**1. Introduction**

Plants' response to stress includes a sharp increase in reactive oxygen, nitrogen, and sulfur species (ROS, RNS, and RSS, respectively) production, which alters the cellular redox balance [1–4]. The damage that is induced by this alteration, and by the excess ROS, is dealt with by the enzymatic and non-enzymatic antioxidant systems [5–7]. The former includes superoxide dismutase (SOD, EC 1.15.1.1), peroxidases (POD, EC 1.11.1.7), catalases (CAT, EC 1.11.1.6), ascorbate peroxidase (APX, EC 1.11.1.11), glutathione reductase (GR, EC 1.6.4.2), monodehydroascorbate reductase (MDHAR, EC 1.6.5.4), and dehydroascorbate reductase (DHAR, EC 1.6.4.2), and the latter includes ascorbate (AsA), reduced glutathione (GSH), phenolics, alkaloids, non-protein amino acids, α-tocopherols, and carotenoids [2,8]. The ascorbate–glutathione (AsA–GSH) cycle removes hydrogen peroxide (H2O2), maintaining redox homeostasis [2]. The production of nitric oxide (NO), the main RNS, is also related to plants' response to stress [9–11]. It acts at the level of the expression of the defense genes that are involved in eliminating ROS [12–14] and plays a key role in the defense mechanisms against different stressors [15], including heavy metals, such as Cd and As [9,16–19]. Heavy metals increase the synthesis of NO [9,16,20], which is involved in activating the antioxidant defense system and in eliminating the excess ROS that is

**Citation:** Espinosa, F.; Ortega, A.; Espinosa-Vellarino, F.L.; Garrido, I. Effect of Thallium(I) on Growth, Nutrient Absorption, Photosynthetic Pigments, and Antioxidant Response of *Dittrichia* Plants. *Antioxidants* **2023**, *12*, 678. https://doi.org/10.3390/ antiox12030678

Academic Editor: Stanley Omaye

Received: 30 January 2023 Revised: 2 March 2023 Accepted: 7 March 2023 Published: 9 March 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

produced, thus contributing to the maintenance of redox homeostasis [9,21–23]. Hydrogen sulfide (H2S) is involved in numerous metabolic processes in plants [24–26], including the response to heavy metal toxicity [20,25,27,28]. The exogenous application of H2S enhances the antioxidant defense system's response, allowing the oxidative stress that is induced by heavy metals to be reduced [29,30]. H2S modulates the activation of the antioxidant system and the gene expression of its components [31,32]. Both NO and H2S act by controlling the AsA–GSH cycle components and the ROS levels, efficaciously removing H2O2 [33]. The levels of GSH are key in the processes of defense against heavy metals, being able to bind to them and also contribute to the biosynthesis of phytochelatins [27,34]. Through the induction of the antioxidant system, the interaction between H2S and GSH lowers ROS production under conditions of heavy metal toxicity [29,35,36]. The increase in NO and H2S, and their interaction, may act on the antioxidant systems that are involved in the stress response, enhancing their activity and the expression of the genes that are involved in these antioxidant defense systems, thus contributing to the elimination of ROS and the maintenance of redox homeostasis [26,32,37].

Thallium (Tl) is a very toxic element for living beings. It belongs to group IIIA of the periodic table, occurring as Tl(I) and Tl(III). The former is the most stable form, and the latter is the most toxic [38]. Tl forms minerals, including silicates and sulfates [39]. It is widely distributed in the environment in low concentrations of between 0.3 and 0.5 μg g−<sup>1</sup> [40]. It is used as a catalyst in alloys, optical lenses, jewelry, low-temperature thermometers, semiconductors, dyes, etc., and its salts as rodenticides and insecticides; although, the WHO recommends against its use due to its great toxicity [40]. It is found in ionic form in drainage waters [41–43], being released from the aqueous waste and soils of disused Pb–Zn and As mines.

Since Tl and K have similar ionic radii, they can be absorbed by plants through the same mechanisms, depending on the plant species [38]. Tl-induced toxicity is commonly a symptom of the replacement of K with Tl [44]. While in *Arabidopsis thaliana* Tl and K have antagonistic effects in that they mutually interfere with each other's absorption [45], in *Biscutella laevigata,* increased K does not inhibit the uptake of Tl and, therefore, it seems that the absorption of Tl would not be carried out through the same systems as K, but it will be carried out through specific transporters [46].

Tl hyperaccumulator plants have been identified in the Brassicaceae family [44]. They accumulate Tl in their leaves and roots, and, to a lesser extent, in their stems and fruits, with a dependence on species and soil type. Thus, in *Brassica juncacea,* the maximum Tl accumulation in the aerial part is in the leaves and the minimum in the flowers [39], whereas in other species, Tl accumulation occurs mainly in the roots, then the leaves, and, to a lesser extent, in the stem [47–49]. Some ferns and aquatic plants of the genus *Lemna* are reported to have a great capacity for Tl accumulation, which would make them interesting for use in Tl phytoremediation processes [42,43].

