*3.1. Effect of Tl on the Growth, Accumulation of Mineral Elements, and Soluble Amino Acid and Protein Content of Dittrichia*

The growth of *Dittrichia* is very similar at all of the Tl concentrations, with little difference between them (Figure 1A, Table 1). On the contrary, the growth that can be observed in the control plants, with 0 μM Tl, is much greater. The plants that were grown under Tl toxicity show a significantly smaller size than the control plants in both the shoot and the root parts (Table 1); furthermore, the leaves appear chlorotic, especially in the venal area and, to a lesser extent, in the interveinal area. The length of the shoots decreases drastically with the Tl concentration to values of 38% and 32% for 10 μM and 100 μM, respectively. The effect of the three concentrations of Tl on the growth of the roots is similar, with an inhibition of 48% with respect to the length of the control roots.

**Table 1.** Effect of Tl on the length, fresh weight (FW), dry weight (DW), and fresh weight percentage (%DW) of the *D. viscosa* plants. The data are the means ± SE from ten independent experiments ± SE, each carried out in triplicate. The different letters indicate significant differences at *p* < 0.05.


The fresh weight of both the shoots and the roots decreases in response to the Tl dose. This weight decreases significantly in the shoots as the Tl concentration increases (73%, 77%, and 82% for 10 μM, 50 μM, and 100 μM Tl, respectively). On the contrary, in the roots, the fresh weight is similar in the different concentrations of Tl that were used, being 75% of the control values. The dry weight decreases in both the shoots and the roots, depending on the concentration of Tl in the medium. The DW/FW percentage increases in the plants that were subjected to Tl toxicity, with the greatest value in the shoots being observed at 100 μM Tl. In the roots, an increase in this percentage can be observed due to the effect of Tl, although this increase is less when increasing the Tl. The RWC (Table S1) shows no alteration in the roots, while it decreases very slightly in the shoots (≈9%).

The absorption and the accumulation of Tl increases with the concentration of Tl in the medium (Table 2). In the roots, the Tl content is positively correlated with that of the medium, but not in leaves, where the accumulation of Tl is similar for 50 μM and 100 μM. The accumulation observed here is much greater in the roots than in the shoots. Thus, in the treatment with 10 μM Tl, the accumulation in the roots is 6× that produced in the leaves, with this accumulation being of greater magnitude with the higher Tl concentrations. This greater capacity for accumulation in the roots is reflected in a higher BF (i.e., BFroots > 350, BFshoots < 100). On the other hand, the value of TFTl is low, decreasing progressively with the increase in Tl in the medium. As the concentration of Tl in the roots increases, its transport capacity decreases (from 0.163 to 0.043 for 10 μM and 100 μM Tl, respectively).

**Table 2.** Tl content in roots and leaves, bioaccumulation factor (BF), and translocation factor (TF) in *D. viscosa* plants. The data are the means ± SE from 10 independent experiments, each carried out in triplicate. The different letters indicate significant differences at *p* < 0.05. nd = not detect.


Table 3 shows the effect of Tl toxicity on the absorption and the accumulation of different macroelements (K, Mg, and Ca) and microelements (Fe, Mn, Cu, and Zn). The K content in the root and shoot samples shows a decreasing trend as the Tl concentration increases, and this decrease is greater in the roots than in the leaves. However, there is a wide variability in the measured data for this microelement. With regard to the value of TFK, it is similar to the control value, except with 10 μM Tl, in which a slight decrease can be observed (Table S2). Similarly to K, the Mg content (Table 3) decreases due to Tl independently of the concentration, and this is more pronounced in the roots than in the shoots. The TFMg values increase (×2) with respect to the control, although they are similar for the three Tl concentrations. The lowest Ca content in the roots is observed in the plants that were grown in 10 μM of Tl, with a decrease of 52%, relative to the control. However, in the roots of the plants that were grown with 50 μM Tl, the decrease is 20%, and with 100 μM Tl, the amount of this element increases (by 28%, relative to the control). In the shoots, the Ca content is not significantly modified.

