Excess Zinc Supply Reduces Cadmium Uptake and Mitigates Cadmium Toxicity Effects on Chloroplast Structure, Oxidative Stress, and Photosystem II Photochemical Efficiency in Salvia sclarea Plants.

Salvia sclarea L. is a Cd2+ tolerant medicinal herb with antifungal and antimicrobial properties cultivated for its pharmacological properties. However, accumulation of high Cd2+ content in its tissues increases the adverse health effects of Cd2+ in humans. Therefore, there is a serious demand to lower human Cd2+ intake. The purpose of our study was to evaluate the mitigative role of excess Zn2+ supply to Cd2+ uptake/translocation and toxicity in clary sage. Salvia plants were treated with excess Cd2+ (100 μM CdSO4) alone, and in combination with Zn2+ (900 μM ZnSO4), in modified Hoagland nutrient solution. The results demonstrate that S. sclarea plants exposed to Cd2+ toxicity accumulated a significant amount of Cd2+ in their tissues, with higher concentrations in roots than in leaves. Cadmium exposure enhanced total Zn2+ uptake but also decreased its translocation to leaves. The accumulated Cd2+ led to a substantial decrease in photosystem II (PSII) photochemistry and disrupted the chloroplast ultrastructure, which coincided with an increased lipid peroxidation. Zinc application decreased Cd2+ uptake and translocation to leaves, while it mitigated oxidative stress, restoring chloroplast ultrastructure. Excess Zn2+ ameliorated the adverse effects of Cd2+ on PSII photochemistry, increasing the fraction of energy used for photochemistry (ΦPSII) and restoring PSII redox state and maximum PSII efficiency (Fv/Fm), while decreasing excess excitation energy at PSII (EXC). We conclude that excess Zn2+ application eliminated the adverse effects of Cd2+ toxicity, reducing Cd2+ uptake and translocation and restoring chloroplast ultrastructure and PSII photochemical efficiency. Thus, excess Zn2+ application can be used as an important method for low Cd2+-accumulating crops, limiting Cd2+ entry into the food chain.

1. Introduction

Increased industrial and agricultural human activities, such as mining and smelting, electroplating, wastewater irrigation, and chemical fertilizers, have resulted in high environmental content of cadmium (Cd2+) [1,2,3]. It is now well recognized that Cd2+, a non-essential element for plants that is not biodegradable in the soil, accumulates in the environment and subsequently becomes toxic to all living organisms [4,5,6,7,8].

Zinc (Zn2+) belongs to a group of eight essential micronutrients that are required for normal plant growth, development, and defense [9,10]. Zinc is involved in various metabolic processes, playing catalytic, regulatory, and structural roles with several crucial functions in the cell [9,10,11,12,13,14,15,16]. It performs a fundamental role in anti-oxidative defense and retains the membranous structure of various cell organelles [14,17]. Zn2+ deficiency or Zn2+ at high concentration in the soil, which occurs in various habitats, ranging from deficient to toxic levels can cause inhibition of numerous plant metabolic processes, reduce plant growth and photosynthesis, and decrease chlorophyll content in the leaf, provoking chlorosis and leaf necrosis [9,11,12,13,14]. The zinc homeostasis mechanism is not global within plants since most plants contain between 30 and 100 μg Zn2+ g−1 dry weight (DW), but some other species are accumulating more than 10,000 μg Zn2+ g−1 DW without showing symptoms of toxicity [18], despite the fact that concentrations above 300 μg Zn2+ g−1 DW are considered toxic to plants [1,9].

Accumulative concentration of Cd2+ in soil is highly alarming due to risk of its entrance into food chain, while foliar Cd2+ concentrations above 100 μg g−1 DW (0.01%) are considered exceptional and a threshold value for Cd-hyperaccumulation [1,19]. Cadmium availability in soils depends upon a large number of aspects such as clay minerals, organic matter, soil pH, cation exchange capacity (CEC), type of fertilizers, and soil Cd2+ content [20,21,22]. Zinc, owing to its chemical similarity with Cd2+, might act as a competitive ion for the binding sites both in the soil and root surfaces for uptake, and/or might interact with Cd2+ within the transport system of plants [22,23]. It is regarded as the principal micronutrient to ameliorate the toxic effect of Cd2+ on plants and to limit its entry into food chain [24]. Foliar application of Zn2+ ameliorated the adverse effects of Cd2+ and decreased grain Cd2+ of wheat grown in Cd2+-contaminated soil [24,25]. In addition, it decreased oxidative stress and increased nutrients, chlorophyll content, and photosynthetic rates, in Cd2+-stressed wheat seedlings [22,24,25]. The addition of Zn2+ to Cd2+-containing soils or nutrient solutions has been concluded to be successful in reducing Cd2+ accumulation in crop plants [22,25,26], but since plant responses vary with genotypes and the dose and duration of the Zn2+ and Cd2+ exposure, it is suggested that more studies are necessary to explore the proper Zn2+/Cd2+ ratios required to reduce Cd2+ toxicity [22].

