International Journal of Molecular Sciences

Article Tolerance of Facultative Metallophyte acaulis to Cadmium Relies on Chelating and Antioxidative Metabolites

Sławomir Dresler 1,*, Maciej Strzemski 2 , Jozef Kováˇcik 3, Jan Sawicki 2, Michał Staniak 2 , Magdalena Wójciak 2, Ireneusz Sowa 2 and Barbara Hawrylak-Nowak 4

1 Department of Physiology and Biophysics, Institute of Biological Science, Maria Curie-Skłodowska University, Akademicka 19, 20-033 Lublin, Poland 2 Department of Analytical Chemistry, Medical University of Lublin, Chod´zki4a, 20-093 Lublin, Poland; [email protected] (M.S.); [email protected] (J.S.); [email protected] (M.S.); [email protected] (M.W.); [email protected] (I.S.) 3 Department of Biology, University of Trnava, Priemyselná 4, 918 43 Trnava, Slovakia; [email protected] 4 Department of Botany and Plant Physiology, University of Life Sciences in Lublin, Akademicka 15, 20-950 Lublin, Poland; [email protected] * Correspondence: [email protected]; Tel.: +48-81-537-5078

 Received: 4 April 2020; Accepted: 15 April 2020; Published: 18 April 2020 

Abstract: The impact of long-term chronic cadmium stress (ChS, 0.1 µM Cd, 85 days) or short-term acute cadmium stress (AS, 10 µM Cd, 4 days) on Carlina acaulis () metabolites was compared to identify specific traits. The content of Cd was higher under AS in all organs in comparison with ChS (130 vs. 16 µg g 1 DW, 7.9 vs. 3.2 µg g 1 DW, and 11.5 vs. 2.4 µg g 1 DW in roots, , and · − · − · − trichomes, respectively) while shoot bioaccumulation factor under ChS (ca. 280) indicates efficient Cd accumulation. High content of Cd in the trichomes from the AS treatment may be an anatomical adaptation mechanism. ChS evoked an increase in root biomass (hormesis), while the impact on shoot biomass was not significant in any treatment. The amounts of ascorbic acid and sum of phytochelatins were higher in the shoots but organic (malic and citric) acids dominated in the roots of from the ChS treatment. Chlorogenic acid, but not ursolic and oleanolic acids, was elevated by ChS. These data indicate that both chelation and enhancement of antioxidative power contribute to protection of plants exposed to long-term (chronic) Cd presence with subsequent hormetic effect.

Keywords: antioxidants; glutathione; heavy metals; phenolic metabolites; terpenoids

1. Introduction Response of plants to cadmium (Cd) stress has been widely studied over the last decades [1,2]. Thousands of experiments with various species proposed many hypotheses regarding the Cd tolerance mechanisms and Cd toxic action in plants. The main complications in unification of plant tolerance mechanisms to Cd are various time of exposure and applied concentrations. Sanità Di Toppi and Gabbrielli [1] pointed out that the major problem of the Cd–plant interaction is the use of a high Cd concentration for a short time, while a common concentration in standard soil is typically less than 1 µM. This implies that most of the papers studied acute stress (high concentrations with short-time exposure), which reflects environmental conditions inadequately. Studies dealing with chronic effect of Cd (long-time, low-concentration) in terrestrial [3,4] or aquatic plants [5,6] including algae [7] are less frequent. At the same time, exposure to low concentrations of metals including Cd may evoke the so-called hormetic effect.

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Cadmium (Cd) is readily absorbed by plants but has no known physiological function. MembersCadmium of the (Cd) Asteraceae is readily absorbedfamily (such by plants as butchamomile has no known and dandelion) physiological also function. readily Members absorb ofconsiderable the Asteraceae amounts family of (such Cd in as the chamomile shoots but and also dandelion) exhibit various also readily metabolic absorb responses considerable [8,9]. amounts Among ofthem, Cd changes in the shoots in non-enzymatic but also exhibit antioxidants various metabolic and chelators responses such [as8, 9ascorbic]. Among acid, them, thiols, changes organic in non-enzymaticacids, and phenols antioxidants are the most and chelatorscommon suchnot only as ascorbic in Cd-exposed acid, thiols, plants organic [10] but acids, also and under phenols abiotic are thestress most more common generally not [11]. only in Cd-exposed plants [10] but also under abiotic stress more generally [11]. Plants belonging to the Carlina genus (Asteraceae(Asteraceae family) contain various biologically active compounds [[12,13].12,13]. Hence,Hence, they they have have been been widely widely used used in folk in medicinefolk medicine [14]. Moreover,[14]. Moreover, some species some ofspecies Carlina, of Carlina, including includingCarlina aculis CarlinaL., aculis are facultative L., are facultative metallophytes, metallophytes, i.e., species i.e., that species tolerate that soils tolerate with elevatedsoils with content elevated of content heavy metals. of heavy Two metals. species, TwoC. species, acaulis and C. acaulisCarlina and vulgaris Carlina, are vulgaris part of, theare florapart of calaminethe flora of areas calamine located areas in metalliferous located in metalliferous sites in Bolesław sites in in Boles Southernław in Poland Southern [15,16 Poland]. [15,16]. The aimaim ofof this this study study was was to investigateto investigate similarities similarities and and differences differences in the in responses the responses of C. acaulis of C. exposedacaulis exposed to low Cdto concentrationslow Cd concentrations over a long over time a (chroniclong time stress, (chronic ChS) orstress, to high ChS) Cd or concentrations to high Cd overconcentrations a short time over (acute a short stress, time AS). (acute The expectedstress, AS). eventual The expected hormetic eventual effect of hormetic low Cd dose effect was of studiedlow Cd atdose the was level studied of various at the metabolites level of various and data metabolit are preciselyes and compared data are with precisely related compared (Asteraceae with family) related or unrelated(Asteraceae (metal family) hyper or unrelated/accumulators) (metal species. hyper/accumulators) species. 2. Results and Discussion 2. Results and Discussion 2.1. Impact of Chronic/Acute Cd Stress on the Growth 2.1. Impact of Chronic/Acute Cd Stress on the Growth Cadmium induces numerous changes in plants, including reduction of growth, modified morphology,Cadmium chlorosis induces/necrosis, numerous etc. changes [1,17]. Larssonin plants, et al.including [18] noted reduction that even of 0.5growth,µM ofmodified Cd can decreasemorphology, areachlorosis/necrosis, while dose above etc. 2.0[1,17].µM Larsson of Cd can et significantlyal. [18] noted reduce that even chlorophyll 0.5 µM content.of Cd can In agreementdecrease leaf with area these while findings, dose above the short-term2.0 µM of Cd 10 canµM significantly Cd stress in reduce the present chlorophyll study resultedcontent. In in visibleagreement necrotic with symptoms these findings, (Figure the S2). short-term Plants cultured 10 µM withCd stress low Cd in concentration the present study (0.1 µ M)resulted showed in significantlyvisible necrotic higher symptoms root biomass (Figure (by S2). ca. Plants 80%) incult comparisonured with withlow Cd the concentration control roots and (0.1 shoot µM) biomassshowed alsosignificantly showed higher increase root (by biomas ca. 20%)s (by but ca. the 80%) difference in comparison was not significantwith the control (Figure 1roots). The and growth shoot stimulatorybiomass also e ffshowedect of the increase low concentrations (by ca. 20%) ofbut non-essential the difference toxic was metals not significant is related to(Figure the so-called 1). The hormeticgrowth stimulatory effect [19]. effect As indicated of the low by Calabreseconcentrations [20], hormesis of non-essential is an adaptive toxic metals compensatory is related process to the inso-called response hormetic to stress effect and [19]. initial As disruption indicated by in homeostasis.Calabrese [20], One hormesis of the is mechanisms an adaptive responsible compensatory for stimulationprocess in ofresponse growth evokedto stress by theand low initial Cd dose disruption could be in an increasehomeostasis. in cell One proliferation of the bymechanisms functional substitutionresponsible for of Znstimulation (by Cd), whichof growth is a cofactorevoked ofby enzymes the low playingCd dose a majorcould rolebe an in increase replication in andcell translationproliferation [21 by]. functional substitution of Zn (by Cd), which is a cofactor of enzymes playing a major role in replication and translation [21].

