The Effects of Foliar Salicylic Acid and Zinc Treatments on Proline, Carotenoid, and Chlorophyll Content and Anti-Oxidant Enzyme Activity in Galanthus elwesii Hook

: Galanthus elwesii Hook. is an important plant species of the Amaryllidaceae family and is used for the medicinal purposes of its valuable bioactive compounds. The present study was conducted to investigate the effects of foliar salicylic acid (SA) and zinc (Zn) treatments on the proline, carotenoid, and chlorophyll content and the anti-oxidant enzyme activity in G. elwesii . The ascorbate peroxidase (APX) enzyme activity, catalase (CAT) enzyme activity, and protein contents were determined with ascorbate oxidation, hydrogen peroxide (H 2 O 2 ), and Bradford experiments, respectively. The plants were treated with three different concentrations of SA (0.5, 1, and 2 mM) and Zn (40, 80, and 120 mM) and were compared with the control. Fresh leaves were harvested in the study. APX (3.99 ± 0.58 EU/mg protein) and CAT (154.64 ± 4.10 EU/mg protein) were obtained from Zn 80 and 120 mM treatments at the highest level, respectively. The proline, chlorophyll b, and carotenoid content increased 12.4, 1.54, and 3.95-fold, respectively, in 0.5 mM SA treatments, when matched with the control group. It was found that increasing doses of SA and Zn increased the content of malondialdehyde (MDA), but this was not at a signiﬁcant level. The total chlorophyll content increased 2.27-fold in Zn 120 mM + SA 2 mM treatment and the chlorophyll content increased 2.41-fold in Zn 40 mM + SA 1 mM treatment.


