Acta agriculturae Slovenica, 119/1, 1–17, Ljubljana 2023 doi:10.14720/aas.2023.119.1.2940 Original research article / izvirni znanstveni članek Tolerance to Zn toxicity in the halophyte Lepidium latifolium L. and the effect of salt on Zn tolerance and accumulation Behzad NEZHADASAD-AGHBASH 1, Tayebeh RADJABIAN 1, 2, Roghieh HAJIBOLAND 3 Received November 28, 2022; accepted January 24, 2023. Delo je prispelo 28. novembra 2022, sprejeto 24. januarja 2023 1 Department of Biology, Faculty of Basic Sciences, Shahed University, Tehran, Iran 2 Corresponding author, e-mail: rajabian@shahed.ac.ir 3 Department of Plant, Cell and Molecular Biology, University of Tabriz, Tabriz, Iran Tolerance to Zn toxicity in the halophyte Lepidium latifolium L. and the effect of salt on Zn tolerance and accumulation Abstract: Halophytes exhibit a high cross-tolerance to multiple stresses that enable them to survive under harsh en- vironmental conditions. We hypothesized that salt treatment in halophytes improves their tolerance against other stressors. To investigate the salt-mediated heavy metal tolerance in halo- phytes, Lepidium latifolium (Brassicaceae) was cultivated in the absence or presence of salt (100 mM NaCl) and excess Zn (200 μM ZnSO4), alone or in combination, for four weeks in the hydroponic medium. Salt treatment ameliorated the reduction of photosynthetic pigments in Zn-stressed plants and decreased Zn accumulation in the young leaves. The activity of peroxidase increased by both Zn toxicity and salt treatments; its maximum activity was achieved under the combination of both treatments associated with a significant reduction in malondialdehyde concentration. The activity of polyphenol oxidase increased by Zn stress alone or in combination with salt, accompanied by ac- cumulation of free and cell wall-bound phenolics and enhanced lignin deposition in the leaves. Our results showed a mitigating effect of salt treatment in Zn-stressed plants through the activa- tion of antioxidant defense and accumulation of phenolic com- pounds including flavonoids. Our results suggest L. latifolium as suitable species for revegetation and rehabilitation of saline soils contaminated with heavy metals. Key words: halophytes; Zn toxicity; Lepidium latifolium; antioxidant defense; phenolics; lignin Toleranca na strupenost Zn pri halofitu Lepidium latifolium L. in učinek soli na toleranco in kopičenje cinka Izvleček: Halofiti imajo veliko navskrižno toleranco na multipli stres, kar jim omogoča preživetje v neugodnih okolj- skih razmerah. Predpostavljamo, da obravnavanje s soljo pri halofitih izboljša njihovo toleranco na druge stresorje. Preu- čevali smo s soljo vzpodbujeno tolerenco na težke kovine pri halofitu Lepidium latifolium (Brassicaceae), gojenem v pri- sotnosti ali odsotnosti soli (100 mM NaCl) in pribitku cinka (200 μM ZnSO4), posamično ali v kombinaciji, štiri tedne v hidroponskem gojišču. Obravnavanje s soljo je zmanjšalo upad vsebnosti fotosinteznih barvil v rastlinah v stresu zaradi cinka in zmanjšalo njegovo akumulacijo v mladih listih. Aktivnost peroksidaze se je povečala v obeh primerih, zaradi toksično- sti zinka in obravnavanja s soljo, in je dosegla največjo aktiv- nost v kombinaciji obeh obravnavanj, kar je bilo povezano z značilnim upadom koncentracije malondialdehida. Aktivnost polifenol oksidaze se je povečala v stresu zaradi cinka samega ali v kombinaciji z obravnavanjem s soljo, kar je bilo poveza- no z akumalacijo prostih ali na celično steno vezanih fenolov in pospešilo odlaganje lignina v listih. Ti rezultati so pokazali blažilni učinek obravnavanja s soljo v rastlinah v stresu zaradi cinka z aktiviranjem antioksidacijske obrambe in akumulacijo polifenolov. Rezultati tudi nakazujejo, da je halofit L. latifolium primerna vrsta za ozelenitev in izboljšanje slanih tal onesnaže- nih s težkimi kovinami. Ključne besede: halofiti; strupenost Zn; Lepidium latifoli- um; antioksidacoijska obramba; fenoli; lignin Acta agriculturae Slovenica, 119/1 – 20232 B. NEZHADASAD-AGHBASH et al. 1 INTRODUCTION Environmental pollutants are rising progressively due to enormous economic development and the rapid growth of agriculture, urbanization, and industrial activ- ities. Heavy metals are the most prevalent contaminants released from natural and anthropogenic sources into the environment and cause soil, air, and water pollution (Tchounwou et al., 2012). Heavy metals may accumulate in high concentrations in the edible part of crop plants, which are considered the primary cause of some diseases in humans and animals (Manara, 2012). Cadmium (Cd), lead (Pb), chromium (Cr), copper (Cu), nickel (Ni), and zinc (Zn) are the most common heavy metals that their accumulation in the environment causes an alarming situation of health problems (Tchounwou et al., 2012). DNA damage, inactivation of enzymes, and carcino- genic effects are predominant complications in humans after exposure to high concentrations of heavy metals (Manara, 2012). Heavy metal toxicity in plants generally occurs through four principal mechanisms: 1) induction of oxi- dative stress through excess generation of reactive oxy- gen species (ROS) and changes in the permeability and integrity of the membranes, 2) changes in folding and ac- tivities of some proteins and enzymes due to the binding of heavy metals to their sulfhydryl groups, 3) competi- tion with micronutrients to participate in cellular func- tions due to having similar physicochemical properties with them and 4) displacement of essential metal ion co- factors in the active sites of enzymes (Dal Corso, 2012). Plants have some mechanisms for coping with heavy metal stress such as avoidance of metal uptake, prevention of their transport into the shoots, activation of defense mechanisms against ROS, and sequestration of heavy metals in the aerial parts through chelation by some organic compounds (Viehweger, 2014). Zinc is an essential micronutrient for higher plants (Hafeez et al., 2013). However, similar to other heavy metals, it is toxic under excess concentrations (Küpper & Andresen, 2016). The toxic effects of Zn depend on its external bioavailable concentration, exposure time, and developmental stage of plants (Balaferj et al., 2020). In- hibition of shoot growth, reduction of root elongation, chlorosis