Rapid Hormetic Responses of Photosystem II Photochemistry of Clary Sage to Cadmium Exposure

Five-day exposure of clary sage (Salvia sclarea L.) to 100 μM cadmium (Cd) in hydroponics was sufficient to increase Cd concentrations significantly in roots and aboveground parts and affect negatively whole plant levels of calcium (Ca) and magnesium (Mg), since Cd competes for Ca channels, while reduced Mg concentrations are associated with increased Cd tolerance. Total zinc (Zn), copper (Cu), and iron (Fe) uptake increased but their translocation to the aboveground parts decreased. Despite the substantial levels of Cd in leaves, without any observed defects on chloroplast ultrastructure, an enhanced photosystem II (PSII) efficiency was observed, with a higher fraction of absorbed light energy to be directed to photochemistry (ΦPSΙΙ). The concomitant increase in the photoprotective mechanism of non-photochemical quenching of photosynthesis (NPQ) resulted in an important decrease in the dissipated non-regulated energy (ΦNO), modifying the homeostasis of reactive oxygen species (ROS), through a decreased singlet oxygen (1O2) formation. A basal ROS level was detected in control plant leaves for optimal growth, while a low increased level of ROS under 5 days Cd exposure seemed to be beneficial for triggering defense responses, and a high level of ROS out of the boundaries (8 days Cd exposure), was harmful to plants. Thus, when clary sage was exposed to Cd for a short period, tolerance mechanisms were triggered. However, exposure to a combination of Cd and high light or to Cd alone (8 days) resulted in an inhibition of PSII functionality, indicating Cd toxicity. Thus, the rapid activation of PSII functionality at short time exposure and the inhibition at longer duration suggests a hormetic response and describes these effects in terms of “adaptive response” and “toxicity”, respectively.


Introduction
Cadmium (Cd) is occurring in soils at low concentrations but can arise to high concentrations as a result of numerous human activities, while being not biodegradable in the soil, it is considered as one of the most toxic elements, and also a non-essential element for plants [1][2][3][4][5][6]. Cadmium is taken up by roots, and its translocation from the roots to the shoots and leaves with subsequent accumulation in the chloroplasts, will eventually disturb photosynthesis [7]. However, many plant species have established numerous special effective mechanisms for Cd detoxification and tolerance [4][5][6]. 6.7 µ g g −1 , decreased to 32.58 ± 1.6, 10.29 ± 0.28, and 88.4 ± 2.6 µ g g −1 , respectively. Total Ca uptake was significantly reduced (58%), with root Ca accumulation to remain almost unaffected, but Ca translocation to shoot-leaves to be significantly reduced (84%) ( Figure  2e). Thus, after Cd exposure, Ca content in roots was 10,122 ± 304 µ g g −1 from 10,005 ± 300 µ g g −1 before Cd treatment, but in the aboveground tissues, decreased to 3820 ± 115 µ g g −1 from that of 23,484 ± 704 µ g g −1 before Cd treatment (Figure 2e). Total Mn ( Figure  2d) and Mg (Figure 2f) uptake decreased (p < 0.05) by 5% and 49%, respectively, after Cd exposure, but while Mg accumulation decreased in both roots (43%) and shoots-leaves (53%) (Figure 2f), Mn accumulation increased in roots (1.6-fold), but its translocation to the shoots decreased (65%) after Cd exposure (Figure 2d). Manganese increased in roots from 62.01 ± 1.8 µ g g −1 before Cd treatment to 98.1 ± 2.9 µ g g −1 , while in aboveground tissues decreased from 64.2 ± 1.9 µ g g −1 before Cd treatment to 22.88 ± 0.69 µ g g −1 after Cd exposure (Figure 2d). Magnesium, the next most negatively affected element after Ca, decreased in roots from 4184 ± 126 µ g g −1 before Cd treatment to 2391 ± 72 µg g −1 after Cd exposure, while in aboveground tissues from 5851 ± 175 µ g g −1 before Cd treatment to 2778 ± 83 µg g −1 after Cd exposure (Figure 2f).   , and Mg (f) content, in µ g g −1 dry weight, of control (con) and 5 days Cd-treated Salvia sclarea aboveground (shoots-leaves) tissues, roots, and whole plants. Error bars are standard deviations (n = 5). Means between the two treatments that are statistically different (p < 0.05) are marked by an asterisk (*).

Chlorophyll a and Chlorophyll b Content after Cadmium Exposure
Chlorophyll a (Chla) content decreased (p < 0.05) in S. sclarea leaves exposed to Cd for 2 and 5 days compared to their respective controls (Figure 3a). The same response pattern was observed in chlorophyll b (Chlb) content of S. sclarea leaves after 2-and 5-days exposure to Cd stress (Figure 3b). These significant chlorophyll content decreases can be explained by the significantly decreased Mg uptake (Figure 2f). , and Mg (f) content, in µg g −1 dry weight, of control (con) and 5 days Cd-treated Salvia sclarea aboveground (shoots-leaves) tissues, roots, and whole plants. Error bars are standard deviations (n = 5). Means between the two treatments that are statistically different (p < 0.05) are marked by an asterisk (*).

Chlorophyll a and Chlorophyll b Content after Cadmium Exposure
Chlorophyll a (Chla) content decreased (p < 0.05) in S. sclarea leaves exposed to Cd for 2 and 5 days compared to their respective controls (Figure 3a). The same response pattern was observed in chlorophyll b (Chlb) content of S. sclarea leaves after 2-and 5-days exposure to Cd stress (Figure 3b). These significant chlorophyll content decreases can be explained by the significantly decreased Mg uptake (Figure 2f). , and Mg (f) content, in µ g g −1 dry weight, of control (con) and 5 days Cd-treated Salvia sclarea aboveground (shoots-leaves) tissues, roots, and whole plants. Error bars are standard deviations (n = 5). Means between the two treatments that are statistically different (p < 0.05) are marked by an asterisk (*).