Introduction
The Amaryllidaceae family comprises the genus Galanthus, which includes over 20 species innately found in Europe and Southwest Asia.These species, commonly known as "snowdrops," possess economic value due to their medicinal properties, decorative appeal, and use in landscaping [1,2].Among them, Galanthus elwesii Hook.stands out as a highly valuable bulbous perennial herbaceous plant with white flowers, cultivated commercially in many countries.The flowers and bulbs of these species contain a diverse array of medicinal compounds, including graciline, galanthamine, hordenine, lycorine, and tazettine [3,4].Lycorine and galanthamine show properties such as anti-cancer [4,5], anti-inflammatory [6], anti-diabetic [7], anti-bacterial, and anti-malarial activity, and acetylcholinesterase and butyrylcholinesterase inhibition [4,8].Lycorine has also demonstrated potential in combating SARS-CoV-2 infection due to its anti-viral activity [9], while galantamine is utilized for treating Alzheimer's disease and other neurological disorders [8,10].Numerous strategies, including plant growth regulators (PGRs) and chemicals, have been investigated to improve the physiological parameters of plants.Plant growth regulators include versatile compounds known for their resident physiological functions in plants.Among these regulators, "salicylic acid and zinc" stands out as a natural growthmodulating compound with the potential to alleviate the negative consequences of abiotic stresses.Research conducted thus far has shown that the composition and content of medicinal compounds in plants are predominantly influenced by genetics, growth and development stages, environment, and cultivation techniques.Among these factors, plant nutrients and hormones have received considerable attention.Zinc (Zn), a micronutrient, plays an active role in various biophysical and biochemical processes in plants, including protein synthesis, growth, development, gene regulation, and enzymatic activation [11].Zn's effectiveness stems from its involvement in the structure of over 300 enzymes belonging to all six enzyme classes [12,13].These enzymes play a crucial role in the cellular defense mechanisms of plants by scavenging elevated levels of free radicals induced by stress [14].Thus, insufficient Zn in plants can impede these essential biophysical and biochemical actions necessary for normal plant functioning and stress detoxification.Inadequate Zn levels have a negative impact on yield and crop quality [15].Salicylic acid (SA) plays a critical role in plant defense against pathogens and can be synthesized through either the phenylalanine or isochorismate pathways [16,17].When externally applied, SA elicits responses similar to those triggered by pathogen exposure or other external stimuli [18,19].This hormone initiates signal transduction pathways that ultimately lead to the transcription of various genes, resulting in the accumulation of defense-related molecules such as steroids, terpenoids, alkaloids, and polyphenols [17].
These secondary metabolites exhibit diverse pharmacological activities, including antidiabetic, anti-asthma, anti-cancer, anti-malarial, anti-viral, and anti-microbial properties.The protective role of medicinal plants against oxidative stress is significant.Various ecological stresses, including the application of elicitors such as SA, induce the production of reactive oxygen species (ROS), such as singlet oxygen (O 2 ), superoxide (O 2 − ), hydrogen peroxide (H 2 O 2 ), and hydroxyl radical (OH), which can cause oxidative damage and cell death.While ROS can be harmful, they also serve as crucial signaling molecules regulating normal plant growth and responses to stress [20,21].To maintain cellular balance and homeostasis, plants employ a complex enzymatic and non-enzymatic anti-oxidant system to control the levels of ROS.Anti-oxidant enzymes such as catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD), along with the ascorbate-glutathione (AsA-GSH) cycle, play essential roles in eliminating ROS, including H 2 O 2 , O 2 − , and OH, generated during stressful conditions [22,23].Moreover, non-enzymatic compounds, such as ascorbic acid, carotenoids, glutathione, flavonoids, phenolics, and α-tocopherol, contribute to cellular protection against the cytotoxic effects of ROS.Phenolics and flavonoids [24], known for their anti-oxidant properties [25,26], often exhibit bioactive functions [27].
Photosynthetic pigments, particularly chlorophyll, are responsible for the vibrant green color seen in plants [28].They play a crucial role in the process of photosynthesis, where they capture and transfer energy from light to the reaction centers, facilitating lightdependent reactions [29].Among the variety of chlorophyll molecules, such as chlorophyll a and b, xanthophylls, and carotenoids, it's worth noting that chlorophyll is often referred to as "green blood" due to its structural resemblance to hemoglobin, a key component in the blood of mammals.The main distinction lies in the central atom, with chlorophyll a and b containing magnesium while hemoglobin contains iron [29].
Recent research has focused on the physiological impact of chlorophylls on human health, particularly in terms of maintaining and preventing chronic diseases [30].Extensive investigations have delved into the beneficial biological properties of chlorophylls and their derivatives [31].Chlorophylls have demonstrated positive effects on inflammation, oxidative processes, wound healing, and the formation of calcium oxalate crystals, among other functions [32].Both in vitro and in vivo studies have highlighted the antioxidant capabilities of chlorophyll and its natural and industrial derivatives, as well as their ability to counteract mutagenic activity, regulate enzymes involved in detoxifying foreign substances, and initiate apoptosis in cancer cell lines [33].Furthermore, they have been found to stimulate the production of mammalian phase 2 proteins, which protect cells from oxidative stress and electrophilic compounds [34].In the realm of animal studies, chlorophyll and its water-soluble salts, known as chlorophyllin, have displayed protective properties against the harmful effects of carcinogens such as aflatoxin [35,36].As a result, there is growing interest in the potential use of chlorophyll-enriched foods as a viable strategy for cancer prevention [30].
When examining the research concerning G. elwesii, it becomes evident that our knowledge about this species is quite limited.The available studies primarily focus on the qualitative and quantitative analysis of bioactive compounds found in G. elwesii specimens collected from their natural habitat [37][38][39][40][41][42][43][44].Some studies have investigated the acetylcholinesterase inhibitory activity and alkaloid enhancement in in vitro culture conditions [45] as well as the effectiveness of G. elwesii extracts against specific microorganism species [46].Regarding cultivation, only two studies have explored G. elwesii, focusing on the distribution of bioactive compounds during different growth and development stages, along with the effects of phosphorus and zinc fertilization on their biological activities [4,47].Therefore, in order to expand our limited knowledge of this species, it is of great importance to reveal the basic physiological parameters that are effective in terms of productivity and quality.Furthermore, the development of cultural practices involving compounds responsible for photosynthesis shows promise in fortifying plant defense mechanisms.Consequently, the goals of the current study encompassed the following objectives: (i) evaluating the impacts of Zn and SAs, (ii) quantifying protein content, (iii) tracking oxidative stress levels, (iv) estimating proline, chlorophyll, and carotenoid levels, and (v) gauging the activities of enzymes responsible for scavenging reactive oxygen species (ROS).

Field Characteristics
A uniform basal dosage of nitrogen, phosphorus, and potassium was applied to all experimental containers.The rates of application were 60:45 and 30 mg kg −1 for nitrogen and phosphorus, respectively.The soil used in the experiment had a sandy loam texture, with a pH of 7.99, electrical conductivity (EC) of 0.49 dS m −1 , 1.2% organic matter content, 0.032% total nitrogen, 4.7 mg kg −1 available phosphorus, and 47 mg kg −1 available potassium [48].The soil parameters were measured using standard methods described by [49][50][51].

