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Research Article
2026
:38;
13912025
doi:
10.25259/JKSUS_1391_2025

Regulation of proline metabolism and antioxidant system by tocopherol and cysteine mitigates salt stress in fenugreek

,
aLaboratory RME – Resources, Materials and Ecosystems, Faculty of Sciences of Bizerte, University of Carthage, Jarzouna, Bizerte 7021, Tunisia
bBiological Sciences Department, Faculty of Science and Arts, King Abdulaziz University, Rabigh, 21911, Saudi Arabia

* Corresponding author: E-mail address: houneida_attia@yahoo.fr (H Attia)

Received: , Accepted: ,
© 2026 Journal of King Saud University – Science - Published by Scientific Scholar
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This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial-Share Alike 4.0 License, which allows others to remix, transform, and build upon the work non-commercially, as long as the author is credited and the new creations are licensed under the identical terms.

Abstract

The effect of exogenously supplied α-tocopherol (200 µM Toc) and cysteine (500 µM Cys) was explored in reducing the NaCl triggered growth alteration in Trigonella foenum-graecum. Salt stress reduced the growth characteristics like height, fresh, and dry weight of the shoot and root; however, the Toc and Cys application alleviated the decline. The application of Toc and Cys mitigated the reduction of chlorophylls and carotenoids due to NaCl stress. The salt stress induced decline in the content of nitrogen and potassium was alleviated by the applied Toc and/or Cys. The content of sodium was less in Toc and Cys treated seedlings. NaCl stress significantly increased the oxidative stress attributes like hydrogen peroxide and lipid peroxidation; however, foliar application of Toc and Cys reduced these parameters and also reduced the activity of lipoxygenase (LOX) and NADPH oxidase, with the highest decline exhibited by plants receiving their combined treatment. Exogenous Toc and Cys differentially regulated the proline metabolism by increasing the activity of pyrroline-5-carboxylate synthetase (P5CS) and γ-glutamyl kinase (γ-GK), and decreasing the activity of proline oxidase (PROX). In addition, salt stress increased the activity of enzymes of the antioxidant system (SOD by 95.51%, APX by 126.21%, MDHAR by 85.68%, DHAR by 99.67%, and GR by 132.06%) and the enzymes of the glyoxylase cycle (glyoxylase I by 104.94% and glyoxylase II by 100.25%), and exogenous Toc and Cys caused further increase in their activities. The content of methionine, glutathione, ascorbic acid (AsA), Toc, and Cys increased due to exogenous Toc and Cys treatments, further increasing the functioning of the antioxidant system. Salt stress increased the endogenous concentrations of nitric oxide (NO) by 98.16% and hydrogen sulfide (HS) by 145.55% however, treatment of Toc and/or Cys to NaCl-treated plants imparted a decline in NO and HS. Hence, the exogenous Toc and/or Cys treatment can be exploited to benefit fenugreek growth and metabolism under salt stress.

Keywords

Glyoxylase
NaCl stress
Osmoregulation
Oxidative stress
Tolerance mechanisms
Trigonella foenum-graecum

1. Introduction

Salt stress is the abiotic factor that affects the growth and yield of plants all over the globe. Salinity is usually considered to occur due to greater accumulation of salts, mainly sodium, chloride, phosphates, etc. This increase in their accumulated concentration can result from excess fertilizer application, use of polluted water for irrigation, improper drainage, etc. (Hassani et al., 2021; Basak et al., 2022). Salinity restricts germination, root growth, and mineral uptake. It has been observed that plants grown under excess salinity exhibit a significant decline in chlorophyll and rubisco activity, photosynthetic attributes, enzyme functioning, mineral assimilation, and yield (Iqbal et al., 2015; Soliman et al., 2020). The main reason for salinity-induced growth and metabolism hindrance includes the increased buildup of toxic ions like Na in the organelles, including chloroplasts, mitochondria, etc. and the subsequent generation of excess reactive oxygen radicals (Fatma et al., 2016; Hasanuzzaman et al., 2014, 2019). These reactive radicals pose significant damage to lipids and proteins, therefore affecting the assembly of cellular membranes and enzyme functioning (Ahanger et al., 2017). To lessen the damage caused by excess salinity, certain mechanisms exist in plants that work to maintain the concentration of reactive radicals at beneficial levels and also contribute to sequester the accumulated toxic ions (Mostofa et al., 2015; Al-Mushhin 2022). The functioning of the antioxidant system, accumulation of osmolytes, and the efficient compartmentation of toxic ions have been reported to assist in mitigating the negative impact of salinity to considerable levels (Mostofa et al., 2015). Tocopherol (Toc) and cysteine (Cys) are important metabolites that actively participate in growth regulation and the mitigation of ill effects of stresses, including salinity (Kim et al., 2022; Akram et al., 2023).