Figure 1. EffectEffect of long-term chronic cadmium stress (ChS, 0.1 µµMM Cd, 85 days) or short-term acute cadmium stressstress (AS,(AS, 1010 µµMM Cd,Cd, 44days) days) on on the the fresh fresh weight weight of ofCarlina Carlina acaulis acaulisorgans. organs. Data Data are are means means ± SE± SE (n (=n 8–10);= 8–10); values values followed followed by by the the same same letter letter are notare significantlynot significantly different different (p < 0.05,(p < 0.05, Tukey’s Tukey’s test). test).

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2.2. Accumulation of Cd and Mineral. Nutrients The contentcontent ofof Cd Cd in in the the plants plants was was significantly significantly aff ectedaffected by itsbyconcentration its concentration and exposureand exposure time (Figuretime (Figure2). The 2). 10 TheµM Cd10 treatmentµM Cd tr (acuteeatment stress) (acute resulted stress) in almostresulted 8-fold in almost higher 8-fold Cd accumulation higher Cd inaccumulation the roots, compared in the roots, to compared the 0.1 µM to Cd the treatment 0.1 µM Cd (chronic treatment stress). (chronic Accumulation stress). Accumulation of Cd in the of abovegroundCd in the aboveground organs (leaves) organs was (leaves) also ca.was 2-foldalso ca. higher 2-fold in higher favor in of favor acute of stress acute with stress even with higher even dihigherfferences differences in trichomes in trichomes (Figure2). (Figure These data 2). indicateThese data that acuteindicate stress that evoked acute higher stress accumulation evoked higher of Cdaccumulation in the Carlina of Cd organs in the probably Carlina due organs to “break” probably of the due protective to “break mechanisms,” of the protective while plants mechanisms, exposed towhile chronic plants stress exposed had time to chronic to adapt stress to the had Cd time presence to adapt by modulation to the Cd ofpresence protective by metabolites,modulation asof mentionedprotective below.metabolites, Despite as lowermentioned absolute below. Cd content, Despite accumulation lower absolute efficiency Cd content, was higher accumulation under ChS ifefficiency expressed was as shoothigher bioaccumulation under ChS if expressed factor (shoot as sh Cdoot amountbioaccumulation as µg per gfactor DW/ solution(shoot Cd Cd amount as µg per as mL;µg per see g [22 DW/solution] for details) Cd with as aµg value per ofmL; ca. see 280. [22] In thefor relateddetails) species with a chamomilevalue of ca. (Asteraceae 280. In the family),related cultivationspecies chamomile with 1.5 µ(AsteraceaeM Cd over 7family), weeks ledcultivatio to shootn bioaccumulationwith 1.5 µM Cd factor over (BAF)7 weeks of only led 145to whileshoot Cdbioaccumulation/Ni hyperaccumulators factor (BAF) from of the only Thlaspi 145 while genus Cd/Ni had shoot hyperaccumulators BAF over 600 or from 1300, the respectively Thlaspi genus [22]. Withhad shoot increasing BAF over exogenous 600 or Cd1300, dose, respectively BAF values [22] typically. With increasing decrease, e.g., exogenous rice exposed Cd dose, to ca. BAF 4.5–18 valuesµM Cdtypically over 42 decrease, days had e.g., shoot rice BAF expo ca.sed 30–15 to ca. [ 234.5–18]. It seemsµM Cd that overC. 42 acaulis daysis had relatively shoot BAF good ca. accumulator 30–15 [23]. ofIt seems Cd at lowerthat C. concentrations acaulis is relatively and survey good accumulator of samples collected of Cd atfrom lower nature concentrations could show and an survey eventual of hazardsamples for collected human from health nature when could used inshow folk an medicine. eventual hazard for human health when used in folk medicine.

Figure 2.2. EEffectffect of long-term chronic cadmium stress (ChS, 0.1 µµMM Cd, 8585 days)days) oror short-termshort-term acute cadmium stressstress (AS,(AS, 10 10µ MµM Cd, Cd, 4 days) 4 days) on theon Cdthe accumulation Cd accumulation in the in roots, the leavesroots, (*leaves leaves (* without leaves trichomes),without trichomes), and trichomes and trichomes of C. acaulis of. C. Data acaulis are. means Data areSE means (n = 5); ± SE values (n = followed5); values by followed the same by letter the ± aresame not letter significantly are not significantly different (p

Cd is a highlyhighly mobilemobile element,element, and its progressiveprogressive accumulationaccumulation in relation to longer exposure time hashas beenbeen observedobserved inin variousvarious speciesspecies [[3,9].3,9]. Owing to greater increase in the root Cd contentcontent under AS, the translocation factorsfactors (TF) leafleaf/root/root or trichometrichome/root/root decreaseddecreased and trichometrichome/leaf/leaf TFTF increased (Table(Table S1).S1). ThisThis phenomenon phenomenon has has not not yet ye beent been reported reported and and we we assume assume that that under under high high Cd concentrationCd concentration (acute (acute stress stress in our in work), our work), Cd can Cd partly can bepartly detoxified be detoxified by storage by in storage the leaf in trichomes the leaf andtrichomes this mechanism and this mechanism of Cd detoxification of Cd detoxification has been proposed has been in proposed several species in several [24–26 species]. Leaf /[24–26].root TF underLeaf/root chronic TF stressunder waschronic ca. 0.2 stress (Table was S1), whichca. 0.2 is (T similarable S1), to related which species is similar (Asteraceae to related family) species such as(Asteraceae chamomile family) cultured such with as 1.5 chamomileµM Cd over cultured 7 weeks wi (TFth ca.1.5 0.13;µM Cd [22 ]),over dandelion 7 weeks cultured (TF ca. with0.13; 30 [22]),µM Cddandelion over 2 weeks cultured (TF with ca. 0.19; 30 µM [9]) Cd or Tanacetum over 2 weeks parthenium (TF ca.cultured 0.19; [9]) with or Tanacetum 35–70 µM parthenium Cd over 40 dayscultured (TF ca.with 0.11–0.20; 35–70 µM [27 Cd]). Owingover 40 todays preferential (TF ca. 0.11–0.20; Cd accumulation [27]). Owing in the to roots, preferentialC. acaulis Cdis accumulation a clear excluder in ofthe Cd. roots, C. acaulis is a clear excluder of Cd. Several mechanisms of the impact of Cd on the uptake of nutrients have been postulated: competition for Ca transporters, inhibition of Fe loading to the xylem, or indirect influence on nutrient movement [28]. Our results showed that the exposure to both Cd treatments resulted in a