Plant Material and Experimental Design
A greenhouse experiment was conducted in Suluova, Amasya, Turkey, from 2021 to 2022.The experimental site was located between 40 • 50 39.3"N and 40 • 50 40" N latitude and 35 • 37 57.3"E and 35 • 37 58" E longitude, at an altitude of 510 m.The focus of the experiment was on "Toros Snowdrop" bulbs, which were planted in disinfected plastic pots with dimensions of 25 cm in diameter and 22.5 cm in depth during the 2021 and 2022 seasons.Each pot contained three bulbs, which were planted at a depth of 5-7 cm, depending on their size, in a mixture of peat and perlite, with a ratio of 2:1, respectively.Observations were made for the emergence of plants from all the pots.Subsequently, individual pot applications were carried out.Zinc (Zn) was sprayed on the plants at four different levels: 0 (control group with no fertilizer), 40, 80, and 120 mM.The Zn was applied using a stock solution of zinc sulfate (ZnSO 4 -0.22%w v −1 ).Similarly, salicylic acid (SA) was sprayed on the plants at four different levels: 0 (water and 1% ethanol), 0.5, 1, and 2 mM.A stock solution of SA (% w v −1 ) was used for this purpose.Each application was made separately.The SA and Zn treatments were applied to the plants through foliar spraying at the beginning of the flowering period.Fresh leaf samples were collected for enzyme activity analysis and stored at −20 • C until further analysis.The experiment was designed using a completely randomized design with six replications.

Bradford Protein Assay
The samples were taken fresh (1 g), ground in liquid nitrogen, and then homogenized in 100 mM KH 2 PO 4 /0.5 mM EDTA pH (7.7) buffer that contained 5 mL of 1% (w v −1 ) PVP.The supernatant was separated from the precipitate by centrifuging at 15,000× g for 20 min at 4 • C in a refrigerated centrifuge.Also, 96 µL of distilled water, 900 µL of Coomassie brilliant blue solution, and the supernatant obtained from the homogenate were placed in a 4 µL cuvette and the absorbance values were read at 595 nm [52].The concentration of protein was determined using ten-point calibration curves of the standards ranging from 1-30 µg mL −1 , with a correlation coefficient of at least 0.9916 (protein: y = 0.041 x + 0.991).

Estimation of Proline Content
The content of proline in the leaf tissues was made according to the Bates [53] method.Briefly, 2 mL of 96% glacial acetic acid and 2 mL of acid-ninhydrin solution were added to the tubes, and the standard solutions were incubated for 1 h in a water bath set at 100 • C.After 1 h, 4 mL of cold Toluene was added to each tube, kept in ice for 10 min, and mixed with a vortex for 20-30 s.Then, absorbance was measured at 520 nm.The concentration of each proline was determined using ten-point calibration curves of the standards ranging from 1-30 µg mL −1 , with a correlation coefficient of at least 0.9935 (proline: y = 0.0379 x − 0.0202).