Cys is a naturally occurring amino acid, the first compound formed in primary sulfate metabolism, and participates in protein synthesis (Takahashi et al., 2011). The formation of the sulfhydryl group makes Cys different from other amino acids. Cys acts as a precursor for several important biomolecules, including cofactors, vitamins, Fe-S clusters, glucosinolates, glutathione, polyamines, thiol-containing proteins, and phytoalexins (Meyer & Hell, 2005; Romero et al., 2014; Ingrisano et al., 2023). Plants accumulating increased Cys content tolerate salinity stress better, as reported by Van Nguyen et al. (2021) in Arabidopsis thaliana. Greater accumulation of Cys has been reported in mustard (Fatma et al., 2016; Xue et al., 2024) and wheat (Qin et al., 2024a) due to NaCl, cadmium, and arsenic stress. Applied Cys has been reported to improve tolerance to chromium in maize by up-regulating antioxidant functioning and the expression of proteins regulating the key metabolic pathways (Terzi & Yildiz, 2021). In Linum usitatissimum, application of Cys prevented the decline in growth and photosynthesis by regulating tolerance mechanisms (Hussein & Alshammari, 2022). On the other hand, Toc is an important vitamin involved in ROS scavenging, antioxidant, maintaining cellular redox, signalling cascade, photoprotection, and tolerance against stresses (Faizan et al., 2023). Vitamin E is fat-soluble, and tocotrienols act as its precursors, which have a strong antioxidant property (Hasegawa et al., 2000). Toc is the most abundant form found in photosynthetic tissues. Toc is mainly synthesized in plastids; however, its accumulation varies with plant species and plant part as well (Hasanuzzaman et al., 2014). Toc quench lipid peroxides and singlet oxygen, coordinate with antioxidants like ascorbic acid (AsA) and also interact with phytohormones, including ethylene, jasmonic acid, abscisic acid, etc. to prevent the damage to plants under stress (Hasanuzzaman et al., 2014; Ghosh et al., 2022). The external supplementation of Toc to Solanum melongena L. lessened the oxidative stress triggered by drought by improving the antioxidant functioning and the osmolyte concentration (Akram et al., 2023). Till date, no research report is available about the interactive role of exogenously applied Cys and Toc on the regulation of tolerance mechanisms under salt stress.

Fenugreek (Trigonella foenum-graecum) is an important legume crop plant that is cultivated worldwide and is consumed as a vegetable or added as a spice to improve the flavor. It has good medicinal importance as anti-carcinogenic, antidiabetic, hypocholesterolemic, and immunological activity. Fenugreek seeds are rich in carbohydrates, amino acids, alkaloids, and other important organic and inorganic substances (Visuvanathan et al., 2022; Ahmad et al., 2023). Globally, the growth and productivity of fenugreek is significantly affected by increasing salinity. In this backdrop, the present study investigated the beneficial role of exogenously applied Cys and Toc (individual and combined) in improving the tolerance mechanism to counteract the salinity-mediated growth decline.

