**2. Potential chemicals involved in abiotic stress responses and their use as biostimulants**

#### **2.1. Reactive short-chain leaf volatiles as potential signaling chemicals**

As described above, chloroplasts are the organelles that are most susceptible to damage under conditions of oxidative stress. Therefore, chloroplasts are also potential sensors of environmental stress, assimilating environmental changes, and transmitting information about the changes to other organelles using infochemicals. Recently, we have found evidence to support the premise that chloroplasts produce signal chemicals that induce gene expression and enhance stress tolerance.

Reactive short-chain leaf volatiles (RSLVs) are a group of C4–C9 straight chain carbonyls characterized by an α,β-unsaturated carbonyl bond (**Figure 2**). They are oxylipins and are derived from PUFAs in the thylakoid membrane. Biologically, plants treated with vaporized RSLVs show an enhanced expression of genes involved in responding to environmental stresses, such as high temperatures and oxidative stress [12]. As this response resembles the acquired thermotolerance inherent in plants as a response mechanism for surviving stress caused by elevated temperatures, plants treated with RSLVs show enhanced thermotolerance. As described later, the discovery of this bioactivity has opened the possibility of the chemical control of plants by volatiles to induce heat stress tolerance.

#### **2.2. Improving crop production by enhancing environmental stress tolerance**

In nature, crop yield is usually reduced by stress related to both biotic and abiotic causes; surprisingly, abiotic stress is the major inhibiting constraint, by up to 70% of potential production, in contrast to 10% for biotic stress (**Figure 3**, reconstituted from [13]). This indicates that crop production is on average only producing 20% of potential yield. Thus, if crop plants were liberated from abiotic stresses, by even only 10% of potential yield, then net crop production would increase by an average of 50%. Achieving this would be dependent on fertilizer-independent crop improvements, based on agriculturally beneficial biostimulants.

**2.3. Use of biostimulants in enhancing tolerance to environmental stress**

abiotic stress, net production would increase 1.5-fold when compared to no treatment being applied.

**2.4. Use of Pyrabactin as an ABA derivative for controlling water use**

are introduced.

practical.

Crop yield has traditionally been improved by the application of fertilizers, pesticides, and irrigation to agricultural fields. Biostimulants are also products that have positive effects on yield by increasing stress tolerance and repairing damage already caused by unfavorable conditions [14, 15]. They can be either natural or synthetic in origin and usually consist of various organic and inorganic components. Naturally derived biostimulants include preparations based on free amino acids, seaweed and fruit extracts, effective microorganisms, humic substances, and chitosan [14, 15]. Synthetic biostimulants include plant growth regulators, phenolic compounds, inorganic salts, essential elements, and other substances with plantstimulating properties. Hereafter, in this chapter, some major biostimulants, in particular chemical biostimulants with the potential to mitigate the effects of environmental stresses,

**Figure 3.** Concept of increase in crop production by using biostimulants. A comparison between the maximum yield recorded in 1975 (potential productivity) and average yield over a period of 50 years (1939–1978, actual productivity) shows that a large proportion of crop production is lost due to abiotic (up to 70%) and biotic (10%) stress (adapted from Table 1 in [13]). As shown in the right panel, if biostimulant treatment were to remove only 10% of the damage due to

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Under drought-stress conditions, plants often produce elevated levels of ABA to reduce transpiration by closing the guard cell aperture, resulting in a reduction in water loss. In order to control water use by plants, ABA derivatives have therefore been developed to activate the ABA receptors. Pyrabactin is representative of these synthetic ABA derivatives that mimic ABA; it activates the ABA receptors needed for improving drought tolerance [16]. Unlike natural ABA, Pyrabactin is not sensitive to light, is easy to synthesize, and relatively inexpensive, and its manufacture for agricultural use is therefore

**Figure 2.** Reactive short-chain leaf volatiles (RSLVs) are signaling chemicals involved in the response to heat and oxidative stresses [12]. They are produced from oxidized polyunsaturated fatty acids, such as linolenic and linoleic acid, in thylakoid membranes through both enzymatic and nonenzymatic mechanisms. The essential chemical structure revealing signal activity is a straight chain carbonyl between C4 and C9, which has an α,β-unsaturated carbonyl bond (indicated by dotted circles). Of these, 2-hexenal is an RSLV produced enzymatically that is also known as a green leaf volatile.

**Figure 3.** Concept of increase in crop production by using biostimulants. A comparison between the maximum yield recorded in 1975 (potential productivity) and average yield over a period of 50 years (1939–1978, actual productivity) shows that a large proportion of crop production is lost due to abiotic (up to 70%) and biotic (10%) stress (adapted from Table 1 in [13]). As shown in the right panel, if biostimulant treatment were to remove only 10% of the damage due to abiotic stress, net production would increase 1.5-fold when compared to no treatment being applied.

