**1. Introduction**

Environmental conditions are rarely ideal and plants are constantly exposed to various types of stress during their life cycle. Stress can be defined as a factor that decreases the rate of physiological processes that negatively affects growth, development and plant productivity [1]. In the context of energy consumption, stress can be observed as a state in which reduced energy production is directed towards stress-defense processes rather than growth and development [2]. In natural conditions, plants are mainly exposed to a combination of different stress-inducing factors that interact with each other and modify their individual effects accordingly. Salt stress is one of the major abiotic factors limiting crop productivity. According to Shrivastava and Kumar [3], more than 50% of lands are affected by salinity, while salinized areas have a tendency to increase by 10% every year. Since almost all food originates from soil, it is more than clear what problem salinization presents to the food supply [4]. In addition to natural salinization, which is the accumulation of dissolved salts in the soil to the levels that interfere with agricultural production and environment, there is also secondary salinization that occurs as a result of anthropogenic influences [5].

**Citation:** Trifunovi´c-Momˇcilov, M.; Stamenkovi´c, N.; Ðuri´c, M.; Miloševi´c, S.; Markovi´c, M.; Giba, Z.; Suboti´c, A. Role of Sodium Nitroprusside on Potential Mitigation of Salt Stress in Centaury (*Centaurium erythraea* Rafn) Shoots Grown In Vitro. *Life* **2023**, *13*, 154. https://doi.org/ 10.3390/life13010154

Academic Editors: Wajid Zaman and Hakim Manghwar

Received: 6 December 2022 Revised: 28 December 2022 Accepted: 3 January 2023 Published: 5 January 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/).

Salt stress disrupts plant homeostasis in two ways. First, high concentrations of salt in the soil prevent water uptake by the roots, while the accumulation of salts in plants, primarily Na+ and Cl<sup>−</sup> ions, further leads to toxic effects [6]. During the initial phase of defense against salt stress, water deficit and osmotic stress causes a decrease in cell division rate in leaves, root and shoot meristems [7]. Osmotic stress also leads to stomatal closure and reduction in photosynthesis efficiency [8]. The next phase of the plant's defense against salt stress occurs due to accumulation of toxic ions, leading to damage of cell membranes' structure and function, inhibition of enzyme activity and finally, plant productivity [9]. Secondary oxidative stress follows immediately after primary stressors, osmotic stress and ion toxicity. Oxidative stress is a complex chemical and physiological phenomenon that occurs as a result of intensive production and accumulation of reactive oxygen species (ROS) which, due to high reactivity, damage proteins, lipids and nucleic acids [10]. By damaging the lipids, the integrity and functions of membranes deteriorate. These fragmentation products can further damage proteins and nucleic acids, thereby interfering with the normal functioning of receptors, enzymes and membrane channels, resulting in cell death [11]. Accordingly, lipid oxidation, also known as lipid peroxidation, is one of the markers of oxidative stress.

Since prolonged exposure to stress leads to cell death, plants have developed numerous mechanisms that enable growth in different stress conditions. Tolerance to salt stress is a complex phenomenon involving numerous regulatory processes such as stomatal opening, changes in hormonal balance, activation of antioxidant defense systems, osmotic adjustment, maintenance of water balance, export of toxic ions or their compartmentalization in vacuoles [12]. Antioxidant defense mechanisms are divided into two groups, non-enzymatic and enzymatic. Both groups of antioxidants are involved in protecting cellular components from oxidation as well as conversion of ROS into less reactive forms. In addition to ascorbic acid, glutathione, tocopherols, polyamines and phenols, proline and glycine betaine are the most important non-enzymatic components [9]. Proline is one of the essential amino acids, with great importance in protein synthesis. The accumulation of proline in plant cell results after different disturbances to the external environment [13]. In addition, proline is known to regulate the expression of genes important for mitochondrial stability, cell division and cell death [9,14,15]. Several different enzymes such as superoxide dismutase (SOD), catalase (CAT), peroxidase (POX), glutathione peroxidase (GPX), glutathione reductase (GR), glutathione S-transferases (GST), ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), and dehydroascorbate reductase (DHAR), act as part of the plant antioxidant defense system [16]. In addition, SOD is considered one of the major enzymatic systems that scavenges stress-generated free radicals in plants while other enzymes such as CAT and POX, work in close synchrony with SOD to prevent the formation of more harmful ROS through the Haber–Weiss reaction.