of young leaves, and in some cases, cell death are the most obvious symptoms of Zn toxicity (Küp- per & Andresen, 2016). Internal detoxification of Zn is achieved through its sequestration in the cytoplasmic compartments particularly in the vacuoles as chelated form with organic molecules, or as free ions (Balaferj et al., 2020). Halophytes adapt to and grow under salinity con- ditions, consequently, they are interesting model species for the study of adaptation and tolerance mechanisms in harsh environments. Numerous physiological and mo- lecular adaptive mechanisms, e.g. the ability to limit the entry of ions into the transpiration stream, ion compart- mentation, and synthesis of compatible osmolytes, have developed in the halophytes, that may confer also toler- ance to toxic concentrations of heavy metals (Van Oos- ten & Maggio, 2014). Halophytes are considered potent candidates for removing heavy metals from soils due to their higher ability for accumulation and phytoremedia- tion (Peng et al., 2022). Lepidium L. is a genus belonging to Brassicaceae and encompasses over 175 species (Mummenhoff et al., 2009). L. latifolium, known as perennial pepperweed or tall white top, is a perennial facultative halophyte na- tive to Asia and part of southeastern Europe (Spenst, 2006). The plants spread through small and large num- bers of seeds or vegetative reproduction and grow in a wide range of habitats. Because of its invasive and near- ubiquitous nature, this species is gaining more attention and is recognized as a global invader (Francis & War- wick, 2007). L. latifolium has also been widely used in traditional medicine as a diuretic and to reduce prostate hyperplasia, is a rich source of antioxidant compounds including phenolics, and is used as a whole herb for bac- terial dysentery, enteritis, and other diseases (Kaur et al., 2013). Physiological studies on L. latifolium have mainly focused on drought resistance, salt tolerance, mineral elements, proteins, and amino acids (Hajiboland et al., 2020). There is no information on heavy metal accumu- lation or tolerance in this species. Priming is a useful strategy for the improvement of the defense responses of plants against stressors. Various chemical compounds are used for priming; however, the effect of low concentrations of salt, as a priming agent has been relatively less investigated (Sako et al., 2020). In most studies, priming agents are applied at the seed ger- mination stage, which may have a short-term effect on the tolerance of plants against stress. The impact of prim- ing treatments in mature plants, however, has attracted much less attention from researchers. Studies on the effect of low concentrations of salt in adult plants on their tolerance to heavy metal toxicity are still scarce. In addition, the effect of salt priming on heavy metal accumulation and tolerance in the halophytes has not been sufficiently addressed. A high cross-tolerance in the halophytes to multiple stresses that enables them to survive under extreme environmental conditions could be, at least partly, mediated by the salt-mediated induc- tion of tolerance mechanisms. Our working hypoth- esis was that tolerance to toxic concentrations of Zn is enhanced through exposure to salt in the halophyte L. latifolium. For evaluation of the physiological response Acta agriculturae Slovenica, 119/1 – 2023 3 Tolerance to Zn toxicity in the halophyte Lepidium latifolium L. and the effect of salt on Zn tolerance and accumulation of plants, the antioxidant defense system, activity of phe- nolics metabolizing enzymes, and lignin deposition were analyzed in addition to the elemental composition under Zn stress in the absence or presence of low salt concen- tration as priming treatment in this species. 2 MATERIALS AND METHODS 2.1 PLANTS CULTURE AND TREATMENTS Seeds of L. latifolium were collected in 2016 from a wild-grown population in Meghan Playa in north central Iran. To obtain sufficient seeds with a high germination rate, the collected seeds were germinated and the young seedlings were cultivated in a private garden until flower- ing and seed set stage. The seeds from these plants were used for this work. Seeds were surface-sterilized with 10  % sodium hypochlorite and sown in plastic containers filled with washed perlite and irrigated with distilled water. After three weeks, the young seedlings were transferred to the light and irrigated with 50  % Hoagland nutrient solu- tion (pH 5.8). Four-week-old seedlings were cultivated in plastic containers filled with aerated 100 % Hoagland solution, and after one week, the seedlings were trans- planted in 2-liter hydroponic pots for starting the treat- ments. Plants were grown in a growth chamber with 16/8  h of light/dark photoperiod at 25/17  °C, relative humidity of 50–60  %, and at a photon flux density of about 400  μmol  m−2  s−1 provided by fluorescent lamps. The nutrient solutions were refreshed weekly. To evaluate the tolerance level of plants to salt and Zn toxicity, a preliminary experiment was designed with 400 mM NaCl and 400 µM ZnSO4 separately in the hy- droponic medium. The concentration of salt and Zn in the culture media was gradually increased by adding 50 mM NaCl and 50 µM ZnSO4 every day. One week af- ter reaching the final concentration of salt and Zn, plants were harvested, and their biomass was determined. In the main experiment, the five-week-old plants were pretreated with 100 mM salt (NaCl) for one week and then Zn (as ZnSO4) treatment at 200 µM was used simultaneously with salt treatment. Both NaCl and Zn were applied increasingly, by 50  mM and 50  μM steps on daily basis, respectively. Plants were grown for four weeks under treatment conditions and then harvested. At harvest, plants were washed with distilled water, blot- ted dry on filter paper, and their fresh mass (FM) was determined. 2.2 BIOCHEMICAL AND ELEMENTAL ANALYSES Shoot parts were separated into young (the second youngest leaf) and old leaves (the second oldest leaf), then were subjected to biochemical and elemental analy- ses. The activity of enzymes was determined in fresh samples immediately after harvest. Other biochemical analyses were carried out after storage at –20  °C for a maximum of six days. Oven-dried samples were weighed and then used for elemental analyses. 