Clary sage (Salvia sclarea L.), a biennial or a perennial 20–130 cm high plant that belongs to the Lamiaceae family, is native to Southern Europe but is being cultivated worldwide in temperate and sub-tropical climates as an ornamental and essential oil-bearing plant [27]. It is used in the aromatic and food industries [28], especially in alcoholic beverages, pastries, gelatins, puddings, frozen desserts, and condiments [27] and for preventing food spoilage due to its antimicrobial properties [29]. Its essential oils present antifungal and cytotoxic activity [30,31]. Recent studies reported analgesic and antidiabetic as well as anti-inflammatory effects [27]. Salvia sclarea is also an economically important plant for phytoextraction and phytostabilization of Cd2+- and Zn2+-contaminated soils, with an increasing interest in cultivation because of this [2,14,27].

We have previously observed that S. sclarea plants show photosynthetic tolerance to Cd2+ toxicity by increasing the photoprotective mechanism of non-photochemical quenching (NPQ) and accelerating the cyclic electron transport around photosystem I (PSI) that protects the function of the photosynthetic apparatus under excess Cd2+ [2]. Nonetheless, after exposure of S. sclarea to Cd2+ for up to 5 days, despite significant levels of Cd2+ in leaves, an enhanced photosystem II (PSII) functionality was observed, with a higher fraction of absorbed light energy to be directed to photochemistry (Φ) and no defects on chloroplast ultrastructure to be noticed [3]. However, in S. sclarea plants exposed to the same level of Cd2+, but for 8 days, an inhibition of PSII functionality was observed, with the photoprotective mechanism of NPQ found to be inefficient in keeping the same fraction of open PSII reaction centers (q) compared with non-treated plants [3]. In contrast, when Salvia sclarea L. was exposed to excess Zn2+ (900 μM) for 8 days, a stimulation of PSI and PSII activity was accompanied by increased synthesis of antioxidants in the leaves that play an important role in Zn2+ detoxification and protection against oxidative stress [14].

In the present study we hypothesized that excess Zn2+ supply to Cd2+-containing nutrient solution would decrease Cd2+ uptake and oxidative stress and would thus ameliorate PSII performance of S. sclarea plants exposed to Cd2+ toxicity. We compared Cd2+ uptake and toxicity effects, with and without Zn2+ supplementation, in order to evaluate the application of excess Zn2+ supply as a possible method to limit Cd2+ entry into the food chain through the medicinal herb clary sage.

2. Materials and Methods

2.1. Plant Material and Growth Conditions

Seeds of Salvia sclarea L. collected from the Rose Valley (Bulgaria) were used for the experiments. After germination in a growth room on soil, for about a month, the seedlings were transferred for about one more month to pots in continuously aerated modified Hoagland nutrient solution that was changed every 3 days and described in detail before [2,8]. The growth room conditions were 14/10 h day/night photoperiod, with 200 ± 20 μmol photons m−2 s−1 photon flux density and 24 ± 1/20 ± 1 °C day/night temperature.