#### **2.3. Use of biostimulants in enhancing tolerance to environmental stress**

Reactive short-chain leaf volatiles (RSLVs) are a group of C4–C9 straight chain carbonyls characterized by an α,β-unsaturated carbonyl bond (**Figure 2**). They are oxylipins and are derived from PUFAs in the thylakoid membrane. Biologically, plants treated with vaporized RSLVs show an enhanced expression of genes involved in responding to environmental stresses, such as high temperatures and oxidative stress [12]. As this response resembles the acquired thermotolerance inherent in plants as a response mechanism for surviving stress caused by elevated temperatures, plants treated with RSLVs show enhanced thermotolerance. As described later, the discovery of this bioactivity has opened the possibility of the chemical control of plants by volatiles to induce heat stress tolerance.

In nature, crop yield is usually reduced by stress related to both biotic and abiotic causes; surprisingly, abiotic stress is the major inhibiting constraint, by up to 70% of potential production, in contrast to 10% for biotic stress (**Figure 3**, reconstituted from [13]). This indicates that crop production is on average only producing 20% of potential yield. Thus, if crop plants were liberated from abiotic stresses, by even only 10% of potential yield, then net crop production would increase by an average of 50%. Achieving this would be dependent on fertilizer-independent crop improvements, based on agriculturally benefi-

**Figure 2.** Reactive short-chain leaf volatiles (RSLVs) are signaling chemicals involved in the response to heat and oxidative stresses [12]. They are produced from oxidized polyunsaturated fatty acids, such as linolenic and linoleic acid, in thylakoid membranes through both enzymatic and nonenzymatic mechanisms. The essential chemical structure revealing signal activity is a straight chain carbonyl between C4 and C9, which has an α,β-unsaturated carbonyl bond (indicated by dotted circles). Of these, 2-hexenal is an RSLV produced enzymatically that is also known as a green leaf

**2.2. Improving crop production by enhancing environmental stress tolerance**

cial biostimulants.

136 Plant, Abiotic Stress and Responses to Climate Change

volatile.

Crop yield has traditionally been improved by the application of fertilizers, pesticides, and irrigation to agricultural fields. Biostimulants are also products that have positive effects on yield by increasing stress tolerance and repairing damage already caused by unfavorable conditions [14, 15]. They can be either natural or synthetic in origin and usually consist of various organic and inorganic components. Naturally derived biostimulants include preparations based on free amino acids, seaweed and fruit extracts, effective microorganisms, humic substances, and chitosan [14, 15]. Synthetic biostimulants include plant growth regulators, phenolic compounds, inorganic salts, essential elements, and other substances with plantstimulating properties. Hereafter, in this chapter, some major biostimulants, in particular chemical biostimulants with the potential to mitigate the effects of environmental stresses, are introduced.

#### **2.4. Use of Pyrabactin as an ABA derivative for controlling water use**

Under drought-stress conditions, plants often produce elevated levels of ABA to reduce transpiration by closing the guard cell aperture, resulting in a reduction in water loss. In order to control water use by plants, ABA derivatives have therefore been developed to activate the ABA receptors. Pyrabactin is representative of these synthetic ABA derivatives that mimic ABA; it activates the ABA receptors needed for improving drought tolerance [16]. Unlike natural ABA, Pyrabactin is not sensitive to light, is easy to synthesize, and relatively inexpensive, and its manufacture for agricultural use is therefore practical.

#### **2.5. Acetic acid**

The external application of acetate enhances drought tolerance in various plant species, such as *Arabidopsis*, maize, rapeseed, rice, and wheat [17]. This effect is related to a novel drought tolerance mechanism in plants involving the acetate-jasmonate signaling pathway, which is regulated epigenetically and conserved in plants. In *Arabidopsis*, exogenous acetic acid promotes JA synthesis and enriches histone H4 acetylation using ON/OFF switching, which is dependent on histone deacetylase HDA6, influencing the priming of the JA signaling pathway for plant drought tolerance. Thus, the external application of acetate to crops is potentially a useful, simple, and low-cost method of enhancing drought tolerance in various plant species.

membrane rigidification can be used to promote tolerance against cold-induced stress. Furuya et al. [25] suggest that a treatment of dimethyl sulfoxide, which is a membrane rigidifier, enhanced the cold acclimation of *Arabidopsis* by activating the MEKK1-MKK2-MPK4 cascade. These results indicate that chemicals modifying lipid fluidity are a possible means of cold