Estimation of Chlorophyll and Carotenoid Content
For this purpose, 0.2 g of fresh leaf sample was weighed on a precision balance, placed in a porcelain mortar, and 0.1 g of magnesium oxide and 0.25 g of fine sand were added to it.The absorbances of the prepared samples were measured at 645, 663, and 480 nanometer wavelengths in a spectrophotometer.The contents of the chlorophyll, chlorophyll-b, total chlorophyll, and carotenoid in the fresh leaves were expressed as mg g −1 fresh weight per plant [54].
Chlorophyll-a, mg g −1 FW = [(12.The malondialdehyde (MDA) level was determined according to Heath and Packer's method [55].To do this, 0.2 g of fresh plant sample was homogenized with 2 mL of 0.1% TCA solution, centrifuged at 10,000× g for 15 min, 0.4 mL of the supernatant was transferred to clean tubes, and 0.5% TBA dissolved in 1.6 mL of 20% TCA was added.The samples, which were kept in a water bath at 95 • C for 30 min, were rapidly cooled and then centrifuged for 15 min.The supernatant was taken, and the absorbance values were read at 532 and 600 nm wavelengths in the spectrophotometer with a blank that consisted of 0.5% TBA dissolved in 20% TCA.The content of MDA in the samples was determined as nmol g FW −1 .
MDA (nmol g FW −1 ) = [(A 532 -A 600 )/155000)]10 6 , (A: absorbance, FW: fresh weight) (µmol g −1 ) MDA = (A 532 − A 600 )/(155 mM Ascorbate peroxidase (APX) activity is generally evaluated by the rate of decreased ascorbic acid.Here, APX activity was stated by measuring the ascorbate oxidation rate at 290 nm in a Shimadzu UV-1800 spectrophotometer and was measured by monitoring the oxidation of ascorbate decrease in absorbance (ε = 2.8 mM −1 cm −1 ) at 290 nm for 3 min.The reaction mixture (1 mL) consisted of 40 mM KH 2 PO 4 buffer (pH 6.0), 1 mM EDTA, 20 mM H 2 O 2 , 2.5 mM L (+) ascorbic acid, and enzyme extract [56].The procedures for the solutions prepared for analysis are detailed below: For the buffer solution (0.04 M KH 2 PO 4 ) 0.544 g of KH 2 PO 4 was weighed and dissolved in 80 mL of distilled water and its pH (7.0) was adjusted.Pure water was added to a final volume of 100 mL.
For 20 mM H 2 O 2 solution, 20 µL of 35.5% H 2 O 2 was taken, and the volume was completed to 10 mL with distilled water.
For 1 mM EDTA solution, 0.029 g EDTA was taken and dissolved in some distilled water and the volume was completed to 10 mL with distilled water.
For 2.5 mM L (+) ascorbic acid solution, 4.4 mg L (+) ascorbic acid was weighed and dissolved in some distilled water and the volume was made up to 10 mL with distilled water.

Catalase (CAT) Activity
Enzymatic activity was determined with a spectrophotometer, depending on the decreased absorbance (ε = 0.0436 mM −1 cm −1 ) value at 240 nm over time, as a result of the interaction of hydrogen peroxide with the enzyme [57].The procedures for the solutions prepared for analysis are detailed below: For the buffer solution (50 mM KH 2 PO 4 ) 0.68 g of KH 2 PO 4 was weighed and dissolved in 80 mL of distilled water and its pH (7.0) was adjusted.Pure water was added to a final volume of 100 mL.
For 120 mM H 2 O 2 solution, 114 µL of 35.5% H 2 O 2 was taken, and the volume was completed to 10 mL with distilled water.

Statistical Analysis
The experiment utilized a random plots design, and the results were reported as mean ± SD (standard deviation).The collected data were initially analyzed using the statistical package for social science (SPSS 22) software, specifically version 22 from Chicago, IL, USA.The analysis involved calculating the mean and the standard deviation and performing an analysis of variance (ANOVA).One-way ANOVA was employed to assess significant differences among the treatments, and Tukey's post hoc test was applied for further evaluation.The confidence level was set at 95% (p < 0.05) for all analyses.