2. Materials and Methods

Healthy seeds of fenugreek (Trigonella foenum-graecum Saudi cultivar) were sterilized by 0.001% HgCl2 for 5 min. Thereafter, seeds were washed five times with distilled water and blotted dry. The seeds were sown in pots containing sand, soil, and compost (4:3:1). Two weeks after germination, 15 uniform plants were maintained in each pot, and pots were divided into two groups. One group of pots was supplied with a normal nutrient solution, while the other was supplied with a modified nutrient solution containing 100 mM NaCl to initiate the salt stress. Composition of the Hoagland nutrient solution used was: 3 mM KNO3, 2 mM Ca(NO3)2, 2 mM MgSO4, 1 mM NH4H3PO4, 50 µM KCl, 25 µM H3BO4, 2 µM MnCl2, 20 µM ZnSO4, 0.5 µM CuSO4, 0.5 µM (NH4)6Mo7O24, and 20 µM Na2Fe-EDTA (Alamer et al., 2025). In both groups (normal and NaCl), pots were foliarly supplied with Toc, Cys, and Toc + Cys onto the foliage using a manual sprayer; however, control pots were maintained without any foliar treatment. Concentration of Toc and Cys used was 200 and 500 µM, respectively, and the volume sprayed was 15 mL per pot. Treatment of salinity of Toc and Cys continued for 4 weeks. For every treatment, there were four pots, and pots were kept and maintained in a greenhouse at 10/14 h light/dark and day/night temperature of 26/15°C; 6-week-old plants were analyzed for different parameters described below.

2.1 Measurement of growth parameters

Different growth parameters were estimated, including the plant height, fresh weight, and dry weight of shoot and root. The height of plants was determined using a scale. Plants were uprooted and separated into root and shoot, and their fresh weight was recorded. However, dry weight was estimated after the tissue was dried in an oven at 60°C for 72 h.

2.2 Estimation of pigments

The photosynthetic pigments (chlorophyll and carotenoids) were extracted by macerating 100 mg of fresh leaf in acetone. Centrifugation of the extract was done at 3000 g for 20 min, and the optical density of the supernatant was recorded at 663, 645, and 480 nm (Lichtenthaler, 1987).

2.3 Determination of oxidative stress parameters

The method described by Velikova et al. (2000) was adopted to determine the hydrogen peroxide, and 100 mg of fresh leaf tissue was homogenized in 0.1% trichloroacetic acid (TCA). The centrifugation was done for 10 min at 10,000 g, and the absorbance of supernatant along with potassium phosphate buffer (pH 7.0) and potassium iodide was taken at 390 nm. The lipid peroxidation was determined according to Heath and Packer (1968), and the malonaldehyde (MDA) formation was measured after reacting the extract with 0.5% thiobarbituric acid. Optical density was taken at 532 and 600 nm.

The activity of LOX was measured according to the method of Doderer et al. (1992) using linoleic acid as substrate. Change in the absorbance was measured at 234 nm. The activity of NADPH oxidase was measured according to Sagi and Fluhr (2001). Assay mixture was Tris-HCl buffer (50 mM; pH 7.5), 3′-[1-[phenylamino-carbonyl]-3, 4-tetrazolium]-bis(4-methoxy-6-nitro) benzenesulfonic acid hydrate (0.5 mM XTT), NADPH (100 µM), and membrane proteins. The absorbance was measured at 470 nm.

2.4 Measurement of the activity of proline metabolizing enzymes and proline content

Among the proline metabolizing enzymes assayed were: pyrroline-5-carboxylate synthetase (P5CS), γ-glutamyl kinase (γ-GK), and proline oxidase (PROX). 500 mg of fresh leaf was homogenised in cold Tris-HCl buffer (100 mM; pH 7.5) in a prechilled pestle and mortar. After centrifuging the homogenate for 30 min at 30,000 g, the supernatant was used for measuring the activity of P5CS, while γ-GK and PROX activities were determined in the pellet. Activities of P5CS and γ-GK were determined according to Hayzer and Leisinger (1980). For P5CS, the optical density was recorded at 340 nm. The assay mixture for measuring the activity of γ-GK contained 50 mM Tris buffer (pH 7.0), ATP, MgCl2, hydroxamate-HCl, L-glutamate, and enzyme. After incubating for 30 min at 37°C, FeCl3 and TCA (prepared in HCl) were added to stop the reaction, and the absorbance was taken at 535 nm. Activity of PROX was measured according to Huang and Cavalieri (1979). Assay mixture contained Tris buffer (50 mM; pH 8.5), MgCl2 (5 mM), 0.5 mM NADP, 0.06 mM phenazine methanosulfate, dichlorophenol indophenol, 1 mM KCN, and 0.1 M proline. Proline triggered the reaction, and optical density was noted at 600 nm for 3 min. For estimating the proline, tissue was extracted in sulphosalicylic acid and centrifuged at 3000 g for 20 min. Glacial acetic acid and nihydrin reagent were added to the supernatant, and the samples were incubated for 1 h at 100°C. Proline was extracted using toluene, and the absorbance was recorded at 520 nm (Bates et al., 1973).