2.2. Cadmium and Cadmium plus Zinc Treatments

Forty-five two-month-old S. sclarea plants were divided into three groups, and each group was subjected to hydroponic culture for 8 more days either (i) with Hoagland nutrient solution alone, which served as control with 5 μM Zn2+, (ii) with Hoagland nutrient solution with 100 μM Cd2+ (supplied as 3CdSO4.8H2O) [2], and (iii) with Hoagland nutrient solution with 100 μM Cd2+ (supplied as 3CdSO4.8H2O) plus 900 μM Zn2+ (supplied as ZnSO4) [14]. All solutions were renewed every 2 days so that nutrient content, Cd2+, and Zn2+ supply remained constant. The duration and the concentration of Cd2+ and Zn2+ treatments were based on our previous experiments with Salvia plants exposed to Cd2+ and Zn2 [3,14]. When S. sclarea plants were exposed for up to 5 days to 100 μM Cd2+, despite significant levels of Cd2+ in the leaves, an enhanced PSII functionality was observed, but when they were exposed to the same level of Cd2+ for 8 days, an inhibition of PSII functionality was noticed [3]. On the other hand, exposure of S. sclarea plants for 8 days to 900 μM Zn2+ enhanced PSII functionality [14]. Thus, in the present study, we used 8 days of exposure to 100 μM Cd2+ plus 900 μM Zn2+ to reveal any mitigative effect of Zn2+ on Cd2+ toxicity.

2.3. Cadmium and Zinc Determination by Inductively Coupled Plasma Mass Spectrometry (ICP-MS)

After 8 days, five plants per group (control, Cd2+-, and Cd2+ + Zn2+-treated S. sclarea) were harvested, separated in roots and aboveground tissues, washed in deionized water, and then dried at 65 °C to constant biomass. Dried milled and sieved samples were further proceeded and digested in a microwave-assisted digestion system as described previously [3]. Samples were analyzed by an inductively coupled plasma mass spectrometry (ICP-MS) model ELAN DRC II (PerkinElmer Sciex, Toronto, ON, Canada) [32]. ICP-MS operational conditions, instrumental settings, calibration solutions, data validation, and validation parameters were as described before [3]. Elemental analysis was performed for Cd2+ and Zn2+.

2.4. Chlorophyll Fluorescence Analysis

Chlorophyll fluorescence measurements were conducted on dark adapted leaves of five plants per group (control, Cd2+, and Cd2+ + Zn2+), using an Imaging PAM M-Series system (Heinz Walz Instruments, Effeltrich, Germany) as described in detail previously [33]. In each leaf, representative areas of interest (AOIs) were selected, and the chlorophyll fluorescence parameters Fo (minimum chlorophyll a fluorescence in the dark-adapted leaf), Fm (maximum chlorophyll a fluorescence in the dark-adapted leaf), Fo’ (minimum chlorophyll a fluorescence in the light-adapted leaf), Fm’ (maximum chlorophyll a fluorescence in the light-adapted leaf), and Fs (steady-state photosynthesis at 900 μmol photons m–2 s–1 actinic light (AL) intensity) were measured [34]. The chlorophyll fluorescence parameters calculated by the Imaging Win V2.41a software (Heinz Walz GmbH, Effeltrich, Germany) from the above parameters were: Fv/Fm (maximum efficiency of PSII photochemistry); Φ (the actual quantum yield of PSII photochemistry); Φ (the quantum yield of regulated non-photochemical energy loss in PSII); Φ (the quantum yield of non-regulated energy dissipated in PSII); the redox state of quinone A (Q), an estimate of the fraction of open PSII reaction centers based on the “puddle” model for the photosynthetic unit (qp = [Fm′ − Fs]/[Fm′ − Fo′]); q (the fraction of open PSII reaction centers that are connected by shared antenna, that is, the so-called “lake” model) [35]; and EXC, the relative excess energy at PSII, (EXC = [Fv/Fm − Φ ]/[Fv/Fm]), according to Bilger et al. [36].

2.5. Determination of Oxidative Damage and Hydrogen Peroxide

The oxidative damage in S. sclarea leaves was estimated by the level of lipid peroxidation according to the method of Hodges et al. [37]. Leaf samples from five plants per group (control, Cd2+, and Cd2+ + Zn2+), after 8 days of treatment, were frozen in liquid nitrogen and stored at −80 °C before the evaluation of malondialdehyde (MDA) content by the reaction with 2-thiobarbituric acid (TBA), as described before [3]. Frozen leaf tissues were homogenized at 4 °C in 1% (w/v) trichloroacetic acid (TCA) and then centrifuged at 14,000× g for 20 min. Absorbance of the supernatant was read at 532 nm using Specord 210 Plus (Ed. 2010, Analytik Jena AG, Germany). Results are expressed as μmol MDA g−1 FW.

Hydrogen peroxide (H2O2) generation was estimated in leaves from the same S. sclarea plants used for MDA evaluation after extraction by homogenization with 50 mM K-phosphate buffer pH (6.5) and reaction with 0.1% TiCl4 in 20% H2SO4, as described before [3].