**Table 3.** Effect of Tl on the K, Mg, Ca, Fe, Mn, Cu, and Zn contents in roots and shoots of *D. viscosa* plants. The data are the means ± SE from ten independent experiments, each carried out in triplicate. The different letters indicate significant differences at *p* < 0.05.


With regard to the absorption and the accumulation of Fe, Mn, Cu, and Zn (Table 3), in the roots there is an increase in the content of Mn, Cu, and Zn, while the content of Fe decreases. While Cu and Zn increase with an increasing Tl dose, the increase in Mn is similar for all of them. The decrease in the Fe content is smaller in response to an increase in the concentration of Tl in the medium (54%, 45%, and 24% for 10 μM, 50 μM, and 100 μM Tl, respectively). In the leaves, Fe decreases with the increase in Tl in the medium and Cu decreases by 30% (independently of the Tl concentration), while Mn and Zn increase. With regard to the values of TF (Table S2), the TFFe increases at 10 μM Tl, but is similar to the control value for the other concentrations. The TFMn increases, while TFCu and TFZn decrease.

Tl induces an increase in the amino acid content, which is greater in the leaves (≈×4.3) than in the roots (≈×1.8) (Table 4). This strong increase does not depend on the dose of Tl that is used. With regard to the protein content (Table 4), in the roots that were subjected to 10 μM of Tl toxicity, there is little difference from the control, with a slight increase being observed; however, with 50 μM and 100 μM, a progressive decrease can be observed, being

by 16% and 41%, respectively. In the leaves, there is a decrease, which is greatest with 10 μM Tl (by 43%, relative to the control).

**Table 4.** Effect of Tl on the soluble amino acid and protein contents in *D. viscosa* plants. The data are the means ± SE from ten independent experiments, each carried out in triplicate. The different letters indicate significant differences at *p* < 0.05.


#### *3.2. Effect of Tl on Photosynthetic Pigment Content and Photosynthetic Efficiency*

Tl toxicity induces a decrease in both chlorophyll a and b, and, consequently, in total chlorophyll (Figure 1A–C). The decrease in these contents is smaller and very similar in 10 μM and 50 μM, with values of 54% and 63% of the control, respectively. For 100 μM Tl, this content is 45% of the control. The chlorophyll a/b ratio (Figure 1D) increases in response to Tl, going from 1.60 to 1.92 for the control leaves and the leaves that were subjected to Tl, respectively. In contrast, the carotenoid content (Figure 1E) shows a clear increase (by around 140%) in the plants that were subjected to Tl, although with no difference between the different concentrations that were used. The decrease in chlorophylls and the increase in carotenoids caused by Tl translate into an increase in the carotenoids/chlorophylls ratio (Figure 2F).

In the Tl treatments, the F0 value increases as the Tl concentration increases (Figure 1G). Thus, the F0 increases by 10% for 10 μM Tl, while with 50 μM and 100 μM Tl the increases are much greater, at 65% and 71%, respectively. The efficiency of photosystem II (Fv/Fm) (Figure 1H) is also strongly affected by the Tl doses, with the decreases being dependent on the increase in Tl, ranging from 10% to 36% for 10 μm and 100 μM Tl, respectively. With regard to the Fv/F0 ratio (Figure 1H), there is a marked decrease with the increase in the Tl concentration that was used. Thus, while in the control plants the Fv/F0 ratio is 3.604, in those that were treated with 100 μM this ratio is 1.026.

