An overview on the modulation of pesticide detoxification mechanism via salicylic acid in the plants

ABSTRACT

The continuous application of pesticides leads to several harmful effects on the ecosystem and get accumulated in the food chain. To regulate the toxicity of pesticides there are several strategies available. In relation to this, the endogenous as well as exogenous role of salicylic acid in pesticide regulation is less overviewed. To regulate the pesticide stress, in presence of salicylic acid, the genes, and proteins related to reduced glutathione (GSH) metabolism, biosynthesis of secondary metabolites, glyoxylate, and dicarboxylate metabolism get upregulated and are found to be more differentially expressed for pesticide detoxification. Salicylic acid regulates pesticide toxicity by activating gene expression of P450, antioxidant enzymes, ABC transporters subfamilies to form a defense network. In this context, the present review tries to comprehend the pesticide detoxification processes involving salicylic acid to regulate the stress caused thereby in plants and further utilize this strategy for wider application.

KEYWORDS:

• Pesticide
• growth
• agriculture crops
• phytohormones
• signaling molecules

1. Introduction

In the modern era, several agricultural production technologies including intensive and multiple cropping systems may intensify the incidence of disease and pathogen dissemination. Therefore, pesticide application is required to control such situations. However, pesticides affect plant biology by affecting numerous physiological traits and altering the plant’s defense mechanisms. The toxic effects of pesticides can be observed in plants in the form of chlorosis, necrosis, stunting, burning, and twisting of the leaves [1]. The absorbed pesticides even impair the plant vigor and also induce a threat to humans and other animals feeding on it [2].

The application of plant growth regulators is an effective method to reduce the toxicity of pesticides in plants. Among the different plant growth regulators, salicylic acid is found to play an important role in regulating biotic and abiotic stress in the plant [3,4]. The mechanism behind the protection through the salicylic acid pretreatment includes the development of stress resistance and the acceleration of growth process recovery. Complex processes like signal transduction, specific information pathways, and intra- or extra-cellular signals are responsible for the mechanistic action of salicylic acid to regulate pesticide stress [5]. Pesticide detoxification occurs in plants through activation, conjugation, and transportation/transformation processes. Salicylic acid facilitates pesticide detoxification by upregulating these processes. Thus, salicylic acid application can be one of the safe and easy methods for reducing pesticide toxicity and accumulation in plants. Yüzbaşıoğlu and Dalyan [6], reported that the salicylic acid (1 mM) pretreatment lowered the amount of H₂O₂ and MDA formation in thiram-treated Solanum lycopersicum Mill. It also increased the photosynthetic pigments, regulated the activity of antioxidative enzymes, and increased the activity and expression of pesticide detoxification enzymes to regulate thiram toxicity. Spormann et al. [7] reported that the growth inhibition due to glyphosate application was found to be improved with the application of salicylic acid (100 µM) in Hordeum vulgare L. Some of the important studies related to salicylic acid-mediated pesticide stress reduction have been given in Table 1. For abiotic stress mitigation the application of salicylic acid leads to transcriptional changes that regulate the level of mRNA to express the level of proteins involved in signaling and metabolic processes [5]. Proteomic and transcriptomic studies have disclosed the role of SA application in the upregulation of the expression of cytochrome genes (OsCYP-1, OsCYP-2, OsCYP-3), and transcripts of Glutathione-s-transferases (GSTs) subfamilies (GSTU19, GSTF10, GSTF9, and GSTF6) to mitigate the pesticide stress [8,9].

Table 1. The different ways to cope the pesticide stress by the exogenous application of salicylic acid.