Chlorophyll a and Chlorophyll b Content after Cadmium Exposure
Chlorophyll a (Chla) content decreased (p < 0.05) in S. sclarea leaves exposed to Cd for 2 and 5 days compared to their respective controls (Figure 3a). The same response pattern was observed in chlorophyll b (Chlb) content of S. sclarea leaves after 2-and 5-days exposure to Cd stress (Figure 3b). These significant chlorophyll content decreases can be explained by the significantly decreased Mg uptake (Figure 2f).

The Efficiency of Photosystem II after Cadmium Exposure
In order to understand how PSII functionality is affected by exposure of plants to Cd, we measured the maximum efficiency of PSII photochemistry (Fv/Fm) ( Figure 4a) and the efficiency of the water-splitting complex on the donor side of PSII (Fv/Fo) [29] (Figure 4b). Both parameters increased (p < 0.05) in S. sclarea plants exposed to Cd stress for 2 and 5 days compared to their respective controls, indicating an enhanced PSII functionality under Cd stress. However, this enhanced PSII functionality up to 5 days exposure had not any significant influence on plant biomass, but 8 days Cd exposure reduced whole plant biomass by 18% (p < 0.05).

The Efficiency of Photosystem II after Cadmium Exposure
In order to understand how PSII functionality is affected by exposure of plants to Cd, we measured the maximum efficiency of PSII photochemistry (Fv/Fm) ( Figure 4a) and the efficiency of the water-splitting complex on the donor side of PSII (Fv/Fo) [29] ( Figure  4b). Both parameters increased (p < 0.05) in S. sclarea plants exposed to Cd stress for 2 and 5 days compared to their respective controls, indicating an enhanced PSII functionality under Cd stress. However, this enhanced PSII functionality up to 5 days exposure had not any significant influence on plant biomass, but 8 days Cd exposure reduced whole plant biomass by 18% (p < 0.05).

Changes in the Quantum Yields and the Fraction of Open Photosystem II Reaction Centers after Cadmium Exposure under Low Light
The quantum efficiency of PSII photochemistry (ΦPSΙΙ) measured at low light (LL, 220 μmol photons m −2 s -1 ) (Figure 5a) increased (p < 0.05) in S. sclarea plants exposed to Cd stress for 2 and 5 days compared to their respective controls, indicating a higher fraction of absorbed light energy to be directed to photochemistry under Cd stress. The concomitant increase in the quantum yield of regulated heat dissipation in PSII (ΦNPQ) (Figure 5b) under 2-and 5-days Cd stress resulted in a significant decrease in the quantum yield of non-regulated energy dissipated in PSII (ΦNO) (Figure 5c), indicating a better use of the absorbed light energy in S. sclarea Cd stressed plants. The fraction of open PSII reaction centers (qP), at LL (Figure 5d), increased (p < 0.05) in S. sclarea plants exposed to Cd stress

Changes in the Quantum Yields and the Fraction of Open Photosystem II Reaction Centers after Cadmium Exposure under Low Light
The quantum efficiency of PSII photochemistry (Φ PSII ) measured at low light (LL, 220 µmol photons m −2 s -1 ) (Figure 5a) increased (p < 0.05) in S. sclarea plants exposed to Cd stress for 2 and 5 days compared to their respective controls, indicating a higher fraction of absorbed light energy to be directed to photochemistry under Cd stress. The concomitant increase in the quantum yield of regulated heat dissipation in PSII (Φ NPQ ) ( Figure 5b) under 2-and 5-days Cd stress resulted in a significant decrease in the quantum yield of non-regulated energy dissipated in PSII (Φ NO ) (Figure 5c), indicating a better use of the absorbed light energy in S. sclarea Cd stressed plants. The fraction of open PSII reaction centers (q P ), at LL (Figure 5d), increased (p < 0.05) in S. sclarea plants exposed to Cd stress for 2 days, while at 5 days exposure remain the same, compared to controls.

Changes in Non-Photochemical Fluorescence Quenching and Electron Transport Rate after Cadmium Exposure under Low Light
Non-photochemical quenching (NPQ) increased (p < 0.05) in plants exposed to Cd stress for 2 and 5 days compared to their respective controls (Figure 5e). Electron transport rate measured at 220 μmol photons m −2 s -1 (Figure 5f) increased (p < 0.05) in S. sclarea plants exposed to Cd stress for 2 and 5 days compared to their respective controls, following the pattern of ΦPSΙΙ (Figure 5a).

Changes in Excess Excitation Energy under Low and High Light after Cadmium Exposure
The excess excitation energy (EXC) at PSII in S. sclarea, at 220 μmol photons m −2 s -1 (LL) after 2 days Cd exposure, decreased (p < 0.05) compared to control, while, after 5 days Cd exposure, it was at the same level with control ( Figure 6a). However, under high light (HL, 900 μmol photons m −2 s -1 ) after 5 days Cd exposure, EXC increased (p < 0.05) compared to control (Figure 6b), indicating that the synergistic effect of Cd stress and HL resulted in a lower efficiency of light energy use by PSII.

Changes in Non-Photochemical Fluorescence Quenching and Electron Transport Rate after Cadmium Exposure under Low Light
Non-photochemical quenching (NPQ) increased (p < 0.05) in plants exposed to Cd stress for 2 and 5 days compared to their respective controls (Figure 5e). Electron transport rate measured at 220 µmol photons m −2 s -1 (Figure 5f) increased (p < 0.05) in S. sclarea plants exposed to Cd stress for 2 and 5 days compared to their respective controls, following the pattern of Φ PSII (Figure 5a).