#### *3.3. Lipid Peroxidation, Production of O2 .*<sup>−</sup> *and SOD Activity, and H2O2, NO, and H2S Content*

Figure 2 shows the response to the treatments with Tl at the level of lipid peroxidation production of O2 .<sup>−</sup> and SOD activity, and H2O2, NO, and H2S content. In the roots, lipid peroxidation increases in response to Tl (Figure 2A). On the contrary, in the leaves, the levels of lipid peroxidation are lower, with similar values for the three concentrations of Tl that were used. The production of O2 .<sup>−</sup> (Figure 2B) also differs between the roots and the leaves, being greater in the former. In the roots, the differences between the treatments are slight and varying, with decreases of 18% and 23% for 10 μM and 100 μM Tl, respectively, but with no significant change for 50 μM Tl. In the leaves however, the O2 .− production increases with increasing Tl concentrations in the medium. The SOD activity, in response to Tl (Figure 2C), increases in both the roots and the leaves, with the latter presenting the greatest values. In both of the organs, the SOD activity increases for all of the concentrations of Tl. For the treatment with 10 μM Tl, the SOD activity increases by ×1.8 and ×1.4 for the roots and the leaves, respectively, and for 50 μM Tl, by ×2.3 and ×1.8, respectively. With 100 μM of Tl, the SOD activity also increases, although at lower values (×1.6 and ×1.4, in the roots and the leaves, respectively). In both of these organs, the lowest and highest Tl concentrations cause similar increases in SOD activity. The H2O2 content (Figure 2D) increases in both the roots and the leaves in response to Tl-induced stress. The H2O2 content in the roots increases from 125 μmol g –1 FW in the controls to values that reach 844 μmol g−<sup>1</sup> FW for 100 μM Tl and shows a strong positive correlation

with the Tl concentration. The relative increases are ×2.5, ×4.8, and ×6.7 for 10 μM, 50 μM, and 100 μM Tl, respectively. A similar response is seen in the leaves, although in this case the levels of H2O2 stabilize at 50 μM Tl, with values for 50 μM and 100 μM being similar. The production of NO (Figure 2E) is much greater in the roots than in the leaves, and it increases in response to the increase in Tl treatments. Thus, 10 μM of Tl induces a ×1.8 increase, with the increases for the two greater concentrations being very similar to each other (×2.5 and ×2.6, respectively). In the leaves, there are also increases in response to Tl toxicity, by very similar factors (≈×1.25) for all of the concentrations. With regard to the production of H2S (Figure 2F), the levels are higher in the leaves than in the roots. In the roots, the greatest increase in H2S content corresponds to the treatment with 10 μM Tl (×1.8). The greater Tl concentrations induce only slight changes in the H2S content, with values that are very similar to the control. In the leaves, there is increased H2S content for all of the concentrations, with the maximum value corresponding to the 100 μM Tl treatment (×3.7, relative to the control).

**Figure 1.** Effect of Tl on photosynthetic pigments and parameters in leaves of *D. viscosa*. Chlorophyll a content (**A**), chlorophyll b content (**B**), total chlorophylls (**C**), chlorophyll a/b (**D**), carotenoids content (**E**), carotenoids/total chlorophylls (**F**), Fo (**G**), and Fv/Fm and Fv/Fo (**H**). The data are the means ± SE from ten independent experiments. The different letters indicate significant differences at *p* < 0.05.

**Figure 2.** Effect of Tl on the lipid peroxidation (**A**), O2. – production (**B**), SOD activity (**C**), and H2O2 (**D**), NO (**E**), and H2S (**F**) content in roots (orange) and leaves (green) of *D. viscosa*. The data are the means ± SE from ten independent experiments. The different letters indicate significant differences at *p* < 0.05.

