*Article* **Aluminum Stress Induces Irreversible Proteomic Changes in the Roots of the Sensitive but Not the Tolerant Genotype of Triticale Seedlings**

**Agnieszka Niedziela 1,\*, Lucyna Domzalska ˙ 2, Wioletta M. Dynkowska 1, Markéta Pernisová 3,4 and Krystyna Rybka 1,\***


**Abstract:** Triticale is a wheat–rye hybrid with a higher abiotic stress tolerance than wheat and is better adapted for cultivation in light-type soils, where aluminum ions are present as Al-complexes that are harmful to plants. The roots are the first plant organs to contact these ions and the inhibition of root growth is one of the first plant reactions. The proteomes of the root apices in Al-tolerant and -sensitive plants were investigated to compare their regeneration effects following stress. The materials used in this study consisted of seedlings of three triticale lines differing in Al3+ tolerance, first subjected to aluminum ion stress and then recovered. Two-dimensional electrophoresis (2-DE) was used for seedling root protein separation followed by differential spot analysis using liquid chromatography coupled to tandem mass spectrometry (LC-MS-MS/MS). The plants' tolerance to the stress was evaluated based on biometric screening of seedling root regrowth upon regeneration. Our results suggest that the Al-tolerant genotype can recover, without differentiation of proteome profiles, after stress relief, contrary to Al-sensitive genotypes that maintain the proteome modifications caused by unfavorable environments.

**Keywords:** acidic soils; abiotic stress tolerance; proteomic studies; two dimensional electrophoresis (2-DE); × *Triticosecale* Wittmack

### **1. Introduction**

Aluminum is the third most abundant element on earth, after oxygen and silicon. Its toxic effect in plants results from the physicochemical properties of common aluminum minerals, presented in the lithosphere as, for example: gibbsite and bauxite (hydroxylated Al-ions), kaolinite, or muscovite (hydrated complexes of aluminum and potassium). All minerals containing aluminum are insoluble at a neutral pH (6.5–7.0); hence, aluminum ions in such soils are biologically passive, non-available and thus non-harmful to plants. In acidic (pH 5.0–6.0) or very acidic (pH 4.0–5.5) soils, aluminum containing minerals can become soluble, releasing hydroxyl complexes of Al-ions in trivalent cationic forms, which are complexed in humus soils but picked up by plant roots from acidic sandy soils [1]. Since acidic soils constitute 30–40% of the world's arable land, with a constantly growing share due to anthropogenic impact, crop plants' aluminum tolerance is one of the features that affect higher/stable yielding in changing environments [2].

**Citation:** Niedziela, A.; Domzalska, ˙ L.; Dynkowska, W.M.; Pernisová, M.; Rybka, K. Aluminum Stress Induces Irreversible Proteomic Changes in the Roots of the Sensitive but Not the Tolerant Genotype of Triticale Seedlings. *Plants* **2022**, *11*, 165. https://doi.org/10.3390/ plants11020165

Academic Editors: Ewa Muszy ´nska, Kinga Dziurka and Mateusz Labudda

Received: 18 November 2021 Accepted: 5 January 2022 Published: 8 January 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Tolerance to Al-ions relies on the inhibition of Al uptake by the roots (external tolerance) and/or on the inhibition of transport to the aerial parts (internal tolerance). In fact, most of the tolerant crop plants are Al-exuders, avoiding the stress by prevention of ion intake into the symplast [3]. Their basic mechanisms are citrate, malate, and/or oxalate secretion into the rhizosphere for chelating Al-ions into non-absorbable complexes as well as a pH increase in the rhizosphere as an effect of plasma membrane H+-ATPase activity, higher in the roots of tolerant plants [4]. The base of the internal tolerance is Al-cation binding by negatively-charged carboxyl groups of the plant cell wall pectins in the root apex. Pectin content and the degree of methylation differentiate the Al tolerance response [5]. Another mechanism for Al tolerance is ion chelation in the cytosol and relocation to leaf vacuoles. Al-ions are present mostly in hydroxylated forms in the apoplast, whereas in symplast, they form complexes with sulfate, phosphate, and organic ligands [2]. When the tolerance mechanisms fail, the Al-ions gradually move inside the cells, misbalancing and eventually blocking ion channels, alternating lipid fluidity, and inducing changes in the cytoskeleton structure by binding to G-proteins and their substrates as well as to ATP-ases and nucleotide polyphosphate groups. Finally, the disruption of DNA synthesis and cell division in the root apex and the lateral roots is accompanied by increased rigidity of the cell walls and DNA double helix, which leads to rapid inhibition (within an hour) of root growth, even with micromolar Al3+ concentrations [2,6]. The biochemical consequences of aluminum intake are an increase in reactive oxygen species, increased fatty acid peroxidation, and inhibition of proton adenosine triphosphatase H+-ATPases. The displacement of Ca2+ in cell membranes contributes to Al3+ accumulation in the apoplast, stimulates callose synthesis, and finally inhibits intercellular transport. The decreasing concentration of Ca2+ in the cytosol alters the pH balance, which in turn interferes with sugar phosphorylases and the deposition of cell wall polysaccharides [2,6,7].

The genetic bases of Al tolerance concern ion chelation and ion transport. The gene families responsible for Al-ion exudation are ALMT (aluminum-activated malate transporter), responsible for malate, and MATE (multidrug and toxin efflux) for citrate exudation [8–10]. The transcriptional expression of ALMT and MATE is controlled by a master zinc-finger transcription factor, STOP1 (sensitive to proton rhizotoxicity 1) [9,11]. Internal Al tolerance is less well characterized. The NRAT1 (Nramp aluminum transporter 1) transporter has been identified as a putative Al transporter involved in rice's internal resistance mechanism, which lowers Al-ion concentrations in the root cell wall, transporting the ions inside the root cells for sequestration in vacuoles [12]. Those genes are conserved in numerous plant crop genomes [13]. The STOP1 transcription factor, in addition to several phytohormones, hydrogen peroxide, and other reactive oxygen species (ROS), take part in the upregulation of genes of the ALMT family or root growth inhibition. In addition, ROS detoxifying enzymes are activated to respond to aluminum ion stress, activating the gene network towards the induction of aluminum tolerance in plant tissues [13].

Hexaploid triticale, a rye–wheat hybrid species of wheat (AABB genomes) and rye (RR genome), is characterized by an intermediate tolerance between wheat considered as an Alsensitive and rye as an Al-tolerant parent. The beneficial influence of the rye genome was confirmed by Quantitative Trait Loci (QTL) localization on the 7R chromosome, explaining up to 36% of the phenotypic variance, including the malate transporter gene [14]. The other loci were found on chromosomes 3R [15,16], 4R, and 6R [15], without a recognized function in the triticale genome. Triticale's tolerance to Al-ions is less than that of rye, suggesting a suppressive effect of the wheat genome on the expression of rye genes.

In the present work, two-dimensional electrophoresis (2-DE) with immobilized pH gradients (IPGs), combined with protein identification by mass spectrometry (MS), was used to detect changes in the proteomes of triticale root tips after Al stress removal. This method has been successfully used to identify proteins involved in various stress responses in plants [17,18]. Al-responsive proteins identified in both monocots [19–22] and dicots [23] were functionally associated with cell division and structure, carbohydrate metabolism, antioxidant system, amino acid metabolism, protein degradation, signal transduction, and transporters [21,24,25]. The upregulation of the enzymes involved in cysteine and methionine metabolism, such as cysteine synthase, S-adenosylmethionine synthase, and O-methyltransferase, is a common response despite Al-ion tolerance [21–23,25,26]. These enzymes are required to maintain methyl cycling and glutathione metabolism, which are important mechanisms that lead Al detoxification [27]. The recent proteomic studies of soybean plasma membrane changes in response to Al-ions revealed about fifty different membrane traffic and transporter proteins [28].

To our knowledge, there are no reports concerning protein identification in triticale roots in the context of aluminum tolerance; however, the triticale root proteome response to drought was studied by Gr˛ebosz et al. [29]. For our comparative proteomic studies, we used the seedling root tips of triticale plants differing in Al tolerance subjected to recovery after the stress treatment.

#### **2. Results**

#### *2.1. Biometric and Biochemical Evaluation of Tested Materials*

The materials used in the studies were selected based on a biometric screening of 232 triticale lines in earlier published experiments [15] (Figure S1). One Al-tolerant line (L198, spring form) and two Al-sensitive lines (L17, spring form and L444, winter form) were chosen for the present studies. Root growth in response to 24 h of Al treatment was inhibited and in sensitive lines the root regrowth remained suppressed 48 h after the stress release, contrary to the roots of tolerant line, which resumed root growth after the stress release (Figure 1). Eriochrome cyanine R dye penetrates damaged or partially damaged root tips. The root regrowth ranged from 0.3 to 2.5 cm for individual seedlings of the tolerant line (Table 1). Additionally, the root apex redox potential of the seedlings released from Al stress was assessed in comparison with the control seedlings as a measure of the dynamics of the response to the Al stress removal [30]. Antioxidant capacity was determined using 2,2-diphenyl-1-picrylhydrazyl anion radical (DPPH\*−), and the cation radical 2,2 -azino-bis(3-ethyl benzothiazoline-6-sulphonic acid (ABTS\*+) [31]. According to one-way analysis of variance (ANOVA), no significant differences (with *p* ≤ 0.05) were detected between the control and stress treated seedlings in the case of reactions with DPPH\*− anion radical, despite a 20% difference in the L444 line between the control and Al-treated samples (Table 1). In the case of reactions with ABTS\*+, 13% higher activity, with statistical importance at *p* ≤ 0.05, was detected after the stress release in the case of the L444 line, and 10% (with no statistical importance) in the case of the L17 line. There were no differences in redox potential between the control and treated roots of the tolerant line L198 (Table 1).

**Table 1.** Root regrowth (cm) and antioxidant potential of triticale root tips from control seedlings and from seedlings 48 h after 16 (ppm) Al treatment. The total antioxidant capacity of tolerant (L198) and sensitive (L444 and L17) genotypes was expressed as the μmol Trolox equivalent antioxidant capacity (TEAC) per mg of root tip tissue.


a,b—statistically different mean values (*p* < 0.05); DPPH\*−—(2,2-diphenyl-1-picrylhydrazyl); ABTS\*+—(2,2 azinobis-(3-ethylbenzothiazoline-6-sulfonic acid).

**Figure 1.** Damaged regions of triticale seedling roots (denoted by arrows) stained with Eriochrome cyanine R after Al-ion treatment prior to recovery. After 48 h recovery, the purple root tips and no rooth regrowth were visible in case of the Al-sensitive lines (L444 and L17 (**A**,**B**), respectively), whereas the dark purple bands on regrown roots were detected in the case of the Al-tolerant line (L198 (**C**)).

#### *2.2. Two-Dimensional Electrophoresis (2-DE)*

The analysis of 2-DE gels revealed approximately 590 spots in each experimental and biological replication of both the control samples and those stress released after the Al-treatment (Table 2). Ninety-five percent of the protein spots were matched and quantified. Isoelectrophocusing using IPG strips of pH 3–10 revealed the protein spots at pI values in the range of 4.0–8.5 and protein masses between 6.5 and 95 kDa (Figure S2A–F, Table 3). In the tolerant line (L198), the proteomes of stress-subjected and control roots were not differentiated according to the established criteria (*p* ≤ 0.01 and difference in spot intensity ≥ two-fold). When the criterion of probability was weakened from 99% to 95%, only four differential protein spots were found (three spots as downregulated and one spot as upregulated). On the other hand, in the root tip proteomes of the Al-sensitive triticale lines, a higher number of differentiated protein spots were found. Regardless of the protein spot intensity, with a *p* ≤ 0.01 probability criterion, in total seventy-one differential protein spots were found in the L17 proteome (23 upregulated, 21 downregulated, 15 induced, and 12 silenced) and forty-three in the L444 proteome (23 upregulated, eight downregulated, three induced, and nine silenced). When the criterion of two-fold difference in spot intensity was added, 14 upregulated and eight downregulated spots were found in the L17 proteome. In L444, upon a double criterion (probability and spot intensity), 18 spots of upregulated proteins, exclusively, were found. For induced or silenced protein spots, a second criterion of spot intensity ≥ 0.2 was decided. In the proteome of L17, nine spots had a relative signal intensity > 0.2 among induced proteins, and three among those silenced, whereas in the proteome of L444 root tips, three induced and nine silenced protein spots were found (Table 2, Figure S3).



The highest, nearly 8-fold, difference in upregulated protein spot intensity was detected for spot #6, and a 5.2-fold decline for #24 were detected in the L17 proteome (Table 3, Figure S2B). In the L444 proteome, a spot identical to spot #6 was induced de novo in response to Al stress with a relative intensity of 0.26, and was numbered as spot #31 (Table 4, Figure S2D). Furthermore, seven of the differential proteins (numbered: 2, 4, 9, 12, 16, 17, and 18 in the proteome of L17 and 3, 8, 13, 14, 15, 19, and 21 in the proteome of L444) showed a more than two-fold increase in signal intensity in the L17 proteome and about a two-fold increase in the L444 proteome (Table 3, Figure S2B,D). Ten spots revealed significant differentiation (with *p* ≤ 0.01) for one of the two lines. Out of the eight protein spots of L17 that were downregulated with two-fold or higher intensities, none differed in this intensity in L444 (Table 3). This number of spots was counted according to the acute cut-off double criterion (probability *p* < 0.01 and intensity difference > 2); however, with weakened criteria and a single cut-off (probability *p* < 0.05), more than 80% of the protein spots were common for L17 and L444 lines. Since the spot identification was performed according to a cut-off by strong, double criteria, the weaker criteria data are not shown in detail (Tables 3 and 4).

#### *2.3. Identification of Differential Proteins*

The identification of differential proteins was carried out using liquid chromatography coupled to tandem mass spectrometry LC-MS-MS/MS system for 25 selected spots, with at least a two-fold change in intensity for at least one of the Al-sensitive lines (Table 3). Differential proteins from the spots marked as identical by the gel analysis software (Image Master 2D Platinum 7.0) were analyzed, except three upregulated protein spots (#2, #16, and #17) that represented both sensitive lines L17 and L444. The spots were extracted from the 2-DE gels followed by the separation of L17 and L444 root tip proteomes. Moreover, six induced and five silenced protein spots, with a signal intensity ≥ 0.2, were identified. In total, out of 36 protein spots, thirty-two represented 23 differentially expressed proteins, whereas three were unassigned (#24 and #25 from L17, and #36 from L444) (Tables 3 and 4). **Table 3.** Identification of differential protein spots from 2-DE gels obtained by separation of seedling root tip proteins extracted from Al-sensitive triticale lines, L17 and L444, after stress release. Protein spots were chosen according to the double criterion of *p* ≤ 0.01 and difference in spot intensity ≥ 2, and for induced or silenced proteins the criterion of relative spot intensity ≥ 0.2 was decided. Protein spots were detected using Image Master 2D Platinum 7.0 software, followed by MS-MS separation and further identification, characterization, and quantitation using Mascot Distiller v. 2.3 software.


<sup>1</sup> MP—matched peptides; <sup>2</sup> DIMBOA—4-hydroxy-7-methoxy-3,4-dihydro-2H-1,4-benzoxazin-2yl beta glucosidase;

\* n.s.—protein spot not significantly changed.

**Table 4.** Aluminum ion-responsive proteins from seedling roots of Al-sensitive triticale lines, L17 and L444, present in roots of control (proteins silenced upon Al3+) or in roots after the stress removal (proteins induced upon Al3+). Double cut-off criterion *<sup>p</sup>* <sup>≤</sup> 0.01 and relative spot intensity <sup>≥</sup> 0.2 were used. The relative intensity of protein spots on the gels are shown.


<sup>1</sup> MP—matched peptides; <sup>2</sup> relative spot intensity on the gel; (−) silenced; (+) induced; <sup>3</sup> DIMBOA—4-hydroxy-7-methoxy-3,4-dihydro-2H-1,4-benzoxazin-2yl beta glucosidase; \* n.s.—protein spot visible on the gel but not changed in a significant manner.

The identified proteins represented ten protein functional groups involved in cell division, protein folding, protein synthesis, stress-related response, metabolic pathways, lignin synthesis, transcription control, protease inhibition, protein degradation, and transport (Tables 3 and 4). The highest upregulation was detected for the protein folding (protein disulfide-isomerase), stress-related response (glutathione S-transferase, oxalate oxidase, 1-Cys peroxiredoxin PER1), and metabolic pathways (flavone O-methyltransferase 1). On the contrary, the downregulated proteins belonged to the cell division (tubulin) and metabolic pathways associated with amino acid metabolism and methylation control (adenosylhomocysteinase) as well as ascorbic acid biosynthesis (phosphomannomutase). The Protein-Protein Interaction Networks analysis (using STRING database) [32] revealed a functional network containing 19 nodes (flavone O-methyltransferase 1, oxalate oxidase, and serpin-Z1C were not connected) with 39 edges (vs. 34 expected) (Figure 2). We discovered two major proteins (1-Cys peroxiredoxin and phosphoglycerate kinase) with nine interactions in the network. Moreover, seven interactions were detected for ubiquitin.

**Figure 2.** Computational prediction of the functional network between differential proteins. The proteins used for analysis are presented in Tables 3 and 4. The number of connecting lines is in proportion to the amount of information about the protein interactions available. The line color indicates the type of interaction evidence. The explanation of symbols used in STRING database annotations are as follows: Traes\_4BS\_7AE61936D.1—oxalate oxidase; GST1—glutathione S-transferase; atp1— ATP synthase subunit alpha, mitochondrial; PER1— 1-Cys peroxiredoxin; Traes\_3B\_FC37FEAEE.2—protein disulfide-isomerase; U2AF65B—splicing factor U2af large subunit B; Traes\_1DS\_C327E495D.1—serpin-Z1C; Traes\_7DL\_6AC3E4622.2 eukaryotic initiation factor 4A; Traes\_1BL\_CB7AE51FA.1—calmodulin; Traes\_1AS\_36865F81C.2 ubiquitin; SHH—adenosylhomocysteinase; Traes\_1AL\_672A850FF.2—phosphoglycerate kinase, cytosolic; GLUD1—DIMBOA 1b, chloroplastic; TUBB3—tubulin beta-3 chain; TUBA tubulin alpha chain; Traes\_3AS\_D1E1079AA1—S-adenosylmethionine synthase; VDAC1 mitochondrial outer membrane porin; FBP—fructose-1,6-bisphosphatase; COR410—dehydrin COR410; rpoB—DNA-directed RNA polymerase subunit beta; OMT1—flavone O-methyltransferase 1; Traes\_2AL81CAF6C30.2—phosphomannomutase.