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Several mechanisms of the impact of Cd on the uptake of nutrients have been postulated: competition for Ca transporters, inhibition of Fe loading to the xylem, or indirect influence on nutrient movement [28]. Our results showed that the exposure to both Cd treatments resulted in a decrease in Ca and Mg root accumulation. Additionally, Cd depleted the content of K and Cu in the leaves (Table1). These results are in agreement with previous studies [ 29] which showed that two Atriplex species exposed to Cd contained significantly lower levels of K and Ca. Interestingly, it was found that leaves exposed to ChS accumulated more Ca than the control or AS-treated plants. The elevated amount of Ca in the leaves under low Cd stress may be a sign of some protective mechanism. The role of Ca in attenuation of Cd toxicity is known [29,30] and several hypotheses have been put forward, including antioxidative protection, competition of Ca for the same channel transporters with Cd, improved antioxidant enzyme activities [31], or even excretion of Ca-Cd crystals through trichomes [25]. Micronutrients (such as Cu or Zn) were rather negatively affect by some of the stresses (Table1) while Mn increased in ChS-exposed plants, indicating a need for further study of Cd–Mn interactions.

Table 1. Accumulation of selected mineral nutrients in the leaves (without trichomes), roots, and trichomes of C. acaulis exposed to long-term chronic cadmium stress (ChS, 0.1 µM Cd, 85 days) or short-term acute cadmium stress (AS, 10 µM Cd, 4 days). Data are means SE (n = 5); values followed ± by the same letter are not significantly different between treatments (p < 0.05, Tukey’s test).

Ca mg g 1 DW Mg mg g 1 DW · − · − leaf root trichomes leaf root trichomes Control 13.1 0.3 b 5.81 0.37 a 11.7 0.9 a 5.53 0.15 a 4.31 0.92 a 2.78 0.15 a ± ± ± ± ± ± ChS 14.6 0.3 a 4.16 0.16 b 13.6 0.5 a 5.58 0.35 a 2.44 0.21 b 3.07 0.23 a ± ± ± ± ± ± AS 13.7 0.4 b 4.12 0.48 b 13.7 0.7 a 4.96 0.22 a 2.41 0.11 b 3.04 0.09 a ± ± ± ± ± ± K mg g 1 DW Fe µg g 1 DW · − · − leaf root trichomes leaf root trichomes Control 85.0 3.0 a 46.5 3.2 a 28.6 2.0 a 79.9 3.3 a 3153 682 a 129.8 6.3 a ± ± ± ± ± ± 116.8 17.6 ChS 74.8 4.4 b 42.4 1.8 a 29.7 3.7 a 72.8 6.7 a 3535 517 a ± ± ± ± ± ± a AS 70.9 1.9 b 45.6 3.9 a 28.2 1.2 a 76.8 3.4 a 3463 325 a 128.5 6.4 a ± ± ± ± ± ± Cu µg g 1 DW Mn µg g 1 DW · − · − leaf root trichomes leaf root trichomes Control 6.13 0.34 a 12.1 1.3 a 6.71 0.85 a 7.02 0.72 b 5.80 0.86 b 7.06 1.40 a ± ± ± ± ± ± ChS 4.82 0.37 b 10.6 1.9 a 4.91 0.47 a 9.92 1.15 a 10.10 0.92 a 7.65 1.42 a ± ± ± ± ± ± AS 4.56 0.53 b 11.8 1.3 a 5.35 0.49 a 7.35 1.03 b 9.13 1.32 a 6.96 0.68 a ± ± ± ± ± ± Zn µg g 1 DW Mo µg g 1 DW · − · − leaf root trichomes leaf root trichomes Control 22.4 2.2 a 64.0 2.9 a 25.2 2.8 a 4.45 0.91 a 11.51 4.00 a 2.60 0.48 a ± ± ± ± ± ± ChS 20.1 0.8 a 28.1 1.7 b 17.6 1.9 b 3.09 0.52 a 8.79 4.03 a 2.58 0.39 a ± ± ± ± ± ± AS 21.5 1.8 a 53.0 6.6 a 22.0 3.1 ab 2.94 0.38 a 11.26 1.79 a 2.39 0.21 a ± ± ± ± ± ±

2.3. Changes in Antioxidants and Chelators Differ. under Acute and Chronic Cd Stress Both AsA and GSH are essential components of the ascorbate-glutathione pathways of ROS removal. Interestingly, both AS and ChS elevated the AsA amount but reduced the GSH content in the shoots, indicating possible reciprocal changes between these antioxidants (Figure3a,b). At the same time, the root contents of these metabolites remained unaffected and indicated more pronounced changes in the photosynthetic tissue. In agreement, ascorbic acid is often elevated by Cd excess but GSH rather decreases during long-term exposure [10]. Lower GSH content in the shoots also confirms its role as a precursor for PCs [32,33], while a more intensive increase in PCs without any impact on the GSH content may indicate more intensive biosynthesis of GSH as a protective mechanism against higher accumulation of Cd in the roots (compared to shoots). Int. J. Mol. Sci. 2020, 21, 2828 5 of 14 Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 5 of 14

Figure 3. EffectEffect of long-term chronic cadmium stress stress (ChS, (ChS, 0.1 0.1 µMµM Cd, 85 days) or short-term acute cadmiumcadmium stress (AS,(AS, 1010µ µMM Cd, Cd, 4 4 days) days) on on (a )(a ascorbic) ascorbic acid acid (AsA); (AsA); (b) glutathione(b) glutathione (GSH); (GSH); and and (c) sum (c) of phytochelatins (PCs) content in the shoots and roots of C. acaulis. Data are means SE (n = 5); values sum of phytochelatins (PCs) content in the shoots and roots of C. acaulis. Data are means± ± SE (n = 5); valuesfollowed followed by the sameby the letter same are letter not are significantly not significantly different different (p < 0.05, (p Tukey’s< 0.05, Tukey’s test). test).

An increasingincreasing AsA AsA level level was was determined determined in the in shoots the shoots of plants of exposedplants exposed to ChS and to ASChS (Figure and 3ASa). (FigureHowever, 3a). the However, highest accumulation the highest accumulation of AsA was observed of AsA inwas the observed ChS shoots. in the It has ChS been shoots. shown It has that been AsA shownprotects that plant AsA cells protects against plant Cd-induced cells against oxidative Cd-induced damage oxidative [34] and damage the age of[34] plants and the a has age significant of plants aimpact has significant on AsA accumulation impact on AsA under accu Cdmulation stress [under35]. Cd stress [35]. Contrary toto AsAAsA and and/or/or GSH GSH accumulation, accumulation, the th contente content of the of sumthe ofsum PCs of in PCs the shootsin the dishootsffered differedbetween between the treatments the treatments (Figure3 c).(Figure PCs are3c). considered PCs are considered as one of theas one major of the Cd major intercellular Cd intercellular chelating chelatingligands and ligands their contentand their is usuallycontent relatedis usually to the relate metald to concentration the metal concentration [36]. In their [36]. review In paper,their review Sanità paper,Di Toppi Sanità and GabbrielliDi Toppi [and1] pointed Gabbrielli out that[1] PCspointe ared theout main that mechanismPCs are the of main plant mechanism cells to cope of with plant Cd cellsstress; to however,cope with they Cd stress; indicated however, that most they investigations indicated that were most focused investigations on acute were Cd stress. focused Our on results acute Cdshowed stress. that Our the results Cd stress showed (both that treatments) the Cd stress significantly (both treatments) elevated the significantly sum of PCs, elevated but the the ChS sum plants of PCs, but the ChS plants had over 2-fold higher PC content than in the AS treatment (Figure 3c). The data showed that even at the lower Cd accumulation in the ChS shoots, the content of PCs was

