, and prevention of senescence. ABA has an important role in the plant response to stress and adaptation. Gibberellic acid (GA) is a plant growth regulator and has a vital role in seed dormancy, formation of floral organs, and lateral shoot growth (Egamberdieva et al. 2017). SA has an important role in plant stress tolerance through modulation of antioxidative enzyme activities. JA is a lipid-derived compound synthesized via the octadecanoid pathway and is important in the development, structure, and flowering of plants (Złotek et al. 2020). CA is a bioprecursor of podophyllotoxin and a huge number of plant substances, including tannins, flavonoids, etc. (Kašparová et al. 2018).

Currently, the use of phytohormones in agriculture and research has been implemented as a strategy to induce the production of secondary metabolites (Egamberdieva et al. 2017). The exogenous application of phytohormones by spraying has resulted in increased production of various significant bioactive compounds in plants (Akram et al. 2020, Wang et al. 2018a). For example, Garcia-Ibañez et al. (2019), applied MeJA, SA, and SA + MeJA to Bimi® plants resulting in a differentiated response to each elicitor, all treatments showed an increased content of GLs in leaves and inflorescences. Table 5.8 shows the result of plant elicitation with phytohormones in the production of bioactive compounds of recent studies.

Table 5.8 Secondary metabolites produced by application of phytohormones to several plant species
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5.3.4 miRNA

MicroRNAs (miRNAs - length of 18 to 28 nucleotides) are noncoding RNAs. The main function of these molecules is to participate in gene expression and regulation at the post-transcriptional level by degrading mRNA or at the translational level by blocking protein biosynthesis at different stages (Fig. 5.1), resulting in regulation of plant development, metabolism, and response to biotic or abiotic stress (Tripathi et al. 2019). In plants miRNAs genes are transcribed by RNA polymerase II producing primary miRNA (Pri-miRNA) which are important for the regulation of genome integrity, primary and secondary metabolism, development, signal transduction, signaling pathways, homeostasis, innate immunity, and environmental stress responses (Vargas-Hernández et al. 2019; Wang et al. 2018b; Samad et al. 2019).

Fig. 5.1
figure 1

Clasification of plant noncoding RNAs (ncRNAs). Linear noncoding RNAs (Linear ncRNAs), circular noncoding RNAs (cncRNAs), HouseKeeping noncoding RNAs (hk ncRNAs), Regulatory noncoding RNAs (Regulatory ncRNAs), long noncoding RNAs (lncRNAs), small RNAs (sRNAs), small interfering RNAs (siRNAs), micro RNAs (miRNAs)

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The regulation mechanism of gene expression by miRNAs is via RNA interference. These small molecules are transcribed as longer precursors in the nucleus and are then further processed into their mature forms (Wang et al. 2018b; Samad et al. 2017). Most plant miRNA genes are located inside intergenic regions between two adjacent genes and are transcriptionally regulated by their promoters and terminators (Hossain et al. 2019). miRNAs were reported to be involved in plant secondary metabolite regulation such as terpenoid, phenolic, fatty acid, flavonoids, phenolic, and nitrogen-containing compound biosynthesis (Liu et al. 2017; Samad et al. 2019).

There is a very close relationship between miRNAs and the transcription factors (TFs), either to be switched “on” or switched “off”. Current research trends have focused on knowing the regulation of miRNA in response to environmental stress and how they interact with transcription factors (Fig. 5.2) (Samad et al. 2019).

Fig. 5.2
figure 2

Stress mechanism and interaction between transcription factors and miRNAs

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The main goal of functional studies on miRNAs has been to understand the biological processes in which the miRNAs are involved. Different technologies have been developed to characterize the function and action mechanisms of these small molecules in various plant materials (Liu et al. 2017). Research on the regulation of miRNAs in Taxus callus cells has been conducted, concluding that miRNAs are capable of direct regulation of secondary metabolism by modulating transcriptional factors (Chen et al. 2020). The expresión level of miRNAs is mostly governed by temperature and radiation (Tripathi et al. 2019). Studies have demonstrated that miRNAs may act as master regulators of flavonoid biosynthesis, e.g., miR156-SPL9 network directly influences anthocyanin production, miR163 targets S-adenosyl-Met-dependent methyltransferases that methylates secondary metabolites and signaling molecules, and miR397 regulates lignin biosynthesis in Arabidopsis and Populus spp (Sharma et al. 2016).

Studies carried out on different plant species indicate that the cellular levels of miRNA have a high regulation control for optimal spatiotemporal regulation of target genes, adding an additional layer of complexity to the signaling processes (Gupta et al. 2017; Sharma et al. 2016). These results suggest that a strategy to induce the production of secondary metabolites may be the use of miRNAs that promote the expression of genes involved in plant biosynthetic pathways.

5.4 Future Perspectives

The use of AE stimuli as a new elicitor in plants has huge sustainable potential. However, this “environmentally friendly” agricultural technology is still under discussion and more studies should be encouraged. Currently, the optimal sound therapy is not known since the effect could differ depending on several factors such as plant model, amplitude, frequency or time and duration of treatment, and application distance among others that are not explained in most studies (Alvarado et al. 2019).