2.6. Chloroplast Ultrastructure Observations

Chloroplast ultrastructure alterations were observed in the same plants that were used for chlorophyll fluorescence measurements after 8 days of treatment. Leaf segments of 0.5 × 1 mm from control, Cd2+-, and Cd2+ + Zn2+-treated plants were fixed with 2% paraformaldehyde plus 4% glutaraldehyde in 0.05 M sodium cacodylate buffer at pH 7.0 as described previously [28]. Fixation took place at room temperature for 5 h and post-fixation for another 3 h and further treatments as described in detail before [3]. Ultrathin sections (80–90 nm) were stained with 2% uranyl acetate and 1% lead citrate and examined in a JEOL JEM 1011 transmission electron microscope equipped with a Gatan ES500W digital camera. Digital electron micrographs were obtained with the DigitalMicrograph 3.11.2 software (Digital Micrograph Gatan, Oxon, UK) [3].

2.7. Statistical Analysis

Statistical analysis was performed by one-way ANOVA analysis followed by post hoc comparisons using Dunn–Šidák correction. Means (± SD) were calculated from five independent biological replicates and were considered statistically different at a level of p < 0.05. Data analysis was performed with IBM SPSS Statistics for Windows version 28.0.

3. Results

3.1. Cadmium and Zinc Accumulation in Leaves and Roots of Salvia sclarea in Response to Cadmium Toxicity with and without Zinc Application

Exposure of S. sclarea plants to 100 μM Cd2+ for 8 days, increased Cd2+ in leaves by 50-fold (p < 0.05) (Figure 1a) and of roots by 7000-fold (Figure 1b), as Cd2+ in the roots reached 34,503 ± 1035 µg g−1 DW vs. 266.3 ± 7.8 µg g−1 DW in the leaves, as also reported previously [2]. Addition of 900 μM Zn2+ to the nutrient solution with 100 μM Cd2+ resulted in almost 50% decreased Cd2+ content in leaves (131.9 ± 3.9 µg g−1 from 266.3 ± 7.8 µg g−1 DW) (Figure 1a) and roots (17,704 ± 531 µg g−1 from 34,503 ± 1035 µg g−1 DW) (Figure 1b).

Figure 1

Changes in Cd2+ accumulation in leaves (a) and roots (b) and Zn2+ accumulation in leaves (c) and roots (d) in µg g−1 dry weight (DW) after eight days treatment of Salvia sclarea plants with control (nutrient solution), Cd2+ (nutrient solution +100 μM Cd2+), and Cd2+ + Zn2+ (nutrient solution +100 μM Cd2+ + 900 μM Zn2+). Error bars are standard deviations (n = 5).

Exposure to Cd2+ enhanced total Zn2+ uptake by 7.0-fold but decreased its translocation to the leaves by 36% (Figure 1c). Zinc content in roots after Cd2+ exposure reached 1228 ± 37 µg g−1 DW, from 89.3 ± 2.7 µg g−1 DW (Figure 1d), while in leaves from 88.1 ± 2.6 µg g−1 DW decreased to 31.8 ± 0.96 µg g−1 DW (Figure 1c). Zinc content in leaves, after Zn2+ supplementation with Cd2+, reached 328.1 ± 9.8 µg g−1 DW (10-fold increase) (Figure 1c), while in roots 39,711 ± 1191 DW µg g−1 dry weight (32-fold increase) (Figure 1d).

3.2. Changes in the Light Energy Utilization in Photosystem II in Response to Cadmium Toxicity with and without Zinc Application

We evaluated the light energy distribution pattern in PSII for photochemistry (Φ) for regulated non-photochemical energy loss—that is, for photoprotective heat dissipation (Φ)—and for non-regulated energy loss in PSII (Φ). The addition of these three fractions is unity [35,38,39,40,41].

In Cd2+-treated S. sclarea plants, the light energy used for photochemistry (Φ) decreased, while the photoprotective energy dissipation as heat (Φ) increased compared with control Salvia plants, with a concomitant decrease in the fraction of non-regulated energy lost (Φ) (Figure 2).