#### *3.4. APX, DHAR, MDHAR, and GR, and Ascorbate and Glutathione Pool*

The effect that Tl has on the enzymes of the AsA–GSH cycle was determined (Figure 3). APX, DHAR, MDHAR, and GR all present behavior that differs between the roots and the leaves. In the roots, the APX activity (Figure 3A) decreases after the stress that is induced by 10 μM Tl but is unaltered by 50 μM or 100 μM Tl. On the contrary, in the leaves, the APX activity increases at the higher Tl concentrations, but is unaffected by 10 μM Tl. The DHAR activity in the roots increases strongly with 10 μM Tl, but the greater concentrations show less activation (Figure 3B). In the leaves, DHAR increases by very similar factors (≈×2.0, relative to the control) in response to all of the Tl concentrations. The MDHAR activity (Figure 3C) also differs in behavior between the roots and the leaves; while in the roots there are slight decreases in this activity at 10 μM, in the leaves there are increases (by between ×2.8 and ×3.7) for all of the Tl concentrations, as was the case for the DHAR activity. The GR activity (Figure 3D) increases in the roots, but this increase is less the greater the Tl concentration (1.8- and 5.4-fold for 10 μM and 100 μM Tl, respectively). In the leaves, the GR activity decreases with 10 μM and 50 μM Tl but is not significantly altered with 100 μM Tl.

**Figure 3.** Effect of Tl on the APX (**A**), DHAR (**B**), MDHAR (**C**), and GR (**D**) activities in roots (orange) and leaves (green) of *D. viscosa*. The data are the means ± SE from ten independent experiments. The different letters indicate significant differences at *p* < 0.05.

The AsA and DHA contents are altered by Tl (Figure 4A,B). Thus, in the roots, the control AsA content is 121 nmol g <sup>−</sup><sup>1</sup> FW, and Tl duplicates (×2.0) this content for 10 <sup>μ</sup><sup>M</sup> and 50 μM Tl, while 100 μM does not alter from the control value. On the contrary, the DHA content decreases by 14–48%, relative to the control, depending on the Tl concentration. In the leaves, the result is very similar, although the contents of both AsA and DHA are greater than those in the roots. The increase in AsA content is 2.23-fold in the presence of 10 μM Tl, and 1.63- and 1.49-fold for 50 μM and 100 μM Tl, respectively. The DHA content decreases for all of the concentrations. The total ascorbate pool (AsA + DHA) (Figure 4C) is unaltered, except with 100 μM Tl, which reduces this pool in both of the organs. The redox state (AsA/DHA) (Figure 4D) is altered, increasing in the roots and the leaves, in response to the Tl concentrations. With regard to the GSH content (Figure 4E), in the roots there is an increase only in those that were subjected to 100 μM Tl, but not for the other concentrations. The GSSG content (Figure 4F) decreases for the two lower concentrations, but not for 100 μM Tl, which produces levels that are similar those of to the control. In the leaves, both the GSH and the GSSG content (Figure 4G) decrease with the Tl concentration, with very similar values for 50 μM and 100 μM Tl. With regard to the glutathione pool, in the roots that were subjected to 10 μM and 50 μM Tl, there is a reduction of 35% of the total content, relative to the control. On the contrary, 100 μM causes a 23% increase (1.23-fold) in the AsA + DHA pool. Whereas, in the leaves, this pool decreases for all of the concentrations of Tl that were used. The GSH/GSSG ratio (Figure 4H) increases in the roots from 1.32 in the control to 4.06, 6.02, and 2.14 for 10 μM, 50 μM, and 100 μM, respectively. In the leaves, this ratio presents a different behavior, decreasing with Tl.

**Figure 4.** Effect of Tl on the AsA (**A**), DHA (**B**), and ascorbate pool (AsA + DHA) (**C**) contents; the AsA/DHA ratio (**D**), the GSH (**E**), GSSG (**F**), and glutathione pool (GSH + GSSG) (**G**) contents; and the GSH/GSSG ratio (H), in *D. viscosa*. The data are the means ± SE from ten independent experiments. The different letters indicate significant differences at *p* < 0.05.

#### **4. Discussion**

In *Dittrichia viscosa*, the concentrations of Tl that were used here cause a decrease in growth and biomass production (Figure S1, Tables 1 and S1), with these results being similar to those that have been observed in other plants [45,47,48,80–82]. The effect of Tl on the dry matter content and RWC is not significant. Only in the shoots is a slight decrease in RWC observed, a result that is very similar to that described by Radi´c et al. [49]. This contrasts with the decreases in RWC that has been observed in plants stressed by Cd [83–85].