#### **3. Discussion**

#### *3.1. Evaluation of the Al Stress Response of Tolerant (L198) and Sensitive (L17, L444) Triticale Lines*

The earliest symptoms of Al toxicity concern the meristematic zone in the root apex [6,7,33], which results in the inhibition of root growth and, finally, in declined crop yield. Such root damages, confirmed by microscopic studies, have been described for many plant species [34,35]. Our experiment, performed in the frame of Al tolerance biometric phenotyping [36] developed for breeding selection purposes, also showed the inhibition of seedling root regrowth (Figure 1). This method enables one to distinguish the Al-tolerant genotypes, without

completely damaging the root meristems [37]. The test developed by Aniol [37] is broadly used in the breeding selection of cereal crops. Depending on the plant species, different concentrations of Al-ions are used [36]. For triticale, which is less tolerant than rye but much more tolerant than wheat, a 16 ppm ion concentration is common and allows for clear differentiation between tolerant, intermediate, and sensitive lines, among which the tolerant forms are in minority [14,36,38]. Recent studies by Szewi ´nska et al. [35], performed in line with the biometric phenotyping method, revealed that after the Al stress release, the epidermal cells of root tips in tolerant rye and triticale seedlings are replaced by new cells and the root growth is maintained [35], and this process is independent on organic acid exudation [39]. In our studies, the roots of Al-tolerant L198 triticale line regrew, and the proteomic data showed identity between the control and root tips recovered after the stress release. In contrast, the root tips of susceptible lines were permanently damaged and roots did not regrow, along with evidenced alterations between the control and stress-treated proteomes, which is in line with the literature data [24].

Additionally, the detected differences in the redox balance in root tips showed complete recovery in the case of the tolerant line after the stress release, and differentiation between the control and stress treated seedlings in the case of the susceptible lines [30]; however, these differences in the present experiment carried out according to the biometric test protocol were found only in the case of line L444 root extract reactions with the ABTS\*+ cation radical (Table 1). The proteomic data were analyzed with double, strong cut-off criterion (probability *p* ≤ 0.01 and difference in intensity ≥ 2), which influenced the small number of identities between the proteomes of L17 and L444. When the criterion was weakened and the intensity ratio was neglected, more than 80% of proteome patterns were common for L17 and L 444 lines (Table 2).

#### *3.2. Annotation of Protein Spots*

The flavone O-methyltransferase 1 (OMT1), with homology to the *Triticum aestivum* enzyme, was the strongest upregulated protein found in triticale root tips of the L17 sensitive line, whereas in L444 it was synthesized de novo. This enzyme catalyzes the sequential O-methylation of tricetin to mono-, di-, or trimethylated derivatives, with tricin, a dimethyl derivative, as a component found in monocotyledonous lignins [40]. Cell wall lignification along with hemicellulose deposition is an important and well-documented mechanism of plant tissue protection against harmful Al-ions, is positively correlated with the inhibition of root elongation, is more strongly expressed in sensitive genotypes [41], and was also detected in our experiment. *Ta*OMT1 was initially considered as a putative caffeic acid O-methyltransferase [42] involved in lignin biosynthesis; however, Zhou et al. [43] documented its low activity in methylation of lignin precursors such as caffeic and 5 hydroxyferulic acids. The next intensity difference was assigned to oxalate oxidase (OXO). Despite the fact that oxalate synthesis and degradation in plant cell walls is not clearly understood [44], it is specified as the enzyme oxidizing the oxalate to CO2 and H2O2. It was reported that an Al-induced increase in OXO was correlated with Al uptake, growth inhibition, damage of the plasma membrane, and disruption of membrane permeability in barley seedling roots [45]. The increased activity of OXO has been observed in the roots of barley [45] and wheat [21]. The increased concentration of H2O2 disturbs cell redox homeostasis, leading to the activation of stress response pathways on the one hand and apoptosis on the other. Delisle et al. [46] concluded that a high level of OXO expression may support trapping the Al-ions in the root cells rather than induction of H2O2-dependent cell death, which was observed in wheat epidermal cells after only 8 h exposure to Al. The other antioxidant enzymes, glutathione-S-transferase (GSH) and 1-Cys peroxiredoxin (PER1), were also found. GSH is known as a universal antioxidant and detoxifier, induced in response to various stresses [47]. It is one of the most common enzymes identified in protein and transcript analyses of different plant species exposed to Al stress, with increased activity in both Al-tolerant and -sensitive genotypes of soybean [23], flax [48], maize [49], *Arabidopsis* [27], and pea roots [50]. However, an unexpected suppression of GSH protein was also observed in tomato (−1.56-fold) and wheat (−2.5-fold) [21,43].

The identified proteins S-adenosylmethionine synthase and adenosylhomocysteinase are enzymes of methyl cycling. S-adenosylmethionine synthase (SAMS) catalyzes the formation of S-adenosylmethionine (SAM) from methionine and ATP. Adenosylhomocysteinase may play a key role in the control of methylations via regulation of the intracellular concentration of adenosylhomocysteine, an inhibitor of SAM-dependent methyl transferase reactions. In earlier proteomic studies, a dynamic induction of SAMS in Al-treated roots of wheat [21], tomato [26], and rice [25] was found. The same analysis showed downregulation of adenosylhomocysteinase in wheat [21]. It was proposed [51] that stimulation of SAM synthesis could be involved in the alteration of the cell wall and polymer structures in roots and/or ethylene-mediated inhibition of root growth. S-adenosylmethionine (SAM) may also serve as an important methyl donor for O-methyltrasferases (OMT), involved in lignin synthesis [52]. Due to the fact, that the synthesis of DIMBOA-Glc requires O-methylation catalyzed by O-methyltransferases with the presence of SAM as methyl donor, we speculate that methyl cycling also plays an important role in DIMBOA synthesis in triticale plants exposed to Al stress. The increase or de novo synthesis of DIMBOA (2,4- dihydroxy-7- methoxy-1,4- benzoxazin-3-one) glucosidases, GLU1b and GLU1c, in protein extracts from Al-sensitive root tips suggests that the hydrolysis of terminal, nonreducing beta-D-glucosyl residues releases the DIMBOA benzoxazinoid, a key defense compound, along with the DIBOA (2,4-dihydroxy-1,4-benzoxazin-3-one), present in major agricultural crops, such as maize and wheat, and biologically active in both the aboveground and underground parts of plants. Poschenrieder et al. [53] documented its role in maize root tip protection by chelating Al-ions in the rhizosphere, and Neal et al. [54] found their attractive function for *Pseudomonas putida* in the maize. The inhibition of root growth entails enhanced cell wall rigidity [7] and changes in the organization of cortical microtubules [55]. A significant decrease in α- and β-tubulins, the main components of microtubules, was observed in proteomes of both sensitive lines. Similar results were obtained for Al-sensitive maize [56] and rice [20] under Al stress. Interestingly, different subunits of tubulin were differentially expressed and changed dynamically in the Al-sensitive soybean [23].

A significant induction of several proteins involved in protein synthesis and degradation was observed as well. Among them, DNA-directed RNA polymerase subunit beta, which catalyzes the transcription of DNA into RNA, as well as splicing factor U2af large subunit B, necessary for the splicing of pre-mRNA, were upregulated. Moreover, we found a high induction of the eukaryotic initiation factor 4A (eIF4A), an ATP-dependent RNA helicase that is a subunit of the eukaryotic translation initiation factor 4F (eIF4F) complex involved in cap recognition, required for mRNA binding to ribosomes [57]. As aluminum stress affects the cellular gene expression machinery, it is evident that molecules involved in nucleic acid processing, including helicases, are likely to be affected in root tips [58]. The two eIF4As/helicases from pea have been shown to play a role in abiotic stress tolerance, especially for salinity and cold stress- [59–61]. The expression of *Pennisetum glaucum* eukaryotic translational initiation factor 4A exhibited superior growth performance and higher chlorophyll retention under simulated drought and salinity stresses compared to the control plants. Abiotic stress usually leads to protein unfolding, misfolding, and aggregation [62]. Protein disulfide isomerase-like proteins (PDIs) catalyze protein disulfide bonds, inhibit aggregation of misfolded proteins, and function in isomerization during protein folding in the endoplasmic reticulum and responses during abiotic stresses [63]. In triticale plants affected by aluminum, PDIs were found to be upregulated in both susceptible lines. PDIs from *Brachypodium distachyon* L., *Brassica rapa* ssp. *pekinensis*, and *Arabidopsis thaliana* were upregulated under abiotic stresses, such as drought or salt, as well as under the influence of abscisic acid (ABA), and hydrogen peroxide (H2O2) an reactive oxygen species, suggesting their involvement in multiple stress responses [62,64,65]. The activation of ubiquitin enzymes illustrates the proteolytic activity in response to Al stress. Ubiquitination

plays a critical role in protein inactivation, the degradation of damaged proteins, and the regulation of several mechanisms related to abiotic stress responses [66]. The enzymes of glycolysis and the tricarboxylic acid TCA cycle were activated as well, showing the influence of Al-ions on these main biochemical pathways. The phosphoglycerate kinase, which catalyzes the ADP-dependent dephosphorylation of 1,3-bisphospho glycerate to 3-bispsphoglycerete in glycolysis, was activated. We also observed upregulation of fructose-1,6-bisphosphatase, a key metabolic enzyme that catalyzes the reversible aldol cleavage of fructose-1,6-bisphosphate into glyceraldehyde-3-phosphate, either in glycolysis or gluconeogenesis and in the Calvin–Benson cycle [67]. Stimulating glycolysis in Al-treated plants may accelerate pyruvate and acetyl CoA production for organic acid synthesis, such as citrate or malate, which serve as Al chelators in the tolerant genotypes [7]. Moreover, acetyl-CoA may be used for the synthesis of malonyl-CoA, an essential substrate of fattyacid synthesis [68]. The regulation of lipid membrane composition and modification of membrane fluidity by changes in unsaturated fatty acid levels is an efficient barrier that prevents metals from entering to the symplasm [69]. The mechanisms of Al tolerance based on increasing the plasma membrane (PM) permeability by binding Al to negative sites on the PM surface of root cells have been well documented for numerous plant species [69–72]. A similar response of sensitive plants in the regeneration phase may suggest that plants still attempt to eliminate aluminum accumulated in root tips.

The other proteins, such as mitochondrial ATP synthase, mitochondrial outer membrane porin, calmodulin, and dehydrin COR410 were upregulated in susceptible triticale lines at 48h after Al treatment, which suggest their important role in the response to Al toxicity. Mitochondrial ATP synthase subunit alpha produces the energy storage molecule adenosine triphosphate (ATP), which is suggested to provide energy for active Al efflux and detoxification [22]. Mitochondrial outer membrane porin is responsible for forming a channel through the cell membrane that allows the passage of small molecules. This protein was upregulated in Al-susceptible triticale lines. The abundance change of these mitochondrion transport-related proteins under Al stress indicates that the ion/metabolite exchange between the mitochondria and cytosol was modulated in the roots to cope with the stress. It was also observed that Al induces calmodulin synthesis, a major sensory molecule that decodes Ca2+ signals in the presence of different biotic and abiotic stresses [73]. Dehydrins (DHNs) play an important protective role in plant cells during dehydration [74]; however, those containing relatively large amounts of reactive residues on their surface exhibit also reactive oxygen species (ROS) scavenging and metal ion binding properties. However, the role of dehydrin in Al stress has not been explained so far, though its documented properties may suggest a positive correlation with Al tolerance.

Our results indicate that seedlings of Al-tolerant genotypes can recover after 16 ppm Al3+ stress relief without differentiation of proteome profiles (according to criteria: *p* ≤ 0.01 and difference in spot intensity ≥ two-fold), contrary to seedlings of Al-sensitive genotypes that maintain the proteome modifications caused by unfavorable environments. Enzymes involved in cell wall lignification were highly induced whereas proteins involved in cell division were strongly downregulated.

#### **4. Materials and Methods**

#### *4.1. Plant Materials*

The experiments were performed using triticale inbred lines differing in aluminum (Al3+) stress tolerance: one Al-tolerant line, L198 (MAH3405 (Milewo) × Matejko), spring form, and two sensitive lines L17 (Gabo × 6944/97), spring form and L444 (MAH3198 × CHD2807/98-7-1), winter form. Seeds were obtained from Plant Breeding Strzelce Ltd., Experimental Station Małyszyn (Poland). The lines were highly homozygotic (F10 generation) and screened for Al tolerance annually in line with our previous and present projects [15,75].

The research was carried out using the common Al tolerance detection method developed by Anioł [37]. Seeds sterilized and germinated for one day to form a 3 mm sprout

were sown on polyethylene nets floated in a tray filled with a base medium of 2.0 CaCl2, 3.25 KNO3, 1.25 MgCl2, 0.5 (NH4)2SO4, and 0.2 NH4NO3, in mM concentrations, and a final pH of 4.5. After three days, the seedlings were transferred for 24 h onto the same medium containing 16 ppm Al3+ ions in the form of AlCl3. Next, after washing of Al3+ ions, seedlings were placed again into the base solution for 48 h to induce root regrowth. The roots of tolerant forms regrow in the opposite to the roots of sensitive forms. The control in this experiment was seedlings grown in medium without Al-ions [37]. The experiment was run in a growth chamber (Pol-Eko Aparatura, ST500 B40 FOT10) at 25 ◦C with a 12 h day/night photoperiod and a light intensity of 40 W·m−2. The seedlings' aluminum tolerance was assessed on the basis of the regrowth rate of roots stained prior to evaluation in 0.1% Eriochrome cyanine R within 10 min (Figure 1). For antioxidant activity estimation and proteomic analysis, the root tips (0.3–0.4 cm) from 7-day old days seedlings, both exposed and non-exposed to Al3+ ions, were excised. The root staining was omitted in this case. The results were based on four independent biological experiments.

#### *4.2. Antioxidant Potential Determination*

Antioxidant potential was determined using two radicals, stable anion radical DPPH\*− (2,2-diphenyl-1-picrylhydrazyl radical) and cation radical ABTS\*+ (2,2 -azino-bis (3-ethylbenzo thiazoline-6-sulphonic acid radical) (Sigma-Aldrich Ltd., Pozna ´n, Poland), and was expressed in (μmol/mg) of Trolox equivalents (6-hydroxy-2,5,7,8-tetramethychroman-2 carboxylic acid) (Sigma-Aldrich Ltd., Pozna ´n, Poland) [31]. A UV-2101PC UV-Vis scanning spectrophotometer (Shimadzu, Kioto, Japan) was used for absorbance measurements. The root tips were mashed into powder in liquid nitrogen, extracted in 80% methanol (MetOH) (100 mg/1 mL) at room temperature for 2h, and centrifuged. The reaction mixture consisted of 200 (μL) of the root extract and 3.2 (mL) of DPPH\*− in 80% MetOH (10 mg/25 mL). Absorbance was measured at 515 nm after 20 min. The ABTS\*<sup>+</sup> cation radical was prepared by oxidation of 7 mM ABTS water solution by 2.45 mM potassium persulfate overnight (16 h), at room temperature in the dark, and then dilution with 80% methanol to absorbance ca. 0.70 at 734 nm. The reaction mixture consisted of 50 μL of the root extract and 3.7 mL ABTS \*+, and the measurement was performed at 734 nm after 6 min.

One-way analysis of variance (ANOVA) was performed with Addinsoft 2020 XL-STAT (New York, NY, USA. https://www.xlstat.com, accessed on 20 December 2021). A Tukey HSD (honestly significant difference) multiple comparison test was used to identify statistically homogeneous subsets at α = 0.05.

#### *4.3. Proteomic Studies*

#### Phenol-SDS Buffer Extraction with Sonication (PSWS)

The phenol extraction of proteins was carried out as described by Hurkman and Tanaka [76]. Root tissue (300 mg) was ground in a mortar in the presence of liquid nitrogen and transferred to a 1.5 mL Eppendorf tube. Proteins were extracted with 3 mL of SDS buffer (30% sucrose, 2% SDS, 0.1 M Tris-Cl, 5% β-mercaptoethanol, and 1 mM phenylmethylsulfonyl fluoride (PMSF), pH 8.0) by triple sonication for 15 s at 60 amps. After sonication, 0.8 mL of Tris buffered phenol was added to the mixture and vortexed for 10 mins at 4 ◦C. The set was centrifuged at 14,000× *g* for 5 min at 4 ◦C, and the phenolic phase was collected and re-extracted with 0.8 mL SDS buffer and shaken for 5 min. Centrifugation was further repeated using the same settings, with the phenolic phase collected and precipitated overnight with four volumes of 0.1 M ammonium acetate in methanol at −20 ◦C. The precipitate obtained by centrifugation at 14,000× *g* for 10 min at 4 ◦C was washed thrice with cold 0.1 M ammonium acetate and finally with cold 80% acetone. The pellet was dried and resuspended in 100 μL of sample buffer (Biorad) and used for further analyses. Protein concentrations were quantified using the Bradford protein assay method, using BSA as a standard.

#### *4.4. Two-Dimensional Electrophoresis (2-DE)*

IPG strips (ReadyStripTMIPG, pH = 3–10, 17cm, Biorad) were passively rehydrated overnight with rehydration sample buffer (7M urea, 2 M thiourea, 4% CHAPS, 0.5% IPG Buffer, 20 mM DTT, 0.002% bromophenol blue) containing 250 μg of isolated protein. First-dimension

Isoelectric focusing (IEF) was conducted using the following parameters: step 1 gradient volt, 1000 V for 60 mins, step 2-gradient volt, 12,000 V for 60 min, step 3-constant volt, 12,000 V for 25,000 volt hours, and step 4-constant volt, 1000 V for 60 min. All steps were performed at 20 ◦C using IEF 100 (Hoefer Scientific Instruments, San Francisco, CA, USA). Following IEF, the strips were reduced with 130 mM DTT in 10 mL of equilibration buffer (29.3% glycerol, 75 mM Tris-Cl, 6 M urea, 2% SDS, pH 8.8) for 15 min and alkylated with 135 mM iodoacetamide in 10 mL equilibration buffer for 15 min. The 2-DE was performed according to the Laemmli [77] protocol in lab cast 1.5 mm 12.5% (w/v) polyacrylamide gels using a Hoefer SE 600 Chroma Vertical Electrophoresis System (Hoefer Scientific Instruments, San Francisco, CA, USA). The following program was implemented: 15 mA/gel for 15 min and 30 mA/gel for 90 min in Tris glycine-SDS running buffer. Three gels, one from each independent biological replication, were used for the identification of differential proteins. The gels were stained with 0.1% (w/v) Coomassie brilliant blue R-250 (Sigma-Aldrich Ltd., Pozna ´n, Poland) overnight, destained, and stored in 5% acetic acid at 4 ◦C for further analysis [78].

#### *4.5. Analysis of 2D PAGE Gel Images*

Stained gels were digitalized, annotated, and analyzed using Image Master 2D Platinum 7.0 software (GE Healthcare). Data were normalized by expressing abundance as relative volume (% vol). A difference in protein expression was accepted when the Student's *t*-test was at a significance level of 99% (*p* ≤ 0.01). Spots were only accepted as present or absent if they were present or missing in all four gels from control or treated material/groups. Moreover, in the case of spots appearing only in the control (silenced) or stressed (induced) roots, only those with a signal intensity value >0.2 were considered as significant. The gels obtained for both NT lines were compared visually for identification of the identical spots showing the highest signal intensity changes.