Figure 2

Changes in photosystem II quantum yields of Salvia sclarea control (nutrient solution), Cd2+ (nutrient solution +100 μM Cd2+), and Cd2+ + Zn2+ (nutrient solution +100 μM Cd2+ + 900 μM Zn2+). Effective quantum yield of photochemistry (Φ), regulated non-photochemical energy loss (Φ), and non-regulated energy loss in PSII (Φ) at 900 μmol photons m−2 s−1 actinic light (AL) intensity. Error bars are standard deviations (n = 5). Columns in each chlorophyll fluorescence parameter with different lowercase letters are statistically different (p < 0.05).

The application of 900 μM Zn2+ in the nutrient solution containing 100 μM Cd2+ increased the fraction of energy used for photochemistry (Φ) and decreased the fraction of heat dissipation (Φ), while there was no difference in the fraction of non-regulated loss (Φ) compared with Cd2+ alone (Figure 2).

3.3. Changes in the Maximum Efficiency of Photosystem II and the Redox State of Quinone A (QA) to Cadmium Toxicity with and without Zinc Application

In Cd2+-treated S. sclarea plants, both the maximum efficiency of PSII (Fv/Fm) (Figure 3a) and the redox state of quinone A (Q), an estimate of the fraction of open PSII reaction centers (qp) (Figure 3b), decreased, while the application of Zn2+ in the nutrient solution increased both of them to the level of control Salvia plants.

Figure 3

Changes in the maximum efficiency of photosystem II (Fv/Fm) (a), and the redox state of quinone A (Q), an estimate of the fraction of open PSII reaction centers estimated at 900 μmol photons m–2 s–1 actinic light (AL) intensity (qp) (b), of Salvia sclarea control (nutrient solution), Cd2+ (nutrient solution +100 μM Cd2+), and Cd2+ + Zn2+ (nutrient solution +100 μM Cd2+ + 900 μM Zn2+). Error bars are standard deviations (n = 5). Columns with different lowercase letters are statistically different (p < 0.05).

3.4. Changes in the Redox State of Plastoquinone Pool Based on the Lake Model and the Excess Excitation Energy in Response to Cadmium Toxicity with and without Zinc Application

The redox state of plastoquinone pool based on the lake model (1 − q) (Figure 4a) and the excess excitation energy (EXC) (Figure 4b), both measured at 900 μmol photons m−2 s−1 actinic light (AL) intensity, increased in Cd2+-treated S. sclarea plants, but both decreased to the level of control Salvia plants with the application of Zn2+ in the nutrient solution.

Figure 4

Changes in the redox state of plastoquinone pool based on the lake model (1 − q) (a), and the excess excitation energy (EXC) (b), estimated at 900 μmol photons m−2 s−1 actinic light (AL) intensity, of Salvia sclarea control (nutrient solution), Cd2+ (nutrient solution +100 μM Cd2+), and Cd2+ + Zn2+ (nutrient solution +100 μM Cd2+ + 900 μM Zn2+). Error bars are standard deviations (n = 5). Columns with different lowercase letters are statistically different (p < 0.05).

3.5. Changes in the Level of Lipid Peroxidation and Hydrogen Peroxide (H2O2) Generation in Response to Cadmium Toxicity with and without Zinc Application

In Cd2+-treated S. sclarea plants, the increased accumulation of malondialdehyde (MDA) (Figure 5a) coincided with the increased generation of hydrogen peroxide (H2O2) (Figure 5b). However, both decreased with the application of Zn2+ in the nutrient solution but remained higher than the level of control Salvia plants (Figure 5a,b).

Figure 5

Changes in the level of lipid peroxidation, assessed by the accumulation of malondialdehyde (MDA) (a), and the generation of hydrogen peroxide (H2O2) (b), in the leaves of Salvia sclarea control (nutrient solution), Cd2+ (nutrient solution +100 μM Cd2+), and Cd2+ + Zn2+ (nutrient solution +100 μM Cd2+ + 900 μM Zn2+). Error bars are standard deviations (n = 5). Columns with different lowercase letters are statistically different (p < 0.05).

3.6. Changes in Chloroplast Ultrastructure to Cadmium Toxicity with and without Zinc Application

Chloroplast of control plants had a typical internal structure, with well-organized grana thylakoids and stroma membranes and with high electron opacity of the stroma (Figure 6a). After 8 consecutive days of Cd2+ application, effects on chloroplasts ultrastructure involved thylakoid disorganization, dilated thylakoid membranes, and the absence of starch grains (Figure 6b). Zn2+ supplementation seemed to alleviate Cd effects to the chloroplasts, with starch grains being present (Figure 7a) and the thylakoid membranes being less dilated (Figure 7b).