Changes in Excess Excitation Energy under Low and High Light after Cadmium Exposure
The excess excitation energy (EXC) at PSII in S. sclarea, at 220 µmol photons m −2 s -1 (LL) after 2 days Cd exposure, decreased (p < 0.05) compared to control, while, after 5 days Cd exposure, it was at the same level with control ( Figure 6a). However, under high

Changes in the Quantum Yields under High Light after Cadmium Exposure
The allocation of absorbed light energy to PSII photochemistry (ΦPSΙΙ) measured at HL in S. sclarea plants exposed to Cd for 5 days decreased (p < 0.05) compared to controls, while the regulated heat dissipation (ΦNPQ) increased (p < 0.05) in such a degree that the non-regulated energy dissipation (ΦNO) in S. sclarea plants exposed to Cd for 5 days did not differ compared to controls (Figure 7).

Changes in Non-Photochemical Fluorescence Quenching, Electron Transport Rate, and the Fraction of Open Photosystem II Reaction Centers under High Light after Cadmium Exposure
Non-photochemical quenching (NPQ), measured at HL, increased (p < 0.05) in S. sclarea plants exposed for 5 days to Cd compared to control plants (Figure 8a), while PSII electron transport rate decreased (p < 0.05) compared to controls (Figure 8b), following the pattern of ΦPSΙI (Figure 7). The fraction of open PSII reaction centers (qP) decreased (p < 0.05) in S. sclarea plants exposed for 5 days to Cd compared to control plants ( Figure 8c).

Changes in the Quantum Yields under High Light after Cadmium Exposure
The allocation of absorbed light energy to PSII photochemistry (Φ PSII ) measured at HL in S. sclarea plants exposed to Cd for 5 days decreased (p < 0.05) compared to controls, while the regulated heat dissipation (Φ NPQ ) increased (p < 0.05) in such a degree that the non-regulated energy dissipation (Φ NO ) in S. sclarea plants exposed to Cd for 5 days did not differ compared to controls (Figure 7).

Changes in the Quantum Yields under High Light after Cadmium Exposure
The allocation of absorbed light energy to PSII photochemistry (ΦPSΙΙ) measured at HL in S. sclarea plants exposed to Cd for 5 days decreased (p < 0.05) compared to controls, while the regulated heat dissipation (ΦNPQ) increased (p < 0.05) in such a degree that the non-regulated energy dissipation (ΦNO) in S. sclarea plants exposed to Cd for 5 days did not differ compared to controls (Figure 7).

Changes in Non-Photochemical Fluorescence Quenching, Electron Transport Rate, and the Fraction of Open Photosystem II Reaction Centers under High Light after Cadmium Exposure
Non-photochemical quenching (NPQ), measured at HL, increased (p < 0.05) in S. sclarea plants exposed for 5 days to Cd compared to control plants (Figure 8a), while PSII electron transport rate decreased (p < 0.05) compared to controls (Figure 8b), following the pattern of ΦPSΙI (Figure 7). The fraction of open PSII reaction centers (qP) decreased (p < 0.05) in S. sclarea plants exposed for 5 days to Cd compared to control plants ( Figure 8c). The quantum yields of PSII photochemistry (Φ PSII ), the regulated heat dissipation (Φ NPQ ), and the non-regulated energy dissipation (Φ NO ), measured at 900 µmol photons m −2 s -1 ; of control (con) and 5 days Cd-treated Salvia sclarea plants. Error bars are standard deviations (n = 6). Means between the two treatments that are statistically different (p < 0.05) are marked by an asterisk (*).

Changes in Non-Photochemical Fluorescence Quenching, Electron Transport Rate, and the Fraction of Open Photosystem II Reaction Centers under High Light after Cadmium Exposure
Non-photochemical quenching (NPQ), measured at HL, increased (p < 0.05) in S. sclarea plants exposed for 5 days to Cd compared to control plants (Figure 8a), while PSII electron transport rate decreased (p < 0.05) compared to controls (Figure 8b), following the pattern of Φ PSII (Figure 7). The fraction of open PSII reaction centers (q P ) decreased (p < 0.05) in S. sclarea plants exposed for 5 days to Cd compared to control plants ( Figure 8c).

Chlorophyll a Fluorescence Images under Low and High Light
Chlorophyll a fluorescence images of the fluorescence parameters ΦPSΙΙ and ΦNO, measured at LL and HL, of control and 5 days Cd-treated S. sclarea plants, revealed a spatial heterogeneity over the whole leaf area ( Figure 9). The heterogeneity was higher under Cd exposure with ΦPSΙΙ values at the center of the leaf and near the main leaf vein to have lower values compared to marginal, while the spatial heterogeneity was even higher under HL and Cd exposure ( Figure 9). The effective quantum yield of PSII photochemistry (ΦPSΙΙ) after 5 days Cd exposure, was higher under LL compared to control, but it was lower under HL compared to control ( Figure 9). The non-regulated energy loss in PSII (ΦNO), under both LL and HL, was lower in 5 days Cd-treated S. sclarea plants compared to controls (Figure 9). At longer duration exposure (8 days) to Cd under LL, the inhibition of PSII functionality that was observed ( Figure 10) resulted in the reduction of whole plant biomass by 18% (p < 0.05). At 8 days exposure of S. sclarea plants to Cd, the lowest Fv/Fm values were found near the midvein, while the lowest ΦPSΙΙ values were at the half leaf area near the base ( Figure 10).

Chlorophyll a Fluorescence Images under Low and High Light
Chlorophyll a fluorescence images of the fluorescence parameters Φ PSII and Φ NO , measured at LL and HL, of control and 5 days Cd-treated S. sclarea plants, revealed a spatial heterogeneity over the whole leaf area ( Figure 9). The heterogeneity was higher under Cd exposure with Φ PSII values at the center of the leaf and near the main leaf vein to have lower values compared to marginal, while the spatial heterogeneity was even higher under HL and Cd exposure ( Figure 9).