The Tl that is absorbed accumulates mostly in the roots [48,49,86,87], with high values of BFTl, and decreases in TFTl with an increasing dose of Tl in the medium (Table 2). This preferential accumulation in the roots may obviate more serious damage to photosynthetic pigments [49].

Tl toxicity produces alterations in the absorption and accumulation of other elements (Table 3). There is a tendency to decrease the K content, although this is not significant. This variability may be due to the great genetic variability that is evidenced in *Dittrichia* [61,62]. Thus, the decline in K content is similar to that described by Buendia-Valverde et al. [88] for *Capsicum annuum* leaves, with decreases in the content at a low Tl concentration and a recovery of control values at a greater concentration. The Tl affects K uptake by the roots, but not its mobility within the plant, with TFK (Table S2) being unaltered and the lower shoot content being due to the decrease in the root concentration rather than any reduced translocation. This effect of Tl on K uptake is a result of their mutual antagonism, since Tl uses K transport systems [34]. Chang et al. [45] also describe the antagonistic effect of Tl and K in *Arabidopsis,* in which Tl interferes with the K uptake. In *Sinapis alba* that are grown in hydroponic culture, the effect of Tl is to reduce the K content in the roots, as well as in the stems and leaves [50]. With regard to Mg, the Tl inhibits its absorption in *Dittrichia*, decreasing the content in the roots. In the leaves, the decrease in the Mg content is less, since there is a significant increase in TFMg. However, despite the increase in TFMg and the lower effect that is observed on the Mg content in the leaves, this does not prevent the decrease in the chlorophyll content. In contrast, in *S. alba*, Holubick et al. [50] describe low Tl concentrations leading to a decrease in Mg in shoots but not in roots, whereas, in *C. annuum,* the changes in the Mg content depend on the variety [88]. With regard to the Ca content, the data show that there is a decrease in its accumulation in the roots, except for those with the highest concentration that was used. The content of this element in the leaves is unaffected, however. Once again, the effects of Tl on the absorption and accumulation of Ca are diverse, depending, as in the case of Mg, on the variety [88] and the accumulating organ [50]. The microelements Fe, Mn, Cu, and Zn behave in different ways. While Fe decreases in both the roots and the leaves [50], Mn and Zn increase in both of these organs, and Cu only increases in the roots but decreases in the leaves. The inhibited growth that is induced by Tl stress may be due to this alteration in the absorption of essential mineral nutrients [89]. Our results show decreases in the absorption of K, Mg, and Fe, and alterations in the absorption and the accumulation of other essential elements, such as Ca, Mn, Cu, and Zn, which have increased presence in both the roots and the leaves, except for Cu, which is below the control levels in the leaves (Tables 3 and S2).

The increase in amino acid content in response to Tl toxicity could be associated with the observed decrease in the protein content, as well as with an accumulation of amino acids in response to this toxicity [90–93]. The increase in the soluble amino acid content and the decrease in the protein content show a clear effect of Tl toxicity (Table 4). Similar results have been described in different plants that were subjected to toxicity, not only of Tl [81], but also of other heavy metals, such as Cu, Cd, and Ni [94–96]. The decrease in protein content could be due to the effect of oxidative damage on protein synthesis, to alterations at the DNA level that would affect protein synthesis, or to the inhibition of the absorption of other elements, such as Mg and K [94]. It could also be due to increases in protease activity [97]. The increase in amino acid accumulation could be due to protein turnover increasing proteolysis, as well as due to the de novo synthesis of osmoprotectant amino acids, which is usually the case for stress-induced proline accumulation [90,91,93]. The amino acids can act on osmoregulation, heavy metal detoxification, and the formation of polyamines and glutathione, secondary metabolites, or the organic nitrogen reserve [92].