#### *4.6. Protein Identification by Mass Spectrometry and Database Search*

To identify the protein content in interesting spots, gel pieces were manually cut out and subjected to a standard procedure during which proteins were reduced with DTT, alkylated with iodoacetamide, and digested overnight with trypsin (Sequencing Grade Modified Trypsin, Promega, Madison, WI, USA). The analyses were made by the Mass Spectrometry Laboratory, Institute of Biochemistry and Biophysics Polish Academy of Science (MS Lab IBB-PAN, Warsaw, Poland). The peptide mixtures were analyzed by liquid chromatography coupled to tandem mass spectrometry (LC-MS-MS/MS) with a classic mass spectrometer and LTQ (linear trap quadrupole ion trap-Orbitrap) (Thermo Electron Corporation, San Jose, CA, USA). Briefly, the peptide mixture was applied to an RP-18 precolumn (nanoACQUITY Symmetry® C18, Waters, Milford, CT, USA) using water containing 0.1% (*m/v*) formic acid (FA) as a mobile phase and then transferred to a nano-HPLC RP-18 column (nanoACQUITY BEH C18, Waters) using an acetonitrile gradient (0–60%, *v/v*, in 120 min) in the presence of 0.05% (*m/v*) formic acid with a flow rate of 0.25 mm<sup>3</sup> min<sup>−</sup>1. The column outlet was directly coupled with the ion source of the spectrometer working in the regime of data dependent MS to MS/MS switch.

After pre-processing the raw data with the Mascot Distiller v. 2.6.1.0 software (Matrix Science, London, UK), the obtained peak lists were used to search the non-redundant protein database of the National Centre for Biotechnology Information (NCBI) using the Mascot search engine (v. 2.5.1, Matrix Science). The taxonomic category selected was *Triticum aestivum*. Only peptides passing a Mascot-defined expectation value of 0.05 were considered

as positive identifications [78]. The functional networks of differentially expressed proteins were constructed using the STRING database [32].

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/plants11020165/s1, Figure S1 The stages of the experiment: (A) triticale seeds germinating on the polyethylene grid tray; (B) 5th days old triticale seedlings; (C) the growth chamber view; Figure S2 Protein separation by 2-DE on gels stained in Coomassie Brilliant Blue (A-F). Proteome of L17 Al-sensitive line control (A) and 48h after Al stress treated (B); proteome of L444 Al-sensitive line control (C) and 48h after Al stress treated (D); proteome of L198 Al-tolerant line control (E) and 48h after Al stress treated (F). The differential protein spots, which were common for both studied Al-sensitive lines, are marked in red on gel pictures of L17 and L444 line (control vs. Al-treated). The differential protein spots, which were characteristic only for one studied Al-sensitive lines, are marked in green on gel pictures of L17 and L444 line (control vs. Al-treated). The Image Master 2D Platinum 7.0 software was used for differential spots identification; Figure S3 Comparison in: (A) number of up/down-regulated and silenced/induced proteins and (B) number of common protein spots according to established criterions (*p* ≤ 0.01 and difference in spot intensity ≥ 2-fold or 0.2 relative intensity of silenced/induced proteins).

**Author Contributions:** Conceptualization, A.N.; methodology, A.N., L.D., W.M.D. and K.R.; investigation, A.N. and L.D.; writing—original draft preparation, A.N.; writing—review and editing, K.R. and M.P.; visualization, A.N, L.D, W.M.D., M.P. and K.R. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Ministry of Agriculture and Rural Development, Poland, as statutory grant of IHAR-PIB No 1-1-03-2-01 and the European Regional Development Fund-Project "SINGING PLANT" (No. CZ.02.1.01/0.0/0.0/16\_026/0008446), which received a financial contribution from the Ministry of Education, Youths and Sports of the Czech Republic in the form of special support through the National Programme for Sustainability II funds.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data available upon request.

**Acknowledgments:** The laboratory equipment was purchased in frame of the grant founded by the National Centre of Research and Development, Poland: grant number NCBR-PBS3/B8/19/2015.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

#### **References**


### *Article* **Copper Tolerance and Accumulation on** *Pelargonium graveolens* **L'Hér. Grown in Hydroponic Culture**

**Antonios Chrysargyris 1, Rita Maggini 2, Luca Incrocci 2,\*, Alberto Pardossi <sup>2</sup> and Nikolaos Tzortzakis 1,\***


**Abstract:** Heavy metal contamination is a major health issue concerning the commercial production of medicinal and aromatic plants (MAPs) that are used for the extraction of bioactive molecules. Copper (Cu) is an anthropogenic contaminant that, at toxic levels, can accumulate in plant tissues, affecting plant growth and development. On the other hand, plant response to metal-induced stress may involve the synthesis and accumulation of beneficial secondary metabolites. In this study, hydroponically grown *Pelargonium graveolens* plants were exposed to different Cu concentrations in a nutrient solution (4, 25, 50, 100 μM) to evaluate the effects Cu toxicity on plant growth, mineral uptake and distribution in plants, some stress indicators, and the accumulation of bioactive secondary metabolites in leaf tissues. *P. graveolens* resulted in moderately tolerant Cu toxicity. At Cu concentrations up to 100 μM, biomass production was preserved and was accompanied by an increase in phenolics and antioxidant capacity. The metal contaminant was accumulated mainly in the roots. The leaf tissues of Cu-treated *P. graveolens* may be safely used for the extraction of bioactive molecules.

**Keywords:** antioxidants; bioaccumulation; copper toxicity; hydroponics; translocation factor

#### **1. Introduction**

Copper (Cu) is an abundant transition metal of the lithosphere that is considered a relevant anthropogenic contaminant, as large amounts of this element have been released into the environment over the past decades [1,2]. In addition to the environmental impact of mining and smelting operations, the extensive application of Cu-containing fertilizers, pesticides and fungicides in agricultural practices has contributed to water body and soil contamination [1–4]; therefore, agricultural soils are particularly exposed to pollution by this contaminant. For example, Chen et al. [5] reported that in China, over 16% of agricultural soil is contaminated by heavy metals, and 2% is polluted by Cu only. Among heavy metals, Cu is often the only contaminant in vineyards, where it is extensively used against downy mildew [1–4]. According to the European Council Directive 86/278/EEC [6] on the protection of the environment, the permitted Cu concentration in agricultural soils amended with sewage sludge is 50–140 mg kg−<sup>1</sup> for pH values in the range 6–7. For uncontaminated soils, Kabata-Pendias and Szteke [7] indicated a Cu concentration range of 1–140 mg kg−1, depending on soil texture; the same authors reported that soil Cu concentrations in the range 25–40 mg kg−<sup>1</sup> may be toxic to plants below pH 5.5, as Cu availability increases with soil acidity.

In nature, Cu commonly exists in the elemental metal form or as Cu<sup>+</sup> or Cu2+ ions, although the oxidation states +3 and +4 can also be found [8]. Due to its redox properties, Cu at low concentration has a fundamental biological role for all living organisms, taking part in several metabolic reactions [9,10]. In higher plants, Cu is an essential micronutrient that is necessary for normal growth and development [11], being involved in mineral nutrition and electron transfer reactions that occur in vital processes, such as respiration

**Citation:** Chrysargyris, A.; Maggini, R.; Incrocci, L.; Pardossi, A.; Tzortzakis, N. Copper Tolerance and Accumulation on *Pelargonium graveolens* L'Hér. Grown in Hydroponic Culture. *Plants* **2021**, *10*, 1663. https://doi.org/10.3390/ plants10081663

Academic Editors: Ewa Muszy ´nska, Kinga Dziurka and Mateusz Labudda

Received: 13 July 2021 Accepted: 6 August 2021 Published: 12 August 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

and photosynthesis, chlorophyll and primary metabolites biosynthesis, or the scavenging of radicals [4]. The Cu concentration range that is considered normal for plants ranges from 2–5 to 30 mg kg−<sup>1</sup> dry weight (DW) [12,13], while at higher concentrations Cu can cause toxicity symptoms [14]. Toxic levels of Cu in plants can impair biochemical reactions, affect gas exchanges, reduce plant growth [15,16]. Plants grown in Cu-polluted soils undergo oxidative stress and accumulate reactive oxygen species (ROS) [17], which induces the activation of antioxidant enzymes and the biosynthesis of antioxidant molecules. Cu toxicity has also been associated with an increased content of proline in plant tissues [18,19].

To counteract the effects of metal toxicity, plants have developed tolerance mechanisms such as metal complexation, storage in vacuoles, precipitation in cell walls, and downregulation of metal transporters via the plasma membrane [20,21]. On the other hand, plant species capable of effectively up taking heavy metals from the soil and accumulating these contaminants in tissues generally show a high translocation rate from roots to shoots. These species can be profitably used for phytoremediation through the removal of toxic metals from the soil, which represents a green and cost-effective strategy for the amelioration of marginal lands [22,23]. According to recent literature [2], about 500 species are currently used for the phytoremediation of metal polluted soils. Conversely, the accumulation of toxic metals by plant species that are employed for human usage represents a serious threat for the consumers safety and has become a health concern worldwide. Medicinal and aromatic plants (MAPs) are typically used in the food, pharmaceutical and cosmetic industries as a natural source of biologically active compounds [24], and are increasingly cultivated on a commercial scale to sustain the expansion of the market demand. Contamination of the plant material is a major health issue concerning the commercial production of MAPs [25]. On the other hand, the physiological markers of plant response to metal-induced stress are often beneficial bioactive secondary metabolites, mainly antioxidants such as phenolic compounds or essential oils constituents [26–29].

The *Pelargonium* genus in the family Geraniaceae comprises several hundreds of aromatic species, distributed worldwide in subtropical and temperate regions [30]. The essential oil from *Pelargonium* spp. is among the top 20 essential oils used all over the world [31] due to its well-known bioactive properties [32–34]. In addition, the pharmacological activity of *Pelargonium* spp. is attributed also to phenolic constituents such as flavonoids and hydroxycinnamic acid-derivatives [35]. *Pelargomium* spp. are tolerant to toxicity by heavy metals and have been successfully applied as hyperaccumulators for several metal contaminants [36], including Cu [37]. Particularly, *Pelargomium graveolens* L'Hér., popularly known as rose-scented geranium, has been reported by several authors as a good candidate for phytoremediation; in addition, the effect of heavy metals on the yield and quality of its essential oil has been widely investigated [38–40]. However, in recent years the pharmacological activity of *P. graveolens* has been increasingly linked also to the leaf content and composition of the pool of antioxidant phenolics [27,41–44], and much less is known about the influence of heavy metals on the concentration of these compounds. Therefore, the aim of the present study was to verify the tolerance of *P. graveolens* to Cu toxicity and to test the hypothesis that Cu-induced stress could stimulate the synthesis of antioxidant phenolic constituents, thus improving the medicinal properties of this species. With these objectives, we evaluated the effects of Cu exposure in *P. graveolens*, in terms of metal translocation to different plant organs, plant growth, and synthesis of bioactive phenolic metabolites.

#### **2. Results**

#### *2.1. Visible Injury and Plant Growth*

The plants appeared healthy during the whole growing cycle. Typical toxicity symptoms, such as leaf chlorosis and necrosis, were not observed in Cu-treated plants. Two-way ANOVA revealed that sampling date (D) significantly (*p* < 0.05; *p* < 0.001) affected the number of leaves produced and total upper fresh biomass, while neither Cu nor the interaction of date x Cu (D x Cu) affected the plant height, leaf number and total upper fresh biomass

and dry matter content (Table 1). Copper concentration in the nutrient solution affected pelargonium growth parameters (Table 1). Plants grown with ≥25 μM Cu in the nutrient solution produced lower number of leaves at 35 DAT (days after transplanting), but this effect did not persist at 49 DAT. Total upper fresh biomass (including leaves, petioles and stems) decreased at the highest Cu (100 μM Cu) levels compared with the plants grown at 25 μM or 50 μM Cu after 35 DAT. Dry matter content at 49 DAT increased in plants grown in ≥50 μM Cu compared to 25 μM Cu and control treatment.

**Table 1.** Effect of increasing copper (Cu) concentration (4–25–50–100 μM Cu+2) in the nutrient solution and sampling date after transplanting (DAT, 35 days and 49 days) on plant height (cm), leaf number, total upper fresh biomass (g plant−1), and biomass dry matter content (%) in pelargonium plants grown hydroponically in perlite.


<sup>Y</sup> At each sampling date, values (*n* = 6) in columns followed by different letters are significantly different, *p* < 0.05, for each plant growth stage. *ns*, \* and \*\*\* indicate non-significant or significant differences at *p* < 5%, and 0.1%, respectively, following two-way ANOVA.

> Looking at the fresh and dry biomass of individual plant organs, it was found that leaves and stems were increased at 35 DAT at 25–50 μM Cu compared to 100 μM Cu (Figure 1). Petiole fresh weight (FW) was also increased at 25–50 μM Cu compared with control or 100 μM Cu. Copper levels did not affect the root FW at 35 DAT. Following 49 DAT, leaf, stem, petiole and root FW were at similar levels (averages of 64.24, 26.13, 60.48 and 14.44 g, respectively), independent of the Cu concentration in the nutrient solution. Petiole dry matter content increased in 25–50 μM Cu, compared to the control treatment at 35 DAT. However, root dry matter content increased in 100 μM Cu compared to 25 μM Cu at 49 DAT.

#### *2.2. Effects on Plant Physiology Attributes*

Plants grown at the high Cu concentration of 100 μM Cu revealed higher stomatal resistance at both 35 and 49 DAT, compared to the control treatment (Table 2). Contrarily, chlorophyll fluorescence as measured by Fv/Fm (representing the maximum quantum yield of PSII), decreased at 100 μM Cu when compared to control and/or 25 μM Cu at 35 DAT and 49 DAT. The content of chlorophylls, as measured by chlorophyll a, chlorophyll b and total chlorophylls, did not change among the treatments at 35 DAT, but decreased at higher Cu levels (i.e., 100 μM Cu) at 49 DAT compared to the control and/or 25 μM Cu.

Two-way ANOVA revealed that sampling date (D) significantly affected stomatal conductivity and chlorophyll fluorescence (*p* < 0.001); copper levels significantly affected stomatal conductance and chlorophyll a (*p* < 0.05), while the interaction of sampling date and copper (D x Cu) did not affect the examined physiological parameters (Table 2).

**Figure 1.** Effect of increasing copper (Cu) concentration (4–25–50–100 μM Cu2+) in the nutrient solution and sampling date after transplanting (DAT, 35 days and 49 days) on the fresh (FW; g plant<sup>−</sup>1) and dry weight (DW; g plant−1) of leaves, stems, petioles and roots respectively, of pelargonium plants grown hydroponically in perlite. Significant differences (*p* < 0.05) among Cu concentrations for each plant tissue are indicated by different letters; ns indicates non-significant. Error bars show SE (*n* = 6).

#### *2.3. Effects on Total Phenols, Flavonoids and Antioxidant Activity*

Two-way ANOVA revealed that sampling dates (35 vs. 49 DAT) significantly effected total phenols and DPPH (2,2-diphenyl-1-picrylhydrazyl) (*p* < 0.01), Cu levels significantly effected ABTS (2,2 -azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (*p* < 0.05) and flavonoids (*p* < 0.01), while the interaction of the sampling date x Cu effected ABTS and flavonoids (*p* < 0.05) and total phenolics (*p* < 0.01). The content of flavonoids and antioxidant activity (as assayed by ferric reducing antioxidant power; FRAP, DPPH, ABTS) revealed their highest values at 50 μM Cu, when compared with control and 100 μM of Cu, and differed significantly also from 25 μM Cu in the case of flavonoids and DPPH at 35 DAT (Figure 2). The content of total phenols and flavonoids, as well as antioxidant activity as assayed by FRAP and ABTS, revealed an increased trend as the Cu level increased at 49 DAT, with significant differences at the high Cu levels compared to the control treatment (Figure 2A–C,E).


**Table 2.** Effect of increasing copper (Cu) concentration (4–25–50–100 μM Cu2+) in the nutrient solution and sampling date after transplanting-DAT (35 days and 49 days) on leaf stomatal resistance (cm s−1), chlorophyll fluorescence (Fv/Fm), chlorophylls (Chl a, Chl b, Total Chl) content (μg g−<sup>1</sup> fresh weight) in pelargonium plants grown hydroponically in perlite.

<sup>Y</sup> At each sampling date, values (*n* = 6) in columns followed by different letters are significantly different, *p* < 0.05, for each plant growth stage. *ns*, \* and \*\*\* indicate non-significant or significant differences at *p* < 5%, and 0.1%, respectively, following two-way ANOVA.

#### *2.4. Plant Stress Indices*

Two-way ANOVA revealed that sampling dates (35 vs. 49 DAT), Cu levels and their interactions significantly affected hydrogen peroxide (H2O2) and malondialdehyde (MDA) levels (*p* < 0.01, *p* < 0.001). Hydrogen peroxide levels increased at 25 μM Cu in comparison to the 50–100 μM Cu, but did not differ from the control at 35 DAT (Figure 3A). Following 49 DAT, H2O2 increased at 100 μM Cu compared to lower Cu levels and/or control. Lipid peroxidation (as assayed by MDA) increased at 50 μM Cu in comparison to higher or lower Cu levels at 35 DAT, while MDA decreased with ≥25 μM Cu compared to the control treatment (at 4 μM Cu) at 49 DAT (Figure 3B).

#### **Figure 2.** *Cont*.

**Figure 2.** Effect of increasing copper (Cu) concentration (4–25–50–100 μM Cu2+) in the nutrient solution and sampling date after transplanting (DAT, 35 days and 49 days) on the leaf content of total phenols, total flavonoids and antioxidant activity in pelargonium plants grown hydroponically in perlite. (**A**) Total phenols, (**B**) total flavonoids, (**C**) FRAP (**D**) DPPH, and (**E**) ABTS. Significant differences (*p* < 0.05) among Cu concentrations at each sampling date are indicated by different letters; ns indicates non-significant. Error bars show SE (*n* = 4).

**Figure 3.** Effect of increasing copper (Cu) concentration (4–25–50–100 μM Cu2+) in the nutrient solution and sampling date after transplanting (DAT, 35 days and 49 days) on the leaf content of hydrogen peroxide (H2O2; (**A**)) and malondialdehyde (MDA; (**B**)) in pelargonium plants grown hydroponically in perlite. Significant differences (*p* < 0.05) among Cu concentrations at each sampling date are indicated by different letters. Error bars show SE (*n* = 4).