2.2.1 Pigments concentration Photosynthetic pigments including chlorophylls (Chl) and carotenoids were extracted from the leaves in ice-cold 80 % acetone in the dark at 4 °C. The absorbance of extracts was determined at 470, 645, and 662 nm, and the concentration of pigments was calculated and ex- pressed based on leaf fresh weight (Lichtenthaler & Wel- burn, 1983). The flavonoids content was determined in the leaf homogenate prepared in an extracting solution containing 2 % AlCl3 in methanol. After centrifugation, the absorbance of supernatants was measured at 415 nm. The total flavonoids concentration was expressed as µg quercetin equivalent per g of FW by drawing a calibra- tion curve with 0-16  mg l–1 concentration of authentic quercetin (Arvouet-Grand et al., 1994). 2.2.2 Antioxidant enzymes assay The total activity of superoxide dismutase (SOD, EC 1.15.1.1) in the plant samples was assayed using the mono-formazan formation test. One unit of SOD activity was defined as the amount of enzyme required for a 50 % inhibition in NBT (ρ-nitro blue tetrazolium chloride) reduction through the monitoring of the changes in the absorbance at 560 nm, compared to the control samples without the enzyme aliquot (Giannopolitis & Ries, 1977). Ascorbate peroxidase (APX, EC 1.11.1.11) activity in the extracts was calculated by determining the decrease in the absorbance at 290  nm for 2  min due to the oxida- tion of ascorbic acid using an extinction coefficient of 2.8  mM–1 cm–1 (Boominathan & Doran, 2002). The activ- ity of CAT in the extracts was estimated by monitoring the decreases in the absorbance of H2O2 at 240 nm for 2 min. One unit of activity was defined as the quantity of enzyme needed to decompose 1 μmol H2O2 per min, us- Acta agriculturae Slovenica, 119/1 – 20234 B. NEZHADASAD-AGHBASH et al. ing the extinction coefficient of 0.28 mM–1 cm–1 (Chance & Maehly, 1954). The activity of peroxidase (POD, EC 1.11.1.7) was assessed in a reaction mixture using guai- acol as substrate, and the enzyme activity was measured at 470 nm using extinction coefficient (26.6 mM−1 cm−1) of tetraguaiacol (Ranieri et al., 2001). The concentration of total soluble proteins was de- termined using the Bradford assay (Bradford, 1976) and bovine serum albumin (BSA, Merck) as the standard. 2.2.3 H2O2, malondialdehyde (MDA), and proline concentrations The concentration of H2O2 in the extracts was deter- mined by recording the absorbance at 390 nm and using the plotted calibration curve in the range of 0-120  µM H2O2 (Harinasut et al., 2003).  The concentration of malondialdehyde (MDA) as a marker of lipid peroxida- tion in the leaf extracts was determined by the absorb- ance at 532 nm due to its reaction with thiobarbituric acid and using a plotted standard curve with 1,1,3,3 - tetraeth- oxypropane (Hodges et al., 1999). For the estimation of proline, leaf samples were extracted in sulfosalicylic acid, and the supernatants were used for the determination of proline according to the method of Bates et al. (1973) and with ninhydrin as a reagent. 2.2.4 Total phenolics concentration and lignin quan- tification Phenolic compounds were extracted in 70 % aque- ous methanol three times, and after centrifugation, the supernatant was used for the determination of soluble phenolics, while cell wall-bound phenolics and lignin were quantified in the pellet. For the release of the cell wall (CW)-bound phenolics, the pellet was washed con- secutively with water and Triton X-100, then after in- cubation with 20 mM NH4-oxalate (70 °C) followed by 100  mM NaOH for 24  h, the phenolics were released from the CW. The concentrations of soluble and CW- bound phenolics were determined using Folin–Ciocalteu reagent at 765 nm and gallic acid as standard (Swain & Hillis, 1959). After air-drying, the residual CW fraction was used for lignin extraction and determination using the acetyl bromide method by recording the absorbance at 280 nm and a specific absorption coefficient value of 8.4 l g –1 cm–1 (Morrison, 1972). 2.2.5 Phenylalanine ammonia-lyase (PAL) and poly- phenol oxidase (PPO) activity Phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) ac- tivity in the leaf and root samples were evaluated as the amount of the formed trans-cinnamic acid at 290  nm and was calculated using its extinction coefficient of 9630  mM–1  cm-1 (Dickerson et al., 1984). Polyphenol oxidase (PPO, EC 1.14.18.1) activity was determined ac- cording to the method described by Casado-Vela et al. (2005). The changes in the absorbance at 334 nm due to the oxidation of pyrogallol were used for the calculation of the PPO activity in the extracts. 2.2.6 Mineral analyses The oven-dried leaf and root samples were used for mineral analyses. The samples were ashed in a muffle fur- nace at 550 °C for 8 h, and after dissolving in 10 % HCl and filtration, were made to volume with distilled water. The concentrations of Na, K, and Ca were determined by flame photometry (PFP7, Jenway, UK), and the stand- ard solutions of the examined elements were used for the construction of the calibration curves. The concentration of Zn in the samples was analyzed by atomic absorption spectrophotometry (AA-6300, Shimadzu, Japan). 2.2.7 Experimental design and statistical analyses This experiment was performed using a complete randomized block design with four independent rep- licates per treatment. Data were presented as mean ± standard deviation (SD). The comparison of means was carried out using SigmaStat 3.5 (Systat Software Inc., USA) with Tukey’s test at p < 0.05. 