The decrease in the chlorophyll content that was observed in *Dittrichia* in response to stress that was induced by Tl toxicity (Figure 1A–D) coincides with results that have been described by other authors, but not the increase that has been observed in the carotenoid content (Figure 1E,F). Thus, Naumann et al. [80] in *Lemna minor* and Mazur et al. [44] in *Sinapis alba* find Tl to induce decreases in both the chlorophyll and the carotenoid content. In *Arundo donax* and *Coix lacryma-jobi*, Pu et al. [48,86] find 50 μg L−<sup>1</sup> Tl to cause a decrease in the chlorophyll content, while lower concentrations have no effect. The increase in the carotenoid content and the carotenoid/chlorophyll ratio that has been observed in *Dittrichia* could be due to the role of carotenoids as ROS scavengers, thus protecting PSII functionality from oxidative damage [11,98–101]. The chlorophyll a/chlorophyll b ratio indicates the degree of appression of the thylakoid membranes in the chloroplast, being inversely proportional to the degree of appression, which, in turn, causes the "light harvesting complexes II" (LHCII) to be more closely connected, increasing the light-gathering capacity, and, therefore, the efficiency of energy transmission. In addition, the increase in the thylakoid appression enhances the electron transfer from photosystem II to the cytochrome b6f complex, thus increasing non-cyclic photosynthetic electron transport [102]. That ratio is lower in control plants than in those that were grown in the presence of Tl, which would mean that the control plants capture light more efficiently. The interaction between Tl and the -SH groups affects the functionality of the enzyme systems that are involved in the biosynthesis and/or degradation of chlorophylls and the stability of the chloroplasts, as described for Sb toxicity [11,102].

The photosynthetic efficiency of control plants and those that were treated with 10 μM Tl are very close to the values that are described as normal by Murchie and Lawson [103]. The higher concentration Tl treatments show greater decreases, indicating an impairment of the photochemical activity. The stress that is induced by Tl toxicity causes an increase in the minimum fluorescence value (F0) (Figure 1G), reflecting an alteration in the photosynthetic apparatus. This reveals that there is excess O2 .<sup>−</sup> production in the chloroplasts, which negatively affects the PSII activity. The F0 fluorescence originates from the chlorophylls that are associated with the antenna complexes. Its increase thus implies a reduced energy transfer from LHCII to PSII, i.e., there is a disconnection of the antennas in response to stress [104,105]. The Fv/Fm ratio represents the maximum PSII quantum yield, which correlates with the net photosynthesis quantum yield. It is indicative of the level of photoinhibition affecting the PSII complexes [106]. Tl toxicity reduces Fv/Fm (Figure 1H) to different degrees depending on the Tl concentration, these results being similar to those that have been described in other plants [44,47,48,86]. Changes in the PSII activity reduce photosynthesis levels [105,107]. In addition, the ratio Fv/F0, which indicates the maximum ratio of the quantum yields of the photochemical and non-photochemical PSII processes, allows the photosynthesis capacity to be estimated. *Dittrichia viscosa* under Tl toxicity conditions show large decreases in this ratio (Figure 1H), which may indicate an interruption of donor-side PSII photosynthesis [108]. These results indicate that Tl toxicity leads to a strong alteration of photosynthetic activity, affecting PSII, as indicated by the increases in F0 and the decreases in the Fv/Fm and Fv/F0 ratios. Recently, Chang et al. [45] have described the negative effect of Tl on the photosynthetic process by decreasing the expression levels of the LCHII subunit genes, thus reducing the aggregation of the LCHII complexes, and, with them, the levels of photosynthesis.