#### *2.5. Copper Content in Plant Tissues*

Two-way ANOVA revealed that sampling date (D) significantly affected AR, BACroots, BAC-stems, TF-leaves, TF-stems, TF-petioles (*p* < 0.001), and BAC-petioles (*p* < 0.05); Cu levels significantly affected BAC-roots, BAC-leaves, BAC-stems, BAC-petioles, TFleaves, TF-stems, and TF-petioles (*p* < 0.001), while the interaction of sampling date and Cu (D x Cu) affected BAC-roots, BAC-stems, BAC-petioles, TF-leaves, TF-stems and TFpetioles (*p* < 0.001) (Table 3). The copper accumulation rate increased at 50 μM of Cu compared with the control at 35 DAT. All bioaccumulation coefficients and translocation factors for leaves, stems, petioles and roots were significantly decreased with ≥25 μM of Cu in the nutrient solution at 35 and 49 DAT (Table 3).

Regarding tolerance index, two-way ANOVA revealed that sampling date (D) significantly affected TI-petiole FW and TI-petiole DW (*p* < 0.001); copper levels significantly affected TI-total biomass, TI-stem FW and TI-petiole FW (*p* < 0.05); while the interaction of sampling date and Cu (D x Cu) affected only the TI-petiole FW (*p* < 0.05) (Table 4). Tolerance index values of plant growth were affected at 35 DAT, as TI increased at 25–50 μM Cu for leaf, stem and petiole FW and as a consequence of the plant total biomass when compared with 100 μM Cu in the nutrient solution (Table 4). Similarly, TI-leaf DW and TI-petiole DW were also increased at 20–50 μM Cu. The TI of leaf number was decreased with ≥25 μM Cu in the nutrient solution at 35 DAT. At 49 DAT, the TI for total biomass was increased with 25 μM Cu in the nutrient solution, while TI-root DW increased at 100 μM Cu when compared to ≤25 μM Cu (Table 4).

#### *2.6. Responses of Other Nutrients*

The accumulation of nutrients in different plant organs (leaves, stems, petioles and roots) under different Cu levels at two sampling periods (35 and 49 DAT) is described in Figures 4 and 5. At 35 DAT, the leaf content of N and K increased in 25 μM Cu and decreased or remained unaffected in ≥50 μM Cu compared to the control (Figure 4A,C). Stem N decreased at 100 μM Cu when compared with 25 μM Cu, however the N level in petioles and roots remained similar in plants grown with different Cu levels in the nutrient solution (Figure 4A). Leaf and stem N levels were similar at 49 DAT in all examined Cu levels in the nutrient solution (Figure 4B). Petiole K increased in 25–50 μM Cu compared to the control (Figure 4C). Phosphorus content in leaves, stems and petioles was unaffected by the Cu levels in the nutrient solution, while P in roots decreased at 50 μM Cu and increased at 100 μM Cu compared to the control treatment (Figure 4E). Increased P levels were found at 49 DAT in roots at 50 μM Cu (Figure 4F). Sodium accumulated more in petioles, compared to leaves, stems and roots, while Na content decreased at high Cu levels (Figure 4G,H).

Copper accumulated in stems, petioles and roots as the Cu concentration increased in the nutrient solution; at 35 DAT, greater effects were observed in roots (2.2-fold increase at 100 μM Cu compared to the control treatment) (Figure 5A). A similar trend was found in Cu accumulation even at 49 DAT, but the increment in roots at 50–100 μM Cu was 6.9-fold greater compared with the control treatment (Figure 5B). Zinc accumulated more in leaves, stems, and petioles at 35 DAT as Cu levels increased in the nutrient solution, whereas Zn content decreased in roots with increasing Cu concentration in the nutrient solution (Figure 5C). However, the reverse was evidenced at 49 DAT, as Zn accumulated in roots following increases of Cu levels in the nutrient solution (Figure 5D).




*D x Cu ns ns ns ns ns ns ns ns ns ns* Y At each sampling date, values (*<sup>n</sup>* = 6) in columns followed by different letters are significantly different, *p* < 0.05, for each plant growth stage. *ns*, \* and \*\*\* indicate non-significant or significant differences at *p*<5%,and0.1%,respectively,followingtwo-wayANOVA.

**49 days**

**25** **50** **100**

*Significance*

*Days (D)*

*Copper (Cu)*

*ns*

\*

 *ns* *ns*

 *ns*

 *ns*

 *ns*

 *ns*

 *ns* \*

\*\*\*

 \* \*

*ns*

*ns*

 *ns*

 *ns*

 *ns*

 *ns*

 *ns*

\*\*\*

*ns*

 *ns*

89.36 ± 15.10 b 106.41 ± 4.20

104.63 ± 6.51 b 103.45 ± 17.39

219.37 ± 77.36 a 94.33 ± 9.80

 72.12 ± 11.84

 72.98 ± 14.42

 91.03 ± 8.03

 92.36 ± 14.09

 96.47 ± 5.56

 85.44 ± 14.69

 100.74 ± 12.76

 102.19 ± 8.24

 83.03 ± 12.44

 91.83 ± 12.71

 92.66 ± 6.20

 79.57 ± 10.38

 94.11 ± 3.02

 86.56 ± 10.47

 98.23 ± 1.84

 89.62 ± 14.77

 98.95 ± 5.91

 87.38 ± 15.64

 103.03 ± 13.16

 115.32 ± 7.77

 93.41 ± 15.21

 91.55 ± 14.43

 101.20 ± 5.87 106.55 ± 13.72 ab

 87.41 ± 12.84

 124.10 ± 1.03 a

 92.56 ± 1.19 b

#### *2.7. Regression Analysis*

Pearson's correlation coefficients were determined between individual pairs of parameters associated with Cu uptake (leaf and root Cu concentrations), leaf antioxidant systems (content of total phenols and flavonoids, antioxidant capacity according to FRAP, DPPH and ABTS assays), and oxidative stress (H2O2 and MDA) (Table 5).

Leaf Cu concentration was positively correlated to the root content of the element at both sampling dates. The correlation coefficients between leaf or root Cu and the biochemical parameters were generally higher at 49 than 35 DAT, and in older plants all correlations were positive except those involving MDA content.

**Figure 4.** *Cont*.

**Figure 4.** Effect of increasing copper (Cu) concentration (4–25–50–100 μM Cu2+) in the nutriment solution and sampling date after transplanting (DAT, 35 days and 49 days) on the content of macronutrients and sodium in different organs of pelargonium plants grown hydroponically in perlite. (**A,B**) Nitrogen–N, (**C,D**) potassium–K, (**E,F**) phosphorus–P, (**G,H**) sodium–Na. Significant differences (*p* < 0.05) among Cu concentrations at each sampling date are indicated by different letters; ns indicates non-significant. Error bars show SE (*n* = 4).

**Figure 5.** Effect of increasing copper (Cu) concentration (4–25–50–100 μM Cu2+) in the nutriment solution and sampling date after transplanting (DAT, 35 days and 49 days) on the content of micronutrients in different organs of pelargonium plants grown hydroponically in perlite. (**A,B**) Copper–Cu, and (**C,D**) zinc–Zn. Significant differences (*p* < 0.05) among Cu concentrations at each sampling date are indicated by different letters; ns indicates non-significant. Error bars show SE (*n* = 4).


**Table 5.** Pearson's correlation table for leaf and root content of copper (Cu), leaf content of total phenols, flavonoids, H2O2 and malondialdehyde (MDA), and leaf antioxidant capacity determined using FRAP, DPPH or ABTS assays, in pelargonium plants grown hydroponically in perlite and exposed to four different Cu concentrations (4–25–50–100 μM Cu2+) in the nutrient solution, sampled at 35 and 49 days after transplanting (DAT).

#### **3. Discussion**

Plant growth and development are regulated by plant physiology, which in turn is tightly linked to both environmental conditions and, in hydroponic cultivation, to the composition of the nutrient solution that is supplied to the plants [45,46]. For evaluation of the effects of Cu toxicity, the choice of appropriate Cu levels depends on the species being tested, the duration of the Cu treatment, and other growing parameters such as the pH of the nutrient solution. In this study, hydroponically grown plants of *P. graveolens* were exposed to Cu concentrations of up to 100 μM. Similar concentrations were tested in several species including Moso bamboo [5], maize [15], or *Carthamus tinctorius* L. [1]. Although higher Cu levels have been reported for turfgrass (120 μM) [16], and particularly for tomato (250 or 350 μM) [47,48], lower concentrations (up to 40 μM) were employed for both tree species [49] or vegetable crops [46].

In our experiments, all growth parameters were, of course, significantly higher at 49 than 35 DAT, with the only exceptions being plant height and total upper dry matter percentage, which were not affected by plant age. At 35 DAT, despite the lower number of leaves in Cu-treated plants than in the control, Cu concentrations up to 50 μM in the nutrient solution increased the fresh biomass production of the aerial part (Table 1); however, a relevant increase in dry matter was observed only for stem tissues, suggesting that the overall effect was partially due to increased water absorption. In contrast, at 49 DAT the fresh weight of the distinct aboveground plant organs did not change across treatments (Figure 1), while the increase in dry mass percentage above 50 μM Cu (Table 1) indicated a lower water content in those tissues. An increase in the percentage of dry matter was observed also in the leaf tissues of tomato plants grown in hydroponics, after 15 days exposure to 100–350 μM Cu concentrations [48]. The detrimental effect of high Cu levels in the root zone on biomass production was observed in several food crops [4], and in MAPs such as *Carthamus tinctorius* [1]. Similarly, in this work both fresh (Table 1) and dry (Figure 1) biomass production were tendentially lower in the 100 μM Cu treatment than the control at both sampling dates, although the difference was never significant. These results showed that *P. graveolens* is a species tolerant to Cu toxicity of up to 100 μM

concentration, consistent with the tolerance indices toward metal stress reported in Table 4. For each plant organ, the values of the latter parameters were generally similar to those of the corresponding control. Toxic Cu concentrations well below 100 μM have been reported in the literature for several species. For example, Reichman et al. [50] reported that the highest Cu concentration in the nutrient solution without negative effects on plant growth was 35 μM for Cu-tolerant populations of *Silene cucubalus*; the no-effect threshold was about 5 μM for Cu-sensitive cultivars of mung bean, sweet potato and wheat, and was below 1 μM in Australian tree species such as ironbark, *Acacia holosericea*, and *Melaleuca leucadendra*.

Plant age significantly influenced both stomatal resistance, which was much higher at 49 than 35 DAT for all the Cu treatments, and photosynthetic efficiency, expressed as chlorophyll fluorescence Fv/Fm, which was lower in older plants. At both sampling dates, increasing Cu concentrations in the nutrient solution interfered with the process of photosynthesis by increasing stomatal resistance and decreasing leaf chlorophyll fluorescence (Table 2). Along with the determination of stomatal resistance, the assessment of chlorophyll fluorescence is a key parameter in the rapid detection of response to physiological stress in higher plants; specifically, the Fv/Fm ratio is a physiological marker of photoinhibition of photosystem II (PSII) induced by stress conditions [51]. It has been reported that excess Cu can impair photosynthetic electron transport particularly at the PSII level, and Cu toxicity has been associated with quenching of variable fluorescence Fv [52]. The values of Fv/Fm reported in Table 2 remained within the typical range for healthy plants, that is 0.75–0.85 [53], and suggested that, despite a significant decline of the indicator at the 100 μM Cu concentration at both sampling dates, the function of the PSII reaction centers was preserved with all Cu treatments. Although plant age did not affect the content of chlorophylls, a significant decrease with increasing Cu concentration was observed at 49 DAT for these pigments. The above data could be reasonably interpreted as early indicators of Cu toxicity that became more severe with the duration of exposure; despite the effects of a possible photosynthetic imbalance this did not translate into a significant biomass decrease, or in the typical visible symptoms of toxicity such as leaf chlorosis [54]. Under impaired photosynthesis, plant metabolism is affected and one possible biochemical process that can be activated is the Mehler reaction, with formation of oxygenated molecules such as H2O2 [55]. This process is consistent with the significant increase in H2O2 concentration that was observed at 49 DAT in plants treated with 100 μM Cu (Figure 3A).

It is generally acknowledged that excess Cu causes oxidative stress in plants [1,2,13,17]. However, due to the time course of the antioxidant response, the levels of stress indicators in plant tissues may undergo fluctuations. This could account for the significant effect of sampling date, Cu concentration, and their interaction on the observed contents of both H2O2 and MDA, and could also explain the contrasting results reported in the literature concerning the levels of ROS or MDA in several plant species [4]. The antioxidant activity of pelargonium at 35 DAT showed the same behavior across the Cu treatments (Figure 2C–E), regardless of the assay used for the determination (FRAP, DPPH or ABTS) and the stimulation of the plant antioxidant response was strictly related to the occurrence of lipid peroxidation, since a similar pattern was observed also for the concentration of MDA (Figure 3B). The content of total flavonoids followed the same trend (Figure 2B), suggesting that this class of compounds could play a key role in the pool of antioxidant molecules of *P. graveolens* that are involved in plant response to excess Cu in the early stages of exposure. Interestingly, the highest values of antioxidant power and flavonoid concentration were obtained with the 50 μM Cu treatment, which also resulted in the highest rate of Cu accumulation in plant tissues (Table 3). On the other hand, a different behavior was observed for total phenols (Figure 2A), whose amount, unlike the content of flavonoids, was affected also by the sampling date. These dissimilarities indicated that, along with flavonoids, other classes of phenolic compounds could contribute significantly to the pool of phenolics of this species.

At 49 DAT, all parameters except DPPH scavenging capacity increased with Cu concentration (Figure 2), suggesting the occurrence of an effective dose-dependent response of the antioxidant system to excess Cu and a central role of phenolic compounds in the plant tolerance to Cu toxicity. These findings showed the effectiveness of high Cu concentrations in the nutrient solution in stimulating the synthesis and accumulation of beneficial antioxidant molecules in the plant tissues of *P. graveolens*. In addition to phenolics, other non-enzymatic antioxidant compounds could have an impact on plant response to Cu toxicity; for example, an increased content of proline has been observed in different species exposed to excess Cu [18,19]. In our experiments, the level of MDA decreased with Cu concentration in the nutrient solution up to 50 μM, showing that Cu-exposed *P. graveolens* could well counteract lipid peroxidation. Likewise, the leaf concentration of H2O2 was effectively controlled in up to 50 μM Cu. In contrast, the increase in the content of H2O2 at 100 μM Cu may indicate a less efficient plant response at this high concentration of the element (Figure 3A,B).

The relationships among the indicators linked to Cu uptake and antioxidant response to Cu treatments are confirmed in the Pearson's correlation table (Table 5). Leaf Cu content followed root content at both sampling dates, and, in younger plants, the content of flavonoids rather than the level of total phenols was strongly correlated with the antioxidant capacity as obtained using either the FRAP, DPPH or ABTS assays. However, at 35 DAT, Cu exposure did not elicit a marked response, as extremely weak relationships were evidenced between Cu levels in the tissues and all other biochemical parameters. The correlation coefficients between the concentrations of Cu and those of H2O2 and MDA also remained low at 49 DAT, suggesting that even older plants could efficiently prevent oxidative stress. On the other hand, higher values of the correlation coefficients were generally evidenced between root or leaf Cu levels and the other biochemical parameters (phenols or flavonoids content, or antioxidant capacity). Therefore, the results of the regression analysis are consistent with an initial antioxidant response to Cu toxicity at 49 DAT.

The TF and BAC indexes are important parameters for the evaluation of plant phytoremediation potential. In hyperaccumulator species, both parameters are greater than 1; in contrast, in our study only the BAC factor was higher than 1, indicating that *P. graveolens* acted as a Cu excluder. According to Saleem et al. [13]. Cu excluders, which have a low potential for metal extraction, could be effectively employed for phytostabilization. The BAC and TF indexes showed significant decreases with increasing Cu concentration in the nutrient solution at both sampling dates (Table 3). At low concentrations (up to 25 μM), young plants accumulated more Cu in root tissues and showed a much larger variation of the root BAC index among the Cu treatments than those sampled at 49 DAT. However, the decrease of root BAC values at both sampling dates indicated a strong inhibition of Cu uptake as the concentration of the element in the nutrient solution increased. A much larger variation was observed for the BAC index of the aerial parts, which decreased more than 10-fold in all aboveground tissues during the whole growing cycle, suggesting a synergistic effect of reduced Cu uptake and reduced element translocation in Cu-treated plants. This outcome was confirmed by lower TF values in the Cu treatments as compared to the control. Although in the latter treatment the TF in the aboveground tissues was higher in older plants, Cu translocation was markedly limited with increasing Cu concentration at both sampling dates. Therefore, the observed tolerance of *P. graveolens* to Cu toxicity was both due to the plant's ability to exclude Cu from the leaf tissues by limiting translocation to the aerial parts, and to an efficient antioxidant system. A similar behavior was observed in *Solanum cheesmaniae* subjected to Cu stress [56]. This effect was further evidenced by the results shown in Figure 5A,B, as Cu accumulated particularly in root tissues independent of the concentration of the nutrient solution. In contrast, the stems were only slightly affected by the 100 μM Cu treatment, and the leaf tissues were totally unaffected.

According to Lange et al. [57], most Cu-tolerant species act as Cu excluders, with very low Cu translocation from root to shoots. Contrarily, Chen et al. [5] reported on 25 plant species identified as Cu hyperaccumulators and provided literature data concerning tolerant and accumulator species, with leaf Cu content ranging from 45 to 596 mg/kg DW and root content ranging from 33 to 3768 mg kg−<sup>1</sup> DW. In plants, Cu uptake is generally dependent on the species, plant organ, concentration in the growing medium, and the time of exposure. For example, according to Adrees et al. [4], maize plants exposed for six days to 100 μM Cu in hydroponics accumulated 1070 and 56 mg kg−<sup>1</sup> DW in roots and shoots, respectively; the same species was reported to accumulate 7790 mg kg−<sup>1</sup> DW in the roots after 15 days treatment with 80 μM Cu. Chen et al. [5] reported that in hydroponically grown *Moso bamboo* with 100 μM Cu in the nutrient solution, the Cu content in leaf and root tissues were, respectively, 24 and 417 mg kg−<sup>1</sup> DW after 15 days, and 91 and 809 mg kg−<sup>1</sup> DW after 30 days exposure. Saleem et al. [13] reported that pot-grown flax accumulated Cu mainly in the root tissues after 35 days cultivation, while the contaminant was accumulated mainly in the shoots in mature plants (105–140 days). In our experiments, despite a dose-dependent Cu accumulation in the roots of up to 468.14 mg kg−<sup>1</sup> DW, Cu content in the leaf tissues remained at the same level as the control both at 35 and 49 DAT, further characterizing *P. graveolens* as a Cu excluder species.