S.No	Plant	Pesticide	Mode and Concentration of SA application	Results	Reference
1)	Beta vulgaris L.	Fomesafen	Foliar treatment (0.025–10 mM)	Exogenous SA alleviated pesticide stress and improved growth, biomass, photosynthesis, and activity of SOD, POD, CAT, PPO enzymes.	Li et al., [71].
2)	Cucumis sativus L.	Clothianidin, Dinotefuran, and, Difenoconazole	Root treatment (10 mg/L)	SA reduced the half-lives of the three pesticides and reduced their absorption in the roots and enhanced translocation from roots to leaves.	Liu [13].
3)	Triticum aestivum	Glyphosate	Foliar treatment (1 mM)	SA triggered antioxidant defense and restored plant growth in pesticide applied plant.	Shopova et al., [72].
4)	Cucumis sativus L.	Thiamethoxam Hymexazol Chlorantraniliprole	Soil treatment(1 mg/Kg) Nutrient solution(1 mg/L)	Exogenous SA promoted the degradation of pesticides, reduced their accumulation in roots and leaves. The alleviating effects of SA were more impactful under hydroponic system than in soil treatment.	Liu et al., [73]
5)	Glycine max Merr	Halosulfuron-methyl	Seed and foliar treatment (0.1 mM)	Pretreatment with SA improved the photosynthetic system, antioxidative defense system, reduced oxidative damage in HSM treated plants.	Li et al., [74].
6)	Oryza sativa	Isoproturon	Foliar treatment (5 mg/L)	SA resisted isoproturon toxicity and accelerated its degradation	Lu et al. [9].
7)	Solanum lycopersicum	Thiram	Foliar treatment (0.01–1 mM)	Regulated antioxidant activity, enhanced pesticide degradation by increasing GST, P450 levels, reduced H2O2 and MDA levels.	Yüzbaşıoğlu and Dalyan [6].
8)	Hordeum vulgare	Glyphosate	Root treatment (100 µM)	SA mitigated the effects of glyphosate on growth, reduced H2O2, AsA levels, increased non-protein thiol content, SOD, CAT, APX, GST enzyme activities.	Spormann et al. [7].
9)	Panax ginseng	Chlorotoluron	Root treatment (5 mM)	SA increased the expression of PgCYP736A12, which is involved in herbicide metabolism	Khanom et al. [38].
10)	Triticum aestivum L.	Fenoxaprop-p-ethyl	Seed treatment (0.5 mM SA)	Reversed the negative effects of the herbicide on antioxidant system, lipid peroxidation, pigment content, and total phenolics	Akbulut et al., [75].
11)	Vigna radiata (L.) Wilczek	Mancozeb, Chlorpyrifos, Metribuzin	Seed treatment (1 mM)	Seed treatment with salicylic acid together with pesticide minimized oxidative stress by improving the enzymatic biochemical parameters.	Fatma et al., [76].
12)	Triticum aestivum, Zea mays, and Brassica napus	Propazine	Foliar treatment (5 mg/L)	SA treatment reduced the accumulation of pesticide, increased chlorophyll content, reduced electrolytic leakage, reduced level of POD, and GST enzymes.	Zhang et al. [70].
13)	Arabidopsis	Isoproturon	Foliar treatment (5 mg/L)	SA application attenuated the toxicity in the plant, differentially expressed genes had its function in the phase I-III of the pesticide detoxification process.	Lu et al. [39].
14)	Oryza sativa ssp. Japonica	Quinclorac	Root treatment (10 mg/L)	Pre-treatment of SA triggered GSH, ALDH, GLO to degrade the herbicide.	Wang et al., [77]
15)	Triticum aestivum	Chlorpyrifos	Foliar treatment (1–16 mg/L)	SA blocked the accumulation of pesticide, regulated the activities of SOD, CAT	Wang and Zhang [78].
16)	Solanum lycopersicum	Glyphosate	seed and foliar treatment (1 mM)	Application of SA prior to pesticide increased chlorophyll content, protein levels, Nitrate reductase activity, reduced level of oxidative stress markers, increased CAT, and APX enzyme activities.	Singh et al., [79].
17)	Vicia faba	Glyphosate	Root treatment, Foliar treatment (50–100 µM)	SA inhibited the seed germination rate, and reduced levels of chlorophyll pigments, affected internal anatomy of chloroplasts, reduced protein, total phenol content, increased MDA production	Fayez and Ali [80].
18)	Oryza sativa	Quinclorac	Root treatment (10 mg/L)	SA increased plant biomass and chlorophyll content, decreased ROS content, modulated antioxidant enzymes and GSH concentration, downregulated ABA genes.	Wang et al., [77].
19)	Pisum sativum	Isoproturon	Soil treatment (1 mM)	SA administration alleviated the oxidative damage, pigment level, and enzyme activities increased to the level of control	Singh et al., [81]
20)	Zea mays	Glyphosate	Root treatment (0.5 mM)	SA treatment caused increase in SOD, POD, APX, GST while GR, GSH, CAT were inhibited.	Akbulut et al., [82].
21)	Helianthus annus	Quizalofop-P-ethyl	Seed treatment (0.5 mM)	Total phenolic, carotenoid, and chlorophyll got improved in SA pretreated plants	Bayram et al., [83].
22)	Triticum aestivum	Isoproturon	Foliar treatment (5 mg/L)	SA application increased the exudation of organic acids from the root, and reduced the level of isoproturon residues in the soil rhizosphere.	Lu et al. [11].
23)	Arachis hypogaea	Fusillade, Basagran	Foliar treatment (1 mM)	Increased content of total protein in herbicide treated plant.	Fayez et al., [84].
24)	Zea mays	Clethodim	Foliar treatment (1 mM)	Improved photosynthesis, gas exchange, increased protein, proline levels.	Radwan and Soltan [85].
25)	Triticum aestivum cv. Yangmai 13	Isoproturon	Foliar treatment (5 mg/L)	SA prevented generation of ROS, decreased antioxidant enzyme activity except for CAT, and prevented accumulation of pesticide	Liang et al., [86].
26)	Vigna mungo	Pendimethalin	Seed treatment (0.5 mM)	Combined application of SA and pendimethalin elevated growth of the seedlings, seed germination, mitotic index, pigment, protein, sugar content, and root length.	Singh et al., [87]
27)	Brassica napus L.	Napropamide	Root treatment (0.1 mM)	Decreased ROS generation, reduced activity of SOD, CAT, APX while increased the activity of POD, GST in pesticide exposed plants.	Cui et al. [48].
28)	Hordeum vulgare	Paraquat	Solution incubation (500 µM)	SA pre-treatment highly increased DHAR, POX, SOD, CAT activity.	Ananieva et al., [88]
29)	Hordeum vulgare	Paraquat	Solution incubation (500 µM)	Improved gas exchange parameters, chlorophyll content, reduced H2O2, MDA, electrolytic leakage, blocked the photosynthetic inhibition.	Ananieva et al., [89].

