1. Introduction
Momordica charantia L. (2x = 2n = 22), popularly called bitter cucumber, balsamic pear, karela, and bitter gourd, is a climbing plant from the Cucurbitaceae family, used both in traditional medicine and in food [
1,
2,
3]. The green bitter cucumber fruit has a diversity of vitamins and minerals: vitamin A, thiamin (B1), riboflavin (B2), niacin (B3), vitamin C, calcium, and iron [
4,
5].
Momordica charantia presents a high number of biologically active chemicals including saponins, triterpenes, flavonoids, proteins, steroidal acids, and alkaloids, thus presenting important medicinal properties, including anticancer, antiobesity, hypotensive, antioxidant, and antihyperlipidemic properties. In Africa, Asia, and Latin America, bitter cucumber has a rich history of use as a hypoglycemic agent; the plant extract has been called vegetable insulin [
6,
7,
8,
9].
Salinity is a problem encountered globally, affecting almost 20% of arable land [
10]. Soil degradation resulting from high salt accumulation is predominantly found in arid and semi-arid regions where rainfall is insufficient to maintain a regular circulation of rainwater through the soil [
11,
12]. Salinity affects plant growth and development through water stress, cytotoxicity due to the excessive uptake of ions such as sodium (Na
+) and chloride (Cl
−), and nutritional imbalance. In addition, salinity is usually accompanied by oxidative stress due to the generation of reactive oxygen species (ROS) [
13,
14,
15,
16].
The occurrence of this abiotic stress triggers cell signaling pathways and cellular responses, such as the accumulation of compatible solutes and the upregulation of antioxidants [
17]. Soil osmotic potential (Ψs) and total water potential (Ψw) are decreased when ions are dissolved in excess in soil water. Plants must keep their internal water potential lower than that of the soil to maintain their water absorption capacity and turgor under such conditions [
18,
19]. The osmotic adjustment of plants exposed to saline stress is mediated by the accumulation of absorbed ions, which at some point reach the toxic level of concentration, but also by osmolytes represented by organic acids, sugars, and synthesized amino acids. Osmolyte accumulation enhances water absorption from saline soils [
20,
21]. When the internal mechanisms of ion exclusion and compartmentalization are overcome, ion toxicity appears, manifested by the chlorosis and/or necrosis of the leaves [
22].
One of the best known osmolytes that acts in the fight against saline stress is Proline (Pro). This amino acid acts as an enzyme protector, free radical scavenger, cellular redox balancer, cytosolic pH buffer, and stabilizer for subcellular structures [
11,
23]. The capture of radicals generated in the biological environment (R
●) takes place in response to abiotic stress. The radical species is taken over by the secondary amine group, forming a molecular compound by eliminating a H
● radical [
24]. Proline metabolism involves the oxidation of NAD(P)H in the cytosol and the reduction of NAD+ in the mitochondria, having an important role as a redox regulator in eukaryotic cells. Proline can act as a pH buffer because it is a compound with mixed functions, one acidic and one basic [
25,
26]. Proline has a chelating effect, by complexing various metal ions [
27,
28]. These biochemical properties make proline an important metabolite for determining plant responses to salt stress. Under optimal conditions, reactive oxygen species (ROS) are constantly generated in the plant at low levels. Under these conditions, they cannot cause damage because they are absorbed by various antioxidant mechanisms [
29,
30]. The balance between ROS generation and ROS scavenging can be affected by abiotic stress factors including salinity [
31]. Reactive oxygen species are presented as hydrogen peroxide (H
2O
2), perhydroxyl radicals (HO
2•), hydroxyl radicals (OH
−), ozone (O
3), singlet oxygen (
1O
2), etc. [
32,
33]. The increased level of ROS is mitigated by proteins and other compounds such as glutathione (GSH) and ascorbate [
34,
35].
L-ascorbic acid (AsA), or vitamin C, is one of the most abundant water-soluble antioxidants in plants and animals. L-ascorbic acid is a non-enzymatic metabolite that plays a vital role in stress perception, redox homeostasis, regulation of oxidative stress, and physio-biochemical responses of plants both under optimal growth and development conditions and under stress conditions [
36]. It is involved in many functions such as the antioxidant defense system, redox signaling, biosynthesis of phytohormones, modulation of gene expression, photoprotection, and regulation of photosynthesis and growth [
37,
38]. There is a consensus on the important role of the relationship between reactive oxygen species (ROS) and AsA in the regulation of cell signaling and metabolic processes. These characteristics make AsA a major plant antioxidant that detoxifies ROS and an important metabolite to be studied under salt stress conditions [
39].