In a very recent paper, Tschinkel et al. [58] reported that the permitted concentration of impurities for drug substances and excipients set by the United States Pharmacopoeia Convention (USP) is 300 mg kg−1. Additionally, according to a recent review from the European Food Safety Authority [59], the maximum residue level (MRL) for Cu compounds (Cu) in leaves and herbs for herbal infusions is 100 mg kg<sup>−</sup>1. These limits are much higher than the Cu concentrations that were found in the leaf tissues of *P. graveolens*, which were below 50 mg kg−<sup>1</sup> DW (Figure 5A,B). Therefore, the leaves of Cu-treated *P. graveolens* plants had higher contents of antioxidants and, at the same time, the same Cu content of the control plants, and may be safely used in the pharmaceutical/herbal industry for the extraction of phenolic compounds and other beneficial constituents such as essential oils. Although the latter were not examined in this work, some authors showed that heavy metals had minimal impact on the quality of *P. graveolens* essential oil, even when the contaminant was partly translocated to the aboveground organs [38,39]. In general, despite a high translocation factor from the root system to the aerial parts being indispensable for species with edible roots, the opposite is preferable for plant species that are used for leaf tissues, like the one examined in this study. Considering the excess of Cu application in agriculture and the consequent contamination of soils and water bodies, selection of MAPs according to their tolerance and potential accumulation in the organs of interest becomes a crucial issue in managing the problem of Cu pollution and preserving the quality of plant materials. It is noteworthy to mention that expanded and unexpanded perlite have some properties that can favor the adsorption of metal ions, including Cu [60]. However, the strong root Cu uptake shown in Figure 5A,B showed that this microelement was available to the plants in all Cu treatments.

Copper had a strong influence on Zn uptake (Figure 5C,D). In the root tissues of younger plants, Zn content was inversely related to that of Cu; this indicated a competitive absorption mechanism for the two micronutrients, in agreement with what reported by Kabata-Pendias and Szteke [7]. The amount of Zn in the aerial parts at 35 DAT increased significantly across the Cu treatments, suggesting that higher Cu levels promoted Zn translocation to the stems. Conversely, Cu and Zn uptake followed the same trend in older plants, despite a much lower Zn accumulation at 49 than 35 DAT. A decrease of Zn uptake in plants exposed to excess Cu has been observed in several species [4]. The content of Na decreased with increasing Cu in all plant organs, especially at 49 DAT, in agreement with what reported by Chrysargyris et al. [49] for the roots of *Mentha spicata*. The opposite trend was observed by other authors in the leaves of *Vicia faba* [61] and the shoots of pistachio seedlings [62]. The addition of Cu to the nutrient solution generally did not have a significant influence on the uptake of the macronutrients N, P and K, which was confirmed the scarce effects observed on the biomass production (Table 1 and Figure 1). The only exception was root P content at 49 DAT, which was higher in Cu-treated plants than in

controls (Figure 4E,F), in contrast with the results reported by Chrysargyris et al. [49] and Eskandari and Mozaffari [62]. According to Adrees et al. [4], although Cu supply generally affects mineral nutrition, the effect of this microelement on the uptake of other mineral nutrients is strongly dose-, time- and species-dependent. In addition, in polluted environments, Cu could interact with other heavy metal contaminants [9,26,51,63]. In general, we observed that the Cu treatments did not impair mineral nutrition and, overall, *P. graveolens* showed a high capacity to grow in Cu-enriched mediums of up to 100 μM.

Further work is necessary to provide a deeper insight into the response of *P. graveolens* to Cu stress. For example, the effects of severe Cu exposure conditions could be investigated through an extension of the growing period beyond 49 DAT, or with Cu concentrations higher than 100 μM in the nutrient solution; additionally, a similar experiment could be carried out in open field, where Cu bioavailability is conditioned by soil properties; finally, the profiling of individual metabolites of interest under Cu stress could help in highlighting the effects of this element on the bioactive properties of *P. graveolens*.

#### **4. Materials and Methods**

#### *4.1. Plant Material and Cultivation Conditions*

*Pelargonium graveolens* L'Hér. plants were selected for the present study, which was implemented at the experimental greenhouse of Cyprus University of Technology, in Limassol, Cyprus. Cuttings of 10 cm length were collected from mother plants (National Agricultural Department, Nicosia, Cyprus) and were grown in peat:perlite (4:1 *v*/*v*) substrate, in plastic seedling trays for 25 days, till roots formation. Plants at the stage of four-to-five leaves were transplanted in pots (one plant per pot; 1.5 L capacity) filled with expanded perlite and placed on plastic trays to achieve proper drainage (see Chrysargyris et al. [64]). Perlite properties have been described previously [65]. Plants were grown in an open (free drainage) hydroponic system and the drainage nutrient solution was available to plants through capillary suction. Plants were sampled at two different growth stages.

Plants were initially grown with the application of a full-strength nutrient solution (electrical conductivity (EC) and pH of 2.1 mS cm−<sup>1</sup> and 5.7, respectively) for 21 days. Nutrient solution composition was: NO3 −-N = 15.00, K = 9.50, PO4 <sup>−</sup>3-P = 1.80, Ca = 4.20, Mg = 1.63, SO4 <sup>−</sup>2-S = 1.55 and Na = 1.85 mmol L−1, respectively; and B = 30.00, Fe = 35.05, Mn = 6.10, Cu = 4.00, Zn = 4.10, and Mo = 0.52 μmol L<sup>−</sup>1, respectively. The described above concentrations were obtained using mineral salts and chelate for iron with ethylenediamine-N-N'bis(2-hydroxy-4-methylphenylacetic) acid (6.5% Fe EDDHMA). After that period, plants were subjected to different Cu levels (treatments) in the nutrient solution, namely (i) 4 μM Cu (control); (ii) 25 μM Cu; (iii) 50 μM Cu; and (iv) 100 μM Cu (in the form of CuSO4). Plants were grown under Cu excess for additional 28 days (in total 49 days after transplanting, DAT). A total of 96 plants were used (4 Cu levels × 2 sampling periods × 12 replicates).

#### *4.2. Plant Growth and Physiological Measurements*

Plant growth and physiological parameters were measured at two sampling periods (35 DAT and 49 DAT) with six replicates per treatment and growth period. Plant height and leaf number were recorded. After harvest, upper fresh and dry biomass parts (leaves, petioles, leaf stem) and roots were measured. Different parts of the plants were separated to evaluate the uptake and translocation of Cu from the roots to upper plant parts and the relevant effects on nutrient accumulation. Individual samples were collected and put at 85 ◦C in a forced-air oven until constant weight was achieved to determine their dry weight.

Leaf stomatal conductance was measured with a ΔT-Porometer AP4 (Delta-T Devices Cambridge, Burwell, Cambridge, UK) [66]. Leaf chlorophyll fluorescence (chlorophyll fluorometer, opti-sciences OS-30p, Hertfordshire, UK) was measured on two fully developed, light-exposed leaves per plant. Following leaf incubation in the dark for 20 min, the Fv/Fm ratio was measured [66]. Leaf chlorophyll was extracted with dimethyl sulfoxide (DMSO) and chlorophyll a (Chl a), chlorophyll b (Chl b) and total chlorophylls (total Chl) were assayed and expressed as μg g−<sup>1</sup> FW [66].

#### *4.3. Antioxidant Activity, Total Phenols and Total Flavonoids Content*

The antioxidant activity of the methanolic leaf plant extracts was determined with four replicates per treatment and sampling date by the assays of 2,2-diphenyl-1-picrylhydrazyl (DPPH) and ferric reducing antioxidant power (FRAP), as previously described by Chrysargyris et al. [67], as well as the 2,2 -azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) assay according to the methodology described by Woidjylo et al. [68]. The Folin–Ciocalteu method was used for determining the total phenols content, as previously described [69] and results were expressed as gallic acid equivalents (mg GAE per g FW). The total flavonoid content was determined according to aluminum chloride colorimetric method [70] and results were expressed as rutin equivalents (mg rutin per g FW).

#### *4.4. Plant Stress Indicators*

Cell damage index of lipid peroxidation in leaves was assessed in terms of malondialdehyde (MDA) content, which was determined by the thiobarbituric acid reaction [71]. Hydrogen peroxide (H2O2) content was measured according to the method of Loreto and Velikova [47]. The results were expressed as nmol MDA or μmol H2O2 per g FW. Four replicates were analyzed for each treatment and sampling date.

#### *4.5. Nutrient Content*

Dried tissue (0.5 g) from leaves, stems, leaf petioles and roots from each treatment (4 biological replications; each replication was a pool of 2 individual plants) at both sampling dates, was subjected to dry ashing at 450 ◦C and acid extraction (2N HCl). The extracts were used for the determination of sodium (Na) and potassium (K) by flame photometry (Lasany Model 1832, Lasany International, Panchkula, India), phosphorus (P) with the molybdate/vanadate method (yellow method) by spectrophotometry (Multiskan GO, Thermo Fischer Scientific, Waltham, MA, USA), zinc (Zn) and copper (Cu) by atomic absorption spectrometry (PG Instruments AA500FG, Leicestershire, UK). Nitrogen (N) was determined using the Kjeldahl method (BUCHI, Digest automat K-439 and Distillation Kjelflex K-360, Flawil, Switzerland) following Chrysargyris et al. [64]. In particular, the measured Cu content in this study refers to total dissolved Cu content, which was almost totally (≥98.21%) available as Cu2+ [49]. Plant nutrient content was expressed in g kg−<sup>1</sup> and mg kg−<sup>1</sup> DW, for macronutrients and micronutrients, respectively.

The Cu accumulation rate (AR), bioaccumulation coefficient (BAC), translocation factor (TF) and tolerance index (TI) of pelargonium were calculated by equations described by Benimeli et al. [72], Amin et al. [2] and Azooz et al. [73], as follows.

The accumulation rate (AR) was calculated as the sum up of Cu concentration in each plant tissue x plant DW divided by the number of days under Cu levels by the total plant DW [72].

Accumulation rate mg per (kg DW x day) =

$$\frac{\left(\left[\text{Cu}\right]\text{ lavee x DW lavee} + \left(\left[\text{Cu}\right]\text{ stem x DW stem} + \left(\left[\text{Cu}\right]\text{ petite} \times \text{DW petile} + \left(\left[\text{Cu}\right]\text{ rot} \times \text{DW root}\right)\right)\right.\right.}{\text{Days x (DW lavee + DW stem + DW petole + DW rot)}}\tag{1}$$

The bioaccumulation coefficient (BAC) was calculated as the ratio of Cu concentration in plant tissue to that of Cu concentration in nutrient solution, according to Amin et al. [2]:

$$\text{Bioaccumulation coefficient} = \frac{\text{Cu concentration in plant tissue (mg/DW)}}{\text{Cu concentration in nutrient solution (mg per L)}} \quad (2)$$

The translocation factor (TF) was calculated as the ratio of Cu concentration in plant tissue to that of Cu concentration in plant roots according to Amin et al. [2]:

$$\text{Translation factor} = \frac{\text{Cu concentration in plant tissue (mg kg DW)}}{\text{Cu concentration in plant root (mg per kg DW)}} \tag{3}$$

Copper tolerance index (TI) was calculated as the quotient of the dry weight of plants grown under copper treated and control conditions according to the following the equations described by Benimeli et al. [72] and Azooz et al. [73], with the following modifications:

Tolerance index (%) = Dry weight of Cu <sup>−</sup> treated plants <sup>×</sup> <sup>100</sup> Dry weight of Cu <sup>−</sup> untreated plants (control) (4)

#### *4.6. Statistical Analysis*

For plant growth and physiological measurements, six samples were used per treatment, whereas chemical composition/antioxidants were recorded from four samples per treatment. The analysis of the data was accomplished with the use of SPSS v. 22.0 program (IBM Corp., Armonk, NY, USA) and the one-way analysis of variance (ANOVA) was carried out for the Cu concentration for each sampling date, while means were compared with the Duncan multiple range test (DMRT) at *p* < 0.05, when significant differences were detected. Results were expressed as mean values and standard error (SE). The two-way ANOVA was also performed, with both Cu concentration and sampling date as the sources of variation. Finally, a regression analysis was applied to the content of Cu in plant tissues and the biochemical parameters associated with antioxidant response and oxidative stress.

#### **5. Conclusions**

Hydroponically grown *P. graveolens* resulted in a species tolerant toward high Cu concentrations in the root zone and the initial symptoms of Cu toxicity. Namely, declines of photosynthesis-related parameters and increases in leaf H2O2 along with considerable Cu accumulation in root tissues were evidenced only at the 100 μM Cu concentration in the nutrient solution. However, the extent of the toxicity symptoms did not have an impact on biomass production; in addition, high Cu levels stimulated plant secondary metabolism, enhancing the production of bioactive antioxidant molecules. Due to low Cu translocation to the aerial organs during the whole growing cycle, this microelement did not reach the leaf tissues, which resulted in suitable plant material for the safe extraction of bioactive compounds. These results show that plant stress from excess Cu does not necessarily preclude the use of MAPs for medicinal purposes, depending on the target organ where the metal accumulates. The outcome of this study showed that the leaves of *P. graveolens* plants exposed to excess Cu could be safely employed for their medicinal properties in herbal or pharmaceutical preparations.

**Author Contributions:** Conceptualization, A.C., R.M. and N.T.; methodology, A.C. and R.M.; software, A.C.; validation, A.C., R.M., L.I., and A.P.; formal analysis, A.C., R.M. and L.I.; investigation, A.C. and R.M.; resources, A.C. and N.T.; data curation, A.C., R.M., L.I., and A.P.; writing—original draft preparation, R.M. and N.T.; writing—review and editing, R.M., L.I., A.P. and N.T.; visualization, R.M. and L.I.; supervision, A.C. and N.T.; project administration, N.T.; funding acquisition, L.I., A.P. and N.T. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research has been co-financed by Cyprus University of Technology and University of Pisa Open Access Author Fund.

**Acknowledgments:** Authors would like to thank Filio Athinodorou and Panayiota Xylia for their technical assistance.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


### *Review* **The Alleviation of Metal Stress Nuisance for Plants—A Review of Promising Solutions in the Face of Environmental Challenges**

**Mateusz Labudda 1, Kinga Dziurka 2, Justyna Fidler 1, Marta Gietler 1, Anna Rybarczyk-Pło ´nska 1, Małgorzata Nykiel 1, Beata Prabucka 1, Iwona Morkunas <sup>3</sup> and Ewa Muszy ´nska 4,\***


**Abstract:** Environmental changes are inevitable with time, but their intensification and diversification, occurring in the last several decades due to the combination of both natural and human-made causes, are really a matter of great apprehension. As a consequence, plants are exposed to a variety of abiotic stressors that contribute to their morpho-physiological, biochemical, and molecular alterations, which affects plant growth and development as well as the quality and productivity of crops. Thus, novel strategies are still being developed to meet the challenges of the modern world related to climate changes and natural ecosystem degradation. Innovative methods that have recently received special attention include eco-friendly, easily available, inexpensive, and, very often, plant-based methods. However, such approaches require better cognition and understanding of plant adaptations and acclimation mechanisms in response to adverse conditions. In this succinct review, we have highlighted defense mechanisms against external stimuli (mainly exposure to elevated levels of metal elements) which can be activated through permanent microevolutionary changes in metal-tolerant species or through exogenously applied priming agents that may ensure plant acclimation and thereby elevated stress resistance.

**Keywords:** abiotic stress; adaptation; priming; defense mechanisms; metallophyte; oxidative stress; phytoremediation; tolerance

#### **1. Introduction**

Rapid industrialization and urbanization, chemicalization of agriculture, and the lack of a proper attitude to the surroundings in which we live are the main causes of unpredictable climate changes, as well as the deterioration of natural environments and ecosystems [1]. As a consequence of such imprudent human domination of the Earth, plants are constantly exposed to a wide array of adverse environmental events, including water deficits, salinity, imbalances in elements (resulting from their deficiency and/or pollution), extremes of temperature, ultraviolet radiation, etc. All the above-mentioned physical and chemical factors, collectively referred to as abiotic stress, may occur singly, sequentially, or simultaneously, and their effects may also act synergistically or additively on plant fitness [2]. Moreover, the effect of each stress factor depends on its intensity and the exposure time of the plants. Despite the impact of such a wide variety of stressors, plant exposure to any of them has one similar outcome, namely the overgeneration of reactive oxygen species (ROS) that are responsible for oxidative damage of cellular components such as proteins, lipids, nucleic acids, carbohydrates, and other metabolites [3–5]. Therefore, as shown in Figure 1, oxidative stress is a secondary but common reaction of plants subjected

**Citation:** Labudda, M.; Dziurka, K.; Fidler, J.; Gietler, M.; Rybarczyk-Pło ´nska, A.; Nykiel, M.; Prabucka, B.; Morkunas, I.; Muszy ´nska, E. The Alleviation of Metal Stress Nuisance for Plants—A Review of Promising Solutions in the Face of Environmental Challenges.

*Plants* **2022**, *11*, 2544. https:// doi.org/10.3390/plants11192544

Academic Editor: Juan Barceló

Received: 26 August 2022 Accepted: 25 September 2022 Published: 28 September 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

to various factors which, by interacting with each other, contribute to disturbances in the optimal growth and development of plants, finally leading to a considerable reduction in their productivity and yield [6,7].

**Figure 1.** A simplified diagram of complex plant responses to stressful environmental stimuli: oxidative burst and its consequences as a universal reaction to different stressors are shown, as well as stressor-dependent reactions leading to plant growth retardation and a decline in productivity.

It should be emphasized that, among the various xenobiotics released to the environment due to anthropogenic activity, heavy metals and metalloids, classified also as metallic trace elements because of their presence at trace concentrations (parts per billion or less than 10 parts per million) in various matrices, have pulled ahead of other wellknown contaminants such as plant protection products and carbon and sulfur dioxides [8]. Environmental pollution with metals is particularly prominent in point source terrains such as metalliferous mines, smelters and foundries, and other metal-based industrial operations. However, among the sources of these most common inorganic contaminants, fossil fuel burning and the use of fertilizers, pesticides, livestock manure, municipal wastes, and sewage should also be mentioned [9]. The problem of heavy metal accumulation may be aggravated by salinity stress, which causes disturbances in the homeostasis of macro- and micro-elements in the soil and facilitates metallic ion uptake by plants [10,11]. Such changes in soil composition are peculiarly important in the case of crops for which a sufficient supply of essential elements has to be ensured, whilst potentially toxic elements should be present only at very low levels. Since heavy metals are now ranked in second place when taking into account the degree of risk they pose to the human population all over the world, in recent years there has been increasing concern about environmental contamination from them [12].