However, there is a further need to find the connection between the salicylic acid application and the signaling response in the plants to get the exact idea about the mechanism of pesticide stress alleviation. Hence, the current review is based upon the objectives like to get information regarding salicylic acid synthesis and signaling as well as its mechanistic action in regulating pesticide stress as an antidote and to explore the involvement of relevant genes and catalytic enzymes responsible for regulating the signaling response to detoxify the pesticide stress. It also includes the exact framework to apply salicylic acid at a wider scale in the natural system for regulating pesticide stress in different edible crops. Overall, it will try to explore the mechanistic action of salicylic acid to reduce the problem of pesticide accumulation in the food chain by reducing its uptake and toxicity.

2. Salicylic acid synthesis and signaling

Plant hormones or phytohormones are produced from various cells of plants and get transported through the vascular system of plants. Salicylic acid (SA) or ortho-hydroxybenzoic acid is an important phytohormone naturally produced in several plants. Wildermuth et al. [10] have reported that different plants have different levels of salicylic acid such as in Nicotiana tabacum, is less than 100 ng/g fresh weight, in Solanum tuberosum, it is up to 10 μg/g fresh weight, and in Arabidopsis thaliana, it varies from 0.25 to 1 μg/g of fresh weight. Salicylic acid is important in defense against pathogen attacks and other stress conditions in plants [11,12,13]. It is synthesized in plants by following two routes (Figure 1).

Figure 1. Biosynthesis (a), Transformation (b), and Induction (c) of Salicylic Acid in the plants. Chorismate is produced from the shikimate pathway in the plastid. The chorismate gets converted into isochorismate with the help of isochorismate synthase (ICS) that is later transported from the plastid to the cytoplasm by EDS5 protein for further processing. Chorismate can also convert into prephenate by chorismate mutase 1 (CM1) and further into arogenate by prephenate aminotransferases (PPA-ATs) and later to phenylalanine by arogenate dehydratase (ADT). Chorismate may get transported into the cytosol by an unknown transporter and convert into prephenate by chorismate mutase II (CM2) and finally into phenylpyruvate by prephenate dehydratase (PDT). The phenylpyruvate is converted into phenylalanine by phenylpyruvate aminotransferase (PPY-AT) enzymes. Further, the phenylalanine is transformed into trans-cinnamic acid (tCA) by phenylalanine ammonia Lyase (PAL). Salicylic acid is then synthesized from benzoic acid or o-coumaric acid with the help of benzoic acid 2- hydroxylase (BA2H). (b) After synthesis SA undergoes chemical modifications and produces different inactive forms like salicyloyl glucose ester (SGE), SA O-β-glucoside (SAG), and methyl salicylate O-β-glucoside (MeSAG). These inactive forms are stored in the vacuole and during the requirement under stress conditions get converted into active forms by hydrolysis. (c): Under stress conditions, the synthesis of SA gets enhanced with the binding of calmodulin (CaM)-with CaM-Binding Protein 60 g (Cbp60g) and its close homolog Systemic Acquired Resistance Deficient 1 (SARD1 or CBP60h). The WRKY transcription factor also to promote the salicylic acid production by translation of ICS1, EDS5 and PBS3 transcripts. The Figure is created using BioRender web server (www.BioRender.com).