Figure 6

Transmission electron microscopy (TEM) images of chloroplasts from control (nutrient solution) (a) and 8-day Cd2+-treated (nutrient solution + 100 μΜ Cd2+) (b) Salvia sclarea leaves. Upon Cd treatment, chloroplasts appeared electronically dense and with swollen thylakoids, while being devoid of starch grains when compared with control. CW: cell wall; sg: starch grain; V: vacuole. Scale bar: 200 nm.

Figure 7

Transmission electron microscopy (TEM) images of chloroplasts from 8-day Cd2++Zn2+-treated (nutrient solution + 100 μΜ Cd2++ 900 μΜ Zn2+) Salvia sclarea leaves. Zn addition to the nutrient solution mitigated Cd effects, with starch grains being present (a), and the thylakoid membranes being less dilated (b). CW: cell wall; sg: starch grain; V: vacuole. Scale bar: 200 nm.

4. Discussion

Salvia sclarea is recognized as a medicinal herb due to its valuable pharmacological properties and numerous health benefits, being also an economically important plant for phytoextraction and phytostabilization of Cd2+- and Zn2+-contaminated soils, with a subsequent increased interest in its cultivation [2,14,27]. Cadmium is greatly injurious to plant growth and almost all human individuals are exposed to Cd2+, mostly through plant-derived food that has reached the threshold for adverse health effects, and therefore, there is a serious demand to lower human Cd2+ intake by development of low Cd2+-accumulating crops [42,43]. In addition to food consumption, medicinal plants, and processed foods, Cd2+ is naturally stored at high concentrations in cigarettes [42,43,44]. Cadmium accumulation in leaf tissues damages the photosynthetic machinery, reducing photosynthetic activity [45] and increasing reactive oxygen species (ROS) accumulation, resulting in oxidative stress, programmed cell death, and necrosis [43,46,47,48,49].

Reactive oxygen species, such as singlet oxygen (1O2), superoxide anion radical (O2−), and hydrogen peroxide (H2O2), produced in plant cells, mainly in the electron transport chain of chloroplasts, are kept in a homeostasis by the antioxidative enzymatic and non-enzymatic systems [50,51,52,53,54]. However, under miscellaneous environmental stress conditions, the absorbed light energy surpasses what it can be used for in photochemistry, developing an increased ROS production that can cause cellular damage by oxidation of DNA, proteins, and lipids, resulting in oxidative stress [34,55,56]. Oxidative stress that is usually assessed by MDA content, a marker of lipid peroxidation [34,57,58], was found to increase under Cd2+ treatment (Figure 5a). However, Zn2+ supplementation to Cd2+-containing nutrient solution decreased lipid peroxidation, which remained higher compared with control values (Figure 5a), suggesting an increased ROS production. Under optimal growth conditions, a homeostasis of ROS is maintained [59,60], while an alteration in this homeostasis activates defense responses [3,59,61,62], but an elevated ROS level is harmful to plants [59,61]. However, ROS are considered to be essential signaling molecules that adjust plant development and the defense responses to various biotic and abiotic stresses [50,60,63,64]. A proper response to a stressor depends primarily on how plants identify the stress signal and respond to initiate a series of signaling cascades for initiation of acclimation mechanisms [65]. Cadmium-induced ROS, such as O2− and H2O2, is attributed to the phytotoxic effect of Cd2+, but ROS can also function as signal molecules in the induction of defense genes against Cd2+ toxicity [46]. The modulation of signal transduction pathways can also protect plants against Cd2+-induced cell death [47,66].

