**1. Introduction**

Soil salinity is a dynamic and globally spreading issue for over one hundred countries [1]. Salinity affects almost all stages of plant development and causes osmotic stress and ionic and nutrient imbalance [1,2]. Halophytes are one of the most suitable models of salt stress tolerance mechanisms, due to their salt resistance [3]. The newest reports indicated that halophytes have considerable potential for the restoration of salt contaminated lands and potential for the phytosanitation and phytoremediation of the soil [4].

The adaptiveness of *T. pannonicum* to salinity habitats are poorly studied and 'adaptive plant strategies' are not explained. Therefore, the experimental model in our studies is the sea aster *Tripolium pannonicum* (Jacq.) Dobrocz. (formerly *Aster tripolium* L.) from the *Asteraceae* family, which grows in the salt marshes and coastal areas of temperate zones and in non-tidal saline areas [5]. The plant is also part of highly specialized habitats: Atlantic salt meadows (1330) and inland salt meadows (1340) listed on the Habitats Directive-Natura 2000 and the European Red List of Habitats [6]. As a plant from the coastal areas, *T. pannonicum* is periodically flooded with sea water while in inland habitats are surrounded by outflows of salty groundwater and are classified as coastal wetland-specific species [7].

**Citation:** Ludwiczak, A.; Ciarkowska, A.; Rajabi Dehnavi, A.; Cárdenas-Pérez, S.; Piernik, A. Growth Stage-, Organ- and Time-Dependent Salt Tolerance of Halophyte *Tripolium pannonicum* (Jacq.) Dobrocz. *Life* **2023**, *13*, 462. https://doi.org/10.3390/life13020462

Academic Editors: Hakim Manghwar, Wajid Zaman and Balazs Barna

Received: 4 January 2023 Revised: 26 January 2023 Accepted: 3 February 2023 Published: 7 February 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/).

This species tolerates long-term flooding, making it more successful than other halophytes that cannot survive long-term flooding [8]. *T. pannonicum* is present also in saline, moist, and nitrogen-rich anthropogenic habitats, e.g., the surroundings of the soda factory in Inowrocław (Poland) with the highest maximum salinity of 140 dS·m−1. However, in natural habitats, such as the nature reserve in Ciechocinek (Poland), salinity can be lower (1.58–38.5 S·m<sup>−</sup>1) [9]. Piernik identified that the ecological growth optima of *T. pannonicum* correspond with very strong saline soils [9]. The variability of the parental habitat's salinity can be the basis of the extreme environmental adaptation of this plant. In addition, the 'adaptive plant strategies' may differ depending on the phase of growth, the plant's organs, and the length of time of exposure to the stress factors and even between and within species [10]. Different halophytes can express different salt stress adaptation strategies that are essential both in the context of their protection/restoration and of the future saline agriculture development [11]. Enhancing ROS and/or osmolyte production and antioxidant defence mechanism improvement are the most documented examples [11,12]. Therefore, studies on the not well-examined endangered halophyte *T. pannonicum* can help to better understand the plant's response to salinity stress and plant-environment relations, especially in the context of extreme climate change and habitat disturbances [13].

The initial growth, occurring at the germination and seedling stages, can influence a plant's capacity to capture resources in later growth when competition for light and soil nutrients becomes more intense [14,15]. Therefore, successful germination and seedling development are crucial steps in the effective growth of a new plant. The salt tolerance of the germination differs between halophytes, therefore it is essential to evaluate the salinity effect on the not well-studied halophyte species. A common effect of abiotic and biotic stressors is an excessive production of the reactive oxygen species (ROS), such as hydrogen peroxide (H2O2) causing cellular oxidative stress and damage of the crucial macromolecules [3,16,17] and osmolytes (such as proline or glycine betaine) [18–20]. The role of these compounds in *T. pannonicum*'s adaptation and tolerance of salinity is not well investigated and the patterns of correlation between these compounds are not documented yet. There is a lack of knowledge of whether the activities of antioxidant enzymes are correlated with the concentration of salinity stress indicators during *T. pannonicum's* adaptation to salinity. There is no evidence as to which organs (roots, stem, or leaves) are most affected by different NaCl concentrations. It is also not clear how the activity of antioxidant enzymes and salinity stress indicators (proline and H2O2) are correlated between organs and related to each other. The studies by Ievinsh et al. indicated a high dispersion of leaf water content, Na+ vs. K+ concentration in water from leaf tissue, and high sodium accumulation with low potassium levels in the leaves of the sea aster but without a dipper biochemical analysis that is crucial for understanding the salinity tolerance and adaptation [7].

Few studies have actually shown plant responses to different durations of salinization in short- and long-term periods. The researchers usually focus only on one type of response to salt stress, short- or long-term [21,22]. Therefore, in our study we want to explain how the short- and long-term NaCl stress act on the activity of antioxidant enzymes and on the concentration of salinity stress indicators. Examining the plant stress response on a wide time scale will allow us to fully understand the adaptation of *T. pannonicum* to extreme and variable soil salinity in the habitat. From the perspective of autecological studies on halophyte adaptation to salinity, the long-term stress effect seems to be even more significant than the short time salinity effect.

The main goal of our research was to determine at which levels (plant growth, organ, time) salinity can modify the stress response of *T. pannonicum*. We performed this autecological study to evaluate the following hypotheses: (1) even though *T. pannonicum* is a halophyte, salinity significantly affects germination and late growth, (2) salinity significantly affects the activity of antioxidant enzymes (APX, POD, and CAT) and salinity stress indicators (hydrogen peroxide and proline) in the root, stem and leaves of *T. pannonicum*, (3) the duration of salinity exposure modifies plant physiological responses. We hope that

the results will provide a novel view to understanding the interactions of individual species with the extreme environment and to recognize the salinity tolerance of this plant.

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

#### *2.1. Seed Collection*

The seeds were collected in November 2019 from the anthropogenic inland saline habitat near a soda factory in the town of Inowrocław (52◦48- N, 18◦15- E). This site represents an industrial saline area in Poland, with salinity associated with waste from the soda production. In this area, the ECe value reached even 140 dSm−<sup>1</sup> (which corresponds to 1400 mM NaCl) [23]. Experiments were conducted at the Nicolaus Copernicus University in Torun, Poland in 2020. The permission to work with the seeds of a protected plant was provided by the regional director of the Environmental Protection in Bydgoszcz (WOP.6400.17.2020JC).