2.1. Isochorismate pathway

In one pathway, chorismate gets converted into isochorismate in the presence of isochorismate synthase (ICS), which thereafter is formed to salicylic acid in the presence of isochorismate pyruvate lyase (IPL) [14]. Rekhter et al. [15] have also observed an alternative route for isochorismate processing in Arabidopsis plants. In this route (Figure 1), isochorismate-9-glutamate gets converted into SA via two steps by avrPphB Susceptible 3 enzyme (PBS3; known as Gretchen Hagen 3.12, GH 3.12) and EPS1 (Enhanced Pseudomonas Susceptibility), an acyltransferase [16]. The locations of both enzymes are different; ICS’s location is in the plastid, and PBS3 is in the cytoplasm. So, the isochorismate (IC) synthesized by ICS activity should be transported from the plastid to the cytoplasm for further processing. For this transportation, ENHANCED DISEASE SUSCEPTIBILITY 5 (EDS5) protein a MATE transporter is needed [17]. In rice, OsWRKY6 (transcription factor) is responsible for activating the OsICS gene, consequently leading to more formation of SA [18].

2.2. Phenylpropanoid pathway

Another pathway is the phenylpropanoid pathway dependent upon the activities of Phenylalanine ammonia-lyase (PAL) and chorismate mutase (CM), responsible for the conversion of prephenate into chorismate, and further into trans-cinnamic acid (tCA) [18] (Figure 1). The AIM1 (abnormal inflorescence meristem1), a multifunctional protein has been also reported in Arabidopsis plant as a key player of PAL pathway and it helps in the conversion of t-CA into benzoic acid (BA). In the last step (Figure 1), the BA is converted into SA, by benzoic acid hydroxylase (BA2H) [18].

2.3. Post-synthesis modifications

After synthesis the SA remains in two main forms: active free form and inactive forms like salicylic glucose ester (SGE), SA O-β-glucoside (SAG), and methyl salicylate O-β-glucoside (MeSAG) (Figure 1). These inactive forms are stored in the vacuole and during the requirement under stress conditions gets converted into active forms by hydrolysis [19]. In the presence of stress conditions, salicylic acid methylation leads to the formation of its more volatile form which showed more membrane permeability.

2.4. Signaling of salicylic acid

The process of SA perception is not fully understood. The most well-studied receptor that acts as a salicylic acid binding protein (SABP) is a non-expresser of pathogenesis-related protein 1; NPR1 (Figure 2). However, it has been reported that not only NPR1, there are other NPR1 paralogs like NPR2, NPR3, and NPR4 that also bind with SA [20]. Along with biotic stress and abiotic stress conditions, changes in the redox status of plant cells NPR1 monomerization occurs, and further the monomeric NPR1 shuttle into the nucleus to interact with transcription factors to regulate the stress condition [21]. Ding and Ding [22], have reported that a higher level of SA induces the monomerization process of NPR1 and further, by directly interacting with TGACG-binding (TGA) transcription factors, it enhances the NPR1-dependent gene expression to manage the stress condition in the plants (Figure 2).

Figure 2. Pesticide application increases the ROS formation that results in increased formation of transcription factors in the nucleus for the detoxification of pesticides. Pesticide detoxification occurs in three phases. The modified pesticide molecule is lastly stored in the vacuole or in the apoplast. The salicylic acid application increases the detoxification of the pesticides and reduces the oxidative damage in plants. The Figure is created using BioRender web server (www.BioRender.com).

The signaling of SA is mediated by two immune systems, pathogen-triggered immunity (PTI) and effectors-triggered immunity (ETI) [23]. The PTI is based upon the pattern recognition receptors (PRRs) that are the parts of plant innate immunity whereas the ETI is activated with the expression of effector resistance proteins (R-protein) known as nucleotide-binding leucine-rich repeats (NB-LRR), to start the defense action [24]. The R protein activates the shikimic acid pathway in the presence of pesticide stress to increase the SA levels to maintain the plasma membrane permeability by maintaining the level of Ca²⁺ in the cytosol (Figure 2). Increased SA interacts with Mitogen-Activated Protein Kinase(s) cascade to regulate ROS and Ca²⁺ levels in the cytosol (Figure 2). Increased SA activates signaling processes to upregulate the transcriptional regulators to start the defense gene expression in presence of stress conditions [25]. SA biosynthesis protects plants by improving the status of antioxidants and compatible solutes that are essential in managing adverse conditions.