3 RESULTS According to the results of the preliminary experi- ment, the growth of L. latifolium plants was decreased under both salinity and Zn toxicity treatments. Reduc- tion of growth upon exposure to 400 mM salt was 49 % and 52 % for the fresh mass (FM) and dry mass (DM) of the shoots, respectively. The reduction of FM and DM of aerial parts under the effect of 400 µM Zn was 50 % and 43 %, respectively (Table 1). Acta agriculturae Slovenica, 119/1 – 2023 5 Tolerance to Zn toxicity in the halophyte Lepidium latifolium L. and the effect of salt on Zn tolerance and accumulation The main experiment was conducted to study salt, Zn, and their interaction effects on the growth, biochem- ical and mineral attributes of the plants as described be- low. 3.1 THE EFFECT OF SALT, ZN, AND THEIR COMBINATION ON PLANTS BIOMASS AND CONCENTRATION OF PIGMENTS Due to a strong reduction of growth in the prelimi- nary experiment, lower concentrations of salt and Zn were applied in the main experiment. According to the obtained results, the shoot DM was decreased by salt, Zn toxicity, and especially under a combination of both treatments (Figure 1A). Root DM decreased significant- ly under Zn toxicity alone or in combination with salt, while it was not significantly affected by salinity as a sin- gle treatment (Figure 1B). The leaf concentration of Chl was decreased by all applied treatments. The highest reduction (90  %) was observed under Zn toxicity without salt and the lowest decline (17 %) was found under salt as a single treatment (Table 2). Unlike Chl, the leaf concentration of carot- enoids increased due to salinity treatment but signifi- cantly decreased by Zn toxicity alone or in combination with salt. The leaf concentration of flavonoids decreased by both salt and Zn toxicity as single stresses, while it was significantly higher than the control plant in the combi- native treatment (Table 2). 3.2 THE EFFECT OF SALT, ZN, AND THEIR COM- BINATION ON THE ACTIVITY OF ANTIOXI- DANT ENZYMES AND CONCENTRATION OF RELATED METABOLITES The activity of SOD decreased by salt treatment alone or in combination with Zn toxicity while remain- ing unaffected under Zn toxicity as a single treatment (Figure 2A). The activity of CAT showed a significant reduction under the combined treatment but remained unchanged under the individual treatments of either salt or Zn toxicity (Figure 2B). The activity of APX, in con- trast, increased by salt treatment alone or in combination with Zn toxicity. The effect of the latter treatment as sin- gle stress on the APX activity was not significant (Figure 2C). The activity of POD was significantly increased by salinity and Zn toxicity; the highest enzyme activity was observed under a combination of these treatments (Fig- ure 2D). The concentration of H2O2 was increased by all ap- plied treatments. The effect of salinity alone or in combi- nation with Zn toxicity was significantly higher than that of Zn toxicity as single stress (Figure 3A). The concentra- tion of MDA increased under salinity and Zn toxicity as single stress. Under the combination of both treatments, however, this parameter did not differ from that in the control plants (Figure 3B). The concentration of proline showed a significant increase under the influence of all applied treatments. The effect of Zn toxicity as a single treatment, however, was higher than that of its combina- tion with salinity; the lowest effect was observed upon salt as single stress (Figure 3C). Reduction of FM Reduction of DM Salinity 49 ± 6 52 ± 8 Zn toxicity 50 ± 13 43 ± 18 Table 1: Decline in biomass production (% over control) in L. latifolium grown with salt (400 mM NaCl) or toxic Zn con- centration (400 µM ZnSO4) for three weeks in a hydroponic medium Figure 1: The biomass of shoots (A) and roots (B) in L. latifo- lium grown for three weeks in the absence or presence of salt (100 mM NaCl) and Zn (200 µM as ZnSO4). Bars indicated by different letters are significantly different (p < 0.05) Acta agriculturae Slovenica, 119/1 – 20236 B. NEZHADASAD-AGHBASH et al. Treatment Chl (a+b) Carotenoids Flavonoids Control –Salt 2.06 ± 0.10 a 0.16 ± 0.01 b 88 ± 5.8 b +Salt 1.70 ± 0.19 b 0.32 ± 0.01 a 66 ± 2.6 c Zn toxicity –Salt 0.19 ± 0.06 d 0.08 ± 0.02 d 78 ± 5.7 bc +Salt 1.32 ± 0.02 c 0.12 ± 0.01 c 147 ± 14.0 a Table 2: The leaf concentrations of chlorophylls (Chl a+b), carotenoids (mg g-1 FM), and flavonoids (µg g–1 FM) in L. latifolium grown for three weeks in the absence or presence of salt (100 mM NaCl) and Zn (200 µM as ZnSO4). Data of each column indi- cated by the different letters are significantly different (p < 0.05) Figure 2: The leaf activity of superoxide dismutase (SOD) (A), catalase (CAT) (B), ascorbate peroxidase (APX) (C), and per- oxidase (POD) (D) in L. latifolium grown for three weeks in the absence or presence of salt (100 mM NaCl) and Zn (200 µM as ZnSO4). Bars indicated by different letters are significantly different (p < 0.05) Acta agriculturae Slovenica, 119/1 – 2023 7 Tolerance to Zn toxicity in the halophyte Lepidium latifolium L. and the effect of salt on Zn tolerance and accumulation 3.3 THE EFFECT OF SALT, ZN, AND THEIR COM- BINATION ON THE ACTIVITY OF PAL, PPO, AND THE CONCENTRATIONS OF PHENO- LICS AND LIGNIN The leaf activity of PAL was decreased by all ap- plied treatments without difference among the three treatments. The root activity of this enzyme, in contrast, remained unchanged or rather increased. The latter ef- fect was observed under Zn toxicity as a single treatment (Figures 4A and 4B). The leaf activity of PPO increased under Zn toxicity alone or in combination with salinity, while salt treatment as a single stress did not affect this parameter (Figure 4C). In the roots, however, all applied treatments increased the PPO activity; the effect of Zn toxicity alone was significantly higher than that of the combinative treatment (Figure 4D). The concentration of free phenolics decreased un- der salt stress, while increased by Zn toxicity treatment, and remained unaffected under the combination of both treatments (Figure 5A). The concentration of CW-bound phenolics, in contrast, increased by salt stress either alone or in combination with high Zn concentration while was not affected by Zn toxicity as a single treatment (Figure 5B). The lignin concentration increased by salt, Zn toxic- ity, and their combination without difference among the three treatments (Figure 5C). 