Int. J. Mol. Sci. 2020, 21, 2828 6 of 14 hadInt. J. overMol. Sci. 2-fold 2020, higher21, x FOR PC PEER content REVIEW than in the AS treatment (Figure3c). The data showed that even6 of at 14 the lower Cd accumulation in the ChS shoots, the content of PCs was higher than in the AS treatment higher than in the AS treatment which involved higher doses of Cd (cf. Figure 2; Figure 3c), which involved higher doses of Cd (cf. Figure2; Figure3c), suggesting that the exposure time, in suggesting that the exposure time, in addition to the applied metal dose, also plays a role. Sun et al. addition to the applied metal dose, also plays a role. Sun et al. [36] suggested that an increasing Cd [36] suggested that an increasing Cd concentration in the nutrient medium elevates PC content but concentration in the nutrient medium elevates PC content but Cd concentration above a critical value Cd concentration above a critical value reduces the content of PCs due to severe toxicity. On the reduces the content of PCs due to severe toxicity. On the other hand there is evidence that even 20 nM other hand there is evidence that even 20 nM of Cd can induces PC in Ceratophyllum demersum [6]. of Cd can induces PC in Ceratophyllum demersum [6]. Organic acids are efficient compounds in detoxification of heavy metals. They are involved in Organic acids are efficient compounds in detoxification of heavy metals. They are involved several mechanisms including (1) reduction of metal availability by chelation with exudates; (2) in several mechanisms including (1) reduction of metal availability by chelation with exudates; intracellular metal chelation; (3) long-distance translocation of metals to compartments with low (2) intracellular metal chelation; (3) long-distance translocation of metals to compartments with low biological activity such as trichomes and the cell wall [37–39]. Organic acids are produced in biological activity such as trichomes and the cell wall [37–39]. Organic acids are produced in response response to Cd by various species of vascular plants [39,40] or algae [34]. In the present work, to Cd by various species of vascular plants [39,40] or algae [34]. In the present work, accumulation of accumulation of citric and malic acids significantly increased (ca. by 2–3-fold compared with citric and malic acids significantly increased (ca. by 2–3-fold compared with control) in both ChS and control) in both ChS and AS shoots but only in the roots in the ChS treatment (Figure 4). It seems AS shoots but only in the roots in the ChS treatment (Figure4). It seems that elevated accumulation of that elevated accumulation of organic acids may be a mechanism for tolerance to chronic Cd stress, organic acids may be a mechanism for tolerance to chronic Cd stress, perhaps through exudation since perhaps through exudation since the ChS treatment resulted in a lower level of Cd in the tissues the ChS treatment resulted in a lower level of Cd in the tissues than in the AS treatment (cf. Figure2; than in the AS treatment (cf. Figure 2; Figure 4). This phenomenon has been described in various Figure4). This phenomenon has been described in various species including crops [41]. species including crops [41].

Figure 4. Effect of long-term chronic cadmium stress (ChS, 0.1 µM Cd, 85 days) or short-term acute Figure 4. Effect of long-term chronic cadmium stress (ChS, 0.1 µM Cd, 85 days) or short-term acute cadmium stress (AS, 10 µM Cd, 4 days) on (a) malic acid; and (b) citric acid content in the shoots and cadmium stress (AS, 10 µM Cd, 4 days) on (a) malic acid; and (b) citric acid content in the shoots roots of C. acaulis. Data are means SE (n = 5); values followed by the same letter are not significantly and roots of C. acaulis. Data are ±means ± SE (n = 5); values followed by the same letter are not different (p < 0.05, Tukey’s test). significantly different (p < 0.05, Tukey’s test). 2.4. Changes of Secondary Metabolites under Various Exposure 2.4. Changes of Secondary Metabolites under Various Exposure Two triterpene acids were detected in the shoots only and their accumulation was significantly enhancedTwo triterpene (approx. 170% acids of were control) detected in the in AS the treatment shoots only (Figure and5 ).their The accumulation concentration was of these significantly acids in enhanced (approx. 170% of control) in the AS treatment (Figure 5). The concentration of these acids in response to heavy metals has only rarely been discussed in the literature, mainly in relation to antioxidant properties, which may also play a role under metal stress [42]. It has been shown that

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Int.response J. Mol. Sci. to 2020 heavy, 21 metals, x FOR PEER has only REVIEW rarely been discussed in the literature, mainly in relation to antioxidant7 of 14 properties, which may also play a role under metal stress [42]. It has been shown that heavy metal stress heavyincreased metal ursolic stress acid increased in Prunella ursolic vulgaris [acid43] andin CdPrunella elicited vulgaris higher accumulation[43] and Cd of oleanolicelicited higher acid in accumulationthe cell cultures of of oleanolicAchyranthes acid bidentate in the[ 44cell] or cultures triterpenoid of Achyranthes saponins in bidentateBacopa monnieri [44] or[ 45triterpenoid]. However, saponinsa negative in e ffBacopaect of Cdmonnieri or Cu stress[45]. However, on the content a negative of triterpene effect hasof Cd also or been Cu observedstress on in the plant content cultures of triterpeneof Centella has asiatica also[ 46been]. Wang observed et al. [44in ]plant suggested cultures that inductionof Centella of asiatica oleanolic [46]. acid Wang accumulation et al. [44] is suggestedrelated to thethat exposure induction time of oleanolic to Cd and acid probably accumula to genetion is expression related to of the 3-hydroxy-3-methylglutaryl exposure time to Cd and probablycoenzyme to A gene reductase expression in this of pathway. 3-hydroxy-3-methylglutaryl coenzyme A reductase in this pathway.

FigureFigure 5. 5. EffectEffect of of long-term long-term chronic chronic cadmium cadmium stress stress (ChS, (ChS, 0.1 0.1 µMµM Cd, Cd, 85 85 days) days) or or short-term short-term acute acute cadmiumcadmium stress (AS,(AS, 1010µ µMM Cd, Cd, 4 days)4 days) on on ursolic ursolic and and oleanolic oleanolic acids acids content content in the in shoots the shoots of C. acaulisof C. . acaulisData are. Data means are meansSE (n ±= SE5); ( valuesn = 5); followedvalues followed by thesame by the letter same are letter not significantlyare not significantly different different (p < 0.05, ± (Tukey’sp < 0.05, test).Tukey’s test).