Janda and Ruelland [14], have observed that in the presence of stress, the expression of Systemic Acquired Resistance (SAR) Deficient 1 (SARD1) and Calmodulin Binding Protein 60 g (CBP60g) transcription factors get enhanced and they bind with the ICT promoter region (GAAATTTTGG) to enhance the transcriptional regulation of ICS pathway (Figure 1). Under different environmental adverse conditions, the Enhanced Disease Susceptibility 5 (EDS1), a lipase-like protein interacts with Phytoalexin Deficient 4 (PAD4) or Sequence Associated Gene (SAG101) to form hetero-complexes to initiate the signaling of salicylic acid [26]. Under stress conditions, the expression of ICS1, EDS5, and PBS3 genes involved in the SA biosynthesis get induced through transcriptional regulation. In the plant system, the SA synthesis gets enhanced with the binding of calmodulin (CaM)-with CaM-Binding Protein 60 g (CBP60g) and its close homolog Systemic Acquired Resistance Deficient 1 (SARD1 or CBP60h) to promote the defense response by transcription of ICS1, EDS5 and PBS3 transcripts against stress condition [27]Figure 1).

With the help of in-vivo and in-vitro DNA-binding assays, including chromatin immunoprecipitation followed by quantitative PCR (ChIP-qPCR) and electrophoretic mobility shift assay, the ICS1 expression is also found to be regulated by WRKY TFs by binding directly to its promoter, at–445 and −460 base pairs upstream [28]. In both kinds of stress whether biotic or abiotic, oxidative stress is a common response. Therefore, the management of ROS homeostasis is the main target to regulate the status of stress in plants. To perceive the environmental stimuli to internal signaling pathways, the mitogen-activated protein kinase (MAPK or MPK) cascade maintains the level of ROS by increasing the synthesis of SA [29]. Under various abiotic stress including pesticide stress, the MAP kinase kinase kinase (MAP3K) gets activated and lead to the phosphorylation of MAP kinase kinases (MKKs) to further activate MAPKs to manage ROS homeostasis [30]. Due to common responses, the signaling pathways can cross-communicate with each other for SA signaling in the presence of both biotic and abiotic stress.

3. Role of salicylic acid in pesticide detoxification

Plants themselves have an intricate system to reduce the toxic effects of pesticides. The pesticide detoxification process in plants comprises several regulatory steps (Figure 2). Pesticides can be converted to more water-soluble and less toxic products through hydroxylation, dealkylation, oxidation, dehydrogenation, desaturation, oxygenation, epoxidation, etc [9,31]. Further, the toxicity gets decreased by conjugating with sugar, amino acid, or glutathione, and finally, the detoxified residue of the pesticide is transported from cytosol to the vacuole and/or apoplast [9]. Salicylic acid plays a significant role in pesticide detoxification at each step by controlling the signaling response of defense-related genes, which are discussed in detail by the following sub-headings:

3.1. Pesticide detoxification via cytochrome P450 mediated by salicylic acid

Cytochrome P450 is a superfamily of membrane-bound heme proteins found in plants and animals. It provides tolerance towards xenobiotic compounds including pesticides [32,33]. They are also involved in the biosynthesis of plant hormones, cell walls, and compounds that induce defense mechanisms or attract pollinators [34]. They catalyze several kinds of oxidation and reduction reactions including hydroxylation, oxygenation, dealkylation, desaturation, and epoxidation among several others. The pesticide detoxification process incorporates cytochrome P450 monooxygenase. The activity of cytochrome P450 monooxygenase comprises cytochrome P450 protein and NADPH-cytochrome P450 reductase (CPR). CPR helps in the transfer of electrons from NADPH to P450 monooxygenases responsible for lowering the toxic property of the pesticide through its oxygenation [35]. The author also observed inhibition in the breakdown of the herbicide 2,4-D by restricting the activity of cytochrome P450 using metyrapone (inhibitor of cytochrome P450). This suggested the crucial role of cytochrome P450 in degrading pesticides. Similarly, Torra et al. [36] have also reported, from studies in different plants, the significance of cytochrome P450 in 2,4-D degradation. The CYP76C subfamily of CYP450 when expressed in the whole plant of Arabidopsis enhanced the tolerance to chlorotoluron and isoproturon herbicides suggesting that the CYP450 gene family provides a suitable background for the evolution of herbicide resistance in plants [37].