Metallic trace elements can spread over long distances through interactions with wind, surface and ground waters, and herbivores. These metals pose a serious threat to our health due to food chain accumulation, dust inhalation, and skin contact which result in cardiovascular, respiratory, and neurodegenerative diseases [13]. They also have a negative impact on the majority of plant species and other living organisms. In the concentrations exceeding the maximum tolerable amount, they cause disturbances in the ultrastructure of cells and affect physiological and biochemical processes such as the biosynthesis of

chlorophylls, photosynthetic capacity, transpiration, nutrient and water uptake, and the activity of enzymes involved in various metabolic pathways, as well as lead to increased ROS formation by direct involvement in redox reactions (in the case of highly reactive metals) or indirectly through depletion of antioxidant pools [5,9,14–17]. All these cellular effects result in morphological changes, such as shortening of shoots and roots, necrotic and chlorotic stains, decreases in leaf number and size, and premature aging [5,18,19]. As a consequence, it leads to limiting the productivity of agricultural crops [20]. The danger of metallic elements lies also in the fact that many of them are dispersed in the environment for a long time and, therefore, they are considered to be persistent [12]. As an example, half-life time varies from 75 to 380 years for cadmium (Cd) and from 1000 to 3000 years for copper (Cu), nickel (Ni), lead (Pb), zinc (Zn), and selenium (Se) in the soils of temperate climates [21].

Recently, research on the mechanisms by which plants recognize and cope with toxic metals and other stressful and dynamic circumstances has undergone a very exciting period leading to significant breakthroughs. The development of knowledge in this field is necessary to relieve the pressure of environmental changes and to ensure global food security for an increasing population, as well as to restore areas degraded by human activity. Since it is well known that plants have developed different adaptation strategies which can occur as a result of adaptation and acclimation, the purpose of this concise review is to indicate what can make the life of stressed plants a little easier, especially in respect to metallic trace elements. We have highlighted only two main possibilities, although many more issues are taken into consideration in the current research. The first one is based on natural defense mechanisms arising through evolutionary changes, the understanding of which enables the development of new strategies to alleviate metal danger for both plants and surroundings (and/or the improvement of those remediation techniques that already exist). The second one refers to the use of novel priming techniques that may provide plants with intracellular acclimation and thereby enhanced stress tolerance. Both of them represent the latest solutions for sustainable, cost-effective, and efficient approaches to environmental challenges.

#### **2. Functional Traits of Plants Developed in Response to Severe External Pressures**

Climate change has caused serious impacts on the ecosystem, including devastating its stability and affecting biodiversity. Plants, as an important component of terrestrial ecosystems, respond to climate change in an all-round way; therefore, changes in the functional traits of plants can be indicative of climate changes. The novel developmental direction of this research is to determine the interrelationships among various indicators based on physiological, biochemical, and ecological plant characteristics and to establish a network indicator system from individual plants and communities towards ecosystem functions.

Since plants are unable to avoid environmental stressors due to their sessile lifestyle, they have evolved effective mechanisms to combat stress which ensure their survival in uncomfortable conditions. Defense response can be attributed to phenotypic plasticity leading to changes within a single organism, that are reversible and result from subsequently occurring, occasional stress events ('priming'), or from chronic exposure to a new environment, to which plant metabolism adjusts ('acclimation') [22,23]. Both of these terms differ from 'adaptation', which describes permanent genotypic changes resulting in phenotypic traits that improve plant fitness or survival over multiple generations [24]. Morphological, anatomical, and physiological adaptations are characterized for metallophytes that have been gradually developed in habitats naturally or artificially enriched with metallic elements. Although in these first conditions metal tolerance may evolve over thousands or even millions of years, on human-influenced metalliferous soils it may be achieved in a relatively short time, i.e., less than 100–150 years [25]. Such genetically altered ecotypes of common species (i.e., pseudometallophytes or facultative metallophytes), as well as genera restricted only to metalliferous soils (i.e., obligate or absolute metallophytes), exhibit a higher toxicity threshold or even slightly beneficial metal effects compared to

their counterparts from unpolluted areas due to a special tolerance mechanism which is not available to non-metalliferous genotypes [26].

#### *2.1. Specific Characteristics of Metal-Tolerant Species and Their Application in Soil Remediation*

Metallophytes utilize several adaptation mechanisms to control the uptake, mobility, and activity of potentially toxic ions in the cell. Firstly, modifications to cell wall components and structure favor the retention of metals and provide a mechanical and chemical barrier against their free penetration into the protoplast [27,28]. Similarly, various membrane transporters belonging to the following families: HMAs (heavy metal ATPases, also known as P-type ATPase), NRAMP (natural resistance-associated macrophage protein), CDF (cation diffusion facilitators), YSL (yellow stripe-like), ABC (ATP-binding cassette), COPT (copper transporter), and ZIP (zinc-regulated transporter, iron-regulated transporter-like protein), play an important role in the regulation of toxic ion influx into the protoplast and organelles, and therefore have been extensively discussed in recent research and numerous review articles [7,29–31]. Subsequently, in the cytosol, harmful ions are effectively detoxified and stored in places safe for metabolism in order to prevent deleterious physiological damage. The important cytoplasmic ligands responsible for the chelation and neutralization of metallic elements include phytochelatins, glutathione (GSH), amino acids, and organic acids [14,26,32]. In turn, metal sequestration may take place in the vacuole, dictiosomal vesicles, or the endoplasmic reticulum [33,34]. From an organismic point of view, ions can be withdrawn into aging leaves and trichomes or drawn outside by secretary glands, which has been observed in *Arabidopsis thaliana* and *A. halleri* [35], *Alyssum montanum* [36], and *Biscutella laevigata* [37].

Other cellular features that make the life of metallophytes easier are related to efficient antioxidant defense systems that confront oxidative stress. Cell redox homeostasis is kept by a synchronous action of various enzymes, such as superoxide dismutase (SOD), catalase (CAT), peroxidases (POD; such as guaiacol peroxidase, GPOX, glutathione peroxidase, GPX, and ascorbate peroxidase, APX), glutathione *S*-transferase (GST), glutathione reductase (GR), and nonenzymatic antioxidants, such as ascorbate (AsA), glutathione (GSH), carotenoids (CAR), α-tocopherols, phenolics, and amino acids such as proline [4]. Although oxidative stress as a reaction to metals is one of the most studied issues recently [3,5,38,39], ROS transformation pathways, as a basis of adaptation to their excess amounts, have not been frequently compared between the representatives of different species sharing the same ecological niches or described for ecotypes of the same species representing different habitats. In this regard, our previous studies on serpentine and calamine ecotypes of *Silene vulgaris* (Caryophyllaceae) and the calamine ecotype of *Alyssum montanum* (Brassicaceae) have shown both species- and ecotype-dependent features [14,18,19,36,40,41]. The response of *S. vulgaris* to metallic elements was mainly related to the activity of antioxidant enzymes during in vitro cultivation on media enriched with Zn, Pb, and Cd at the same concentration as in the post-industrial habitat of the calamine ecotype [41]. In turn, the response of *A. montanum* was associated with the transformations of phenolic compounds, which, in the metallicolous ecotype, led to the synthesis of phenolic acids with a high ability to ROS scavenging and, in the non-metallicolous ecotype, to the synthesis of other compounds not involved in alleviating oxidative stress [19]. Interestingly, the common reaction of the metallicolous *S. vulgaris* and *A. montanum* individuals was the activity of GPX. Nevertheless, the importance of this enzyme in particular ecotypes differentiated both calamine specimens from the serpentine ones. In the former, the increased activity of this enzyme correlated with the increased accumulation of phenylpropanoids which, acting together, contribute to the formation of a lignified cell wall preventing the easy penetration of ions into the protoplast; whereas GPX activity in the serpentine ecotype provided only ROS neutralization [36,41]. Curiously, we have also found that the exposure of the calamine ecotype of *S. vulgaris* to the concentration of metallic elements reflecting their level in the zinc–lead substrate resulted in a significant increase in the efficiency of all analyzed components of the antioxidant apparatus. As a consequence, the studied

ions stimulated the growth of calamine specimens, which was manifested in accelerated growth and biomass accretion [40,41]. It is therefore likely that the trace element ions, at the doses which the calamine ecotype has adapted to in the selection process, play a pivotal physiological role, perhaps even as micro- or ultra-elements.

The above-mentioned mechanisms guarantee a high propensity of metallophytes to take up metallic trace elements; however, tolerant species, ecotypes, or particular populations differ in their degree of accumulation and the element distributions in their organs, even if they grow on the same soil. Furthermore, the enhanced ability of metal-tolerant species to accumulate one metal does not mean that other ions will be stored with the same intensity and distributed over the organs in a similar way [32]. As proposed by Baker [42], plants appearing in metal-enriched environments can be divided, on the basis of the relationship between ion content in tissue and soil, into:


The amazing biology and behavior of metallophytes, in respect to metal accumulation and detoxification, make them to useful in various phytoremediation techniques. It is plant-based, environmentally friendly, non-invasive, and low-cost technology which is applied to remediate contaminated soils by accumulation, immobilization, or degradation of these pollutants [44]. In phytoextraction, which constitutes the most popular method of phytoremediation relying on the total removal of contaminants from the environment, hyperaccumulating plants may work the best due to their ability (about 100–1000-fold higher than in other plants) regarding effective uptake and translocation of metallic elements [45]. Many studies have demonstrated the phytoextraction potential of metallophytes from various genera, such as *Alyssum murale* [46], *Arabidopsis halleri* [35], *Biscutella laevigata* [37], and *Stackhousia tryonii* among others [47]. Nowadays, phytoextraction achieves two goals at once. It is not only exploited to clean up soil, but also to mine metal (so-called phytomining), mainly in places where the use of conventional methods for ore exploitation is economically unprofitable. As an example, the cultivation of Ni-hyperaccumulators *Alyssum corsicum* and *A. murale* allows the extraction of about 400 kg of Ni per hectare [48]. In turn, metal excluders are excellent candidates for phytostabilization, which is aimed at reducing metal mobility in the soils in order to prevent them leaching deeper into the ground water and to also prevent the dust blowing into the atmosphere [44]. Despite metal stabilization, this technique involves the permanent establishment of a vegetative cover, which performs anti-erosive and soil-forming functions. Currently, some studies have indicated that the recovery of vegetation on heavy metal polluted terrains should be performed by native metalliferous species, which spontaneously occur on degraded areas and are thus better adapted to local ecological conditions than introduced ones [49]. Such an approach was first used in the 1960s, when Zn-Pb tolerant populations of *Agrostis tenuis, A. stolonifera, Anthoxanthum odoratum, Festuca rubra*, and *F. ovina* were investigated [50]. Recently, the potential of native metal-tolerant species for revegetation has been successfully verified for *Agropyron smithii* and *Artemisia tridentate* [51], *Lygeum spartum* [52], *Achillea wilhelmsii* [53], *and Matthiola dagestanica* and *Draba stylaris* [46]. The usefulness of metallophytes for revegetation and the phytostabilization of Zn-Pb rich soils in the Olkusz Ore-bearing Region, one of the biggest industrial areas in Poland, has been also proven in our earlier studies for *Biscutella laevigata* [49,54], *Dianthus carthusianorum* [49], *Gypsophila fastigiata* [55], and *Silene vulgaris* [56]. Undoubtedly, phytoremediation combined with the biological reclamation of

destroyed or degraded ecosystems may constitute a new and safe opportunity for humans to positively interact with the environment.

#### *2.2. Relationship between Chosen Metal Tolerance Traits and Other Stresses*

Besides evolving metal tolerance, metallicolous species or their ecotypes were coselected for tolerance to other adverse site conditions because soils contaminated with heavy metals are often salinized and dry [49]. Therefore, metallophytes share tolerance mechanisms with other specialized groups of plants, which makes their biology even more interesting.

Apart from activation of the antioxidant defense system constituting the basic response to various types of stress, metal-tolerant species exhibit specific adaptations that ensure a high degree of resistance to salinity and drought as well, both of which may cause a lack or deficiency of water for plants. The increased resistance to water deficit in metallophytes may result from their ability to accumulate toxic ions in large amounts. Since metallic ions can be preferentially accumulated within epidermal leaf cells, reduced cuticular transpiration can be achieved [47]. Furthermore, one hypothesis justifying reasons for metal (hyper)accumulation postulates that elements stored within cells overcome the effect of water constraints by acting as an osmolyte [45]. Confirmation of the osmoregulatory role of metals can be found in a study conducted by Bhatia et al. [47], who proved that Ni content in the shoots of *Stackhousia tryonii*, a Ni hyperaccumulator, increased significantly as the soil moisture levels decreased. Another excellent osmolyte that also accrues during metal stress is proline. This important amino acid contributes to maintaining water balance and cell turgor through osmotic regulation, and also contributes to the stability of cell membranes by preventing electrolyte leakage, which in turn can help during water deficit [57,58]. Besides osmoregulation function, proline also acts as a metal chelator and an antioxidative defense molecule which prevents oxidative burst due to ROS scavenging, thus mitigating a wide array of adverse effects from toxic ions [4,41,59]. Subsequent features of metallophytes which may provide simultaneous protection against water losses or their better adjustment to drought are related to morpho-anatomical structure. Leaves of metal-tolerant species often have reduced transpiration surfaces, are less numerous, narrower, thicker, and waxy, and possess a limited number of stomata and increased mesophyll cell size [17,60,61]. Furthermore, plants from metalliferous areas, probably in response to dry substrate, may produce deeper roots covered with dense root hairs; however, root architecture and size do not form a rule enabling metal-tolerant individuals to be distinguished from non-tolerant ones [32].

Metallophytes also show some similarities with halophytes, natives of saline soils mostly rich in sodium (Na+) and chloride (Cl−) ions. Both these specialized groups of plants possess specific and more common functional mechanisms of tolerance towards numerous stresses, which refer not only to strong antioxidant defense systems and the synthesis of compatible solutes, but also to ion sequestration and detoxification pathways [62]. The vacuolar compartmentalization of salt and heavy metals through the enhanced activity of membrane transporters is one of them [63]. Nevertheless, it has not been fully explained if the same proton pumps are involved in this process, although the role of vacuolar H+-ATPase was proven to protect against salt and Cd stress in the halophyte *Tamarix hispida* [64]. Moreover, halophytes, similarly to metal-tolerant species, are able to excrete excess deleterious ions from photosynthetically active tissues on leaf surfaces by different structures, such as salt glands, bladders, and trichomes; however, they are not specific to salt alone, as other toxic ions can be also removed in this way [62]. As an example, *Armeria maritima* ssp. *halleri*, an obligate metallophyte, can remove Cu ions via salt glands [65], while *Limoniastrum monopetalum*, a halophytic plant, uses these structures to excrete salt, Cd, and Pb as a detoxification mechanism [66]. On the other hand, all the above-mentioned mechanisms indicate clearly that the adaptation of halophytes for survival in the presence of high salt concentrations may also confer their tolerance to metallic elements. For this reason, halophytes can be good candidates for the phytoremediation

of heavy metal polluted soils. These specimens with exclusion ability, rapid growth, and deep root systems can form dense vegetation cover and therefore be utilized for the purposes of phytostabilization. *Atriplex halimus* [67], *Cochearia anglica, C. x hollandica, C. danica*, *C. py-renaica* [63], and others are good examples (Table 1). Among halophytes, species that are able to accumulate both heavy metals and salt in extraordinary amounts in the shoots without suffering phytotoxic effects can be also found. One of the most effective in removing toxic ions seems to be an annual halophyte, *Chemopodium botrys*, which accumulates several times more Cd than *Noccaea caerulescens*, a well-known hyperaccumulator of Cd and Zn [68]. The study of Mazharia and Hoameed [68] indicated that the total amount of Cd removed by shoots of *Ch. botrys* was 120 g/ha; whereas the average Cd extraction ability of *N. caerulescens* may stay at a level of about 35 g/ha. Such salt/metal-accumulating species are extremely important for the decontamination of metal polluted saline soils, although recent findings also encourage their use for reclamation of purely saline soils, mainly in arid and semiarid regions [69]. Some more examples of halophytic species and their potential usefulness in particular soil phytoremediation methods are shown in Table 1, whilst the HALOPH database, which is available at http://www.sussex.ac.uk/affiliates/halophytes/ (accessed on 3 August 2022), presents probably the largest collection of halophyte examples for various applications. In turn, more aspects of halophyte responses to metallic elements (including common and specific mechanisms of metal and salt tolerance in this group of plants), their potential utilization for the phytoremediation of metal-contaminated soils, and their relevance to the phytodesalination of saline lands have been broadly discussed in some recently published reviews and books [62,70–72].


**Table 1.** Examples of the usefulness of halophytes in particular phytoremediation techniques for the removal of various metallic trace elements.

#### **3. Chemical and Physical Agents for Enhancing Plant Resistance to Abiotic Stress**

Until now, many different techniques developed by humans have been applied to improve plant tolerance to abiotic stress factors. Some of them are based on conventional breeding; however, they have many limitations, such as being time-consuming, possessing the possibility of transferring numerous undesirable genes along with desirable ones, and having no guarantee of obtaining a particular gene combination responsible for better resistance [77]. Other techniques are related to plant biotechnology and genetic engineering, but these last options are unacceptable in many countries and remain in the laboratory experiments phase [78,79]. As an alternative, increasing attention is being paid to the priming process, i.e., short-lasting pre-exposure of plants to a variety of exogenously applied agents in order to induce a rapid and/or effective defense response to subsequently occurring stress [23,80]. There are many different types of priming methods, which are generally classified into chemical, physical, and biological methods, depending on the source of priming agents. Thus, plants can be primed by natural or synthetic chemical

compounds (e.g., phytohormones), by physical factors such as (non-)ionizing radiations, and by colonization with beneficial microorganisms such as bacteria and mycorrhizal fungi [81]. Moreover, priming can be applied to various organs and at various stages of the plant life cycle. The most frequently used is seed priming, which provides faster and more uniform seed germination, ensures efficient nutrient and water uptake, releases photo- and thermo- dormancy, as well as improves seedling vigor in relation to their further growth and yield under both optimal and adverse conditions [82–84]. Less often, priming concerns seedlings, young plants, or their parts although they show significantly greater tolerance to different abiotic stresses than untreated ones [85,86]. Importantly, priming acts on the phenotypic level without any permanent DNA modification, and therefore its effects can be reversed [22]. Moreover, its performance can vary in respect to plant species, temperature, priming duration, priming agents, and their concentration [81].

Currently, priming seems to be the most promising approach for the mitigation of abiotic stress due to various possibilities regarding application. In the present review, we have briefly summarized the latest achievements in the techniques which have attracted the greatest interest recently. Their types, and the general mode of action discussed in this text, are shown in Figure 2.

#### *3.1. Chemical Priming Agents*

Chemical priming is one of the most popular strategies and one which has some advantages. One of them is versatility, since chemical compounds work in a broad number of species and improve tolerance to multiple stress types. Furthermore, chemical agents might be applied directly to selected plant tissues/organs, or during specified developmental stages, in order to minimize growth inhibition [87]. It is a good technique, especially for producing tolerant plants when more conventional methods are difficult to perform [88]. On the other hand, little is still known about the impact of priming agents on ecosystems and their persistence in the environment, although it is anticipated that their application may be a widespread tool in agriculture in the near future. The mode of action for three main groups of chemical priming agents, the ones that are most frequently used, is presented below on the basis of the latest scientific achievements.