3.4 THE EFFECT OF SALT, ZN, AND THEIR COM- BINATION ON THE CONCENTRATION OF ELEMENTS IN THE LEAVES AND ROOTS As expected, Zn was accumulated in the leaves and roots of plants treated with a high Zn concentration. In the young leaves, the concentration of Zn was signifi- cantly lower under the combinative treatment than that under Zn as a single treatment. The effect of salt on the reduction of Zn concentration, however, was not ob- served in the old leaves and roots (Figure 6). Also, as anticipated, Na was accumulated in the young and old leaves and roots upon exposure to salt in the medium. Leaf accumulation of Na in plants treated with a combination of salt and Zn toxicity was signifi- cantly higher than that in plants grown with salt as a sin- gle treatment. The opposite was observed in the roots; Na concentration in this organ was lower under combinative treatment compared to that under single salt treatment (Table 3). The concentration of K decreased significantly by salt treatment in the old and young leaves and in the roots. Treatment of plants with toxic Zn concentration caused also a decrease in the K concentration of old leaves and roots but did not affect this parameter in the young leaves. Reduction of K concentration was also ob- served under the combination of Zn stress and salt treat- ments. However, the effect of combinative treatment on Figure 3: The leaf concentration of hydrogen peroxide (H2O2) (A), malondialdehyde (MDA) (B), and proline (C) in L. latifo- lium grown for three weeks in the absence or presence of salt (100 mM NaCl) and Zn (200 µM as ZnSO4). Bars indicated by different letters are significantly different (p < 0.05) Acta agriculturae Slovenica, 119/1 – 20238 B. NEZHADASAD-AGHBASH et al. the leaf K concentration was less than the effect of single salt stress (Table 3). The effect of salt and Zn toxicity treatments on the Ca concentration was dependent on the plant organ. The presence of salt in the medium increased Ca concentra- tion in the roots and did not influence it in the young leaves while decreasing it in the old leaves. Treatment with toxic Zn concentration led to an increase in the Ca concentration of the young leaves but resulted in its re- duction in the old leaves and roots. The effect of com- binative treatment in the reduction of Ca concentration was observed only in the old leaves and roots (Table 3). 4 DISCUSSION Studies on the stress tolerance in Brassicaceae spe- cies, particularly its halophyte members, attract the atten- tion of plant scientists because of the possibility for com- parison of these species with the model plant, Arabidopsis thaliana (L.) Heynh.. Based on these studies, Schrenkiella parvula (Schrenk) D.A.German & Al-Shehbaz (Hajibo- land et al., 2018), Cakile maritima Scop. (Debez et al., 2004), and Thellungiella salsuginea   (Pall.) O.E.Schulz (syn. Eutrema salsugineum (Pall.) Al-Shehbaz & War- wick) (Gao et al., 2008) are the most salt-tolerant Bras- Figure 4: The activity of phenylalanine ammonia-lyase (PAL) (A and B) and polyphenol oxidase (PPO) (C and D) in the leaves and roots of L. latifolium grown for three weeks in the absence or presence of salt (100 mM NaCl) and Zn (200 µM as ZnSO4). Bars indicated by different letters are significantly different (p < 0.05) Acta agriculturae Slovenica, 119/1 – 2023 9 Tolerance to Zn toxicity in the halophyte Lepidium latifolium L. and the effect of salt on Zn tolerance and accumulation sicaceae species, which are able to grow even under salt concentrations of about 400 mM. The tolerance mecha- nisms in halophytes of this family include avoidance of excessive uptake of Na and maintenance of a proper ra- tio of K/Na in the cytosol, accumulation of organic os- molytes (including proline), activation of defense against ROS as well as changes in the levels of plant hormones (Van Zelm et al., 2020). To expand our knowledge of the mechanisms of salt tolerance and to find other model plants within Brassicaceae, i.e. Arabidopsis-related model species (ARMS, Arbelet-Bonnin et al., 2019), more inves- tigations are necessary particularly on the halophytes of this family. In this study, L. latifolium, a facultative halo- phyte species was investigated for salt tolerance and its interaction with plant response to Zn toxicity. Figure 5: The leaf concentration of free phenolics (A), cell wall (CW)-bound phenolics (B), and lignin (C) in L. latifolium grown for three weeks in the absence or presence of salt (100 mM NaCl) and Zn (200 µM as ZnSO4). Bars indicated by dif- ferent letters are significantly different (p < 0.05) Figure 6: Zn concentration in the young leaves (A), old leaves (B), and roots (C) of L. latifolium grown for three weeks in the absence or presence of salt (100 mM NaCl) and Zn (200 µM as ZnSO4). Bars indicated by different letters are significantly different (p < 0.05) Acta agriculturae Slovenica, 119/1 – 202310 B. NEZHADASAD-AGHBASH et al. 4.1 THE EFFECT OF SINGLE AND COMBINATIVE TREATMENTS OF SALT AND ZN TOXICITY ON PLANT BIOMASS In comparison to glycophytes, halophytes have generally higher resistance against not only salt but also other soil-derived abiotic stressors, such as drought and heavy metal toxicity (Lokhande & Suprasanna, 2012). Mechanisms such as heavy metal exclusion, reduction of their mobility in soil (MacFarlane & Burchett, 2002), restriction of shoot-root translocation (Mejías et al., 2013), internal detoxification and sequestration, acti- vation of the antioxidant system (Sharma et al., 2016), and even excretion of heavy metals through salt glands and trichomes (Lokhande & Suprasanna, 2012) all con- tribute to a higher heavy metal tolerance in halophytes. In addition, salt treatment mitigates heavy metal toxic- ity effects in some halophytes. The biomass of Sesuvium portulacastrum (L.) L. under Cd toxicity (Ghnaya et al., 2007) and growth of Mesembryanthemum crystallinum L. (Kholodova et al., 2005) and Spartina densiflora Brongn. (Mahon & Carman, 2008) under Zn toxicity were higher under simultaneous application of salt and heavy metal toxicity compared to single heavy metal stress. In our study, however, the effect of Zn toxicity was not mitigat- ed by simultaneous treatment with salt suggesting that the ameliorative effect of salt on heavy metal toxicity in the halophytes is not common and is likely dependent on the species and the heavy metal. To the best of our knowledge, the effect of salt on heavy metal toxicity has not been investigated in the Brassicaceae halophytes, but in the glycophytes of this family including Brassica napus L. and Brassica juncea (L.) Czem. the combined treat- ment of salt and Cd had a higher inhibitory effect on the growth and photosynthesis of these species compared to single treatments (Shah et al., 2011). 