Nitric oxide (NO) is a paramagnetic molecule with an unpaired π∗ electron, which can easily diffuse through membranes [17]. Initially, NO was considered an air pollutant that inhibits plant growth and denatures DNA, damages lipids, and decreases intensity of photosynthesis and respiration [18]. Today, however, it is known that NO is an important molecule in redox signaling, participates in the control of numerous physiological processes and plays an important role in establishing resistance to pathogens and regulating the plant's response to abiotic stress [19–21]. Sodium nitroprusside (SNP), a common NO donor, plays diverse roles in plant growth and development. Numerous studies have confirmed the protective role of SNP during salt stress conditions in tomato [22], cucumber [23], orange [24], cotton [25], alfalfa [26], apple [27], wheat [28] and lentil [29] plants.

Centaury (*Centaurium erythraea* Rafn) is medicinal plant that is widely used in traditional medicine as an antidiabetic, antipyretic, antiflatulent and detoxifying agent [30]. Various bioactive compounds isolated from the aerial part of centaury have shown different therapeutic properties [30–36]. Among the species belonging to the *Centaurium* genus, centaury is the plant species to which the greatest attention has been paid during recent years. The first and most important reason is the relatively easy manipulation of this plant species, which makes it an excellent model system for studying genetic transformation, secondary metabolites and salt stress physiology [37–40]. Moreover, centaury has recently shown to possess great developmental plasticity and the ability to induce somatic embryogenesis in root and leaf cultures [41]. A previous report described the salinity-stress response of centaury shoots and roots grown in vitro [38]. In this work, we investigated whether exogenous application of SNP can alleviate the effects of stress caused by NaCl in centaury shoots grown in vitro.

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

#### *2.1. Plant Material, Culture Conditions and Experimental Design*

Mother stock cultures of centaury plants were used as the primary plant material. The centaury shoots were cultured in vitro, on half-strength MS medium ( <sup>1</sup> <sup>2</sup>MS, [42]) solidified with 0.7% agar and supplemented with 3% sucrose as well as 100 mg L−<sup>1</sup> *myo*-inositol. The medium was adjusted to pH 5.8 with NaOH/HCl and autoclaved at 121 ◦C for 25 min. All in vitro cultures were grown at 25 ± 2 ◦C and a 16/8 h light/dark photoperiod ("Tesla" white fluorescent lamps, 65 W, 4500 K; light flux of 47 μmol s−<sup>1</sup> m−2). During the threeweek long pretreatment, the centaury shoots were first placed on four types of <sup>1</sup> <sup>2</sup>MS nutrient media containing different SNP concentrations (0, 50, 100 or 250 μM). After pretreatment, the centaury shoots were transferred to fresh <sup>1</sup> <sup>2</sup>MS nutrient media supplemented with NaCl (0 or 150 mM) and/or SNP (0, 50, 100 or 250 μM) and cultured for one week (Figure 1). All experiments were repeated three times.

**Figure 1.** Schematic of experimental design including different SNP pretreatments and NaCl and/or SNP treatments.

#### *2.2. Quantification of Photosynthetic Pigments*

Isolation of total chlorophyll (*Chl*) and carotenoids was accomplished from the leaves collected from the bottom part of the centaury rosette after four weeks of cultivation. Total *Chl* and carotenoid content were extracted using 96% ethanol as proposed by Lichtenthaler [43] and previously described in detail by Trifunovi´c-Momˇcilov et al. [39]. The absorbance of the photosynthetic pigments was measured using a UV–visible spectrophotometer (Agilent 8453, Life Sciences, Santa Clara, CA, USA).

#### *2.3. Estimation of Oxidative Stress Biomarkers*

The level of lipid peroxidation was measured as malondialdehyde (MDA) concentration by the procedure described by Heath and Packer [44], while H2O2 concentration was determined as described by Velikova et al. [45]. In both assays, 0.1% trichloroacetic acid was used and detailed protocols were previously described by Trifunovi´c-Momˇcilov et al. [38]. The spectrophotometric determination of MDA and H2O2 were measured using an ELISA Micro Plate Reader (LKB 5060–006, Winooski, VT, USA).

#### *2.4. Estimation of Nonenzymatic Antioxidants*

Free proline content was determined by the ninhydrin reaction which consists of the reaction of proline and ninhydrin reagent (2,2-dihydroxyindane-1,3-dione) resulting in a yellow reaction product [46]. Proline extraction and measurement was performed according to a modified method by Carillo and Gibon [47] and described in detail by Trifunovi´c-Momˇcilov et al. [38].