ChSChS considerably stimulatedstimulated (over (over 9.4-fold 9.4-fold for for shoot shoot and and 3.2-fold 3.2-fold for root, for comparedroot, compared with control) with control)mainly themainly accumulation the accumulation of chlorogenic of chlorogenic acid in both acid organs, in both while organs, the ASwhile treatment the AS induced treatment an inducedincrease inan theincrease content in of the this content acid which of this was acid not which significantly was not aff significantlyected (Figure affected6a). The (Figure accumulation 6a). The of accumulation3,5-dicaffeoylquinic of 3,5-dicaffeoylquinic acid was significantly acid elevatedwas signif inicantly the shoots elevated only (byin the approx. shoots 2.5-fold) only (by (Figure approx.6b). 2.5-fold)This increase (Figure in both 6b). phenolicThis increase acids underin both the phen ChSolic treatment acids under was related the ChS to higher treatment TPC andwas antioxidant related to highercapacity TPC in theand roots, antioxidant compared capacity to the in control, the roots, and significantcompared to elevation the control, of TPC and in significant the shoots comparedelevation ofto TPC AS (Figure in the S3).shoots In compared an earlier to work, AS Kov(Figureáˇcikand S3). In Klejdus an earlier [8] observedwork, Ková significantčik and elevationKlejdus [8] of observedchlorogenic significant acid in the elevation related speciesof chlorogenic chamomile acid (Asteraceae in the related family) species after prolongedchamomile exposure (Asteraceae even family)to a low after Cd prolonged dose (3 µM), exposure indicating even that to a chlorogenic low Cd dose acid (3 µM), has probably indicating more that generalchlorogenic antioxidative acid has probablyaction. The more negative general eff ectantioxidative of multi-heavy action. metal The stressnegative on chlorogeniceffect of multi-heavy acid accumulation metal stress has alsoon chlorogenicbeen observed acid in Carlinaaccumulation vulgaris hasplants also collected been observed from metalliferous in Carlina areas vulgaris [15]: plants the authors collected found from that metalliferousplants inhabiting areas heavy [15]: metal the pollutedauthors found areas accumulatedthat plants lessinhabiting soluble phenolicsheavy metal and polluted flavonoids areas and accumulatedexhibited lower less antioxidant soluble phenolics capacity and than flavonoids plants from and non-polluted exhibited lower areas. antioxidant capacity than plants from non-polluted areas.

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Figure 6. EffectEffect of long-term chronic cadmium stress (ChS, 0.1 µMµM Cd, 85 days) or short-term acute cadmium stressstress (AS,(AS, 1010µ µMM Cd, Cd, 4 4 days) days) on on (a ()a chlorogenic) chlorogenic acid acid and and (b )( 3,5-dicab) 3,5-dicaffeoylquinicffeoylquinic acid acid in the in theshoots shoots and and roots roots of C. of acaulis C. acaulis. Data. Data are meansare meansSE ± (SEn = (n5); = values5); values followed followed by theby the same same letter letter are notare ± notsignificantly significantly diff erentdifferent (p < (0.05,p < 0.05, Tukey’s Tukey’s test). test).

2.5. Principle Principle Component Analysis The PCA of the obtained variables, especially fr fromom the shoots (Figure 77a),a), clearlyclearly separatedseparated thethe individuals intointo threethree groups groups according according to to the the experimental experimental treatments. treatments. The The first first factor factor explained explained 37% 37%and 35%and of35% total of variability total variability for the shoots for the and shoots roots, an respectively,d roots, respectively, while the second while factor the explainedsecond factor 17% explainedfor the shoots 17% and for 15% the forshoots the roots and (Figure15% for7a,b). the Thisroots means (Figure that 7a,b). both factorsThis means explained that approx.both factors 54% explainedand slightly approx. more than 54% 50% and of slightly the total more variance than for 50% the of shoots the total and roots,variance respectively. for the shoots In the and case roots, of the respectively.shoots, the first In factorthe case facilitated of the separationshoots, the of first both factor Cd-stressed facilitated groups separation of plants fromof both the Cd-stressed control, and groupsfactor 1 of was plants positively from the correlated control, with and GSH,factor K,1 was and Cupositively and negatively correlated correlated with GSH, with K, bothand Cu organic and negativelyacids, 3,5-dica correlatedffeoylquinic with acid, both AsA, organic PCs, Cd, acids, and triterpenes.3,5-dicaffeoylquinic On the other acid, hand, AsA, the PCs, shoot Cd, biomass and wastriterpenes. strongly On correlated the other with hand, factor the 2. shoot Factor biomass 2 was alsowas partially strongly determined correlated wi byth the factor Cd,ursolic, 2. Factor and 2 wasoleanolic also partially acid variables, determined whose by high the contentsCd, ursolic, were and noted oleanolic in the acid shoots variables, of the ASwhose plants high (Figure contents7a). wereIn the noted roots, in factor the shoots 1 distinguished of the AS plants the control (Figure and 7a). ChSIn the plants roots, (Figure factor 71b). distinguished The first component the control wasand positivelyChS plants determined (Figure 7b). by The the contentfirst component of phenolic was compounds, positively organicdetermined acids, by and the biomass content and of phenolicnegatively compounds, correlated withorganic Zn, acids, Ca, and and Mg. biomass In turn, and factor negatively 2 separates correlated the AS with roots–individuals Zn, Ca, and Mg. with In turn,high Cd,factor Mo, 2 Fe,separates Cu, and the partially AS roots–individuals AsA and PC content. with high Cd, Mo, Fe, Cu, and partially AsA and PC content.

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FigureFigure 7. 7. ScaledScaled scatter scatter plot plot of of principal principal component component analysis analysis of of selected selected secondary secondary metabolites metabolites (ChA—chlorogenic(ChA—chlorogenic acid; acid; CQA—3, CQA—3,5-dica5-dicaffeoylquinicffeoylquinic acid; acid; UA—urso UA—ursoliclic acid; acid; OA—oleanolic OA—oleanolic acid; acid; TPC—totalTPC—total phenolic phenolic content),content), thiol-peptidesthiol-peptides (GSH—glutathione; (GSH—glutathione; PCs—phytochelatins), PCs—phytochelatins), organic organic acids acids(MA—malate (MA—malate acid; CA—citrateacid; CA—citrate acid), antioxidant acid), anti capacityoxidant (ABTS),capacity biomass, (ABTS), macro- biomass, and microelementsmacro- and microelementsin the shoots (ina) andthe shoots roots ( b().a) Theand lengthroots ( ofb). lines The showslength aof correlation lines shows between a correlation original between data and originalfactor axes. data and factor axes. 3. Materials and Methods 3. Materials and Methods 3.1. Plant. Material, Growth Conditions, and Experimental Design 3.1. Plant. Material, Growth Conditions, and Experimental Design The achenes of Carlina acaulis L. (Asteraceae, voucher specimen no. 2005A) were obtained from theThe Botanical achenes Garden of Carlina of Maria acaulis Curie-Skłodowska L. (Asteraceae, voucher University specimen in Lublin. no. 2005A) They were were obtained germinated from on thethe Botanical surface of Garden garden of soil Maria and Curie-Sk 10-day oldłodowska seedlings University were transferred in Lublin. into They polyethylene were germinated pots filled on thewith surface garden of soil.garden After soil 28 and days 10-day of soil old cultivation, seedlings thewere plants transferred were carefully into polyethylene washed with pots distilled filled withwater garden and transferred soil. After into28 days pots (oneof soil plant cultivation, per pot) withthe plants 0.5 L ofwere half carefully strength Hoagland’swashed with hydroponic distilled watermedium and [ 40transferred]. After 5 daysinto ofpots acclimation, (one plant the per plants pot) were with divided 0.5 L intoof half three strength groups (15Hoagland’s plants per hydroponictreatment): medium (1) control [40]. plants After (continual 5 days of hydroponicacclimation, cultivationthe plants were without divided addition into ofthree Cd), groups (2) chronic (15 plantsstress /perChS treatment): (85 days of (1) cultivation control plants with (continual 0.1 µM Cd), hydroponic and (3) acute cultivation stress/ ASwithout (4 days addition of exposure of Cd), to (2) chronic stress/ChS (85 days of cultivation with 0.1 µM Cd), and (3) acute stress/AS (4 days of exposure to 10 µM Cd): total cultivation in hydroponics was 90 days in all treatments (see time axes