#### 3.1.1. Phytohormonal Priming

As has been shown in recent years, exogenous application of phytohormones may increase the metabolic status of plants in response to various abiotic and biotic stresses. In this respect, **abscisic acid** (ABA) is one of the more promising priming agents. The effectiveness of ABA lies in both reducing the ROS pool and activating non-enzymatic and enzymatic ROS scavenging. In research conducted by Saha et al. [89], seedlings of two rice genotypes were pre-treated with 10 μM ABA for 24 h and then exposed to arsenite (As (III)). In contrast to the untreated control, seedlings of both ABA-primed genotypes had reduced accumulation of superoxide anion (O2 •−) and hydrogen peroxide (H2O2) under arsenite toxicity. Furthermore, lipid oxidative damage, measured by 2-tribarbituric acid reactive substances (TBARS), was reduced by 25% and 48% under metal stress for ABA-treated individual genotypes compared to non-pre-treated ones. Mitigation of oxidative stress in primed seedlings was associated with higher concentrations of total glutathione, non-protein thiols, cysteine, and phytochelatins, as well as the increased activity of glutathione reductase [89]. Similarly, previous studies by Rehman et al. [90] and Leng et al. [16] demonstrated that Cd inhibited plant growth parameters; whereas the application of ABA (10 μM ABA) on seedlings considerably counteracted the Cd-caused negative effect and improved the root length, plant height, and biomass of shoots and roots of mung bean. Also in this case, the enhanced growth of Cd-stressed individuals sprayed with ABA was due to modification of the antioxidant defense systems. Interestingly, it is supposed that leaves-applied ABA can be then transferred to roots in order to regulate the response of the whole plant to metal stress [90–92]. ABA may also act positively on growth and physiological parameters under alkaline stress via effective control of ROS homeostasis, as found for alfalfa seedlings in which the enhanced activity SOD and POD was observed [93]. Furthermore, a significant increase in Ca2+ and Mg2+ content, as well as higher Ca2+/Na+ and Mg2+/Na+ ratios, was noticed in primed seedlings under alkaline conditions. In addition, genes encoding some important proteins involved in the sequestration of Na<sup>+</sup> in vacuoles, i.e., vacuolar Na+/H+ exchanger (NHX) and vacuolar H+-PPase (AVP), which might help in neutralization of its excess amount, were overexpressed in primed seedlings [93].

Recent studies showed that, as well as ABA, priming with **gibberellins** (GAs) has a positive effect on plant growth under stress conditions. A study by Ahmad et al. [39] showed that foliar application of GA3 (1 μM) on chickpea seedlings resulted in the increased activity of antioxidant enzymes (SOD, CAT, GST), which provided effective ROS scavenging and reduced membrane disruption, thus ensuring tolerance to Cd stress. The better response of plants treated with GA3 to Cd presence can be also attributed to the reduced uptake and translocation of toxic ions, as well as increased accumulation of nutrient minerals (Ca, Na, Mg, K, Cu, P, Fe) [39]. This could possibly be achieved through the regulation of H+-ATPase activity, as shown for soybean [94]. The advantageous impact of GA3 application on morpho-physiological parameters and stress mitigation was also determined in *Lolium perenne* under Ni and Cd exposure and in *Lepidium sativum* under As treatment [95,96].

The latest articles also indicate the beneficial role that priming with **salicylic acid** (SA) has regarding plant tolerance to abiotic stresses; however, in the case of this phytohormone, seed priming seems to be the most effective technique. It has been recently found that the soaking of wheat seeds in SA at a concentration of 100 μM for 24 h results in significant improvements in germination rate and growth parameters in the presence of chromium (Cr) and Zn due to the prevention of ROS imbalance associated with the increase in the concentration of non-enzymatic antioxidants, mainly AsA and GSH [97]. In turn, SAprimed sunflower seeds exposed to Zn showed better germination properties because the exogenous SA application modulated the endogenous profile of the phytohormones [98]. It was noted that concentrations of SA and GA were increased, while ABA accumulation was inhibited as a result of the overexpression of genes related to SA and GA biosynthesis and the decrease in the expression of ABA-related genes that occurred in combination

with a simultaneous increase in the expression of genes engaged in the catabolism of this phytohormone. Additionally, the role of SA in metal stress mitigation may also result from the upregulation of genes encoding proteins related to ion transport, such as heavy metal ATPases and metal tolerance protein (MTP), the overexpression of which provided a reduced accumulation of Zn in sunflowers [98] and Cr in tomato [99]. In these latter examples, seed soaking or foliar spraying with SA at a concentration of 0.5 mM ameliorated growth and the physiological reaction to Cr, in respect to chlorophyll biosynthesis and photosynthetic efficiency, through modulation of the ascorbate-glutathione (AsA-GSH) cycle that contributes to a decline in ROS accumulation and lipid peroxidation [99].

It is well known that the exogenous application of auxin, especially **indole-3-acetic acid** (IAA), or its precursors improves growth and development of plants; however, its role in the mitigation of metal stress is not fully understood, and the physiology of these tolerance mechanisms remains largely unknown. Nevertheless, a study by Mir et al. [85] revealed that foliar application of IAA (at a dose of 10 nM) on *Brassica juncea* plants under Cu stress significantly mitigated adverse responses due to the activation of cell division and elongation, as well as lateral root formation in which this phytohormone is involved. Furthermore, in *B. juncea* plants sprayed with IAA, effective ROS scavenging was observed which, together with improved photosynthesis and chlorophyll fluorescence parameters, sugar metabolism, and N, P, and K content, led to biomass accretion [85]. In turn, priming with indole-3-butyric acid (IBA), an IAA precursor, provided antioxidant protection through the stimulation of glutathione peroxidase activity and greater accumulation of nitric oxide (NO) that effectively reduced the elevated level of superoxides and organic peroxides in the root cells of barley seedlings under Cd stress [100].

#### 3.1.2. Nanoparticle Priming

Nanotechnology is an emerging field with potentially wide-ranging applications in agriculture. The use of nanoparticles (NPs) in plant production, as well as in enhancing plant growth under stressful conditions, including those related to environmental pollution with heavy metals, has increased significantly in recent years [101,102]. Several studies on the seed priming of various plant species with **zinc oxide NPs** (ZnO NPs) have been published, with results indicating the beneficial effects of this NP on germination and growth. Wheat seeds primed with ZnO NPs (at a concentration of 10 mg/L) exhibited better germination rates and vigor index values compared to untreated seeds. In seeds primed with ZnO NPs, increased α-amylase activity was observed that could facilitate the efficient mobilization of starch reserves. Moreover, in plants 30 days after seed priming, increased photosynthetic pigment content (chlorophyll *a*, chlorophyll *b*, and total chlorophylls) and improved photosynthetic efficiency compared to untreated plants were determined. Additionally, the use of nanopriming had a positive effect on redox homeostasis in wheat plants [103]. ZnO nanoparticles, sodium selenite (Na-selenite), sodium selenate (Na-selenate), and their combinations as priming agents for direct-seeded rice seeds were also investigated [84]. It was observed that all tested combinations of the priming agents (10 μmol ZnO-NPs; 50 μmol Na-selenite; 50 μmol Na-selenate; and the following combinations at the mentioned concentrations: Na-selenite + Na-selenate; ZnO-NPs + Na-selenite; ZnO-NPs + Na-selenate; ZnO-NPs + Na-selenite + Na-selenate) resulted in the early emergence of seedlings with increased vigor compared to the control. Furthermore, in the field experiment, all tested combinations improved the plant growth parameters and yield, which was the result of increased photosynthetic pigments, increased phenol and protein content, and the increased uptake of nutrients such as N, P, and K [84]. Salam et al. [80] showed that priming maize seeds with ZnO NPs nanoparticles (500 mg/L for 24 h) significantly improved plant growth, biomass, and photosynthesis efficiency under cobalt (Co) stress. In this case, priming also caused a reduction in ROS accumulation and lipid peroxidation due to increased antioxidant activity in maize shoots. Additionally, priming with ZnO NPs reduced the toxic effect of Co by reducing its absorption. More importantly, the ultrastructures of cell organelles, guard cells, and stomatal aperture were

stabilized and able to reduce the adverse effects of Co stress. In turn, the study by Zafar et al. [86] showed the effect of seed priming and the foliar application of Zn NPs (0.1–0.3%) on spinach salinity tolerance. It was found that external use of ZnNPs enhanced the growth of spinach plants, as well as improved biochemical parameters under stress conditions compared to untreated plants. Seed soaking and foliar application of ZnNPs provided a decline in H2O2 content accompanied by the activation of enzymatic and non-enzymatic antioxidant defense systems, as well as simultaneous accumulation of osmolytes.

The positive effect of priming was also demonstrated in the case of **titanium dioxide NPs** (TiO2 NPs). Shah et al. [104] investigated the effect of seed priming with TiO2 NPs on the germination and growth of maize seedlings under salinity conditions. Priming with TiO2 NPs (60 ppm) resulted in improved germination percentage and energy, improved seedling vigor index values, increased root and shoot length, and improved fresh and dry weights of seedlings. Moreover, priming increased the activity of antioxidant enzymes and ROS scavenging capacity. This experiment showed that priming with TiO2 NPs reduced the adverse effects of salinity stress in maize seedlings, as evidenced by a reduction in membrane lipid peroxidation and the relative electrolyte leakage level.

Recently, the effect of priming sunflower seeds with **sulfur NPs** (S NPs) on the cellular defense of seedlings against manganese (Mn) toxicity was also investigated. Priming with S NPs (50 and 100 μM) had a significant impact on reducing oxidative damage caused by excess H2O2, which was reflected in decreased lipid peroxidation. In primed seedlings, the values of these parameters under Mn stress were similar to those observed in seedlings growing under the control conditions [105].

#### 3.1.3. Priming by Reactive Chemical Species

A significant amount of research has confirmed that the pre-treatment of plants or seeds with low concentrations of reactive oxygen, nitrogen, and sulphur species (such as H2O2, sodium nitroprusside (SNP), one of the donors for NO, or sodium hydrosulfide (NaHS), a donor for hydrogen sulphide (H2S)) strengthens their resilience to later stress events [106–109]. Improved resistance to abiotic stress may be due to the fact that, at low concentration, these compounds can act as a stress signal transduction which induces stress acclimation and alleviates abiotic stress injury [87]. They play a significant protective role, mainly due to the induction of tolerance to oxidative stress caused by drought, salinity, temperature, or metal toxicity [107,110]. On the other hand, too high a concentration of these reactive chemical species results in oxidative burst and damage to cellular compounds [87].

The exogenously sourced H2O2 (at a concentration ranging from 100 to 500 μM) has the potential to counteract the toxicity of metallic trace elements in a number of plants, and its mode of action was briefly summarized in some review articles, such as those written by Hossain et al. [111] and Cuypers et al. [112] which discussed H2O2 interaction with signaling components (e.g., transcription factors, phytohormones, mitogen-activated protein kinases) as well as its involvement in the regulation of ROS homeostasis and gene expression during metal stress. Based on various studies, it can be assumed that the positive effects of H2O2 priming prior to metal exposure include the reduced accumulation of ROS accompanied by an enhanced activity of antioxidant enzymes, such as SOD, CAT, GPX, APX, and GST, as well as elevated levels of reduced forms of non-enzymatic antioxidants such as GSH and AsA [111]. This may be related to proactive protection of the thiol groups present in proteins that are particularly exposed to oxidation under stressful conditions [113]. Besides suppressing oxidative damage, the accumulation of GSH plays a role in metal detoxification in the cytosol through direct ion binding to thiol groups of its cysteine residues and acts as a precursor of metal-chelating phytochelatins [112]. Indeed, the reduced translocation of Cd ions from root to shoot was demonstrated in *Oryza sativa* cultivars pre-treated with H2O2 [114]; whereas an opposite result was obtained for Cr in *Brassica napus* seedlings in which foliar application of H2O2 increased metal movement from roots to aerial organs [115].

Interestingly, more and more recent studies concern the simultaneous application of H2O2 and other compounds in order to explore their cumulative role in metal stress resilience. As an example, the combination of H2O2 with 24-epibrassnolide (EBL), an effective byproduct from brassinolide biosynthesis, provided tolerance and helped *Solanum lycopersicum* plants to cope well with Cu stress [58]. The positive morpho-physiological response of tomato to Cu treatment was related to a decreased accumulation of these metallic ions in the roots and shoots. Such an effect resulted from the complementary action of both applied molecules, since H2O2 may affect the absorption and transport of excess Cu ions to above-ground organs due to Cu precipitation at the root surface and preferentially affect the uptake of Ca; whereas EBL improves the accumulation of K, Ca, Fe, and Mg, which are translocated to younger leaves to minimize oxidative damage in photosynthetic machinery [58]. Although, in the study conducted by Nazir et al. [58], H2O2 and EBL were implemented through distinct modes, i.e., root dipping and foliar spraying, respectively, they both minimized ROS content (H2O2 and O2 •−) and electrolyte leakage in Cu-stressed plants by modulating the activities of antioxidant enzymes (CAT, POD, SOD) and providing osmotic adjustment through increased storage of proline. In turn, Verna and Prasad [116] investigated the involvement of H2O2 and NO when applied jointly in the regulation of Cd toxicity in cyanobacteria (from genera of Nostoc and Anabena). Their findings demonstrated the synergistic action of both molecules towards the improved growth and enhanced tolerance of cyanobacteria to Cd. In this case, H2O2 and NO reduced the intracellular content of Cd through an increased secretion of exopolysaccharides, which make a slimy physical barrier against ion penetration into the protoplast. Furthermore, tested cells were characterized by a well-operating antioxidant defense system, and ROS homeostasis was provided by the enhanced activity of antioxidant enzymes and the endogenous content of reactive nitrogen species that indirectly responded to the balancing of antioxidants in order to cope up with Cd stress [116].

Taking into account other abiotic stresses, research by dos Santos Araújo et al. [117] showed that H2O2 promoted salt tolerance in maize by protecting chloroplast ultrastructures, as reflected in more efficient photosynthetic performance. Furthermore, plants treated with 15 mM H2O2 and then exposed to salinity showed increased accumulation of metabolites, such as arabitol, glucose, asparagine, and tyrosine, which may contribute to the maintenance of osmotic stability and reductions in oxidative stress [117]. The role of H2O2 in salt stress prevention can also be attributed to ion homeostasis. After priming, a decline in Na+ and Cl<sup>−</sup> content in the leaves of sunflowers was observed during salinity stress, as well as positive control of K+ and NO3 − uptake [109]. In turn, the beneficial activity of H2O2 and NO towards drought stress was noticed by Habib et al. [107]. Despite stress conditions, pre-treated wheat plants exhibited increased growth and grain yield as a result of osmolyte storage and the effective functioning of an antioxidant defense mechanism, leading to a reduced accumulation of H2O2 and membrane lipid peroxidation [107]. Drought stress effects on agronomic features of plants were also minimized in the case of *Oryza sativa* after both seed soaking and foliar spraying with H2O2 [118]. Regardless of the application form, rice plants pre-treated with this molecule showed improved yield components such as tiller numbers, number of panicles, number of filled grains, filled grain weight, and harvest index [118].

The beneficial role of **exogenously applied NO**, used in the form of donor compounds (mainly SNP) due to its gaseous nature, has also been well-documented. In experiments that involve increasing stress tolerance, NO is applied the most frequently via foliar spraying [119] or seed soaking [120] at a concentration of 50 μM to 200 μM. Similar to other reactive chemical species, NO can prevent the spread of oxidative stress in cells. As an example, SNP enhanced the activity of enzymatic antioxidants and the AsA-GSH cycle in soybean cultivars under Cu stress [121]. The alleviation of Co stress by foliar-applied SNP in *Lactuca sativa* var. *capitata* resulted in a notable reduction in H2O2 and malonyldialdehyde (MDA) content, enhanced accumulation of photosynthetic pigments, and biomass accretion that was accompanied by the better nutritional status of plants [122]. In turn, Basit et al. [120] studied the impact of SNP under Cr stress on rice seedlings. It has been shown that seed priming improved carbon assimilation and minimized oxidative damage, since NO-treated plants were characterized by lower accumulation of oxidative markers (such as H2O2, O2 •−, and MDA) and electrolyte leakage as compared to control plants. Consequently, their morphological traits were also improved [120]. It was also proven that NO stimulated seed germination and counteracted the inhibitory effect of Cd and Pb (and salinity as well) on the root growth of *Lupinus luteus.* Additionally, in this case, the increased activity of antioxidant enzymes, mainly SOD which is responsible for the neutralization of O2 •−, was correlated with a decreased level of ROS [123]. Although it would appear most likely that NO modulation of antioxidant enzyme activities and phenol and flavonoid production provides stress amelioration, Hassanein et al. [124] observed the opposite tendency in *Lupinus albus* subsp. *termis* in response to SNP and Ni treatment. Therefore, it was postulated that NO may act as an antioxidant molecule, interacting directly with ROS and giving rise to a number of reactive nitrogen species and their derivatives, which are rapidly degraded to nitrite and nitrate [124]. This is in accordance with the study by He et al. [125] which found that, regardless of aluminum presence, SNP significantly suppressed the generation of O2 •− and H2O2 by mitochondria in peanut root tips.

The addition of NO (pre-sowing and foliar) can also minimize the adversaries of salinity stress, not through the activation of antioxidant machinery, but mainly due to osmotic adjustment and Na ion homeostasis. As an example, NO-increased tolerance in broccoli was associated with higher amounts of proline and glycine betaine keeping water potential in cells below the external solution under stress conditions [57]; whereas, in wheat, the antagonistic uptake of toxic Na<sup>+</sup> with key mineral elements, such as N, K, and Ca, reduced the deleterious effects of salt [119]. Importantly, Alnusairi et al. [119] showed that the application of NO may dismiss salt stress-mediated ravaging by the overexpression of genes encoding both antiporters that are responsible for excluding Na ions from the cytosol to outside the plasma membrane or inside the vacuole (SOS1/NHX1), and aquaporin (AQP) as well as osmotin (OSM-34) which are involved in the maintenance of proper plant–water relations. In turn, the protective effect of exogenous NO under drought stress may be dose-dependent. Majeed et al. [108] found that a foliar spray of 100 μM of SNP markedly improved water status and chlorophyll content and alleviated drought-induced oxidative damages through increased antioxidant enzyme activities (CAT, APX, SOD) in maize hybrids. Moreover, an exogenous supply of SNP increased nitrite and nitrate reductase activities and upregulated GR, GST, and GPX compared to plants not supplied with SNP [108]. In contrast, higher SNP doses (150 and 200 μM) intensified the toxic effects of oxidative stress through increased MDA, H2O2, and NO content and inhibited the enzymatic activities of antioxidants.