The mechanism of pesticide resistance can be aggravated with the application of salicylic acid by regulating the expression of subfamilies of CYP450 protein. Yüzbaşıoğlu and Dalyan [6], observed the expression level of P450 to regulate thiram toxicity in tomato plants pre-treated with salicylic acid (1 mM). It has been observed that with the pre-treatment of salicylic acid, the relative expression level of P450 increased by 4–8 folds over to the control plants. Even after 11 days of thiram application, the application of salicylic acid could be able to increase the expression level of P450 by 50% more than the control plant. This suggests the role of salicylic acid in the enhancement of the pesticide detoxification mechanism. Khanom et al. [38] have reported that the mRNA levels of PgCYP736A12 (a subfamily of CYP 450) increased three-fold in the leaves of ginseng with the application of 5 mM salicylic acid, and further, this gene has been transferred to Arabidopsis plant and this transgenic line further showed enhancement in the tolerance against chlortoluron (3 µM). By using ultra-performance liquid chromatography-time of fight tandem-mass spectrometer/mass spectrometer (UPLC-TOF-MS/MS) Lu et al. [39] detected that the transcripts of CYP gene (AT3G28740) get induced in the presence of salicylic acid (5 mg/L) to regulate the toxicity of 2,4,6-trinitrotoluene, and munition hexahydro-1,3,5-trinitro-1,3,5-triazine in Arabidopsis. These studies provide information about the involvement of CYP450 in pesticide degradation in plants and the importance of salicylic acid in uplifting the action of CYP450.

The role of salicylic acid in pesticide degradation has also been studied in yeast cells by regulating the CYP genes [9]. It has been observed that transforming the three novel CYP genes (OsCYP-1, OsCYP-2, and OsCYP-3) into the yeast cells treated with salicylic acid (5 mg/L) showed upregulation in the degradation of isoproturon (IPU). The OsCYP-1, OsCYP-2, and OsCYP-3 transformed yeast cells showed enhancement in IPU degradation, respectively by 9.4%, 8.1%, 9.3% at 24 h, 13.0%, 12.5%, 13.6% at 48 h, and 16.3%, 14.7%, 17.8% at 72 h compared to the wild strains after the salicylic acid treatment. These findings convey the message that by mediating the expression of CYP members, salicylic acid helps in pesticide detoxification.

3.2. Pesticide detoxification by conjugation mediated by salicylic acid

The pesticide degradation by the CYP members makes the binding of sulfhydryl groups (GSH), sugars, amino acids, and organic acids to the pesticides more conducive [40]. The addition of these polar groups accelerates the further detoxification of the pesticides in the plant cell [41]. Wang et al. [42] have analyzed the importance of GSH against the pesticide stress in the presence of salicylic acid (10 mg/L). It lead to the binding of the pesticide and ultimately reduction in their level of toxicity. GSH functions as an antioxidant molecule and plays a role in enzymatic and non-enzymatic protection against ROS formation. The conjugation with GSH particularly plays an important role in regulating pesticide stress in plants. GSH replaces halogen, phenolate, or alkyl sulfoxide groups from the pesticides and hence decreases their toxic property. Hence, the increase in the GSH content protects the cells against the stress induced by pesticides. For pesticide detoxification glutathionylation mediated by GSTs is a common pathway [43–46]. The enzyme GST increases the rate of conjugation step of pesticide molecules with the GSH [47]. Salicylic acid has also been reported to regulate pesticide toxicity by regulating the GST enzyme [48]. The author reported that after exposure to napropamide (8 mg/kg), the activity of GST in Brassica napus gets enhanced suggesting its role in the detoxification of the herbicide. Pre-treatment with salicylic acid (0.1 mM) is reported to increase the activity of GST, indicating the importance of salicylic acid in enhancing resistance against herbicide toxicity in plants. Yüzbaşıoğlu and Dalyan [6], analyzed the expression of GST1, GST2, and GST3 genes to interpret their role in thiram detoxification. The author found that gene expression of GST2 was relatively higher than GST1, and GST3 in the thiram-treated plants, and pre-treatment with salicylic acid (1 mM) elevated the expression of GST1, GST2, and GST3 by 3 to 4 folds. However, the specificity and capacity of different isoenzymes of GST differ among different pesticides [49]. It has been observed that at low doses of Chlorothalonil (CHT), GST2 transcripts elevated while at its high concentration, transcript levels of GST1, and GST3 were increased. Mezzari et al. [50] also reported that the Arabidopsis plants treated with acetochlor, and metolachlor showed an increased transcriptional response of AtGSTF2, AtGSTU1, and AtGSTU24, which increased the activity of GSTs. Csiszár et al. [51] have reported that the application of salicylic acid (10⁻⁴ M) to modulate the toxic response in S. lycopersicum by inducing the expression of the GST gene family SlGSTT2, SlGSTT3, and SlGSTT4. Similarly, Li et al. [52] have also observed an increase in the expression of GST1, and GST2 in Triticum aestivum for regulating the stress response, with the application of salicylic acid (0.5 mM).