With regard to ROS, by accumulating the Tl preferentially in the roots (Table 2), there is an increase in ROS production, which can cause the lipid peroxidation that is observed in the roots (Figure 2A). This behavior is similar to that described in *Dittrichia* plants in response to stress induced by Sb toxicity [11]. The increase in lipid peroxidation has also been described in response to Tl-induced stress [49,81]. On the contrary, Sirova and Vaculik [109] describe an increase in lipid peroxidation in leaves due to Cd toxicity, but not in roots, since a large part of the Cd is translocated to the leaves, provoking oxidative stress that would damage the membranes. Yao et al. [55] describe a decrease in lipid peroxidation in rice roots that were subjected to Tl(I) toxicity. In *Dittrichia*, TFTl decreases progressively with increasing Tl (Table 2), and this, together with the increase in SOD activity, may prevent increases in lipid peroxidation in leaves [110]. In the roots, the accumulation of Tl is very high, inducing strong increases in the production of O2 .<sup>−</sup> and H2O2, and, although the SOD activity increases, damage by lipid peroxidation occurs (Figure 2). The stress induced in *Dittrichia* by Tl leads to increases in the content of O2 .<sup>−</sup> and H2O2 and in the SOD activity (Figure 2B–D), which is a response that is similar to that described in other plants [49,81]. On the contrary, while decreases in O2 .<sup>−</sup> and H2O2 content have also been observed, the SOD activity increases [55]. However, the toxicity that is induced by Cd and As induces increases in the production of O2 .<sup>−</sup> and H2O2, as well as in SOD activity [9,23,85,111]. NO and H2S perform signaling functions, both under physiological conditions and under stress conditions, intervening at the level of nitration, nitrosation, and persulfidation [112,113]. Tl induces increases in the NO content in *Dittrichia* (Figure 2E), similar to those that have been described in response to toxicity by heavy metals such as As and Sb [10,11,16,114]. However, a decrease in the NO content in As toxicity has also been described [85,115,116]. These contradictory results could be explained by the magnitude of the stress, the plant age, the organ, or the treatment duration [116,117]. Increases in the H2S content (Figure 2F) have also been described in response to toxicity by Sb [11], Cd [9,85], and Cr [118]. The amount of H2S is greater in leaves than in roots, possibly because of the greater production of H2S in chloroplasts than in cytoplasm [119].

AsA and GSH are involved in the activation of the antioxidant systems of plants against stress [120]. In addition, they maintain cellular redox homeostasis [121]. Our results show that Tl toxicity induces alterations in APX, DHAR, MDHAR, and GR activities (Figure 3), as well as increases in AsA content and decreases in DHA, GSH, and GSSG (Figure 4). The AsA content in the roots increases, and that of DHA decreases, thus raising the AsA/DHA ratio (Figure 4A–D). The increase in AsA may be due to the increase in DHAR and GR and a decrease in the APX activity (Figure 3A,B,D), with an increase in H2O2 content. However, the MDHAR activity decreases (Figure 3C) despite it being the main enzyme responsible for AsA levels. In roots, although there is an increase in NO, the H2S levels only increase significantly with 10 μM of Tl, whereas the greater concentrations barely raise this content. In leaves, as in roots, the AsA content increases and the DHA content decreases, with consequent increases in the AsA/DHA ratio. Both DHAR and MDHAR increase, which raises the AsA content, since APX, although increasing in activity, does so to a lesser extent. The increases that are observed in the levels of NO and H2S in leaves may increase the APX activity, which is modulated by both S-nitrosation and by persulfidation [122,123]. Espinosa-Vellarino et al. [11] describe Sb toxicity in *Dittrichia* leading to increased AsA and decreased DHA in leaves, with a decrease in both in the roots. On the contrary, decreases in AsA content have been described due to the toxicity of Tl [74], Cd, and As [85,124]. Hasanuzzaman et al. [125], also with Cd toxicity, describe decreases in AsA and increases in DHA.