Various hydroponic culture studies have been carried out on the absorption, accumulation, and toxicity of Tl [46,50,51]. In *Sinapis alba* under these conditions, most of the Tl is transported to the leaves [50]. In *Silene latifolia* and *Biscutella laevigata*, low Tl concentrations induce a hormetic increase in biomass in response to slight toxic stress [46,51–53]. This contrasts with the results of the in vitro culture of *Arabidopsis,* which show a decline in biomass [45]. Although Tl(I) is not a redox metal, it can alter photosynthetic electron transport, leading to increased ROS and MDA [49]. The oxidation of pigments, the alteration of complexes, and the disappearance of the grana occur in the discolored foliar areas [44]. Chang et al. [45] describe the inhibition of gene expression of LCH II subunits. Tl toxicity induces oxidative stress [44], with increases in H2O2 and in SOD, APX, and POX activities [49,54]. These enzymes are also positively regulated at the gene expression level under Tl toxicity [44]. The effect differs between the ionic forms, with Tl(III) inducing greater ROS production than Tl(I) [55]. Pu et al. [48] describe a decrease in the SOD activity and an increase in POX, while Liu et al. [56] describe increases in both of these activities. This activity in response to heavy metal toxicity can vary with exposure time and can

also inhibit the synthesis of these enzymes and alter their assembly [57]. Tl has a strong affinity for the amino imino and the sulfhydryl groups of proteins and other biological macromolecules, an example being glutathione, which decreases in its reduced form (GSH) due to oxidation or to the formation of complexes with Tl (Tl(SG)3) [27,34]. There has yet to be evidence for the participation of NO and H2S in these defense responses. *Dittrichia viscosa* (L.) Greuter is a species belonging to the Asteraceae family that develops in a wide range of soils and climatic conditions in the Mediterranean basin. *Dittrichia* can colonize poor soils and also those that are degraded due to anthropic activities, such as mining, with high concentrations of heavy metals and metalloids [58]. *Dittrichia* are capable of absorbing and accumulating large amounts of Cd, Cu, Fe, Ni, Pb, Zn, As, and Sb [11,58–64]. The participation of the components of the AsA–GSH cycle, as well as NO and H2S, in the Sb accumulation process has recently been reported [11,63]. However, this capacity varies depending on the different genotypes or populations, which shows great genetic plasticity [60–62]. In accordance with this, *Dittrichia* can be considered a good candidate to carry out phytoremediation processes for heavy metals and metalloids, despite not being a hyperaccumulator plant [62]. The present study was therefore aimed at determining the involvement of ROS, NO, H2S, and the antioxidant systems in *Dittrichia* plants' response to Tl, and the morphophysiological alterations that areinduced by the toxicity of this element.

### **2. Materials and Methods**

#### *2.1. Plant Materials, Growth Conditions, and Treatments*

The seeds of *Dittrichia viscosa* (L.) Greuter obtained from "Semillas Silvestres, SL" were surface sterilized for 15 min in 10% sodium hypochlorite solution (40 g L−1), rinsed several times with distilled water, and, before their germination, were imbibed in distilled water, aerated, and agitated for 2 h at room temperature. After imbibition, the seeds were germinated in a plastic container (30 × 20 × 10 cm) filled with a sterilized perlite mixture substrate wetted with Hoagland solution and were kept at 27 ◦C in the dark for 48 h. After germination, the seedlings were cultivated for 5 days at 27 ◦C and 85% relative humidity, with a light/dark photoperiod of 16 h/8 h, and a constant illumination under photosynthetic photon flux density of 350 μmol m−<sup>2</sup> s−<sup>1</sup> during the day.

After 7 days, the plants were grown in hydroponic culture in lightweight polypropylene trays (20 × 15 × 10 cm; 4 plants per container) and the same environmental conditions (except for relative humidity, 50%). The plants were cultivated in a basal nutrient solution composed of the following: 4 mM KNO3, 3 mM Ca(NO3)2 4H2O, 2 mM MgSO4 7H2O, 6 mM KH2PO4, 1 mM NaH2PO4 2H2O, 10 μM ZnSO4 7H2O, 2 μM MnCl2 4H2O, 0.25 μM CuSO4 5H2O, 0.1 μM Na2MoO4 2H2O, 10 μM H3BO3, and 20 μM NaFeIII-EDTA. After 10 days in hydroponic culture, we started our assay with Tl. For the Tl treatment, the basal solution was supplemented with Tl (I) sulfate (Tl2SO4) in final concentrations of 0 μM (control), 10 μM, 50 μM, and 100 μM Tl. Each cultivation solution was adjusted to pH 5.8, continuously aerated, and changed every 5 days. The plants were exposed to the Tl treatments for 7 days.

The plants of each treatment were divided into roots and shoots, which were washed with distilled water, dried on filter paper, and weighed to obtain the fresh weight (FW). Half of the roots and shoots from each Tl treatment were dried in a forced-air oven at 70 ◦C for 24 h to obtain the dry weight (DW) and the subsequent analysis of the concentration of Tl. The other half of the fresh roots and leaves were used for the biochemical analyses. The relative water content (RWC) of the leaves was determined at the time of harvest from fresh material in accordance with the method described by Smart and Bingham [65]. Leaf disks were collected from the different treatments, and their FWs were determined. They were then immersed in distilled water for 1 h, dried externally with filter paper, and weighed again to obtain the turgid weight (TW). Finally, they were oven-dried at 70 ◦C for 24 h and weighed to obtain the DW. The RWC was calculated as RWC% = (FW − DW)/(TW − DW) × 100.

## *2.2. Determination of Tl and Mineral Content*

The plant material (roots and leaves) of the control and Tl treatments was harvested and rinsed with distilled water. After 24 h of drying at 70 ◦C, the root and leaf material was crushed in a marble ceramic mill. The Tl, K, Mg, Ca, Fe, Mn, Cu, and Zn content was measured by inductively coupled plasma-mass spectrometry (ICP-MS, model NexION 300, PerkinElmer) in accordance with Lehotai et al. [66]. The bioaccumulation factor (BF) was calculated from the ratio between the concentration of the element in the roots or leaves and that present in the hydroponic solution, and the de translocation factor (TF) was calculated from the ratio between the concentration of the element in the leaves and in the roots.