Chlorophyll a Fluorescence Images under Low and High Light
Chlorophyll a fluorescence images of the fluorescence parameters ΦPSΙΙ and ΦNO, measured at LL and HL, of control and 5 days Cd-treated S. sclarea plants, revealed a spatial heterogeneity over the whole leaf area ( Figure 9). The heterogeneity was higher under Cd exposure with ΦPSΙΙ values at the center of the leaf and near the main leaf vein to have lower values compared to marginal, while the spatial heterogeneity was even higher under HL and Cd exposure ( Figure 9). The effective quantum yield of PSII photochemistry (ΦPSΙΙ) after 5 days Cd exposure, was higher under LL compared to control, but it was lower under HL compared to control ( Figure 9). The non-regulated energy loss in PSII (ΦNO), under both LL and HL, was lower in 5 days Cd-treated S. sclarea plants compared to controls (Figure 9). At longer duration exposure (8 days) to Cd under LL, the inhibition of PSII functionality that was observed ( Figure 10) resulted in the reduction of whole plant biomass by 18% (p < 0.05). At 8 days exposure of S. sclarea plants to Cd, the lowest Fv/Fm values were found near the midvein, while the lowest ΦPSΙΙ values were at the half leaf area near the base ( Figure 10). The effective quantum yield of PSII photochemistry (Φ PSII ) after 5 days Cd exposure, was higher under LL compared to control, but it was lower under HL compared to control (Figure 9). The non-regulated energy loss in PSII (Φ NO ), under both LL and HL, was lower in 5 days Cd-treated S. sclarea plants compared to controls (Figure 9). At longer duration exposure (8 days) to Cd under LL, the inhibition of PSII functionality that was observed ( Figure 10) resulted in the reduction of whole plant biomass by 18% (p < 0.05). At 8 days exposure of S. sclarea plants to Cd, the lowest Fv/Fm values were found near the midvein, while the lowest Φ PSII values were at the half leaf area near the base (Figure 10).

Lipid Peroxidation and Hydrogen Peroxide (H2O2) after Cadmium Exposure
The final product of lipid peroxidation, malondialdehyde (MDA) content ( Figure  11b), increased with increased exposure time to Cd. The same pattern was observed in H2O2 generation (Figure 11a). This trend was also obvious in the histochemically detected H2O2 production of Salvia sclarea leaves (Figure 12). After 5 days exposure to Cd, the increased H2O2 production was detected mainly in the leaf midveins near the basal leaf area, while after 8 days exposure, the highly increased H2O2 was not identified in the midveins but it was noticed to spread to the whole leaf ( Figure 12).

Lipid Peroxidation and Hydrogen Peroxide (H2O2) after Cadmium Exposure
The final product of lipid peroxidation, malondialdehyde (MDA) content ( Figure  11b), increased with increased exposure time to Cd. The same pattern was observed in H2O2 generation (Figure 11a). This trend was also obvious in the histochemically detected H2O2 production of Salvia sclarea leaves (Figure 12). After 5 days exposure to Cd, the increased H2O2 production was detected mainly in the leaf midveins near the basal leaf area, while after 8 days exposure, the highly increased H2O2 was not identified in the midveins but it was noticed to spread to the whole leaf ( Figure 12). This trend was also obvious in the histochemically detected H 2 O 2 production of Salvia sclarea leaves (Figure 12). After 5 days exposure to Cd, the increased H 2 O 2 production was detected mainly in the leaf midveins near the basal leaf area, while after 8 days exposure, the highly increased H 2 O 2 was not identified in the midveins but it was noticed to spread to the whole leaf ( Figure 12).

Chloroplast Ultrastructure after Cadmium Exposure
Leaves from control plants exhibited rather electronically dense mesophyll chloroplasts (Figure 13a) that showed a typical internal membrane structure with well-organized grana and stroma thylakoids (Figure 13a). Chloroplasts, in both 2 days (Figure 13b) and 5 days (Figure 13c) Cd-treated plants, did not show any noticeable structural disruption having a similar appearance to the control, with the 5 day Cd-treated plastids to appear more electronically dense (Figure 13c). However, after 8 days exposure to Cd, chloroplasts appeared even more electronically dense and their thylakoids were swollen (Figure S1b). In control, 2 days and 5 days Cd-treated plants, starch grains were noticeable in chloroplasts, which were absent in 8 days Cd-treated chloroplasts ( Figure S1b).

Chloroplast Ultrastructure after Cadmium Exposure
Leaves from control plants exhibited rather electronically dense mesophyll chloroplasts (Figure 13a) that showed a typical internal membrane structure with well-organized grana and stroma thylakoids (Figure 13a). Chloroplasts, in both 2 days (Figure 13b) and 5 days (Figure 13c) Cd-treated plants, did not show any noticeable structural disruption having a similar appearance to the control, with the 5 day Cd-treated plastids to appear more electronically dense (Figure 13c). However, after 8 days exposure to Cd, chloroplasts appeared even more electronically dense and their thylakoids were swollen ( Figure S1b). In control, 2 days and 5 days Cd-treated plants, starch grains were noticeable in chloroplasts, which were absent in 8 days Cd-treated chloroplasts ( Figure S1b).

Chloroplast Ultrastructure after Cadmium Exposure
Leaves from control plants exhibited rather electronically dense mesophyll chloroplasts (Figure 13a) that showed a typical internal membrane structure with well-organized grana and stroma thylakoids (Figure 13a). Chloroplasts, in both 2 days (Figure 13b) and 5 days (Figure 13c) Cd-treated plants, did not show any noticeable structural disruption having a similar appearance to the control, with the 5 day Cd-treated plastids to appear more electronically dense (Figure 13c). However, after 8 days exposure to Cd, chloroplasts appeared even more electronically dense and their thylakoids were swollen (Figure S1b). In control, 2 days and 5 days Cd-treated plants, starch grains were noticeable in chloroplasts, which were absent in 8 days Cd-treated chloroplasts ( Figure S1b).