Total polyphenol content was determined using the Folin–Ciocalteu test (FC test) based on reaction of polyphenols from plant tissues and Folin–Ciocalteu reagents forming a blue-colored complex that can be spectrophotometrically quantified. This method was previously described by Singleton et al. [48]. The plant material (200 mg) was homogenized in liquid nitrogen and extracted with 96% ethanol. The homogenate was incubated for 60 min at room temperature and then centrifuged for 15 min. The supernatant was further mixed with the FC reagent solution, which was previously prepared by adding distilled water to the FC reagents in a volume ratio of 2:1. The reaction mixture was quickly vortexed and 20% Na2CO3 was added. After 90 min at room temperature in darkness, the absorbance was measured at 765 nm. In this assay, gallic acid was used as a phenol standard.

The antioxidant activity in the centaury shoots was determined after evaluation of stable DPPH radical concentrations. The samples were prepared using the same method as the FC test. In the reaction with antioxidants, the DPPH radical is converted to a non-radical form through reduction by hydrogen ions. After homogenization and centrifugation of the supernatant, methanol and DPPH reagent solution were added. The reaction mixture was incubated at room temperature in the dark. After 60 min, the degree of reduction of the DPPH radical was estimated through an absorbance measurement at 520 nm. The scavenging capacity of the DPPH radical was calculated using the following equation: (%) = [1 − (A1 − A0)] × 100 where A1 is the absorbance of the sample and A0 is the absorbance of the blank reaction.

For the spectrophotometric determination of all nonenzymatic antioxidants, an ELISA Micro Plate Reader (LKB 5060–006, Winooski, VT, USA) was used.

#### *2.5. Estimation of Enzymatic Antioxidants*

Centaury shoots were homogenized in potassium phosphate extraction buffer containing insoluble polyvinylpolypyrrolidone, dithiothreitol and phenyl methyl sulfonyl fluoride. The homogenate was centrifuged at 4 ◦C for 5 min and the protein content was determined from the supernatant according to Bradford [49] using bovine serum albumin as the standard. The quantification of SOD, CAT and POX was also performed.

SOD activity was determined spectrophotometrically using a modified method from Beyer and Fridowich [50]. The reaction mixture contained potassium phosphate buffer, ethylenediaminetetraacetic acid, methionine, nitroblue tetrazolium chloride (NBT) and riboflavin. The reaction mixtures were added to the samples, which were then illuminated for 1–2 min and the absorbance was measured at 540 nm. One unit of SOD activity is the amount of sample required for 50% inhibition of NBT photoreduction and is presented as the specific activity (U/mg). SOD activity was spectrophotometrically detected using an ELISA Micro Plate Reader (LKB 5060–006, Winooski, VT, USA).

CAT activity was determined spectrophotometrically using the method from Aebi [51]. This method is based on monitoring the kinetics of the consumption of H2O2, which can be detected by measuring the absorbance (at 240 nm) of the reaction mixture consisting

potassium phosphate buffer, H2O2 and enzyme extract. One unit of CAT activity is defined as the amount of enzyme required to degrade 1 μM of H2O2 in 1 min and is indicated as μM min−<sup>1</sup> mg protein−<sup>1</sup> (U/mg protein).

POX activity was determined spectrophotometrically using the method from Kukavica and Veljovi´c-Jovanovi´c [52]. The reaction mixture contained potassium phosphate buffer and pyrogallol as the enzyme substrate. The POX-catalyzed oxidation of pyrogallol to purpurogallin in the presence of H2O2 was monitored by absorbance determination at 430 nm. Enzyme activity is indicated as μM min−<sup>1</sup> mg protein−<sup>1</sup> (U/mg protein). The absorbances of the CAT and POX reactions were measured with a UV–visible spectrophotometer (Agilent 8453, Life Sciences, USA).

### *2.6. Statistical Analysis*

The effect of different SNP pretreatments/treatments on the biochemical parameters of centaury shoots, after four weeks of culture, were evaluated using standard two-factor analysis of variance (ANOVA). All analysed parameters were measured using three biological samples per treatment. In addition, the absorbances of all supernatants were measured in triplicate for each sample. The results are presented as mean ± SE. The comparisons between the mean values were made using a Fisher LSD (the least significant difference) post-hoc test, calculated at a confidence level of *p* ≤ 0.05.