Results and Discussion
3.1.Soluble Protein (SP), Proline, Chlorophyll, and Carotenoid Content Soluble protein content obtained from the G. elwesii samples treated with Zn and SA are shown in Table 1.SP was higher in the Zn 80 mM + SA 1 mM (2.17 ± 0.60 µg/g) and Zn 0 mM + SA 2 mM (2.16 ± 0.24 µg/g) treatments as compared to the control (1.84 ± 0.50 µg/g) treatment.The lowest SP was noted in the Zn 40 mM + SA 0.5 mM (1.72 ± 0.18 µg/g) treatment.When evaluated in general, significant changes were obtained in the amount of protein in all applications except the Zn 40 mM + SA 0.5 mM (1.72 ± 0.18 µg/g) treatment, while the SA treatments were found to be more effective than the Zn treatments.The results of the Zn and SA treatments to the leaves of G. elwesii on the content of proline, total chlorophyll, chlorophyll a, chlorophyll b, and carotenoid are given in Table 1.The highest proline was obtained from the SA 0.5 mM (2480 ± 0.78 µg/g) treatment when compared to the control (200.3 ± 0.11 µg/g).The lowest proline content was recorded in the Zn 80 mM (176.5 ± 0.44 µg/g) treatment.When Table 1 is reviewed, it is seen that SA 0.5 mM treatment increased the proline content 12.38-fold compared to the control.However, the proline content of Zn 80 and 120 mM showed a toxic effect and decreased by 11.75% and 36.90%,respectively, when compared to the control.The data show that a dose of SA 0.5 mM has a significant curative effect on the proline content of G. elwesii.The highest total chlorophyll Zn 120 mM + SA 2 mM was obtained from (2.81 ± 0.50 mg/g) administration compared to the control (0.78 ± 0.11 µg/g-Figure 1).The highest content of chlorophyll a was obtained from the treatment of Zn 40 mM + SA 1 mM (0.58 ± 0.10) when compared to the control (0.24 ± 0.08 mg/g).Chlorophyll b content was determined at the highest SA 0.5 mM (0.54 ± 0.05 mg/g) compared to the control (0.35 ± 0.06 mg/g).The highest content of carotenoid was detected in the SA 0.5 mM (0.57 ± 0.08 mg/g) treatment compared to the control (0.14 ± 0.01 mg/g).According to scientists, the accumulation of more than normal proline by the cell is used as a stress indicator, and for this reason, proline increase is usually discovered in plants exposed to biotic and abiotic stresses and has adapted to be a protective mechanism [58].A study conducted with Chrysanthemum balsamita L. reported that Zn treatments decreased the content of proline compared to the control group, and the lowest content of proline was obtained from 1 g L −1 Zn treatments [59].In a study that was conducted in Triticale, it was reported that the Zn fertilizers increased the content of proline, but this increase was not at a significant level [60].Behtash [61] reported that only zinc treatments did not have a significant effect on proline content, but the content of proline decreased by 337% at a dose of 10 mg L −1 Zn.According to Salim [62], Zn and chitosan treatments increased the proline content.As a result of these studies, it was revealed that the tolerance limits of plants to heavy metal toxicity vary depending on the plant species and metal species, and the content, usefulness, severity, and type of damage, as well as the damage formation process [63].Carotenoid and chlorophyll b play important protective roles in photosynthetic organisms by scavenging singlet oxygen and anion superoxide.Carotenoids are isoprenoid-based molecules that are essential for light-harvesting processes and protection against photo-oxidative damage.They have a high ability to quench singlet oxygen, which is a physical phenomenon.In addition, carotenoids can interact with radical species to form carotenoid-radical intermediates, which can then be converted into other compounds.Chlorophyll b also plays a role in scavenging ROS, as it is part of the photosynthetic electron transport chain that helps to reduce the production of ROS.Both carotenoids and chlorophyll b are important antioxidants that help protect photosynthetic organisms from oxidative damage caused by ROS [64].In a study conducted with Chrysanthemum balsamita L., it was reported that Zn treatments increased the content of chlorophyll compared to the control, but this increase was not at a significant level [59].In a study that was conducted in Triticale, it was reported that Zn fertilizers increased the content of chlorophyll [60].In another study, it was reported that Zn treatments improved the total chlorophyll, chlorophyll a, and chlorophyll b content, but decreased the carotenoid content.This study showed that only Zn treatments decreased the total content of chlorophyll when compared to the control, but increased the content of chlorophyll b; however, this increase was not at a significant level.Also, as can be seen in Table 1, the SA 0.5 had a curative effect on the content of chlorophyll b and carotenoids.