Int. J. Mol. Sci. 2020, 21, 2828 10 of 14

10 µM Cd): total cultivation in hydroponics was 90 days in all treatments (see time axes in Figure S1). The culture solutions were continuously aerated, evapotranspiration loss was replenished daily with dH2O, and the medium was renewed every 14 days to prevent micronutrient deficiency. Cadmium was added in the form of Cd(NO ) 4H O (Sigma-Aldrich, St. Louis, MO, USA). Cultivation was 3 2· 2 carried out in a growth chamber at 18/25 ◦C (night/day) under light-emitting diodes at photosynthetic 2 1 photon flux density of 90 µmol m− s− and relative humidity of 60–65%. Plants from all treatments were harvested after total 90 days of cultivation in hydroponics. Plants were separated into shoots and roots and the fresh weight was determined. Five individual plants were powdered in liquid nitrogen and stored at 80 C for determination of organic acids, phytochelatins (PCs), glutathione − ◦ (GSH), and ascorbic acid (AsA). Parallel aliquots were dried at room temperature for determination of secondary metabolites and at 70 ◦C to constant weight for quantification of Cd and mineral nutrients. Two parallel repetitions of the whole experiments were performed to verify the responses of the main parameters including growth changes, Cd accumulation, and thiol content.

3.2. Determination of Cd and Mineral. Nutrients Cd and mineral nutrients were determined in trichomes and parenchyma separately. The separation of trichomes from parenchyma was achieved in several steps. First, dry shoots were milled, then the trichomes were separated from parenchyma by shaking (WL-2000 shaker, JWElectronic, Warsaw, Poland) on a perforated plate with pore size 1.5 mm. These processes were repeated until clean trichomes fraction was obtained without visible parenchyma particles. The roots, trichomes, and leaves without trichomes were digested in 5 mL of a mixture of HNO3:H2O (2:8 v/v) in a microwave digestion apparatus (TOPwave, Analytick Jena AG, Jena, Germany). The resulting clear solutions were transferred into volumetric flasks and filled up to 25 mL with deionized water. The amounts of all elements were measured using ICP-OES PlasmaQuant PQ 9000 Elite (Analityk Jena AG, Jena, Germany). Effective plasma power was 1300 W and the plasma, auxiliary, and nebulizer argon flow rate were 12.0, 0.5, and 0.6 L/min, respectively. Attenuated axial direction of measurement for Ca, K, Mg and axial direction for Cd, Zn, Mn, Fe, Mo, Cu were applied. Each sample was measured in three replicates.

3.3. Measurement of Organic Acids, Ascorbic Acid, and Thiols The organic acids, AsA, and thiols were analyzed in the plant tissue using Agilent 7100 Capillary Electrophoresis (Agilent Technologies, Santa Clara, CA, USA): the organic acids (malic and citric acids) according to the method proposed by [40], total AsA content following [35], and thiols (phytochelatins and glutathione) after monobromobimane derivatization [33].

3.4. HPLC of Triterpene and Phenolic Acids A total of 0.5 g of dried plant material was extracted three times with 100% methanol (3 1.5 mL) × in an ultrasonic bath for 30 min. The extracts were combined, filtered through 0.22 nylon filters, and filled up to 5 mL in a volumetric flask. High performance liquid chromatography analyses were performed on a VWR Hitachi Chromaster 600 chromatograph (Merck, Darmstadt, Germany) with a PDA detector (Merck, Darmstadt, Germany) and EZChrom Elite software (ver. 3.18, Merck, Darmstadt, Germany). The RP18e LiChrosper 100 column (Merck, Darmstadt, Germany) (25 cm 4.9 mm i.d., × 5 µm particle size) was used to separate triterpenic acids—oleanolic and ursolic acid. Other technical details are the same as reported previously [12]. Chlorogenic and 3,5-dicaffeoylquinic acids were analyzed using C18 reversed-phase column Kinetex (Phenomenex, Torrance, CA, USA) (10 cm × 4.0 mm i.d., 2,6 µm particle size) according to previous work [47]. The identity of compounds was confirmed by comparison of the retention time and spectral similarity with standards. Int. J. Mol. Sci. 2020, 21, 2828 11 of 14

3.5. Quantification of Total Phenolic Content and Antioxidant Capacity Analyses were performed in the same methanolic extract used for determination of triterpene and phenolic acids. The total (soluble) phenolic content (TPC) expressed as mg of gallic acid equivalents per gram of air dry weight of plants was measured using the Folin–Ciocalteu reagent as described previously [9]. The antioxidant capacities expressed as mg of trolox equivalents per gram of air dry weight were measured using free radical ABTS/2-azino-bis-3ethyl-benzthiazoline-6 sulphonic acid [48].

3.6. Statistical Analysis Samples from five individual plants were assessed for each treatment, parameter, and organ (n = 5). One-way ANOVA followed by a Tukey’s post-hoc test was used to evaluate the significance of differences (p < 0.05) between treatments. Principal component analyses (PCA) were performed separately for shoots and roots based on all studied parameters. All statistical analyses were carried out using Statistic ver. 13.3 software (TIBCO Software Inc. 2017, Palo Alto, CA, USA).

4. Conclusions The present study demonstrated some different physiological responses of Carlina acaulis to chronic (long time/low concentration) and acute (short time/high concentration) Cd stress. Although mineral nutrients were typically negatively affected by Cd, chronic stress had fewer negative effects and even stimulated root growth, probably due to lower endogenous accumulation of Cd. At the same time, ascorbic acid and phytochelatins were more elevated in the shoots but the content of organic (malic and citric) acids increased in the roots of plants from chronic treatment. In combination with the strongly elevated chlorogenic acid content in this treatment, both chelation and enhancement of antioxidative power are expected to contribute to protection in the plants exposed to the long-term Cd stress. On the contrary, the role of triterpene acids in chronic or acute stress tolerance was not immediately apparent.

Supplementary Materials: Supplementary materials can be found at http://www.mdpi.com/1422-0067/21/8/2828/ s1. Author Contributions: S.D., M.S. (Maciej Strzemski), and J.K. conceived the experiments and the experimental design; S.D. did project administration; S.D. B.H.-N. and M.S. (Maciej Strzemski) cultivated the plants; S.D. and B.H.-N. performed LMWOA, GSH, AOC, AsA, and PC analysis; M.S. (Michał Staniak) and M.W. analyzed the secondary metabolites; I.S. and J.S. analyzed the elements; S.D. conducted statistical analysis and data visualization; S.D. prepared the original manuscript draft; J.K. was involved in the experimental ideas and critically revised the manuscript. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Acknowledgments: This work was supported by the Botanical Garden of Maria Curie-Skłodowska in Lublin (Poland). We would like to thank Gra˙zynaSzymczak and Mykhaylo Chernetskyy for providing achenes of Carlina acaulis. The authors thank Anna Zo´nfor the English language editing. Conflicts of Interest: The authors declare no conflict of interest.