Aromatic amino acids are important components in the metabolic network of plants, supporting vital processes such as macromolecule synthesis, environmental adaptation, and stress responses [
40,
41]. Amino acids are the precursors of several hormones and various primary and secondary metabolites, which are essential for plant growth and combating abiotic stress. Phenylalanine (Phe), tyrosine (Tyr), and tryptophan (Trp) are aromatic amino acids (AAAs) characterized by the presence of an aromatic ring in their structures. In addition to their essential function in protein synthesis, AAAs serve as fundamental precursors for a diverse range of primary and secondary aromatic metabolites in plants. The synthesis of these metabolites occurs via the shikimate pathway, thus representing a significant contribution to the ability of plants to respond and adapt to biotic and abiotic stress [
42]. The carbon skeletons required for the synthesis of aromatic amino acids are provided by the shikimate pathway, which is characterized by a significant flux. The estimates indicate that more than 30% of the total fixed carbon is directed through this pathway [
43]. The role of these amino acids in the resistance of plants to abiotic stress conditions represents the premise of their study. The aim of this study was to emphasize the species
Momordica charantia’s response to salt stress and to find some genotypes that were resistant to this abiotic stress by examining certain metabolites.
2. Materials and Methods
2.1. Materials
Ferric chloride (FeCl3)—Sigma Aldrich (Darmstadt, Germany); potassium chloride (KCl)—Sigma Aldrich; nitric acid (HNO3)—Sigma Aldrich; hydrochloric acid (HCl 37%)—Merck (Darmstadt, Germany); potassium hexacyanoferrate(III) (K3[Fe(CN)6])—Roth (Schoemperlenstr, Germany); L-ascorbic acid (C6H8O6)—Sigma Aldrich; ninhydrin (C9H6O4)—Merck; sulfosalicylic acid (C7H6O6S)—Chemical Company (Iași, Romania); glacial acetic acid (C2H4O2)—Merck; acid phosphoric (H3PO4)—Merck; toluene (C7H7)—Merck; L-proline (C5H9NO2)—Sigma Aldrich; ammonium hydroxide NH4OH—Chemical Company.
2.2. Methods of Characterization
UV-Vis spectra were recorded on a Specord 210 Plus AnalitikJena spectrophotometer, in cuvettes with a diameter of 1 cm; Fourier-transform infrared (FTIR) spectra in transmission mode (400–4000 cm−1 spectral range, 2 cm−1 resolution, with the accumulation of 32 scans, room temperature) were recorded on a Bruker Vertex 70 FT-IR spectrometer. Studying bonding in the mid-IR range (400–4000 cm–1) reveals numerous significant and practical bonds. The most basic and popular kind of FTIR experiment is the transmission-mode experiment. The amount of infrared beam transmitted through the sample is measured using a detector. The pure medium’s spectrum is first gathered and utilized as a guide in order to acquire the sample spectrum. The sample’s spectrum is then obtained by gathering its spectra in the medium and comparing it to the reference. Once the functional groups are identified, the chemical structure of the analyzed substance can be deduced.
2.3. Procedure for Obtaining Plant Material
Five genotypes of bitter cucumbers were used for the experiment, of which two were Romanian varieties (Brâncusi and Rodeo) and three were experimental lines (Line 1, Line 3, and Line 4). The seedlings were obtained from the greenhouse of the Research Institute for Agriculture and the Environment (ICAM) belonging to the “Ion Ionescu de la Brad” University of Life Sciences, Iași, Romania. A total of three treatments were applied, and their application began when the plants reached phenophase 201 of the BBCH scale, corresponding to the appearance of the first lateral shoot. The second treatment was applied in phenophase 501 of the BBCH scale, corresponding to the appearance of the first flower, and the third treatment was applied in phenophase 701 of the BBCH scale, corresponding to the appearance of the first fruit. The treatments involved different concentrations of saline solutions (M—control plants treated with 0 mM NaCl; V1—plants treated with 100 mM NaCl; and V2—plants treated with 200 mM NaCl). The analyses were carried out on the bitter cucumber fruits harvested 7 days after the application of the third saline treatment. The plants were treated with an amount of 300 mL of saline solution per treatment. The procedure for growing the plants was similar to that presented in our previous work on bitter cucumber [
44].
2.4. Procedures for the Determination of Different Metabolites
2.4.1. Procedure for the Determination of Proline
In total, 1 g of plant tissue was triturated in 5 mL sulfosalicylic acid 3%; after trituration, the mixture was centrifuged at 5000 rpm for 20 min. Then, 2 mL of the supernatant was pipetted and added over 2 mL of acidic ninhydrin solution (prepared by dissolving 1.25 g of ninhydrin in 30 mL of glacial acetic acid (AcAc) and 20 mL of 6M phosphoric acid, at 60 °C) and 2 mL of glacial AcAc. The resulting mixture was subjected to thermal treatment at 100 °C for 1 h. After the heat treatment, the solution was left to cool to room temperature, after which it was placed in an ice bath. Afterwards, the mixture was extracted with 4 mL of toluene. A reaction time of 1h and temperature of 100 °C were required to ensure the decarboxylation and addition reaction of the tetrahydropyrrole ring.