4.2 THE EFFECT OF SINGLE AND COMBINATIVE TREATMENT OF SALT AND ZN TOXICITY ON THE CONCENTRATIONS OF LEAF PIG- MENTS An antagonistic effect of toxic heavy metal concen- trations on the uptake and utilization of Fe, the main el- ement for Chl biosynthesis, has been well documented (Leiková et al., 2017). In addition, impairment of bio- chemical reactions under heavy metal toxicity leads to the formation of excess excitation energy and genera- tion of ROS that in turn cause instability of the thyla- koid membranes and destruction of Chl (Riyazuddin et al., 2021). In L. latifolium, the leaf concentration of Chl was decreased upon exposure to excess Zn by up to 90 %. This was probably the consequence of both reductions in biosynthesis and the rise of its destruction. Carotenoids play important roles in the stability of chloroplast membranes and protect photosynthetic ap- paratus against damages caused by excess excitation en- ergy (Uarrota et al., 2018). A significant increase in the leaf carotenoids observed in salt-treated L. latifolium in- dicates that some protection mechanisms are triggered under these conditions leading to a reduction of injury to the chloroplasts similar to that reported for other halophytes, Arthrocnemum macrostachyum (Moric.) Pi- irainen & G.Kadereit, Sarcocornia fruticosa (L.) A.J.Scott (Ghanem et al., 2021), and Nitraria retusa (Forssk.) Asch. (Boughalleb & Denden, 2011). However, a salt-mediated increase in the leaf carotenoids has not been observed in all halophytes, as in Salicornia europaea L. salt treatment reduced this parameter (Aghaleh et al., 2009). Contrary to salt treatment, leaf carotenoids significantly decreased under Zn toxicity in our study, which may increase the vulnerability of photosynthetic apparatus to ROS-in- duced damages under these conditions. In Halimione portulacoides (L.) Aellen the amounts of carotenoids increased under Zn toxicity (400  mM) (Cambrollé et al., 2012) while in another halophyte, Avicennia marina Treatment Na K Ca Young leaf Control –Salt 0.6 ± 0.2 c 43 ± 0.5 a 35 ± 3 b +Salt 6.5 ± 1.6 b 32 ± 2.4 c 38 ± 7 b Zn toxicity –Salt 1.5 ± 0.7 c 41 ± 2.8 a 49 ± 2 a +Salt 15 ± 2.8 a 35 ± 1.6 b 37 ± 9 b Old leaf Control –Salt 2.3 ± 1.0 c 68 ± 2.0 a 66 ± 7 a +Salt 18 ± 3.0 b 34 ± 1.0 d 44 ± 9 c Zn toxicity –Salt 2.4 ± 0.6 c 53 ± 5.0 b 58 ± 6 b +Salt 42 ± 6.0 a 41 ± 3.6 c 45 ± 3 c Root Control –Salt 1.1 ± 0.3 c 91 ± 8.7 a 62 ± 11 b +Salt 14 ± 2.0 a 44 ± 7.9 c 154 ± 13 a Zn toxicity –Salt 1.2 ± 0.1 c 78 ± 2.6 b 44 ± 9 c +Salt 6.5 ± 1.2 b 38 ± 1.7 c 23 ± 5 d Table 3: Concentrations (mg g-1 DM) of Na, K, and Ca in the young leaves, old leaves, and roots of L. latifolium grown for three weeks in the absence or presence of salt (100 mM NaCl) and Zn (200 µM as ZnSO4). Data of each column within each organ indicated by different letters are significantly different (p < 0.05) Acta agriculturae Slovenica, 119/1 – 2023 11 Tolerance to Zn toxicity in the halophyte Lepidium latifolium L. and the effect of salt on Zn tolerance and accumulation (Forssk.) Vierh. the amount of this pigment decreased under excess Cu and Zn, but remained unchanged under the Pb toxicity (MacFarlan & Burchett, 2002). The leaf concentrations of Chl, carotenoids, and flavonoids were significantly higher under the combina- tion of salt with Zn toxicity compared to the single Zn treatment in L. latifolium, likely as the consequence of the effect of salt treatment on the stimulation of protection mechanisms in the chloroplasts. Application of low con- centrations of salt protects the structure and function of chloroplasts; ascorbate plays a central role in this prim- ing effect because Arabidopsis mutants lacking ascor- bate show a considerable disruption of photosynthesis under salt stress (Acosta-Motos et al., 2017). Similarly, salt priming decreased high temperature-induced dam- age to the photosystem II in Atriplex centralasiatica Iljin (Qiu & Lu, 2003). In Suaeda salsa (L.) Pall. salt priming increased the quantum yield of photosystem II and in- creased the amount of unsaturated fatty acids (Cheng et al., 2014). Flavonoids are low-molecular-weight polyphe- nolic metabolites not only involved in ROS scavenging, but also as chelating molecules, bind to heavy metals and thus, play a role in internal detoxification (Keilig & Lud- wig-Müller, 2009; Samanta et al., 2011). Under the com- bination of salt and Zn treatments, the leaf concentration of flavonoids was two-fold higher than that found under control and single-stress conditions in L. latifolium. En- hanced flavonoids level under combinative treatment was associated with significantly lower MDA concentra- tion suggesting their contribution to the prevention of membrane damage under these conditions. 4.3 THE EFFECT OF SINGLE AND COMBINATIVE TREATMENTS OF SALT AND ZN TOXICITY ON THE FUNCTION OF THE ANTIOXIDANT DEFENSE SYSTEM Higher constitutive or stress-induced antioxidant defense is one of the most important mechanisms in halophytes to cope with various environmental stresses (Sruthi et al., 2017). SOD is involved in scavenging su- peroxide radicals as one of the most damaging ROS in cells (Sruthi et al., 2017). In this study, however, the ac- tivity of SOD decreased under both single and combina- tive salt and Zn stresses. Reduction in the activity of SOD under salinity has also been reported in other halophytes such as Gypsophila oblanceolate Barkoudah (Sekmen et al., 2012) and Salvadora persica L. (Rangani et al., 2016). Unlike SOD, the activity of APX was significantly in- creased by salt treatment alone or in combination with Zn toxicity, which is in agreement with many reports on the effect of salinity on the APX activity in the glycophytes, e.g. Arabidopsis thaliana and halophytes, e.g. Cakile mar- itima (Ellouzi et al., 2011) and Sesuvium portulacastrum (Ben Amor et al., 2020). The activity of CAT was not af- fected by the single salt and Zn toxicity stress and signifi- cantly decreased under combinative treatments. The lack of any response to low concentrations of salt (100 mM) was similar to another report in the euhalophyte, Salva- dora persica, where the activity of CAT did not change under 250  mM salt, but increased under 500  mM salt treatment (Rngani et al., 2016). Unlike CAT and APX, the activity of POD increased by all three applied treat- ments and could probably be considered