Six distinct sub-families (Phi, Zeta, Tau, Theta, Lambda, and dehydroascorbate reductase) of the plant GST genes have been identified that help in the detoxification of pesticides [48]. The Phi (GSTFs) and Tau (GSTUs) sub-families are reported to take part actively in detoxifying the pesticides in several plants [8]. The GSTFs transcripts are reported to be strongly induced in stress conditions and also after the application of phytohormones [53,54]. Further, the author identified the roles of these two sub-families in salicylic acid signaling to bring the defense response in the plant. The author characterized the response of the GST family towards SA application by gene-specific proteomic and transcript analysis against stress conditions. With the application of SA (0.1 mM), an increase in the transcript and protein abundance of four GST subfamilies (GSTU19, GSTF10, GSTF9, and GSTF6) have been reported that are involved in the pesticide detoxification. Among all the subfamilies GSTU19 (tau GST protein) accounts for more than 98% induction in the presence of the salicylic acid to provide the tolerance against the stress condition [55]. It suggests the importance of different sub-families of the GST enzyme to reduce pesticide toxicity by binding with salicylic acid.

Similarly, along with the GST, the glycosyltransferase (GTs) enzyme plays a significant role in promoting the conjugation of sugar with exogenous chemicals like pesticides in plants [56]. It reduces the level of pesticide toxicity by increasing substrate metabolism [57]. It has been reported that the overexpression of the GT gene helps in conferring tolerance against isoproturon (IPU), and acetochlor in transgenic rice plants [40]. Lu et al. [11,58] reported an increase in the glycosylation of isoproturon degradation products in wheat plants treated with SA (5 mg/L). Further, with the help of UPLC-MS the relative accumulation of IPU and its derivatives has been measured that showed a reduction in the accumulation of IPU in different plant parts compared to the control [11].

Besides glycosylation, pesticides may also undergo condensation reactions wherein amino/carboxyl groups are attached to the pesticide molecule. The condensation reaction between xenobiotics and amino acids mainly depends on the enzymes and co-factors taking part. As many as 13 conjugates have been reported to form with the metabolites of isoproturon and amino acids such as serine, alanine, glutamate, tryptophan, aspartic acid, and threonine [9]. Similarly, in soybean, Peterson et al. [59] identified conjugation of the herbicide 2,4-D with glutamic or aspartic acid. However, so far, only a limited number of studies have been reported on the condensation of pesticides in plants.

Processes such as acetylation, and methylation of pesticides are also responsible for reducing the toxicity of pesticides by making them more water-soluble. Acetylation is catalyzed by acetyltransferases utilizing the acetyl-coenzyme A. Lu et al. [9,24] have reported the importance of acetyltransferases in the reduction of the toxic impact of pesticides. The process of methylation is catalyzed by methyltransferases (MT) where S-adenosyl L-methionine (SAM) acts as the methyl donor [60,61]. The methylation process attenuates the polarity of a precursor metabolite causing a reduction in its excretion or mobility in the cells [62]. The importance of methylation of pesticides in plants for their detoxification has been studied by Liscombe et al. [60]. Lu et al. [24] have also confirmed that the activity of methyltransferase enzymes is responsible for providing tolerance against atrazine by identifying two O-Methyltransferase loci (LOC_Os04g09604 and LOC_Os11g15040) in rice plants.

Two other conjugation processes: hydroxylation and dealkylation are also involved in reducing the toxicity of the pesticides. It has been observed that in response to salicylic acid signaling also these processes are significantly able to reduce the IPU toxicity [9]. They have observed that mutant of rice plant (Ospal) which is unable to synthesize salicylic acid synthesizing enzyme, accumulated more IPU as compared to the wild one. Further, with the help of liquid chromatography-time of fight tandem-mass spectrometer/mass spectrometer (LC-Q-TOF-MS/MS) analysis, 32 IPU metabolites and conjugates were differentially detected between the Ospal mutant and wild-type. Here also, hydroxylation, dealkylation, and methylation processes are involved in the transformation of the different IPU metabolites. It suggests the involvement of salicylic acid in reducing the accumulation of pesticides in the plant [9].

3.3. Pesticide detoxification via compartmentalization mediated by salicylic acid

To provide the resistance against xenobiotics including pesticides, the ABC transporter subfamilies: pleiotropic drug resistance (PDR), multidrug resistance (MDR), and multidrug-resistant associated proteins (MRP) play a significant role in pesticide detoxification and their compartmentalization [63,64]. In the presence of pesticides the expression of genes that encode ABC transporters and their subfamilies get induced [24,65]. It is the largest protein family and most diverse i.e. present from bacteria to humans to provide tolerance towards foreign chemical substances in yeast, animal, and plant cells. These membrane transporters are prominently located in the tonoplast and the plasma membrane. The transportation and compartmentalization of the modified xenobiotic compounds in plants are carried out by ABC-type transporters.