2.4.2. Procedure for the Determination of Ascorbic Acid
The solution to be analyzed was prepared by mixing 1 mL of FeCl3 solution (2 × 10−3 M), 1 mL of K3[Fe(CN)6] solution (2 × 10−3 M), 1 mL of KCl solution (0.1 M), 1 mL HCl solution (0.01 M), and 1 mL sample. The solution was prepared by appropriately diluting a stock solution of AsA (1 g/L) with a solution of HNO3 (0.01 M). To analyze the AsA from the fruits of Momordica charantia, the following procedure was followed: After crushing 1 g of fruit, 15 mL of HNO3 (0.01M) was added, and the resulting mixture was heated at 90 °C for 15 min. The mixture was then cooled and centrifuged at 2000 rpm for 5 min. Then, 1 mL of the supernatant was diluted with 9 mL of HNO3 (0.01 M). From the final solution, 1 mL was used for the analysis. Three tests were performed for each analyzed sample.
2.4.3. Procedure for Identification of Aromatic Amino Acids in Proteins
A total of 0.77 g fruit was thermally treated at 60 °C with 10 mL of distilled water for 20 min. From the resulting extract, 2 mL was pipetted and subsequently treated with 0.5 mL HNO3 65% at 60 °C for 15 min. After approximately 3 min of treatment, the solutions of interest turn yellow. After another 15 min of heat treatment, the solutions were cooled to room temperature; later, 1 mL of 20% ammonia solution was added to neutralize the acid, so the color was slightly intensified. The solutions of interest were subjected to centrifugation at 2000 rpm for 10 min; 100 µL was pipetted from the supernatant and diluted with 3 mL of distilled water, and the absorbance of the solutions was read at 305 nm.
For the studied metabolites, three replications were made for each variant (M, V1, V2) of each genotype.
2.5. Statistical Analysis
The statistical tests performed in this paper were the Kolmogorov–Smirnov test, the two-way ANOVA test, the Student’s t-test, and the Pearson correlation coefficient.
The Kolmogorov–Smirnov test was performed to determine the normal distribution of the data used for the two-way ANOVA tests. This test was conducted online using the following link:
https://www.socscistatistics.com/tests/kolmogorov/default.aspx (accessed on 20 December 2023) Two-way ANOVA tests were performed to analyze the simultaneous impact of two factors on the studied metabolites [
45]. The two factors analyzed were the saline concentration and genotypes. H0: there are no significant differences between metabolite values in the control, 100 mM, and 200 mM treated variants. This assumes that there is no influence of the tested factors (saline concentration and genotype), nor of their interaction, on the metabolite values (proline, vitamin C, and aromatic amino acids).
Student’s t-tests were performed to make comparisons between variants (M, V1, and V2) and identify statistical differences between them. To perform the Student’s statistical t-tests, the data obtained from the five genotypes were separated into three columns that represented data from the control variant, the variant treated with 100 mM NaCl, and the variant treated with 200 mM NaCl. For each metabolite studied, the t-test was performed using all the data obtained from the analyses; thus, the results of all analyzed genotypes are summarized.
Two-way ANOVA and Student’s
t-test were performed using the statistical functions in Excel software. The Pearson correlation coefficient was performed to measure the degree of linear correlation between the studied metabolites. It is a number between −1 and 1 that measures the strength and direction of the relationship between two variables [
46]. It was performed using Excel and represented using OriginPro 2021, v:9.8.0.200 software.
5. Conclusions
After the Pro content was determined, a series of geotype differences were identified in the biosynthesis capacity of this amino acid both in the control variants, which were not treated with sodium chloride, and in those that were subjected to stress saline.
Line 4 showed the highest Pro differences between the control and treated variants, which underlines its struggle for resistance to osmotic stress. In contrast, the variety Rodeo and Line 3 registered the smallest differences between the control and the group of plants treated with saline solutions. This can be attributed to their adaptation to salt stress conditions.
After the AsA content of bitter cucumber fruits was analyzed, a significant difference was observed between the control and saline-treated varieties in Lines 1 and 4, indicating the responsiveness of plants to salt stress.
Although the studied genotypes showed a tendency to increase the amount of vitamin C in proportion to the concentration of the applied treatments, the Brâncusi variety showed lower values in the case of the V2 variant, which shows the inclusion of the plants in the biphasic model proposed by Munns, being the only variant that passed from the osmotic stress phase to the toxic stress phase, from this point of view being the least resistant genotype to saline stress.
Lines 1 and 4 that were exposed to salt stress synthesize large amounts of vitamin C compared to the control, being indicated to be cultivated in soils with moderate salinity. This will lead to an increase in the amount of vitamin C in the fruit, since the salinity helps to improve the antioxidant properties of the fruit.
Apart from Line 3, which showed a slight increase, the analysis of absorbances of aromatic amino acids showed a general trend of decreasing values, inversely proportional to the increase in applied NaCl concentrations.
The results of the analyses showed that each of the five genotypes examined are affected by salt stress in a distinct way. Consequently, these genotypes react differently to stress. The accumulation of preferred metabolites depending on the genotype and the degree of adaptation to salt stress can be indicated by weak correlations between the accumulation of the studied metabolites and negative correlations between them, according to the Pearson correlation coefficient analysis.
The Brâncusi variety, which showed symptoms associated with excess ions, was the weakest genotype in terms of resistance to abiotic stress.
Compared to the control variant, Line 3 plants that were exposed to salt stress showed the least variation in the biosynthesis of the studied metabolites, indicating a good resistance to salt stress.