as an indicator of the effect of treatments. Peroxidases are a large group of enzymes oxidizing a wide array of substrates using H2O2 (Veitch, 2004). Several reports showed a consistent increase in the activity of POD by salt stress both in the glycophytes and in halophytes (Yang et al., 2010; Ellouzi et al., 2011). The highest activity of POD was observed under combinative treatment associated with a reduction of MDA to the levels observed in control plants. Overall, our results demonstrated that antioxidant enzymes re- spond differently to the applied treatments depending on enzyme and stress factors, and suggest the different con- tributions of each enzyme in the defense of plants against salt, heavy metals, and their combinations. H2O2 plays a dual role in plant stress response: it acts as a signaling molecule at the nanomolar or low micromolar range of concentration (per g FM) while at the millimolar level damages the molecular structure of proteins, lipids, and nucleic acids (Černý et al., 2018). The range of cytosolic H2O2 concentration in our plants (200-400  nmol g–1 FM, Figure 3A) was highly relevant to its signaling function. Furthermore, the ability of salt treatment in the induction of H2O2 signal was higher than that of Zn toxicity, leading in turn to a higher H2O2 level in the combinative treatment (Figure 3A). This may be one of the mechanisms for the ameliorative effect of salt on Zn stress in this work, as was also reflected in the levels of biochemical stress markers under combinative treatment. The effect of salt on the induction of H2O2 signaling and stress tolerance has been observed in other halophytes (Ellouzi et al., 2011). Interestingly, H2O2 sign- aling in a halophyte (L. latifolium) is characteristically different from its glycophyte relative (Lepidium sativum L.) in the timing and magnitude of induction (Hajibo- land et al., 2020). Accumulation of MDA, as the final product of per- oxidation of poly-unsaturated fatty acids in the mem- branes, is one of the most common effects of heavy metal toxicity in plants (Manara, 2012). An increase in the MDA content has been reported in Atriplex rosea L. and Arabidopsis hortensis L. grown in soils contaminated with Ni and Zn (Kumari et al., 2019) or in Acanthus ilici- Acta agriculturae Slovenica, 119/1 – 202312 B. NEZHADASAD-AGHBASH et al. folius L. under Cd stress (Shakira & Puter, 2019). In our work, the MDA concentration increased by Zn toxicity as a single treatment, while was reduced to the level of that in control plants under the combination of Zn with salt treatment. This may indicate higher protection of cell and plastid membranes in the combinative treatment, as was also reflected in the higher concentration of leaf pig- ments under these conditions compared with Zn toxicity as a single treatment (Table 2). The protective role of salt under heavy metal stress has also been observed in the halophyte Atriplex halimus L. against Cd and Cu toxici- ties as a reduction of MDA accumulation in the roots un- der the combination of heavy metals with salt treatments (Bankaji et al., 2016). Accumulation of proline driven by different en- vironmental stresses is a well-documented response in plants (Hayat et al., 2012). The major function of pro- line under salt stress is osmotic regulation, which along with other compatible organic osmolytes, e.g. polyols and glycine betaine, confronts the osmotic component of salt stress (Siddique et al., 2018). The function of proline, however, is not limited to an osmotic role but it contrib- utes to a wide range of protective functions, including stimulation of antioxidant defense enzymes, role in the stability of protein structures, and redox homeostasis (Hayat et al., 2012). Under heavy metal stress, proline protects cells against toxicity damage (Siddique et al., 2018). In the halophyte Acanthus ilicifolius, proline con- centration increased in response to Cd toxicity (Shackira & Puthur, 2019) and in Mesembryanthemum crystallinum under excess Zn concentration (Kholodova et al., 2005). The expression of the proline biosynthetic gene (P5CS) was increased under Cr toxicity and its combination with salt in Chenopodium quinoa Willd. that was associated with proline accumulation in this species (Guarino et al., 2020). In our work, the leaf concentration of proline increased under salt stress, particularly by Zn toxicity treatment. Although the proline level was lower under combinative treatment compared to single Zn stress, it remained still higher than that found under salt stress and was about 9-fold higher than the proline concentra- tion of control plants. As an indicator of stress (Ashraf & Harris, 2004), lower proline concentration in the combi- native treatment could be likely the result of mitigation of the Zn toxicity stress, as was also reflected in the lower MDA content and higher amounts of leaf pigments. In agreement with our findings, the proline concentration in the halophyte Kosteletzkya pentacarpos (L.) Ledeb. increased under Cd toxicity, while decreased under the combination of salt and Cd stress (Zhou et al., 2019). 4.4 THE EFFECT OF SINGLE AND COMBINATIVE TREATMENTS OF SALT AND ZN TOXICITY ON THE CONCENTRATIONS OF PHENOLICS, LIGNIN, AND THE ACTIVITY OF RELATED ENZYMES Phenolic compounds possessing an aromatic ring with one or more hydroxyl substituents are contributed to ROS scavenging and stabilization of membranes and other cell structures (Moura et al., 2010). An enhanced synthesis and accumulation of phenolic compounds un- der the toxicity of heavy metals has been extensively re- ported (Ghori et al., 2019). In Kandelia obovata Sheue, Liu & Yong, Cd toxicity caused phenolics accumulation accompanied by a significant increase in PPO activity (Chen et al., 2019). Similarly, in Matricaria chamomilla L., the concentration of phenolic compounds increased under Ni toxicity associated with an increase in the activ- ity of PAL but a decrease in the activity of PPO (Kováčik et al., 2009). In our work here, the concentration of free phenolics increased under Zn toxicity treatment (Figure 5A), indicating a probable role for phenolics in the in- creasing plants’ resistance against Zn toxicity. In addi- tion to their antioxidant and protective function, a metal chelating capacity of