Several methods have been developed to remove or stabilize soil heavy metals and to reduce their accumulation in crops [67,68,69]. Mechanisms that interfere with Cd2+ uptake and accumulation have been shown to reduce Cd2+ toxicity [22,24,25,66,70,71]. Zinc, melatonin, and salicylic acid application are alleviating Cd2+-induced toxicity by inhibition of ROS overproduction [22,72,73]. Application of Zn2+ to a nutrient solution containing Cd2+ increased the fraction of energy used for photochemistry (Φ) compared with Cd2+ alone, but Φ remained lower compared with control Salvia plants (Figure 3). However, the fraction of non-regulated loss (Φ) decreased in both Cd2+ alone and Cd2+ + Zn2+-treated plants, compared with control Salvia plants (Figure 2). Overexcitation of PSII increases the probability of the formation of the triplet chlorophyll state (3Chl*) from the singlet excited state (1Chl*) through intersystem crossing, producing single oxygen (1O2) [38,74,75,76,77,78,79,80]. This non-regulated energy loss in PSII is reflected by Φ [38,75,76,78,80]. Thus, Cd2+ alone, and Cd2+ + Zn2+-treated plants displayed less ROS production, as 1O2 (Figure 2), but increased total ROS due to increased H2O2 generation, compared with control plants (Figure 5b).

In Cd2+-treated plants, hydrogen peroxide (H2O2) generation increased by 48% compared with control leaves (Figure 5b), while the accumulation of MDA, indicating the degree of oxidative stress causing lipid peroxidation, increased by 42% (Figure 5a). The lower increase in the degree of oxidative stress may be attributed to the decrease in ROS production as 1O2, assessed by the decreased fraction of non-regulated loss (Φ) (Figure 2). Reactive oxygen species are formed by energy transfer (1O2) and electron transport (H2O2) simultaneously, and it seems that their action interferes with their signaling pathways, sometimes to antagonize each other [3]. It has been often shown that hydrogen peroxide diffuses through leaf veins to act as a long-distance molecule activating stress defense responses under biotic and abiotic stresses in plants [52,59,60,76,78]. The increased H2O2 generation in Cd2+ + Zn2+-treated plants (Figure 5b) seems to have triggered a defense response that lowered Cd2+ toxicity effects on PSII photochemistry by increasing the fraction of energy used for photochemistry (Φ) (Figure 2), restoring the PSII redox state (Figure 3b) and the maximum PSII efficiency (Fv/Fm) (Figure 3a), suggesting beneficial effects of ROS production [59]. Under Cd2+ treatment alone, when the PQ pool (monitored by the 1 − q) was highly reduced (Figure 4a), an excess excitation energy (EXC) (Figure 4b) was observed in Cd2+-treated Salvia plants. High excess excitation energy, and therefore an imbalance between energy supply and demand, results in overproduction of ROS [3,39,79,80], having as a consequence oxidative stress, which was detected by the increased MDA content, representing the degree of lipid peroxidation (Figure 5a).

A dose-related negative impact of Cd2+ that raises ROS concentration, inducing oxidative damage and inhibition of photosynthetic function, has been extensively reported [81,82,83,84,85,86,87,88]. However, stimulation of the photosynthetic rate by Cd2+ at low concentrations has also been described, with a concomitant altered ROS homeostasis [3,89,90]. Plants employ several enzymic and non-enzymatic antioxidative systems to remove the different types of ROS, thus diminishing possible cell damage [52,76,89,90]. The alteration of ROS homeostasis has been suggested to be the mechanism of Cd2+-induced hormetic response of photosynthesis in medicinal herbs [3,90]. An enhanced PSII photochemistry, indicating an “over-compensation” response to Cd2+ exposure was corelated with Cd2+ disruption of ROS homeostasis [3], validating the declaration of Carvalho et al. [91] that Cd2+ can also be considered a beneficial element as well as a toxic one. However, in our experiment, in Cd2+-treated plants, the light energy used for photochemistry (Φ) decreased (Figure 2), and at the same time, the accumulation of MDA, indicating the degree of oxidative stress, increased (Figure 5a). Insufficient supply of energy for photochemistry decreases photosynthesis efficiency, which is detrimental to plant growth [92]. Zinc application to the nutrient solution, together with Cd2+, resulted in a 10-fold increase in Zn2+ in the leaves, which increased the fraction of energy used for photochemistry, compared with Cd2+ alone (Figure 2), while it decreased oxidative stress by 17%, also compared with Cd2+ alone (Figure 5a). Zinc, as a component of antioxidant enzymes, is essential for scavenging H2O2 and O2− [93]. Zinc application reduces Cd2+ accumulation, which depends greatly on the crop cultivar, and increases leaf Zn2+ content and at the same time decreases MDA concentration [94,95,96,97,98]. Application of selenium, silicon, melatonin, and salicylic acid have been also found to alleviate Cd2+-induced toxicity by inhibition of ROS overproduction [72,73,99,100,101,102,103].