Many studies have indicated the significant role of **H2S** priming in the response of plants to various abiotic factors [126]. It has been reported that pre-treating seedlings or a mature plant with NaHS as a H2S donor may increase the tolerance of the plant upon following exposure to heavy metals such as Pb, Ni, and As [96,127]. Although many studies have assessed the positive effect of the pre-treatment of seedlings or mature plants with H2S in relation to enhancing plant tolerance, few studies have employed H2S for seed priming. Valivand et al. [128] reported that seed priming with Ca2+ and NaHS influenced the induction of cross-adaptation in seedlings under Ni stress. The authors reported that seed priming with H2S and Ca2+ triggered signaling pathways, which resulted in the systemic accumulation of dormant stress memory in embryo cells in seeds. Upon subsequent exposure to Ni ions, stress memory was activated and primed plants showed enhanced tolerance-related responses, e.g., enhanced AsA-GSH cycle activity, redox homeostasis, and expression of phytochelatin genes [128]. In turn, Zanganeh et al. [127] reported that pre-treatment with NaHS, applied separately and together with SA, reduced Pb toxicity and improved Fe homeostasis in maize plants. The mechanism of their action was related to modulation of the glyoxalase system consisting of enzymes detoxifying methylglyoxal, which is a potent reactive cytotoxin capable of a complete disturbance of cellular roles, including oxidation of lipids and proteins [127,129].

Christou et al. [130] studied the effect of NaHS (100 μM for 48 h) on the tolerance of strawberry plants to subsequent exposure to salinity. Pre-treatment of roots resulted in increased leaf chlorophyll fluorescence, stomatal conductance, and leaf relative water content, as well as lower lipid peroxidation levels. Additionally, synthesis of NO and H2O2 in leaves was reduced and high ascorbate and glutathione redox states were maintained. The observed positive changes correlated with the stimulated gene expression of antioxidant enzymes (cytosolic APX, CAT, MnSOD, GR), enzymes involved in ascorbate and glutathione biosynthesis (glutamylcysteine synthetase; L-galactose dehydrogenase; glutathione synthetase), a transcription factor (DREB), and salt overly sensitive (SOS) pathways (SOS2-like, SOS3-like, SOS4) [130]. Hydrogen sulfide pre-treatment (500 μM NaHS for 72 h) also mitigated growth inhibition and regulated root architecture under salt stress in *Malus hupehensis* seedlings, not only through the activation of antioxidant defense (mainly CAT and POD activities), but also through maintaining the balance of water (by proline accumulation) and Na+/K+ (by higher uptake of K than Na ions) as well [131]. Under drought conditions, H2S may improve tolerance by regulating stomatal closure and reducing water loss thanks to ABA synthesis and signaling, which was noticed in *Oryza sativa* seedlings together with an increase in endogenous H2S production and antioxidant capacity [132]. However, an innovative approach in the use of H2S as a priming agent is its application in combination with NO. In this respect, NOSH is a novel hybrid synthetic compound that simultaneously releases NO and H2S. Antoniou et al. [133] demonstrated that NOSH synthetic compounds provide significant protection in *Medicago sativa* plants against drought stress. This protection appears to be achieved through a coordinated modification of improved physiological performance, reactive oxygen/nitrogen species homeostasis, and transcriptional regulation of defense-related pathways [133].

#### *3.2. Physical Priming*

Priming with physical factors includes a number of methods, especially those related to radiation. Among them, both non-ionizing radiations, such as UV radiation, microwaves, magnetic field radiation, and sonication, and ionizing radiations, i.e., X-ray radiation and γ-radiation, can be distinguished [134]. Physical priming is considered to be an accessible, affordable, and eco-friendly technique which brings beneficial effects on seed parameters, the metabolic activities of plants, and plant development and growth [135]. It has the advantage, over chemical priming, that it does not pollute the environment, which is an important aspect in agriculture, especially if the contemporary injudicious application of chemical compounds during food production is taken into account [136]. Therefore, until recently, physical treatment was successfully employed in crops, mainly for stimulating seed germination and seedling establishment since these stages are considered to be the most critical stages in the life cycle and ultimately determine field production. This aspect of physical priming application has been widely discussed over the past few years [6,134–137].

Despite increasing understanding of the effects of physical priming performance on plants under optimal conditions, data in the literature on its application to alleviating stress nuisance are still limited, especially in respect to metal toxicity. Thus, in the present review we have focused on the latest achievements that are related mostly to drought and salinity, during which physical priming strengthens antioxidant response. It is therefore likely that physical treatment of plants subsequently exposed to excess amounts of metallic trace elements will bring comparable responses. Nevertheless, the mode of the physical agent's actions in plants under metal stress is also mentioned whenever the most recent studies were available.

#### 3.2.1. Priming with Non-Ionizing Radiation

**Ultraviolet (UV)** radiation is a type of electromagnetic radiation with a vibration frequency between 30 PHz and 750 THz, photon energy between 3 and 124 eV, and a wavelength between 10 and 400 nm, which is shorter than visible light but longer than X-ray radiation. UV radiation is divided into UV-A, UV-B, and UV-C, with UV-A radiation being the least harmful to living organisms and UV-C the most [134]. Both seed and seedling UV-B priming, applied for 45 min at 4 kJ/m2 intensity, was shown to effectively alleviate oxidative stress and its resulting damage by significant reductions in superoxide, H2O2, and MDA content in stress-sensitive rice variety (*Oryza sativa* cv. Aiswarya) seedlings under stressful conditions caused by NaCl, PEG, and UV-B treatments [138]. The study also demonstrated that UV-B priming led to significant increases in glutathione and ascorbate contents, SOD, CAT, and APX activity, gene expression levels, photosystem activities, foliar gas exchange parameters, and, finally, in mitochondrial activity. The increases were the most pronounced in seedlings subjected to NaCl stress. Similar results were reported for UV-B primed seeds for two varieties of rice: Neeraja and Vaisakh. Additionally, reductions in leaf osmolarity level, increases in proline, total sugar, and free amino acids content, and induced expression levels of stress-related proteins (Hsp90 and Group 3 late embryogenesis abundant proteins) under NaCl and PEG stress were observed [139]. The observed differences were significantly higher in the tolerant variety (Kanchana) than in the sensitive one (Aiswarya) and were also reported by Thomas et al. [138], who found that UV-B priming at low doses (4 and 6 kJ/m2) led to increased levels of flavonoids and anthocyanins, the increased activity of phenylalanine ammonia lyase, and increased levels of cuticular wax in rice seedlings under UV-B, NaCl, and PEG stress. The UV-C seedling priming of a cumulative dose of 10.2 kJ/m<sup>2</sup> was also found to significantly reduce leaf spot disease severity in strawberry plants due to induced accumulation of pathogenesis-related proteins, terpenes, phenolic compounds with triggered ROS, and antioxidant enzymes, while also inducing plant hormone synthesis [140]. Moreover, the transgenerational effect of UV-B priming was shown by the rice seedlings of the drought-tolerant Vaisakh variety being characterized by the increased expression of genes encoding antioxidant enzymes and stress-related proteins in F0 generation, with even more of an increase in the F1 generation after re-priming. This resulted in better protection against PEG stress [141]. The UV-B priming protection against UV-B stress was proven to be related to the UV RESISTANCE LOCUS (UVR8) pathway in *Arabidopsis thaliana*, since 14-day old seedlings without UVR8, primed for 10 min with UV-B at 35 μW/cm2, did not acquire UV-B resistance [142].

**Microwave radiation** is a form of electromagnetic radiation with a frequency ranging between 300 MHz and 300 GHz [143]. Physical seed priming with microwave radiation at 2.45 GHz for a short time had stimulatory effects on seed germination, seedling growth, and biomass accumulation in different cereals, such as barley, rice, and wheat [144]. A study by Bian et al. [145] proved that treatment of *Fagopyrum tataricum* with microwaves with a power of 300 W and a frequency of 2.45 GHz for 75 s optimally increased the activity of antioxidant enzymes (SOD, CAT, POD, and APX), leading to the increase in the total reduction potential of plants and the ability of the seedlings to neutralize radicals such as 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS), O2 •−, and •OH [145]. Microwaves have been used as factors for improving the resistance of crops to a number of stress factors. Maswada et al. [146] applied microwave priming prior to sowing two *Triticum aestivum* genotypes, Giza 168 and Gharbiya. The results of the conducted experiments proved that microwave priming (with 700 W of power, a variable frequency of 2.45 GHz, and a wavelength of 125 nm with a power intensity of 126 mW/cm2) improved wheat resistance to drought, increasing both the yield and growth parameters through improvement of tissue water content and a reduction in membrane permeability. Furthermore, osmotic adjustment and decreased H2O2 accumulation through increasing proline content and ROS scavenging activity were also observed [146]. In turn, Farid et al. [147] showed that microwaves can also help to alleviate heavy metal stress in *Brassica napus*. Pre-saw treatment of the genotype Faisal Canola (RBN-03060), with microwaves with a frequency of 2.45 GHz for 30 s, led to a greater ability to grow and biomass accretion for plants treated with Ni. Heavy metal-stressed plants also showed a higher concentration of photosynthetic pigments, including chlorophyll *a* and *b* and

carotenoids, and higher antioxidant enzyme activity (SOD, POD, APX, CAT), which was associated with a reduction in ROS (H2O2) content and the oxidative damage caused by them (MDA, electrolyte leakage). Moreover, it was shown that microwave priming resulted in greater accumulation of Ni from the soil, especially in roots, stems, and leaves [147].

**Magnetic fields** can be used for priming in several variants: as alternative magnetic field (AMF), electromagnetic field (EMF), pulsed magnetic field (PMF), static magnetic field (SMF), and sinusoidal magnetic field (SSMF) priming. All of those techniques were used in research on crops, and their application improved the germination and vigor of plants, as well as the response to unfavorable environmental factors, although SMF is the most common one [148]. Mohammadi and Roshandel [149] applied SMFs of 90 mT, 200 mT, and 250mT on *Hyssopus officinalis* plants for 5 min. The best effect was obtained at 200 mT. In response to drought stress, the plants subjected to magnetopriming showed higher dry matter content, total chlorophyll and phenol content, and a higher reduction capacity (DPPH, O2 •− scavenging) resulting from, among other factors, higher CAT, APX, and GPX activity. At the same time, magnetopriming led to a reduction in oxidative damage to biological membranes, which reduced electrolyte leakage [149]. Kataria et al. [150] applied an SMF of 200mT on soybean for 1 h, which resulted in increased resistance to salinity. Plants subjected to magnetopriming showed greater leaf area, leaf mass, photosynthetic activity, and nitrogenase activity than plants subjected to salinity stress only. However, the content of H2O2 and AsA, and the activity of antioxidant enzymes, was reduced due to magnetopriming. These changes resulted in higher biomass accumulation, yield, and harvest index values for soybean under both the saline and non-saline conditions [150]. Baghel et al. [151] showed that the use of a 200 mT SMF for 1 h on *Zea mays* plants reduced their susceptibility to salinity. Magnetopriming increased the content of photosynthetic pigments, as well as increasing photosynthesis parameters such as the quantum yield of PSII photochemistry (Fv/Fm), electron transport per leaf CS (ETo/CSm), the density of reaction centers (RC/CSm), and the performance index (PI). Moreover, the maize leaves showed lower H2O2 accumulation, which proves the reduction in oxidative stress. These changes resulted in better plant growth and increased maize yield under salinity conditions [151].

**Ultrasound priming** involves treating plants or seeds with the energy of acoustic waves with a frequency greater than 20 kHz [134]. Xia et al. [137] applied high-intensity ultrasound (HIU) with a frequency of 28 kHz and a power of 17.83 W/cm<sup>2</sup> for 5 to 30 min on brown rice seeds. Ultrasound priming led to both an increase in starch content and a simultaneous reduction in the size of grains and in the content of reducing sugars. Moreover, the accumulation of free amino acids, γ-aminobutyric acid, antioxidants, and proline (as stress-responsive secondary metabolites) may also have potentially positive effects on plant response to adverse environmental factors [137]. Dashab and Omidi [152] primed *Brassica napus* with ultrasound at 40 kHz and 59 kHz with a power of 60, 80, and 100 W for 2, 4, 6, 8, and 20 min. Depending on the combination of those parameters, different physiological effects were achieved. The greatest increase in seed germination was observed with 40 and 59 kHz at 100 W for 2 min of exposure, while an increase in vigor and seedling weight was observed with 59 kHz at 100 W. At 40 kHz with 80 W and an exposure time of 8 min, an increase in the content of photosynthetic pigments was determined [152]. In turn, Rao et al. [135] treated canola cultivars Youyanzao18 and Zaoshu104 for 1 min with ultrasound at a frequency of 20 kHz in order to reduce susceptibility to Cd stress. It has been shown that ultrasound, depending on the Cd dose, can improve such parameters as germination, shoot and root length, and fresh mass. Moreover, in the Youyanzao18 cultivar, ultrasound priming increased the activity of SOD, POD, CAT, and APX, as well as the increased content of proline, GSH, and soluble protein. This translated to a reduction in MDA content, which indicates less oxidative damage to biological membranes in response to Cd. In both cultivars, ultrasound increased pods per plant, seeds per pod, and rapeseed yield. Importantly, the accumulation of Cd in all parts of the plant decreased [135]. Similarly, Chen et al. [153] demonstrated that ultrasonic vibration can help wheat seedlings eliminate an excess amount of ROS resulting from Cd and Pb treatment, as well as improve the

biosynthesis of molecules and division of cells, leading to biomass accretion despite the metal stress.

#### 3.2.2. Priming with Ionizing Radiation

**Gamma (**γ**) radiation** is a high-energy type of ionizing radiation capable of penetrating and interacting with living tissues, whose absorbed dose is expressed in units of Gray (Gy). Usually, Cobalt-60 is used for this type of priming [144]. When Hussein [83] used 5, 10, and 20 Gy gamma radiation on barley plants, it was shown that both lower doses improved plant growth and yield, while the highest one (20 Gy) increased shoot growth and tiller number; however, only at the lowest radiation dose (5 Gy) was an increase in the content of photosynthetic pigments observed. Gamma radiation enhanced the accumulation of phenols, flavonoids, free amino acids, and antioxidant enzymes (APX, POD, CAT), but it also elevated H2O2 content. Moreover, it led to a reduction in the content of sugars and proline [83]. Researchers have proven that low doses of γ-rays not only modify redox homeostasis, but they also change the protein pattern and the metabolic profile in plants, leading to improved growth and yielding. As an example, a study by Hanafy and Akladious [154] showed that a dose of 100 Gy improved growth and yield for *Trigonella foenum-graecum* plants, as well as the content of soluble proteins in leaves and the content of phenols and flavonoids. Moreover, a significant rise in the content of AsA, α-tocopherol, retinol, and proline was observed. In turn, the highest dose of radiation (400 Gy) caused a decrease in the content of all tested parameters and induced changes in the DNA profile that consisted of the appearance and disappearance of polymorphic bands [154]. Pradhan et al. [155] used gamma radiation at a dose of 10 Gy on the microalgae *Chlamydomonas reinhardtii* (which is considered to be a model organism for studying the effects of heavy metals on photosynthetic organisms) and exposed it to Cd stress. As a result of Cd treatment, redox homeostasis was disturbed due to a decline in antioxidant enzyme activity and in the content of photosynthetic pigments. Consequently, cell death was induced and growth was minimized. On the contrary, the application of γ-radiation had positive effects on the mentioned parameters, and cell growth and biochemical synthesis were not injured. As a consequence, an increased resistance to toxic Cd ions was achieved [155].

**X-rays** are characterized by a wavelength ranging from 0.01 to 10 nm of the electromagnetic spectrum, which corresponds to frequencies ranging from 30 to 30,000 PHz and energies oscillating from 120 eV to 120 keV [144]. Currently, there are very few new studies concerning the effects of this type of priming on plants; however, in 2019, Rezk et al. [82] used X-rays in doses from 0 to 100 Gy on two genotypes of okra (*Hibiscus esculentus*), genotypes of Hassawi and Clemson. It was shown that radiation doses up to 5 Gy improved plant morphological parameters, the content of photosynthetic pigments, the activity of antioxidant enzymes (CAT, SOD, APX), and the content of low-molecular weight antioxidants (AsA, GSH, anthocyanins). In contrast, higher doses of radiation (at levels above 5 Gy) had the opposite effect, and plants treated with this type of priming showed greater lipid peroxidation caused by the increased concentration of ROS (mainly H2O2 and O2 •−) [82]. This confirmed the previous discoveries of Al-Enezi et al. [156] regarding the influence of X-rays on date palm (*Phoenix dactylifera* cv. Khalas). In this case, the inhibitory impact of radiation on seed germination was noticed even at a dose of 0.25 Gy, and a graduated increase in X-ray dose up to 15 Gy contributed to further reductions in germination; however, at the same time, an increase in root length was observed. A similar stimulatory effect was found for the leaf length of the date palm plants, but it concerned only X-ray doses between 0.05 and 0.25 Gy [156]. It can be summarized that only low doses of this type of radiation may improve plant growth parameters, but little is still known about its ameliorative actions under various stress conditions and further research is therefore required.

#### **4. Concluding Remarks**

In the present review, we have briefly discussed the adaptative traits of metallophytes, whose application may be an antidote to environmental pollution with heavy metals, perceived as one of the most dangerous factors for all living organisms. The amazing biology of metallophytes, especially in respect to metal detoxification and accumulation, as well as tolerance to drought and salinity, make them applicable for the phytoremediation and reclamation of chemically degraded areas which, after returning to their original state before contamination, can be reused for different goals. Furthermore, deeper insight into plants with evolutionarily developed tolerance mechanisms, may help to obtain specimens with ideal survival levels and fertility under stressful conditions. It seems to be particularly important to take into account that the majority of plants do not exhibit tolerance to abiotic stresses developing as a result of severe selection pressure due to the complexity associated with the inheritance of adaptive traits. Therefore, to combat the most important global problems, including metal pollution, drought, and salinity, through biological methods and to provide sustainable agriculture and food security for continuing global population growth, increasing attention is being given to priming strategies which make plants capable of responding more effectively and more rapidly to stress. Since priming offers a large variety of priming factors, doses, and application forms, the diversified morphological and biochemical responses of plants can be observed. Thus, the chemical and physical treatment for stress amelioration requires extensive future research for the elaboration of specific protocols in respect to optimal dosage and duration of exposure, which certainly vary between genotypes and environmental conditions. Furthermore, further understanding of both the mode of actions of particular priming agents and the mechanisms underlying the better performance of primed plants can lead to combined usage of various priming methods, preferably with synergistic effects that would allow a reduction in the dose of each agent compared to the dose used individually. Undoubtedly, the joint knowledge gathered here clearly indicates that all priming agents contribute to the scavenging of excess amounts of ROS via efficiently operating antioxidant machinery and thus put oxidative mitigation at the core of enhanced tolerance to various stressors.

**Author Contributions:** Conceptualization, E.M.; formal analysis, E.M.; writing—original draft preparation, M.L., K.D., J.F., M.G., A.R.-P., M.N., B.P., I.M. and E.M; visualization, M.G. and E.M.; supervision, E.M.; funding acquisition, M.L., K.D. and E.M. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