Discussion
Exposure of S. sclarea plants to 100 µM Cd for 5 days in hydroponics resulted in a high Cd uptake with a 2400-fold increase at the whole plant level but with Cd ions to be retained almost exclusively in the roots (Figure 1b) and only 53.3 µg g −1 to be translocated to the aboveground tissues (Figure 1a). Low Cd accumulation in leaves may represent a tolerance mechanism that protects the photosynthetic equipment against additional oxidative stress [48][49][50]. In the tolerant plant species, the excess heavy metals in roots play a significant role by sequestrating and detoxifying the extreme amount of heavy metal in order to protect the delicate aboveground photosynthetic tissues [51,52]. Since leaf Cd contents greater than 5-10 µg g −1 have been characterized toxic to most plants [53][54][55], it seems that S. sclarea could have kept Cd concentration in the photosynthetic tissues in non-toxic forms. This could be done by depositing it in the vacuoles of leaf epidermal cells [56] and/or by complexation with cellular ligands [7, [55][56][57]. Hyperaccumulators can accumulate Cd to levels above 100 µg g −1 of shoot dry weight, without showing any toxicity symptoms [7,58]. Our results agree with those of He et al. [59] that roots of Cd tolerant plants (non-hyperaccumulators) retain considerably higher Cd concentrations than the aboveground parts, and only minor Cd is translocated to the aerial parts. In comparison to the above-ground tissues, S. sclarea roots showed a higher bioaccumulation ability of Cd with translocation to shoots-leaves to be restricted.
Cadmium uptake is affected by Ca levels because Cd competes for Ca channels [7,60,61] and the low Ca content of the hydroponic solution may enhance Cd uptake [62], resulting in enhanced Cd and decreased Ca in many plant species [7,61], as we also observed in S. sclarea experiments, with Ca being the most affected element (58% total uptake decrease, Figure 2e). Likewise, in Oryza sativa seedlings exposed to Cd, uptake of Ca was decreased, and Ca content in both roots and aboveground parts was significantly reduced [63]. Magnesium was the next most negatively affected element after Ca in our experiments, but low Mg status has been associated with increased Cd tolerance [64][65][66][67][68], indicating that plants regulate nutrient concentrations to mitigate Cd toxicity [68].
Antagonistic effects of Cd with Fe [50,61,69] and Zn [57,70] have been frequently reported. However, in S. sclarea exposed to 100 µM Cd for 5 days, total Zn, Cu, and Fe uptake increased but their translocation to the aboveground parts decreased possible due to translocation barriers. It seems that Cd uptake in S. sclarea is not taking place through the Fe or Zn pathway, while conditions that lead to increased Cd uptake in plants may also favor increased Fe uptake [7]. Cd treatment has been frequently mentioned that it increases Fe retention in roots but obstructs its translocation to shoots, thus reducing Fe concentrations in aboveground parts [59,71,72]. In rice, Cd has been shown to be taken up predominantly via the Mn pathway [73,74] but this was not the case in S. sclarea, since Mn was the less negatively affected element.
Cadmium contamination of soil has become a serious environmental alarm as it is estimated that around 30,000 t of Cd is released annually into the environment with a consequence to the food chain and a threat to human health [75]. The use of plants for heavy metals elimination from pollutant soils and water is a technique known as phytoremediation [76]. In phytoremediation, plants that absorb heavy metals from soils and translocate them to the harvestable shoots are used for phytoextraction, while those that stabilize metal contaminants through accumulation in the root zones are used for phytostabilization [75,77]. Plant species with high bioconcentration factor but relatively low translocation factor (<1) may be considered as potential phytostabilizers [78]. Salvia sclarea exhibited high accumulation capacity for Cd, and by limiting its translocation from roots to shoots, it may be considered a potential phytostabilizer that can be used in heavy metal contaminated environments. Other plant species that have been proposed as Cd phytostabilizers are Iris lactea [75] and Sesuvium portulacastrum [79,80].
Despite the significant levels of Cd in leaves, a higher fraction of absorbed light energy was directed to photochemistry (Φ PSII ) under 2-and 5-days Cd stress, with a concomitant increase in Φ NPQ that resulted in a significant decrease in Φ NO ( Figure 5). The non-regulated energy loss in PSII (Φ NO ) encompasses internal conversions and intersystem crossing, which results in singlet oxygen ( 1 O 2 ) creation via the triplet state of chlorophyll ( 3 chl*) [29,[81][82][83]. To optimize photosynthesis and growth under stressful conditions, plants have evolved a variety of mechanisms against photodamage and photoinhibition [84,85]. Non-photochemical quenching is the key photoprotective process that dissipates excess light energy as heat and protects photosynthesis [81,[86][87][88][89][90]. Thus, the increased nonphotochemical quenching of photosynthesis (NPQ) altered ROS homeostasis through a decreased 1 O 2 formation. Consequently, in S. sclarea plants exposed to 100 µM Cd, ROS homeostasis could be regulated by NPQ in such a way so that plants can cope with Cd stress [19,22,26,27].
The potential PSII efficiency of S. sclarea plants exposed to 100 µM Cd estimated by the maximum efficiency of PSII photochemistry (Fv/Fm) (Figure 4a) and the efficiency of the water-splitting complex on the donor side of PSII (Fv/Fo) (Figure 4b) [29,91] indicated an enhanced PSII functionality under Cd stress. In accordance, S. sclarea plants exposed to 100 µM Cd show an increased capacity to keep quinone (QA) oxidized, thus, to have a higher fraction of open PSII reaction centers (q P ) compared to controls (Figure 5d). In other words, S. sclarea plants exposed to 100 µM Cd show a low PSII excitation pressure associated with toxicity tolerance mechanisms [92,93]. High excitation pressure defines excess energy and consequently a disproportion between energy resource and requirement [94]. This discrepancy leads to an increase in the energy transmitted from chlorophyll to oxygen, resulting in 1 O 2 generation [95]. Control S. sclarea plants that show increased excess excitation energy (EXC) at PSII (Figure 6a), show also increased 1 O 2 creation via 3 chl*, compared to plants exposed to Cd for 2 days (Figure 5c). In contrast to 1 O 2 generation that decreased under 2 and 5 days Cd exposure (Figure 5c), H 2 O 2 production after 5 days Cd exposure increased compared to control (Figure 11a), being detected mainly in the leaf midveins near the basal leaf area (Figure 12), while after 8 days exposure, increased more ( Figure 11a) and was noticed to spread to the whole leaf ( Figure 12). Thus, since ROS are formed by energy transfer ( 1 O 2 ) and electron transport (H 2 O 2 ) simultaneously, it appears likely that their action interferes with the signaling pathways sometimes to antagonize each other. It has been frequently shown that hydrogen peroxide disperses through leaf veins to act as a long-distance molecule, triggering the stress defence response in plants [20,25,27,83,89].
A basal level of ROS is needed for optimal growth (control) [20,25], with a low increased level of ROS to be beneficial for triggering defense responses (5 days Cd exposure), and a high level of ROS (8 days Cd exposure) to be out of the boundaries and harmful to plants [20,34]. Photosystem II responses to short time Cd exposure of S. sclarea can be described as a hormetic response (Figure 14), representing an "over-compensation" response to a disruption in homeostasis [42]. Despite the significant levels of Cd in leaves, a higher fraction of absorbed light energy was directed to photochemistry (ΦPSΙΙ) under 2-and 5-days Cd stress, with a concomitant increase in ΦNPQ that resulted in a significant decrease in ΦNO ( Figure 5). The non-regulated energy loss in PSII (ΦNO) encompasses internal conversions and intersystem crossing, which results in singlet oxygen ( 1 O2) creation via the triplet state of chlorophyll ( 3 chl*) [29,[81][82][83]. To optimize photosynthesis and growth under stressful conditions, plants have evolved a variety of mechanisms against photodamage and photoinhibition [84,85]. Non-photochemical quenching is the key photoprotective process that dissipates excess light energy as heat and protects photosynthesis [81,[86][87][88][89][90]. Thus, the increased non-photochemical quenching of photosynthesis (NPQ) altered ROS homeostasis through a decreased 1 O2 formation. Consequently, in S. sclarea plants exposed to 100 μM Cd, ROS homeostasis could be regulated by NPQ in such a way so that plants can cope with Cd stress [19,22,26,27].
The potential PSII efficiency of S. sclarea plants exposed to 100 μM Cd estimated by the maximum efficiency of PSII photochemistry (Fv/Fm) (Figure 4a) and the efficiency of the water-splitting complex on the donor side of PSII (Fv/Fo) (Figure 4b) [29,91] indicated an enhanced PSII functionality under Cd stress. In accordance, S. sclarea plants exposed to 100 μM Cd show an increased capacity to keep quinone (QA) oxidized, thus, to have a higher fraction of open PSII reaction centers (qP) compared to controls (Figure 5d). In other words, S. sclarea plants exposed to 100 μM Cd show a low PSII excitation pressure associated with toxicity tolerance mechanisms [92,93]. High excitation pressure defines excess energy and consequently a disproportion between energy resource and requirement [94]. This discrepancy leads to an increase in the energy transmitted from chlorophyll to oxygen, resulting in 1 O2 generation [95]. Control S. sclarea plants that show increased excess excitation energy (EXC) at PSII (Figure 6a), show also increased 1 O2 creation via 3 chl*, compared to plants exposed to Cd for 2 days (Figure 5c). In contrast to 1 O2 generation that decreased under 2 and 5 days Cd exposure (Figure 5c), H2O2 production after 5 days Cd exposure increased compared to control (Figure 11a), being detected mainly in the leaf midveins near the basal leaf area (Figure 12), while after 8 days exposure, increased more ( Figure 11a) and was noticed to spread to the whole leaf ( Figure 12). Thus, since ROS are formed by energy transfer ( 1 O2) and electron transport (H2O2) simultaneously, it appears likely that their action interferes with the signaling pathways sometimes to antagonize each other. It has been frequently shown that hydrogen peroxide disperses through leaf veins to act as a long-distance molecule, triggering the stress defence response in plants [20,25,27,83,89].
A basal level of ROS is needed for optimal growth (control) [20,25], with a low increased level of ROS to be beneficial for triggering defense responses (5 days Cd exposure), and a high level of ROS (8 days Cd exposure) to be out of the boundaries and harmful to plants [20,34]. Photosystem II responses to short time Cd exposure of S. sclarea can be described as a hormetic response (Figure 14), representing an "over-compensation" response to a disruption in homeostasis [42].