It has been reported that in the vacuole the xenobiotics including pesticides get transported in their glutathione form (GS-X) [66]. The transportation of GS-X in the vacuole is confirmed to be mediated by the ABC transporter rather than through the proton motive force by using vanadate (an inhibitor of ABC-mediated transport processes). Frelet – Barrand et al. [67] reported that the subfamily of ABC transporters such as AtMRP2 and AtMRP3 are involved in transporting the glutathione and glucuronide conjugates of 1–chloro–2,4–dinitrobenzene to protect the cell from its toxicity. Similarly, Qiao et al. [68] have also observed that under four pesticides (ametryn, AME; bentazone, BNTZ; fomesafen, FSA; mesotrione, MTR) stress there were a large number of differentially expressed genes (DEGs) related to ABC transporters in rice plants to enhance their transportation and detoxification processes. The molecular docking emphasized the response of ABC transporters. The four ABC transporters OsABCB5, LOC_01g50100 for AME; OsABCG41, LOC_02g32690 for BNTZ; OsABCG48, LOC_11g37700 for FSA; OsABCG32, LOC_01g24010 showed high expression levels and act as pesticide binding-proteins [68].

Further, with the exogenous supplementation of salicylic acid (5 mg/L), Lu et al. [39] have identified that there was an enhancement in the degradation of IPU in Arabidopsis by comparing with their mutant lines pal-1, pal-2, and eps-1 (unable to synthesize salicylic acid). It has been observed that the application of salicylic acid (200 µM) induced the genes responsible for the PDR-subfamily of ABC transporters in soybean plants [69]. The subtractive suppression hybridization approach in soybean (Glycine max cv. Williams 82) revealed the transporter gene GmPDR12 to be induced by salicylic acid. Salicylic acid controls the transcriptional activation of GmPDR12, to regulate stress responses [69]. This gene was reported to express in response to the salicylic acid, and methyl jasmonate and starts accumulating shortly (nearly 30 min.) after salicylic acid application (200 µM). The RNA sequencing study has revealed that some of the differentially expressed genes (DEGs) related to ATP-binding cassette transporters (ABCs) get upregulated with the exogenous application of SA. So, by regulating the expression of different subfamilies of ABC transporters, salicylic acid signaling helps in pesticide detoxification. However, reports describing the function and mechanism of ABC transporters concerning pesticides are very few [70].

4. Conclusion

The application of pesticides are one of the major threats to the agricultural system. The accumulation of pesticides in the plants may lead to physiological hindrances and other health issues for consumers. The plants induce several signaling molecules including phytohormones to cope with such an adverse condition. With the help of overviewed studies, it has been observed that for foliar application the level of salicylic acid commonly applied in the range of 50 µM to 1 mM in most crops could act as a strong and potential tool in alleviating the toxic impact of pesticides. It affects metabolic functioning and secondary metabolite production to regulate the level of toxicity under pesticide stress. The exogenous application of salicylic acid may further modulate the stress resistance in the plant by regulating the expression of P450, antioxidative machinery, and ABC transporters subfamilies. In addition, with molecular dissection and interdisciplinary studies the specific pathway for pesticide detoxification could be further understood and its application could be promoted at a wider scale for sustainable agriculture.

Author contributions

The authors A Kumar and PK Yadav have equally contributed to writing the manuscript under the guidance of author A Singh. Another author, S Singh, has helped to finalize the manuscript’s figure and format.

Acknowledgments

The authors are grateful to the Head, Department of Botany, Institute of Science, Banaras Hindu University, Varanasi, for providing the necessary facilities. The Banaras Hindu University is also thankfully acknowledged for providing financial assistance to Dr. A. Singh as PI under the IoE seed grant scheme (Dev. IoE Scheme No. 6031). The authors P.K. Yadav and A. Kumar are also thankful to the UGC Non-Net Fellowship scheme (R/Dev./Sch./25437) and the CSIR–UGC, New Delhi (UGC-Ref. No.974/CSIR-UGC NET DEC.2017) repectively, for providing financial assistance.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Correction Statement

This article has been republished with minor changes. These changes do not impact the academic content of the article.

Additional information

Funding

The work was supported by the BHU Non-Net [25437]; IoE, B.H.U. [6031]; University Grant Commission, New Delhi [UGC-Ref.No. 974/CSIR-UGC NET DEC. 2017].