In the roots, the GSH content does not change with 10 or 50 μM of Tl, but increases with 100 μM Tl, while that of GSSG decreases with 10 and 50 μM and remains at the control levels with 100 μM of Tl. The GSH/GSSG ratio increases with Tl (Figure 4H), and, although there is greater GR activity, the GSH content does not increase in absolute terms, although it does so relative to GSSG. The increased DHAR might consume the GSH that is produced by the GR, or this GSH might be used for other reactions, such as the direct elimination of ROS, phytochelatin synthesis, or direct binding to Tl, which has a high affinity for -SH groups [27,34]. In leaves, GSH and GSSG decrease, as does the GSH/GSSG ratio, which could be due to increased DHAR activity and decreased GR activity. These alterations are similar to those that are described by Hasanuzzaman et al. [125], with a decrease in the content of GSH and redox pool, an increase in APX activity, decreases in DHAR and MDHAR, and variable behavior of the GR. In *Dittrichia* under Sb toxicity [11], there are similar alterations in the leaves, although in the roots, the GSH content also decreases, as well as that of GSSG. The decrease in the GSH content in the leaves contrasts with the increase that has been described in response to Zn toxicity [126]. Alterations in the AsA/DHA and GSH/GSSG ratios are fundamental as a protective mechanism of the cell against ROS [127]. In our case, Tl produces an increase in the AsA/DHA ratio (Figure 4D) in both the roots and the leaves, and in the GSH/GSSG ratio (Figure 4H) in the roots, but with this ratio remaining constant, or slightly lowered, in the leaves. Tl induces an alteration of the cellular redox state. These results show the great capacity of *Dittrichia* to

activate antioxidant defense systems, from amino acids, carotenoids, antioxidant enzymes, and AsA, maintaining a reduced state of the main antioxidants, especially in the leaves, in response to Tl stress.

#### **5. Conclusions**

In *Dittrichia*, the stress induced by Tl led to a major reduction in plant biomass, with reductions in the size and FW, altered the DW/FW ratio, but hardly affected RWC, strongly increased the amount of soluble amino acids, and decreased that of proteins. At the level of nutrient absorption and accumulation, it induced a major decrease in K, Mg, and Fe contents in both the roots and the leaves. On the contrary, Ca, Mn, and Zn accumulated to a greater extent in both of the organs. While chlorophylls decreased, carotenoids increased, possibly to act as a protection system by reducing the photosynthetic efficiency. With regard to stress markers, an increase in lipid peroxidation was observed, especially in the roots. The production of O2 .−, H2O2, and NO was greater in the roots than in the leaves, increasing in both of the organs in response to Tl toxicity, except for O2 .<sup>−</sup> production in roots, which presented fluctuations. There was also greater H2S production, especially in the leaves. The activities of SOD, APX, DHAR, MDHAR, and GR increased, except for APX in the roots and GR in the leaves. The components of the AsA–GSH cycle were also affected, with AsA increasing while DHA, GSH, and GSSG decreased. These Tl toxicity-induced alterations modified the AsA/DHA and GSH/GSSG redox status. The effects of Tl could be related to its high -SH group binding affinity, altering the functionality of proteins and the cellular redox state.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/ 10.3390/antiox12030678/s1, Figure S1: *Dittrichia viscosa* plants subjected to Tl toxicity for 7 days; Table S1: Effect of Tl toxicity on the relative water content (RWC) in roots and leaves of *D. viscosa* plants; Table S2: Effect of Tl toxicity on the translocation factor of K (TFK), Mg (TFMg), Ca (TFCa), Fe (TFFe), Mn (TFMn), Cu (TFCu), and (TFZn) of *D. viscosa* plants.

**Author Contributions:** All authors contributed to the manuscript. Conceptualization: F.E. and I.G. Funding acquisition: F.E. and I.G. Data curation, investigation, validation, and writing—original draft: F.E., A.O., F.L.E.-V. and I.G. Writing—review and editing: F.E. and I.G. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was made possible thanks to the Junta de Extremadura for the support given to the Research Group FBCMP (GR21112) and for the research project IB18074.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this article will be made available without any reservation.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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