It is well supported that Cd2+ negatively influences chloroplast structure, with observed defects that include thylakoid membrane dismantling, increased presence of plastoglobuli, starch grain decrease, and plastid envelope rupture [104]. The above are also observed in hyperaccumulator or Cd2+-tolerant plants when a threshold of Cd2+ concentration and duration treatment is surpassed [3,105,106]. In Cd2+-treated plants, chloroplast structure is compromised, with Cd2+ causing thylakoid membrane disruption and starch grain loss (Figure 6b). Increased ROS production (Figure 5b) could lead to a noticeable increase in membrane damage, indicated by the elevated MDA levels (Figure 5a), and the subsequent changes in chloroplast ultrastructure could specify dysfunctions in metabolism, indicating defects of the photosynthetic parameters (Figure 2). The observed decrease in photosynthetic parameters can, in turn, be associated with reduced carbon fixation [107] and consequently a lower number of starch grains [106]. In Cd2+ + Zn2+-treated plants, Cd2+-related toxic effects are ameliorated, with starch grains being present and the thylakoid membranes being less affected (Figure 7a,b). Generally, it has been stated that exogenous Zn2+ application alleviates oxidative damage induced by Cd2+ application (Figure 5a) since Zn2+ improved the function of the antioxidant systems [108,109], a notion mirrored in the chloroplast structure as well (Figure 7a,b).

Exposure of S. sclarea plants to 100 μM Cd2+ for 8 days resulted in 266 µg g−1 DW Cd2+ concentration in the leaves (Figure 1a). Addition of Zn2+ resulted in 50% decreased Cd2+ content in leaves (132 µg g−1 DW) (Figure 1a), and thus, it can be regarded as the principal element for ameliorating the toxic effects of Cd2+ on plants and limiting its entry into the food chain [24]. The average daily intake of Cd2+ via food in European countries and North America is 15-25 μg, while in Japan it is commonly 40–50 μg, but it may be considerably higher in Cd2-polluted areas [110]. Zinc content in S. sclarea leaves, after Zn2+ supplementation with Cd2+, reached 0.328 mg g−1 DW (Figure 1c), while daily Zn2+ intake must not exceed 40 mg per day for adults [111]. Proper Zn2+ biofortification, however, requires specification of plant species and soil type [111,112].

Our data on chlorophyll a fluorescence analysis as well as previous data indicated that Cd2+ exposure results in a partial inactivation of PSII reaction centers and inhibition of PSII functionality, disturbing electron transport in the oxygen-evolving complex [6,7,8,113,114,115]. Chlorophyll a fluorescence analysis is a promising technique for quick detection of photosynthetic efficiency, permitting short- or long-term abiotic or biotic stress impact on the mechanisms of PSII functionality to be revealed [116,117,118], while the use of chlorophyll fluorescence imaging analysis permits the detection of spatiotemporal heterogeneity at the total leaf surface [119]. Additional studies will be performed to elucidate defense mechanisms, including antioxidants, PSI-dependent cyclic electronic transport, and kinetics of oxygen-evolving reactions, which will provide more detailed information on the role of excess Zn in the increased Cd tolerance of Salvia sclarea.

5. Conclusions

Exposure of the medicinal herb Salvia sclarea to 100 μM Cd2+ for 8 consecutive days enhanced total Zn2+ uptake but decreased its translocation to the leaves and resulted in an inhibition of PSII functionality, with the photoprotective mechanism of the dissipation of the excess energy as heat to be ineffective in keeping the redox state of quinone A (Q) oxidized at the same level as non-treated plants. Application of Zn2+ effectively mitigated Cd2+-induced toxicity on Salvia sclarea by reducing Cd2+ uptake, together with its translocation to the leaves, while it mitigated oxidative stress that restored partially the chloroplast ultrastructure. Excess Zn2+ ameliorated PSII photochemistry by increasing the fraction of energy used for photochemistry (Φ) and restoring the PSII redox state and maximum PSII efficiency (Fv/Fm), while it lowered the excess excitation energy at PSII (EXC). We conclude that excess Zn2+ reduced Cd2+ uptake and translocation and restored chloroplast ultrastructure and PSII photochemical efficiency. Our results show that Zn2+ application on clary sage, by decreasing Cd2+ uptake to half, can be regarded as a fundamental method to limit Cd2+ entry into the food chain.