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Nitrophenolates are biostimulants and are already being manufactured commercially in Japan under the name Atonik, a synthetic product composed of three phenolic compounds: sodium *p*-nitrophenolate (0.3%), sodium *o*-nitrophenolate (0.2%), and sodium 5-nitroguaiacolate (0.1%), together with water. Atonik has been used successfully for many years in the cultivation of most globally important crops. Its mode of action is still not understood but might be involved in hormone regulation, nutrient uptake, and nitrogen metabolism [26]. Atonik therefore stimulates plant growth and development and contributes to enhancing biomass accumulation, increasing water uptake, protecting against drought, and mitigating stress due

As described in Section 2.1, RSLVs potentially act as signaling chemicals involved in heat and oxidative responses. A representative RSLV, 2-hexenal, is a green leaf volatile that induces gene expression in response to heat and oxidative stresses [12] and thus enhances thermotolerance in plants. Terada et al. suggest that this effect is partly explained by transpiration being sustained at higher temperatures [27]. The field use of 2-hexenal is being progressed commercially in Japan. 2-hexenal is a volatile; its vaporization from a tablet form by sublimation has enabled the effective concentrations for use in closed greenhouses to be determined. A preliminary examination showed that its application in greenhouses improved the production of crops such as tomato, strawberry, and cucumber in the summer (unpublished data),

Historically, the use of pesticides, irrigation, and fertilizers, especially chemical fertilizers, has proven highly successful in increasing crop yields and thus in meeting the demands of increasing population levels. However, recent climate change is having an adverse impact on crop production, and therefore, more efficient methods of crop production need to be established. The use of genetically modified organisms (GMOs) is undoubtedly a solution to combat losses in plant production caused by global environmental changes. However, GMO is limited to major crops, and its use is also either strictly restricted or not even permitted legally in several countries. Therefore, chemical control of abiotic stress tolerance is required

suggesting that its use as a biostimulant is effective in overcoming heat stress.

**3. Perspective: toward an integrated chemical control against** 

as an alternative solution for ensuring unrestricted agricultural production.

adaptation in plants.

**2.8. Nitrophenolates**

to noble metals.

**2.9. Use of RSLVs as biostimulants**

**environmental stress**

#### **2.6. Nonprotein amino acids and derivatives**

The nonprotein amino acid β-aminobutyric acid (BABA), a potent inducer of resistance to infection by various pathogens [18], exerts its functions via priming of the SA-dependent defense mechanisms in Arabidopsis [19]. In other cases, BABA acts through potentiation of the ABA-dependent signaling pathways [20]. As both pathways can contribute to water stress tolerance, BABA is also able to protect Arabidopsis against abiotic stress, such as drought and high salinity [21], although BABA is a rare amino acid in plants [18]. This result suggests that BABA can be used as a biostimulant to protect plants from drought and salinity stress when it is based on ABA-dependent but not on SA-dependent defense mechanisms.

Glycine betaine is a major organic osmolyte that accumulates in various plant species in response to stresses such as drought and salinity [22]. It is an endogenous osmolyte produced by two enzymes: choline monooxygenase converts choline to betaine aldehyde, which is then catalyzed by betaine aldehyde dehydrogenase to form glycine betaine. As an osmolyte, glycine betaine is considered to have positive effects on the enzyme and membrane integrity in plants growing under stressful conditions; its role as a biostimulant has been subjected to field tests, and it is already being produced commercially. However, although many plant species show a significant increase in growth and final crop yield under conditions of environmental stress when treated with glycine betaine, others do not. Thus, the most useful and economic application of these compounds requires further investigations in order to determine the most effective concentrations and number of applications, as well as the most responsive growth stage(s) of the plant.

#### **2.7. Controlling cold tolerance by modifying membrane fluidity**

There is a close correlation between the chilling sensitivity of plants and the level of unsaturated fatty acids in the phosphatidylglycerol (PG), a phospholipid found in the thylakoid membranes of the chloroplasts [23]. When glycerol-3-phosphate acyltransferase, a key enzyme in determining the extent of unsaturated fatty acids in PG, is overexpressed, then increases in the relative levels of saturated and monounsaturated fatty acids in PG have been shown to increase the sensitivity of tobacco plants to low temperatures during the growth of young seedlings and maturation of reproductive organs [24]. As increases in the unsaturation of fatty acids result in decreases in biomembrane rigidification, then chemicals that enhance membrane rigidification can be used to promote tolerance against cold-induced stress. Furuya et al. [25] suggest that a treatment of dimethyl sulfoxide, which is a membrane rigidifier, enhanced the cold acclimation of *Arabidopsis* by activating the MEKK1-MKK2-MPK4 cascade. These results indicate that chemicals modifying lipid fluidity are a possible means of cold adaptation in plants.