Lipid Peroxidation and Antioxidant Enzyme Activity
The results of Zn and SA treatments to the leaves of G. elwesii on the content of MDA are given in Table 2.The highest content of MDA was obtained from the Zn 40 mM + SA 2 mM (1.84 ± 0.28 nmol/g) treatment compared to the control (1.05 ± 0.10 nmol/g).As can be understood from the related Table, the Zn 40 mM + SA 2 mM treatment increased the content of MDA 1.75-fold compared to the control.The treatments did not have a significant effect on the content of MDA.Behtash [61] reported that only Zn treatments did not have a significant effect on MDA content, but the content of MDA decreased by 27%, at a dose of 10 mg L −1 Zn.As a result of these studies, it was revealed that the tolerance limits of plants to heavy metal toxicity vary depending on the plant species and metal species and the content, usefulness, severity, and type of damage, as well as the damage formation process [63].The antioxidant enzyme activities of G. elwesii leaves were determined using the APX and CAT enzyme activity methods.The results showed that the highest APX enzyme activity was obtained from the Zn 120 mM treatment (3.99 ± 0.58 EU/mg), followed by the Zn 40 mM + SA 0.5 mM (3.52 ± 0.18 EU/mg) treatment and the Zn 120 mM + SA 0.5 mM (2.86 ± 0.48 EU/mg) treatment.The lowest APX enzyme activity was recorded in the control (0.16 ± 0.62 EU/mg).This suggests that Zn treatment can significantly increase the APX enzyme activity in G. elwesii leaves (Table 2).When Table 2 is reviewed, it is seen that the treatment of Zn 120 mM increased the APX enzyme activity 25.5-fold compared to the control.The highest CAT enzyme activity was obtained from the treatment of Zn 80 mM (154.6 ± 4.10 EU/mg), which was 13.1-fold higher than that of the control (11.76 ± 0.15 EU/mg).However, it was also found that the CAT enzyme activity decreased by 30.16% in the plots where Zn 120 mM was applied.This suggests that high concentrations of Zn can be toxic to G. elwesii leaves and can lead to a decrease in CAT enzyme activity.Previous studies have shown that Zn can act as a cofactor of many antioxidant enzymes, which can help reduce the accumulation of ROS in plant tissues [65,66].Our results are consistent with these findings, and they suggest that Zn treatment can be an effective way to increase the antioxidant capacity of G. elwesii leaves.However, it is important to note that high concentrations of Zn can be toxic to plants.Aery and Sarkar [67] reported that Zn can inhibit the activity of the CAT enzyme, which can lead to increased oxidative stress in plants.Our results suggest that this is also the case for G. elwesii leaves and that high concentrations of Zn should be avoided.The activation of enzymatic defense systems in plants allows them to control ROS production and reduce oxidative damage.For example, a study by Chakhchar [68] found that zinc stress significantly induced the enzymatic defense system (APX and CAT) of snowdrop plants.This induction was further enhanced by the exogenous application of SA.Similarly, Imran [69] reported that SA improved the activity of the enzymatic defense system in mung bean seedlings by increasing the activity of CAT, POD, and APX under cadmium stress conditions.Increased enzymatic antioxidant activity, along with higher levels of proline, speeds up the detoxification of H 2 O 2 , which helps to alleviate oxidative damage.This has been shown in several studies, including Chakhchar [58] and Faraz et al. [70].SA also lessens the effects of oxidative stress brought on by Zn by further accelerating the antioxidant machinery in plants.This has been reported in several studies, including [71][72][73].Exogenous application of salicylic acid (SA) improved the tolerance of chia seedlings under zinc stress by protecting them against oxidative damage.This was evident from the decreased levels of malondialdehyde (MDA), a marker of oxidative damage.Several studies have shown that SA can mitigate zinc-induced damage by lowering reactive oxygen species (ROS) levels and bolstering the antioxidant defense systems in plants.For example, Sharma et al. [74] found that SA treatment decreased ROS levels and increased the activities of antioxidant enzymes in chickpea (Cicer arietinum) seedlings under zinc stress.Aydin et al. [75] reported similar findings in cucumber (Cucumis sativus) seedlings.Marichali et al. [76] and Yahaghi et al. [77] also showed that SA treatment protected alfalfa (Medicago sativa L.) seedlings from zinc-induced oxidative damage.In our study, we found that SA treatment at 0.8 mM significantly decreased MDA levels and increased the activities of antioxidant enzymes in chia seedlings under zinc stress.These results suggest that SA may have protective effects against zinc-induced oxidative damage by enhancing antioxidant defense and osmoprotectant capacity.

Lipid Peroxidation and Antioxidant Enzyme Activity
The results of Zn and SA treatments to the leaves of G. elwesii on the content of MDA are given in Table 2.The highest content of MDA was obtained from the Zn 40 mM + SA 2 mM (1.84 ± 0.28 nmol/g) treatment compared to the control (1.05 ± 0.10 nmol/g).As can be understood from the related Table, the Zn 40 mM + SA 2 mM treatment increased the content of MDA 1.75-fold compared to the control.The treatments did not have a significant effect on the content of MDA.Behtash [61] reported that only Zn treatments did not have a significant effect on MDA content, but the content of MDA decreased by 27%, at a dose of 10 mg L −1 Zn.As a result of these studies, it was revealed that the tolerance limits of plants to heavy metal toxicity vary depending on the plant species and metal species and the content, usefulness, severity, and type of damage, as well as the damage formation process [63].The antioxidant enzyme activities of G. elwesii leaves were determined using the APX and CAT enzyme activity methods.The results showed that the highest APX enzyme activity was obtained from the Zn 120 mM treatment (3.99 ± 0.58 EU/mg), followed by the Zn 40 mM + SA 0.5 mM (3.52 ± 0.18 EU/mg) treatment and the Zn 120 mM + SA 0.5 mM (2.86 ± 0.48 EU/mg) treatment.The lowest APX enzyme activity was recorded in the control (0.16 ± 0.62 EU/mg).This suggests that Zn treatment can significantly increase the APX enzyme activity in G. elwesii leaves (Table 2).When Table 2 is reviewed, it is seen that the treatment of Zn 120 mM increased the APX enzyme activity 25.5-fold compared to the control.The highest CAT enzyme activity was obtained from the treatment of Zn 80 mM (154.6 ± 4.10 EU/mg), which was 13.1-fold higher than that of the control (11.76 ± 0.15 EU/mg).However, it was also found that the CAT enzyme activity decreased by 30.16% in the plots where Zn 120 mM was applied.This suggests that high concentrations of Zn can be toxic to G. elwesii leaves and can lead to a decrease in CAT enzyme activity.Previous studies have shown that Zn can act as a cofactor of many antioxidant enzymes, which can help reduce the accumulation of ROS in plant tissues [65,66].Our results are consistent with these findings, and they suggest that Zn treatment can be an effective way to increase the antioxidant capacity of G. elwesii leaves.However, it is important to note that high

