International Journal of Molecular Sciences Article Tolerance of Facultative Metallophyte Carlina 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 Plant 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 (Asteraceae) 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, leaves, 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 plants 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. Int. J. Mol. Sci. 2020, 21, 2828; doi:10.3390/ijms21082828 www.mdpi.com/journal/ijms Int. J. J. Mol. Mol. Sci. Sci. 20202020,, 2121,, x 2828 FOR PEER REVIEW 2 of 14 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, leaf 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). Int. J. Mol. Sci. 2020, 21, 2828 3 of 14 Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 3 of 14 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).