References

1. Sanità Di Toppi, L.; Gabbrielli, R. Response to cadmium in higher plants. Environ. Exp. Bot. 1999, 41, 105–130. [CrossRef] 2. Clemens, S. Molecular mechanisms of plant metal tolerance and homeostasis. Planta 2001, 212, 475–486. [CrossRef] 3. Arduini, I.; Masoni, A.; Mariotti, M.; Ercoli, L. Low cadmium application increase miscanthus growth and cadmium translocation. Environ. Exp. Bot. 2004, 52, 89–100. [CrossRef] 4. Kováˇcik,J.; Klejdus, B.; Štork, F.; Hedbavny, J. Physiological responses of Tillandsia albida (Bromeliaceae) to long-term foliar metal application. J. Hazard. Mater. 2012, 239–240, 175–182. [CrossRef] Int. J. Mol. Sci. 2020, 21, 2828 12 of 14

5. Andresen, E.; Kappel, S.; Stärk, H.-J.; Riegger, U.; Borovec, J.; Mattusch, J.; Heinz, A.; Schmelzer, C.E.H.; Matoušková, Š.; Dickinson, B.; et al. Cadmium toxicity investigated at the physiological and biophysical levels under environmentally relevant conditions using the aquatic model plant Ceratophyllum demersum. New Phytol. 2016, 210, 1244–1258. [CrossRef][PubMed] 6. Andresen, E.; Mattusch, J.; Wellenreuther, G.; Thomas, G.; Abad, U.A.; Küpper, H. Different strategies of cadmium detoxification in the submerged macrophyte Ceratophyllum demersum L. Metallomics 2013, 5, 1377–1386. [CrossRef][PubMed] 7. Perreault, F.; Dionne, J.; Didur, O.; Juneau, P.; Popovic, R. Effect of cadmium on photosystem II activity in Chlamydomonas reinhardtii: Alteration of O–J–I–P fluorescence transients indicating the change of apparent activation energies within photosystem II. Photosynth. Res. 2011, 107, 151–157. [CrossRef][PubMed] 8. Kováˇcik,J.; Klejdus, B. Dynamics of phenolic acids and lignin accumulation in metal-treated Matricaria chamomilla roots. Plant. Cell Rep. 2008, 27, 605–615. [CrossRef][PubMed] 9. Kováˇcik,J.; Bujdoš, M.; Ketzer, P.; Babula, P.; Peterková, V.; Krenn, L. Dandelion is more tolerant to cadmium than to nickel excess. Chemosphere 2019, 224, 884–891. [CrossRef] 10. Kováˇcik,J. Role of low molecular weight compounds in cadmium stress tolerance. In Cadmium Tolerance in Plants; Hasanuzzaman, M., Vara Prasad, M.N., Nahar, K., Eds.; Academic Press: Cambridge, MA, USA, 2019; pp. 281–318, ISBN 978-0-12-815794-7. 11. Chrysargyris, A.; Papakyriakou, E.; Petropoulos, S.A.; Tzortzakis, N. The combined and single effect of salinity and copper stress on growth and quality of Mentha spicata plants. J. Hazard. Mater. 2019, 368, 584–593. [CrossRef] 12. Strzemski, M.; Wójciak-Kosior, M.; Sowa, I.; Rutkowska, E.; Szwerc, W.; Kocjan, R.; Latalski, M. Carlina species as a new source of bioactive pentacyclic triterpenes. Ind. Crops Prod. 2016, 94, 498–504. [CrossRef] 13. Strzemski, M.; Wójciak-Kosior, M.; Sowa, I.; Agacka-Mołdoch, M.; Dr ˛aczkowski,P.; Matosiuk, D.; Kurach, Ł.; Kocjan, R.; Dresler, S. Application of Raman spectroscopy for direct analysis of Carlina acanthifolia subsp. utzka root essential oil. Talanta 2017, 174, 633–637. [CrossRef][PubMed] 14. Strzemski, M.; Wójciak-Kosior, M.; Sowa, I.; Załuski, D.; Verpoorte, R. Historical and traditional medical applications of Carlina acaulis L.—A critical ethnopharmacological review. J. Ethnopharmacol. 2019.[CrossRef] 15. Strzemski, M.; Wójciak-Kosior, M.; Sowa, I.; Załuski, D.; Szwerc, W.; Sawicki, J.; Kocjan, R.; Feldo, M.; Dresler, S. Carlina vulgaris L. as a source of phytochemicals with antioxidant activity. Oxid. Med. Cell. Longev. 2017, 2017.[CrossRef][PubMed] 16. J˛edrzejczyk-Koryci´nska,M. Floristic diversity in calamine areas of the Silesia-Cracow Monocline. Biodiv. Res. Conserv. 2006, 3–4, 340–343. 17. Gallego, S.M.; Pena, L.B.; Barcia, R.A.; Azpilicueta, C.E.; Iannone, M.F.; Rosales, E.P.; Zawoznik, M.S.; Groppa, M.D.; Benavides, M.P. Unravelling cadmium toxicity and tolerance in plants: Insight into regulatory mechanisms. Environ. Exp. Bot. 2012, 83, 33–46. [CrossRef] 18. Larsson, E.H.; Bornman, J.F.; Asp, H. Influence of UV-B radiation and Cd2+ on chlorophyll fluorescence, growth and nutrient content in Brassica napus. J. Exp. Bot. 1998, 49, 1031–1039. [CrossRef] 19. Poschenrieder, C.; Cabot, C.; Martos, S.; Gallego, B.; Barceló, J. Do toxic ions induce hormesis in plants? Plant. Sci. 2013, 212, 15–25. [CrossRef] 20. Calabrese, E.J. Evidence that hormesis represents an “overcompensation” response to a disruption in homeostasis. Ecotoxicol. Environ. Saf. 1999, 42, 135–137. [CrossRef] 21. Aina, R.; Labra, M.; Fumagalli, P.; Vannini, C.; Marsoni, M.; Cucchi, U.; Bracale, M.; Sgorbati, S.; Citterio, S. Thiol-peptide level and proteomic changes in response to cadmium toxicity in Oryza sativa L. roots. Environ. Exp. Bot. 2007, 59, 381–392. [CrossRef] 22. Kováˇcik,J. Hyperaccumulation of cadmium in Matricaria chamomilla: A never-ending story? Acta Physiol. Plant. 2013, 35, 1721–1725. [CrossRef] 23. Yu, H.; Guo, J.; Li, Q.; Zhang, X.; Huang, H.; Huang, F.; Yang, A.; Li, T. Characteristics of cadmium immobilization in the cell wall of root in a cadmium-safe rice line (Oryza sativa L.). Chemosphere 2020, 241, 125095. [CrossRef][PubMed] 24. Salt, D.E.; Prince, R.C.; Pickering, I.J.; Raskin, I. Mechanisms of cadmium mobility and accumulation in indian mustard. Plant. Physiol. 1995, 109, 1427–1433. [CrossRef][PubMed] Int. J. Mol. Sci. 2020, 21, 2828 13 of 14