PEF and MF in food, horticulture, and biotechnology asthe postharvest processes have increased substantially during the last few years (Xi-ran and Ting 2017; Gilani et al. 2017; Rusakova et al. 2017). Generally, it can be noted that the application of PEF and MF as pretreatment is a novel method to improve plant development due to a higher production of ROS and an associated activation of antioxidant defense systems, such as POD, SOD, and CAT. However, the effect of electromagnetic sources is plant and treatment specific. It is difficult to have a strong conclusion in the analyzed experiments in this chapter since the experimental details often were incomplete, including an insufficient description of equipment and treatment conditions. It is recommended to consider the description of parameters, such as electric field strength (V/m), frequency (Hz), magnetic flux density (T), electric current (A), PPFD, distance of light source to plant, light decay among the experimental source, photoperiod, light wavelength, the duration of application, and plant or food complete physical description (Dannehl 2018; Alvarado et al. 2019).

Nanoparticles in agriculture have shown benefits related to physiological and growth parameters. Moreover, the new knowledge related to its effect on secondary metabolism has opened a field of possibilities in the production of bioactive compounds with high commercial value using NPs. However, as with many elicitors, there is still a lack of knowledge about the type and size of nanoparticle and appropriate concentrations to use depending on the species of interest. Furthermore, a large part of the existing nanoparticles has not yet been studied and it is also necessary to continue generating knowledge to understand the molecular mechanisms of elicitation with NPs.

Metal ions have been proposed as suitable elicitors of secondary metabolism in cell cultures (Rudrappa et al. 2004), since they can make more efficient the tissue culture techniques during the obtention of valuable secondary metabolites. Many studies have proposed the use of different chemical metal species to enhance bioactive compounds in plants. This tool is very attractive in the sense that, in addition to producing these types of compounds, it can become an environmental remediation technique. Also, the idea of producing specialized metabolites for later extraction eliminates the risks associated with the potential health risks to the consumers.

Among the study of new agricultural tools in the last decade, VOCs stands out for being considered as an eco-friendly, cheap, and effective alternative. Even genetically modified plants with altered VOC emission and synthetic formulations of plant VOCs are been developed as a promising technology for agriculture and horticulture (Rakshit et al. 2020). However, there are still many unknowns, especially about the mode of action of VOCs and about the molecular and biochemical mechanisms related to eliciting responses of interest in plants.

The application of phytohormones in different phenological stages of plants, including in the postharvest stage, induces the production of secondary metabolites and is an effective strategy that uses defense mechanisms to mimic stress caused by various environmental factors. This method can be used at the agronomic level to improve the quality of plant products by increasing the content of bioactive molecules. Defense plant response due to different types of stress depends on the type of crosstalk (positive or negative) between the hormone signaling pathways rather than on the individual contributions of each hormone (Verma et al. 2016). This suggests that for future perspectives it should be taken into account that the results of elicitation will depend on the synergy of the used phytohormones, the concentration, the application conditions, and the type of cultivation. For example, there is a balance between SA and JA to regulate biotic stress in tomato (Verma et al. 2016). In the same way, SA and gibberellins have been used as elicitor and biostimulant to enhance the production of steviol glycosides in stevia, producing tall plants with a greater number of leaves and a larger stem diameter (Vazquez-Hernandez et al. 2019).

Endophytic microbes have been shown to be able to promote plant growth, induce tolerance and production of bioactive compounds (Lata et al. 2018). Endophytic microbes generally reside in tissues and plants without causing symptoms. However, they activate plant defense system and induce secondary metabolites production with potential pharmaceutical use (Jalgaonwala and Mahajan 2014). Therefore, the inoculation of plants with this type of microorganism is a strategy that can be applied to induce and/or increase the production of natural bioactive compounds in plants of commercial interest.

Fungi elicitation is a popular practice among horticultural producers due to the demonstrated increase in crop yield and the production of bioactive compounds. The ability of plant roots to uptake nutrients from the substrate is enhanced by this type of elicitation, with a positive influence on phytohormone production and gene expression reprogramming. Fungi are used as biocontrol treatments, bioremediation agents, and as biostimulants, which can contribute to the development of sustainable agriculture. In the same way, mineral nutrients play an important role in the growth, development, and yield of crops (Nath and Tuteja 2016). Nutrient management is a common agricultural practice, especially in soilless cultivation and controlled systems in order to guarantee high crop yield. On the other hand, consumers demand natural products with high nutritional quality. Both objectives are feasible to be induced by the nutrition of the crops. Therefore, the management of nutrients during the production cycle allows a balance between growth and accumulation of bioactive compounds.

The use of specific miRNAs to enhance the production of metabolites is a novel technique that is still under research and discussion among the scientific community. Research has focused on the identification of the regulatory mechanisms of these molecules that will lead to the design of strategies for direct manipulation, identification, and understanding of the spatial and temporal expression scheme of miRNAs. Innovative tools (e.g., bioinformatics) can be used to predict, by means of algorithms, the modulation of miRNA molecules which are part of a very complex regulatory network of secondary metabolites. Research in this area could help to understand how biosynthetic pathways are modified by these small molecules that have particular target genes and that can influence metabolic plant bioengineering, generating technology to induce the production of secondary metabolites.