Figure 14.
Overview of the hormetic response of photosystem II photochemistry to Cd exposure. Hormesis [96] is defined as the stimulatory effect of short exposure times of toxic constituents, e.g., Cd on a biological factor (photosystem II photochemistry), of a particular organism (S. sclarea). The hormetic effect is defined by an inverse U-shaped biphasic curve [8,34] in which short exposure time has a stimulatory effect; however, at longer exposure time, a toxic effect is evident.
Although excess Cd accumulation is detrimental to plants, different strategies of Cd tolerance and accumulation are adopted by plants [97]. Tang et al. [98] described a stimulation of plant growth, increase of photosynthesis, and an up-regulation of the related genes in Sedum alfredii exposed to 5 µM Cd. Similar results with stimulation of growth when Noccea caerulescens was exposed to 100 µM Cd were reported by Lombi et al. [99]. A stimulatory effect of Cd on the photosynthetic apparatus of Arabidopsis halleri was also described recently [33]. Małkowski et al. [36] reported a stimulation of the photosynthetic rate by Cd only at low concentrations, whereas at higher Cd concentrations, there was a significant decrease compared to controls. Nevertheless, other studies have shown a dose dependent negative impact of Cd that increases with the generation of ROS and oxidative damage and the inhibition of photosynthetic rate to follow [5,[100][101][102][103][104][105][106]. On the other hand, protection to stress through ROS production [25,107] has been shown that it can be regulated by NPQ in such a way so that plants can cope with stress [22,26,27].
A negative impact of Cd on photosynthesis has been assigned to decreases in chlorophylls; ascribed to Cd-induced damage in chloroplasts' ultrastructure [108,109]. However, in our experiment, the significant chlorophyll content decreases in S. sclarea leaves exposed to Cd for 2 and 5 days (Figure 3a) cannot be attributed to chloroplasts' ultrastructure destruction but rather to the significantly decreased Mg uptake (Figure 2f). However, Mg content in the leaves after 5 days Cd exposure (2778 ± 83 µg g −1 ) remained higher than the adequate range limit (2000 µg g −1 ) [110].
Cadmium has been reported to alter chloroplast ultrastructure, reduce photosynthesis [68,106,111], and inactivate enzymes involved in CO 2 fixation [60]. Ultrastructural changes that are observed in Cd exposed leaves of sensitive plants (thylakoid dismantling, increase of lipid droplets, etc.) [112][113][114][115] are similar to those occurring at leaf ageing [116]. In Cd tolerant species, the only ultrastructural alteration observed in leaves was a reduction of starch grains in chloroplasts [50,102,117], which may be due to disorders in the photoassimilate transport or to nutrient deficiency [50,118], a phenomenon also observed after 8 days exposure to Cd ( Figure S1). In S. sclarea Cd-treated plants, an increase in deposited electron-dense material was observed, as reported also by Mizushima et al. [50], but no other noticeable alteration in 2-and 5-days Cd-treated plastids was detected, further consolidating the chlorophyll fluorescence imaging results. Thus, a hormetic response of PSII photochemistry to short term Cd exposure was observed, indicating an "over-compensation" response to Cd disruption in homeostasis, justifying the statement of Carvalho et al. [8] that Cd can be regarded from a toxic element, a beneficial one. Hormesis research data and data on priming (preconditioning), an expression of hormesis [16,34,[119][120][121], indicate that stimulatory response detection of the low-dose or short-time exposure is highly dependent on the study strategy, including dose range and the number with duration exposure and endpoint selected [13,14,16,34,52].
Exposure of S. sclarea plants to a combination of Cd and high light (900 µmol photons m −2 s -1 ) resulted in an inhibition of PSII functionality (Figures 7 and 9), while the increased NPQ (Figure 8a) was inefficient to keep the same number of open reaction centers PSII (q P , Figure 8c) compared to control plants. Dissipation of excess light energy as heat (NPQ) under environmental pressure conditions is effective only if it is regulated so as to maintain the same fraction of open reaction centers as in unstressed conditions [88,89,[122][123][124][125], as was observed under low light exposure of S. sclarea plants to Cd, with even an increased fraction of open reaction centers to occur (Figure 5d). Thus, the combination of Cd and high light points out to Cd toxicity. The same conclusion is reached [126] at longer duration exposure (8 days) to Cd at LL, with an inhibition of PSII functionality to be observed ( Figure 10).