phenolics as a mechanism for the internal detoxification of heavy metals has been docu- mented (Michalak, 2006). The capacity of particular phe- nolics such as cinnamic acid, ferulic acid, gallic acid, and naringenin for Zn chelation has been demonstrated both in vivo and in vitro (Fedenko et al., 2022). In addition to free phenolics, low molecular weight phenolic acids that are bound to various CW compo- nents have several important roles including responses to stresses (Wallace & Fry, 1994). The concentration of CW- bound phenolics was higher in the salt-treated plants ei- ther as single stress or in combination with Zn toxicity (Figure 5B). The carboxylic groups of CW-bound pheno- lics have a high affinity for heavy metals and the formed complexes show high stability constants (McDonald et al., 1996). This mechanism may contribute to Zn de- toxification in our work, particularly in the combinative treatment with an enhanced concentration of CW-bound phenolics. The composition of plant CWs is modified under bi- otic and abiotic stresses (Gall et al., 2015). The biosynthe- sis of lignin, as one of the major components of the CW, increases under various stresses, including salinity and heavy metal toxicity (Moura et al., 2010). ROS produced under stress in the apoplasts participate in lignin syn- thesis via various enzymes including POD and PPO (Ali Acta agriculturae Slovenica, 119/1 – 2023 13 Tolerance to Zn toxicity in the halophyte Lepidium latifolium L. and the effect of salt on Zn tolerance and accumulation et al., 2006). In Arabidopsis thaliana, salt treatment up- regulates laccase- and POD-encoding genes leading to lignification in salt-stressed plants (Chun et al., 2019). In Tamarix hispida Willd., the expression of genes involved in the lignin biosynthesis, i.e. S-adenosyl methionine synthase (SAM synthase) and catechol-O-methyltrans- ferase (COMT) were increased under salt stress (Han et al., 2022). Interestingly, there is a relationship between the extent of lignification and salt tolerance, so that leaf lignification under salt stress was higher in the halophyte L. latifolium than that was found in its glycophyte rela- tive, L. sativum (Hajiboland et al., 2020). In this work, the lignin concentration was increased by all applied treat- ments, especially by Zn toxicity indicating its contribu- tion to the adaptation of plants to stress. An increase in the lignin content mediated by Zn toxicity has also been observed in Thlaspi caerulescens J.Presl & C.Presl, fol- lowing the upregulation of the related biosynthetic genes (Van De Mortel et al., 2006). Interestingly, the lignin dep- osition under Zn toxicity was higher in Thlaspi caerule- scens compared with its glycophyte relative, Arabidopsis thaliana with higher sensitivity to Zn toxicity, indicating again a relationship between the higher ability to lignin synthesis and tolerance to not only salt (Hajiboland et al., 2020) but also to Zn toxicity (Van De Mortel et al., 2006). 4.5 THE EFFECT OF SINGLE AND COMBINATIVE TREATMENTS OF SALT AND ZN TOXICITY ON THE ACCUMULATION OF ELEMENTS It has been observed that salt treatment results in higher accumulation of Zn in B. juncea (Novo et al., 2014) and Brassica rapa L. (Zeiner et al., 2022) because of salt-mediated increase in the mobility of Zn in the soil and within plants and enhancement of the root-shoot translocation of Zn in these glycophyte species (Novo et al., 2014). In the present work, the application of salt reduced Zn accumulation in the aerial parts of L. latifo- lium. Although there is no information about other halo- phytes of this family, similar results have been obtained for Cd in the halophytes from Aizoaceae (Carpobrotus rossii (Haw.) Schwantes) in that application of salt de- creased Cd concentration in plant’s aerial parts (Cheng et al., 2018). A relatively low concentration of Na in the leaves (6-18 mg g–1 DM) was in agreement with the halophytic behavior of L. latifolium. It has been reported that under relatively low salt concentrations (50-150  mM), halo- phytes are more successful in controlling the Na uptake than glycophytes, and thus, do not accumulate much Na under these conditions (Munns, 2005). Salt treatment significantly reduced the concentration of K and Ca simi- lar to that observed in other halophytes, e.g. Plantago cor- onopus L. (Koyro, 2006). Zn toxicity treatment increased the leaf Na concen- tration in salt-treated plants by about 2.3 fold. A simi- lar increase was observed in K concentration however, it was a ‘concentration effect’ because the K content (mg plant–1) was rather decreased by Zn treatment (data not shown). In Kosteletzkya virginica (L.) C.Presl ex A.Gray Zn toxicity increased Na and decreased K concentration in the aerial parts (Han et al., 2012). Damages to mem- branes under Zn toxicity are likely the mechanism for an increase in the Na concentration in Zn-stressed plants that may be in turn prevented the mitigating effect of the combinative treatment on the biomass in our plants. 5 CONCLUSIONS In contrast to available reports on the ameliorative effects of salt on heavy metal tolerance in the halophytes, in this study, a growth improvement was not observed under the combination of salt and Zn toxicity compared with excess Zn as single stress. This may suggest that the mitigating effect of salt on heavy metal stress is related to the heavy metal and/or the halophyte species. Neverthe- less, the improvement of leaf pigments and, reduction of MDA and Zn accumulation in the leaves under the com- bination of both treatments showed that salt treatment is still able to stimulate the defense mechanisms in plants for protecting membranes and photosynthetic pigments against excess Zn concentrations. Lepidium latifolium plants grow under diverse envi- ronmental conditions, have a high growth rate, and pro- duce considerable biomass with perennial habit. These properties make this species a suitable candidate for revegetation and rehabilitation of saline soils contami- nated with heavy metals. 6 ACKNOWLEDGMENTS The authors would like to express their gratitude to the Council of Shahed University for financial support during the course of this research for the plant physiol- ogy Ph.D. thesis. 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