25. Choi, Y.-E.; Harada, E.; Wada, M.; Tsuboi, H.; Morita, Y.; Kusano, T.; Sano, H. Detoxification of cadmium in tobacco plants: Formation and active excretion of crystals containing cadmium and calcium through trichomes. Planta 2001, 213, 45–50. [CrossRef] 26. Isaure, M.-P.; Fayard, B.; Sarret, G.; Pairis, S.; Bourguignon, J. Localization and chemical forms of cadmium in plant samples by combining analytical electron microscopy and X-ray spectromicroscopy. Spectrochim. Acta Part. B At. Spectrosc. 2006, 61, 1242–1252. [CrossRef] 27. Hojati, M.; Modarres-Sanavy, S.A.M.; Enferadi, S.T.; Majdi, M.; Ghanati, F.; Farzadfar, S.; Pazoki, A. Cadmium and copper induced changes in growth, oxidative metabolism and terpenoids of Tanacetum parthenium. Environ. Sci. Pollut. Res. 2017, 24, 12261–12272. [CrossRef] 28. Matraszek, R.; Hawrylak-Nowak, B.; Chwil, S.; Chwil, M. Interaction between cadmium stress and sulphur nutrition level on macronutrient status of Sinapis alba L. Water Air Soil Pollut. 2016, 227, 355. [CrossRef] 29. Nedjimi, B. Heavy metal tolerance in two Algerian saltbushes: A review on plant responses to cadmium and role of calcium in its mitigation. In Plant Nutrients and Abiotic Stress Tolerance; Hasanuzzaman, M., Fujita, M., Oku, H., Nahar, K., Hawrylak-Nowak, B., Eds.; Springer Singapore: Singapore, 2018; pp. 205–220, ISBN 978-981-10-9044-8. 30. Kováˇcik,J.; Dresler, S. Calcium availability but not its content modulates metal toxicity in Scenedesmus quadricauda. Ecotoxicol. Environ. Saf. 2018, 147, 664–669. [CrossRef] 31. Wang, C.Q.; Song, H. Calcium protects Trifolium repens L. seedlings against cadmium stress. Plant. Cell Rep. 2009, 28, 1341–1349. [CrossRef] 32. Zenk, M.H. Heavy metal detoxification in higher plants-A review. Gene 1996, 179, 21–30. [CrossRef] 33. Dresler, S.; Hawrylak-Nowak, B.; Kováˇcik,J.; Pochwatka, M.; Hanaka, A.; Strzemski, M.; Sowa, I.; Wójciak-Kosior, M. Allantoin attenuates cadmium-induced toxicity in cucumber plants. Ecotoxicol. Environ. Saf. 2019, 170, 120–126. [CrossRef][PubMed] 34. Kováˇcik,J.; Klejdus, B.; Babula, P.; Hedbavny, J. Ascorbic acid affects short-term response of Scenedesmus quadricauda to cadmium excess. Algal Res. 2017, 24, 354–359. [CrossRef] 35. Dresler, S.; Maksymiec, W. Capillary zone electrophoresis for determination of reduced and oxidised ascorbate and glutathione in roots and leaf segments of Zea mays plants exposed to Cd and Cu. Acta Sci. Pol. Hortorum Cultus 2013, 12, 143–155. 36. Sun, Q.; Ye, Z.H.; Wang, X.R.; Wong, M.H. Cadmium hyperaccumulation leads to an increase of glutathione rather than phytochelatins in the cadmium hyperaccumulator Sedum alfredii. J. Plant. Physiol. 2007, 164, 1489–1498. [CrossRef] 37. Kutrowska, A.; Szelag, M. Low-molecular weight organic acids and peptides involved in the long-distance transport of trace metals. Acta Physiol. Plant. 2014, 36, 1957–1968. [CrossRef] 38. Hawrylak-Nowak, B.; Dresler, S.; Matraszek, R. Exogenous malic and acetic acids reduce cadmium phytotoxicity and enhance cadmium accumulation in roots of sunflower plants. Plant. Physiol. Biochem. 2015, 94, 225–234. [CrossRef] 39. Dresler, S.; Wójciak-Kosior, M.; Sowa, I.; Stanisławski, G.; Bany, I.; Wójcik, M. Effect of short-term Zn/Pb or long-term multi-metal stress on physiological and morphological parameters of metallicolous and nonmetallicolous Echium vulgare L. populations. Plant. Physiol. Biochem. 2017, 115, 380–389. [CrossRef] 40. Dresler, S.; Hanaka, A.; Bednarek, W.; Maksymiec, W. Accumulation of low-molecular-weight organic acids in roots and leaf segments of Zea mays plants treated with cadmium and copper. Acta Physiol. Plant. 2014, 36, 1565–1575. [CrossRef] 41. Fu, H.; Yu, H.; Li, T.; Zhang, X. Influence of cadmium stress on root exudates of high cadmium accumulating rice line (Oryza sativa L.). Ecotoxicol. Environ. Saf. 2018, 150, 168–175. [CrossRef] 42. Do Nascimento, P.G.G.; Lemos, T.L.G.; Bizerra, A.M.C.; Arriaga, Â.M.C.; Ferreira, D.A.; Santiago, G.M.P.; Braz-Filho, R.; Costa, J.G.M. Antibacterial and antioxidant activities of ursolic acid and derivatives. Molecules 2014, 19, 1317–1327. [CrossRef] 43. Wu, Z.; Guo, Q.; Wang, Q.; Zhou, L.; Zhang, Z.; Zhang, L.; Huang, T. Effects of lead, copper and cadmium stresses on growth and inherent quality of Prunalla vulgaris. Zhongguo Zhongyao Zazhi 2010, 35, 263–267. [PubMed] 44. Wang, Q.J.; Lei, X.Y.; Zheng, L.P.; Wang, J.W. Molecular characterization of an elicitor-responsive 3-hydroxy-3-methylglutaryl coenzyme A reductase gene involved in oleanolic acid production in cell cultures of Achyranthes bidentata. Plant. Growth Regul. 2017, 81, 335–343. [CrossRef] Int. J. Mol. Sci. 2020, 21, 2828 14 of 14

45. Gupta, P.; Khatoon, S.; Tandon, P.K.; Rai, V. Effect of cadmium on growth, bacoside A, and bacopaside I of Bacopa monnieri (L.), a Memory Enhancing Herb. Sci. World J. 2014, 2014, 824586. [CrossRef][PubMed] 46. Kim, O.T.; Kim, M.Y.; Hong, M.H.; Ahn, J.C.; Hwang, B. Stimulation of asiaticoside accumulation in the whole plant cultures of Centella asiatica (L.) Urban by elicitors. Plant. Cell Rep. 2004, 23, 339–344. [CrossRef] 47. Sowa, I.; Paduch, R.; Strzemski, M.; Zieli´nska,S.; Rydzik-Strzemska, E.; Sawicki, J.; Kocjan, R.; Polkowski, J.; Matkowski, A.; Latalski, M.; et al. Proliferative and antioxidant activity of Symphytum officinale root extract. Nat. Prod. Res. 2018, 32, 605–609. [CrossRef] 48. Re, R.; Pellegrini, N.; Proteggente, A.; Pannala, A.; Yang, M.; Rice-Evans, C. Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radic. Biol. Med. 1999, 26, 1231–1237. [CrossRef]

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