Plant Material and Growth Conditions
Seeds of Salvia sclarea L. used for the experiments were collected from the Rose Valley (Karlovo, Bulgaria). After germination on soil in a growth room for about a month, the seedlings were transferred to pots containing continuously aerated modified Hoagland nutrient solution (described in detail before) [6]. The nutrient solution was adjusted to pH 6.0 and changed every 3 days. The growth room conditions were 24 ± 1/20 ± 1 • C day/night temperature, 14/10 h day/night photoperiod with photon flux density 200 ± 20 µmol photons m −2 s −1 .

Cadmium Treatment
Two-month-old S. sclarea plants in the hydroponic culture experiments were subjected to 0 or 100 µM Cd (as 3CdSO 4 8H 2 O) for a period up to five days. The pots containing only Hoagland nutrient solution served as the control, while all solutions were renewed every two days.

Determination of Elemental Concentration by Inductively Coupled Plasma Mass Spectrometry (ICP-MS)
After 5 days treatment with 0 (control) or 100 µM Cd, Salvia plants were harvested, separated in roots and aboveground (shoots-leaves) tissues, washed three times in deionized water, and then dried at 65 • C to constant biomass, milled and finally sieved. Dried sieved samples of 0.3 g were transferred in 10 mL quartz vessels with 65% (v/v) nitric acid (Suprapur, Merck, Darmstadt, Germany) and 30% (v/v) hydrogen peroxide (Suprapur, Merck, Darmstadt, Germany) in 3:1 ratio. Digestion was carried out in the microwave assisted digestion system Ethos One (Milestone Srl, Sorisole, BG, Italy). The process run out in 3 stages: ramp time-20 min to reach 200 • C and 1500 W; hold time-30 min at 200 • C and 1500 W; cooling-30 min. The next step was the quantitative transfer of digested samples into polypropylene tubes and dilution with demineralized water (Direct-Q 3 UV, Merck, Darmstadt, Germany). All prepared samples were diluted immediately prior to inductively coupled plasma mass spectrometer (ICP-MS) analysis. Samples were analyzed in an ICP-MS model ELAN DRC II (PerkinElmer Sciex, Toronto, Canada) [127]. ICP-MS operational conditions, instrumental settings calibration solutions, data validation, and validation parameters are given in Appendix A. Elemental analysis was performed for Cd, Cu, Ca, Mg, Mn, Fe, and Zn.