#### **2.8. Nitrophenolates**

**2.5. Acetic acid**

**2.6. Nonprotein amino acids and derivatives**

138 Plant, Abiotic Stress and Responses to Climate Change

growth stage(s) of the plant.

The external application of acetate enhances drought tolerance in various plant species, such as *Arabidopsis*, maize, rapeseed, rice, and wheat [17]. This effect is related to a novel drought tolerance mechanism in plants involving the acetate-jasmonate signaling pathway, which is regulated epigenetically and conserved in plants. In *Arabidopsis*, exogenous acetic acid promotes JA synthesis and enriches histone H4 acetylation using ON/OFF switching, which is dependent on histone deacetylase HDA6, influencing the priming of the JA signaling pathway for plant drought tolerance. Thus, the external application of acetate to crops is potentially a useful, simple, and low-cost method of enhancing drought tolerance in various plant species.

The nonprotein amino acid β-aminobutyric acid (BABA), a potent inducer of resistance to infection by various pathogens [18], exerts its functions via priming of the SA-dependent defense mechanisms in Arabidopsis [19]. In other cases, BABA acts through potentiation of the ABA-dependent signaling pathways [20]. As both pathways can contribute to water stress tolerance, BABA is also able to protect Arabidopsis against abiotic stress, such as drought and high salinity [21], although BABA is a rare amino acid in plants [18]. This result suggests that BABA can be used as a biostimulant to protect plants from drought and salinity stress when

Glycine betaine is a major organic osmolyte that accumulates in various plant species in response to stresses such as drought and salinity [22]. It is an endogenous osmolyte produced by two enzymes: choline monooxygenase converts choline to betaine aldehyde, which is then catalyzed by betaine aldehyde dehydrogenase to form glycine betaine. As an osmolyte, glycine betaine is considered to have positive effects on the enzyme and membrane integrity in plants growing under stressful conditions; its role as a biostimulant has been subjected to field tests, and it is already being produced commercially. However, although many plant species show a significant increase in growth and final crop yield under conditions of environmental stress when treated with glycine betaine, others do not. Thus, the most useful and economic application of these compounds requires further investigations in order to determine the most effective concentrations and number of applications, as well as the most responsive

There is a close correlation between the chilling sensitivity of plants and the level of unsaturated fatty acids in the phosphatidylglycerol (PG), a phospholipid found in the thylakoid membranes of the chloroplasts [23]. When glycerol-3-phosphate acyltransferase, a key enzyme in determining the extent of unsaturated fatty acids in PG, is overexpressed, then increases in the relative levels of saturated and monounsaturated fatty acids in PG have been shown to increase the sensitivity of tobacco plants to low temperatures during the growth of young seedlings and maturation of reproductive organs [24]. As increases in the unsaturation of fatty acids result in decreases in biomembrane rigidification, then chemicals that enhance

it is based on ABA-dependent but not on SA-dependent defense mechanisms.

**2.7. Controlling cold tolerance by modifying membrane fluidity**

Nitrophenolates are biostimulants and are already being manufactured commercially in Japan under the name Atonik, a synthetic product composed of three phenolic compounds: sodium *p*-nitrophenolate (0.3%), sodium *o*-nitrophenolate (0.2%), and sodium 5-nitroguaiacolate (0.1%), together with water. Atonik has been used successfully for many years in the cultivation of most globally important crops. Its mode of action is still not understood but might be involved in hormone regulation, nutrient uptake, and nitrogen metabolism [26]. Atonik therefore stimulates plant growth and development and contributes to enhancing biomass accumulation, increasing water uptake, protecting against drought, and mitigating stress due to noble metals.

#### **2.9. Use of RSLVs as biostimulants**

As described in Section 2.1, RSLVs potentially act as signaling chemicals involved in heat and oxidative responses. A representative RSLV, 2-hexenal, is a green leaf volatile that induces gene expression in response to heat and oxidative stresses [12] and thus enhances thermotolerance in plants. Terada et al. suggest that this effect is partly explained by transpiration being sustained at higher temperatures [27]. The field use of 2-hexenal is being progressed commercially in Japan. 2-hexenal is a volatile; its vaporization from a tablet form by sublimation has enabled the effective concentrations for use in closed greenhouses to be determined. A preliminary examination showed that its application in greenhouses improved the production of crops such as tomato, strawberry, and cucumber in the summer (unpublished data), suggesting that its use as a biostimulant is effective in overcoming heat stress.