Conclusions
In this research, the impact of treating G. elwesii species' leaves with SA and Zn on various aspects, such as antioxidant enzyme activity and proline, chlorophyll, and carotenoid content, was examined.The outcomes showed a remarkable 25.5-fold rise in APX enzyme activity with Zn treatment at 120 mM and a 13.1-fold increase in CAT enzyme activity with Zn treatment at 80 mM.Interestingly, APX enzyme activity displayed a linear growth pattern across all tested Zn doses.While CAT enzyme activity notably increased with Zn treatment up to a dose of 80 mM, the highest dose of Zn (120 mM) combined with SA (2 mM) led to a substantial 30.16% decline in CAT enzyme activity compared to the control group.Moreover, SA treatments at 2 mM resulted in a reduction of 68.19% and 70.73% in APX and CAT enzyme activities, respectively.Particularly noteworthy is the significant augmentation in proline content due to SA treatments in G. elwesii species when compared to the control.The most substantial increase of 12.4-fold was observed in response to the treatment with 0.5 mM SA.Interestingly, only Zn treatments were associated with a decrease in total chlorophyll content compared to the control group.However, they also prompted an increase in chlorophyll b content, though this increase did not reach statistical significance.Additionally, a dosage of 0.5 mM SA displayed a therapeutic effect on chlorophyll b and carotenoid content.
The results show that the use of SA and Zn treatments can effectively increase antioxidant enzyme activity, which is responsible for plant defense, and the contents of proline, chlorophyll b, and carotenoids, which are responsible for yield and quality, in G. elwesii.Furthermore, SA treatments exhibited a noteworthy boost in proline, chlorophyll b, and carotenoid content, particularly at a dosage of 0.5 mM SA.

Figure 1 .
Figure 1.Effect of different doses of zinc and salicylic acid applied to snowdrop plants on total chlorophyll (mg/g FW), chlorophyll a (mg/g FW), and chlorophyll b (mg/g FW), a f * = mM b a, b, c…=Letters such as a, b, c, d, e, f indicate statistical difference between the data.

3Figure 1 .
Figure 1.Effect of different doses of zinc and salicylic acid applied to snowdrop plants on total chlorophyll (mg/g FW), chlorophyll a (mg/g FW), and chlorophyll b (mg/g FW), a f * = mM b a, b, c. . .= Letters such as a, b, c, d, e, f indicate statistical difference between the data.

70 h 9 .
500 ± 0.56 n a The results are expressed as means ± standard deviation (n = 6); b EU = equivalent; c FW = fresh weight; d Zn = Zinc; e SA = Salicylic acid; f * = mM; g a, b, c. . .= Letters such as a, b, c, d, e, f indicate statistical difference between the data; h Means followed by the same letter in the same column do not differ statistically at p ≤ 0.05 according to the Tukey test.

Table 1 .
Effect of different doses of zinc and salicylic acid applied to snowdrop plants on the soluble protein (µg/g FW), proline (µg/g FW), and carotenoid content (mg/g FW).

Table 2 .
The effects of zinc and salicylic acid at different doses applied to the snowdrop plant on the malondialdehyde content (nmol/g FW), ascorbate peroxidase activity (EU/mg protein), and catalase activity (EU/mg protein).