Measurements of Chlorophyll a and Chlorophyll b Content
Chlorophyll a (Chla) and chlorophyll b (Chlb) content was determined according to Lichtenthaler [128]. Leaf tissue (50 mg) was homogenized with 10 mL ice-cold 80% (v/v) acetone and centrifuged at 5000× g for 5 min at 4 • C. The absorbance of the supernatant was measured at 646. 8 [128]. The mean values were averaged from three independent treatments with 2 repetitions for each treatment and are presented as mg g −1 FW.

Chlorophyll Fluorescence Imaging Analysis
Chlorophyll fluorescence measurements were conducted on dark adapted (20 min) leaves of S. sclarea plants, treated for 2 and 5 days with 0 (control) or 100 µM Cd, using an Imaging PAM M-Series system (Heinz Walz Instruments, Effeltrich, Germany) as described in detail previously [129]. Two light intensities were used for measurements of photosynthetic efficiency of S. sclarea leaves, a LL, similar to the growth light (220 µmol photons m −2 s −1 ), and a HL (900 µmol photons m −2 s −1 ). In each leaf, representative areas of interest (AOIs) were selected so as to have measurements of the whole leaf area. The definitions of the five main chlorophyll fluorescence parameters (Fo, Fm, Fo , Fm , and Fs) measured by the Imaging PAM M-Series system are presented in Table S1, while a typical modulated fluorescence trace showing how the main five parameters are formed is presented in Figure S2. The chlorophyll fluorescence parameters calculated from the five main parameters with their definitions are described in Table 1. Representative results are also shown as color-coded images of Fv/Fm after dark adaptation and of Φ PSII and Φ NO , after 5 min illumination with 220 µmol photons m -2 s -1 (LL) or/and 900 µmol photons m -2 s -1 (HL).

Determination of Oxidative Damage
Leaf samples were frozen in liquid nitrogen and stored at −80 • C for analysis of hydrogen peroxide (H 2 O 2 ) and malondialdehyde (MDA) content. The level of lipid peroxidation in S. sclarea leaves of control, and 5-and 8-days Cd-treated plants was measured as malondialdehyde (MDA) content determined by the reaction with 2-thiobarbituric acid (TBA), according to the method of Hodges et al. [130]. Hydrogen peroxide (H 2 O 2 ) was extracted by homogenization with 50 mM K-phosphate buffer pH (6.5) and determined as described by Hossain et al. [131] after reaction with 0.1% TiCl 4 in 20% H 2 SO 4 .
The histochemically detection of H 2 O 2 in leaves was performed as described by Daudi and O'Brien [132] by staining with 1% 3,3 -diaminobenzidine (DAB) solution. DAB is oxidized by H 2 O 2 in the presence of some heme-containing proteins to generate a dark brown precipitate. This precipitate is exploited as a stain to detect the presence and distribution of hydrogen peroxide in plant tissues.

Leaf Ultrastructure Observations by Transmission Electron Microscopy
In order to study leaf ultrastructure alterations after 2 and 5 days of Cd treatment, leaves from both Cd-treated and untreated plants were excised and segmented with a razor blade into small pieces of 0.5 × 1 mm. Leaf segments were fixed with 2% paraformaldehyde plus 4% glutaraldehyde, in 0.05 M sodium cacodylate buffer, pH 7.0 solution [129]. After a 5 h fixation at room temperature, the samples were washed with a 0.05 M sodium cacodylate buffer and post-fixed for another 3 h in a similarly buffered 2% osmium tetroxide solution (Agar Scientific, Essex, UK). Afterwards, samples were dehydrated in an acetone series, treated with propylene oxide, and embedded in Durcupan ACM resin (Fluka Chemie AG, Buchs, Switzerland). Ultrathin sections (80-90 nm) were cut in a ULTROTOME III TYPE 8801A ultramicrotome (LKB, Stockholm, Sweden), equipped with a glass knife, collected on nickel grids. The sections were stained with 2% uranyl acetate and 1% lead citrate and examined in a JEOL JEM 1011 (JEOL, Tokyo, Japan) TEM, equipped with a Gatan ES500W (Gatan, Pleasanton, CA, USA) digital camera. Digital electron micrographs were obtained with the DigitalMigrograph 3.11.2 (Gatan, Pleasanton, CA, USA) software according to the manufacturer's instructions.

Statistical Analyses
Mean values were calculated from three independent treatments (biological replicates). Statistically significant differences among the means were determined using one-way analysis of variance or two-way ANOVA. Means (±SD) were considered statistically different at a level of p < 0.05.

Conclusions
Although surplus Cd accumulation is detrimental to most plants, different strategies of Cd tolerance and accumulation are adopted by different plant species [97]. When clary sage was exposed to Cd for a short time, tolerance mechanisms were triggered, with PSII photochemistry to be enhanced, without any defects to chloroplasts, as observed by transmission electron microscopy ( Figure 13). However, exposure to a combination of Cd and high light (Figures 7 and 9), or longer duration exposure to Cd alone (8 days), resulted in an inhibition of PSII functionality ( Figure 10) and [126], pointing out to Cd toxicity. Thus, an activation of PSII function at short time exposures and an inhibition at longer duration suggests a hormetic response (Figure 14), and describes these effects in terms of "adaptive response" and "toxicity", respectively.