2.5 Measurement of antioxidant enzyme activities

The antioxidant enzymes were extracted by macerating the fresh 500 mg leaf tissue in 100 mM phosphate buffer (pH 7.8), having polyvinyl pyrolidine (1%), EDTA (0.1 mM), and PMSF (0.1 mM). Centrifugation of the extract was carried out at 12,000 g for 15 min, and the supernatant was used for assaying the antioxidant enzyme activities. Activity of superoxide dismutase (SOD) was measured using the Bayer and Fridovich (1987) method, and absorbance was noted at 560 nm. Ascorbate peroxidase (APX) activity was determined according to Nakano and Asada (1981), and the change in absorbance was noted at 290 nm. For determining glutathione reductase (GR) activity, glutathione-dependent oxidation of NADPH was measured at 340 nm for 2 min (Foyer and Halliwal 1976). The method described by Nakano and Asada (1981) was used to measure the dehydroascorbate reductase (DHAR) activity, while the method of Hossain et al. (1984) was used for measuring monodehydroascorbate reductase (MDHAR) activity. Protein was determined by Lowry et al. (1951).

2.6 Determination of nitric oxide (NO) and hydrogen sulfide (HS)

The content of NO was measured following Zhou et al. (2005). Briefly, the fresh 500 mg leaf tissue was extracted in acetic acid buffer (50 mM; pH 3.6) and centrifuged at 11,500 g for 15 min. A pinch of charcoal was used to neutralize the supernatant, followed by the addition of Griess reagents. The optical density was noted at 540 nm, and the calculation was done using the standard curve of sodium nitrite. The method of Xie et al. (2014) was employed to estimate the HS. Fresh leaf tissue was extracted in 20 mM Tris-HCl buffer (pH 6.8) and 10 mM EDTA. Centrifugation was done for 15 min at 12,000 g, and 1% zinc acetate (w/v) was added to the supernatant. After 30 min, dimethyl-p-phenylenediamine and ferric chloride were added, and the absorbance was noted at 670 nm. Calculations were done from the standard curve of NaHS.

2.7 Estimation of elements

For the estimation of nitrogen, the micro-Kzeldahl method of Jackson (1973) was followed, and plant samples were digested in sulfuric acid; however, Na and K were estimated using a flame photometer.

2.8 Assay of glyoxalase I and glyoxylase II activities

For extracting the glyoxylase enzymes, fresh tissue was homogenized in extraction buffer (pH 7.0) containing potassium chloride, ascorbate, β-mercaptoethanol, and glycerol. Homogenate was centrifuged for 15 min at 11,500g, and the supernatant was used as the enzyme for assaying the activities of glyoxylase enzymes. For measuring the activity of glyoxylase I, the method described by Hasanuzzaman et al. (2011) was followed, and the absorbance was noted at 240 nm. The method described by Principato et al. (1987) was employed to assay the activity of glyoxylase II by measuring the formation of reduced glutathione (GSH) at 412 nm.

2.9 Determination of nonenzymatic antioxidants/redox components

Among the redox components estimated, including the determination of GSH, Cys, AsA, Toc, and methionine, were also determined in normal and stressed plants. Standard methods were employed for the estimation of AsA (Mukherjee and Choudhuri, 1983), GSH (Ellman, 1959), Cys (Gaitonde, 1967), Toc (Backer et al., 1980), and methionine (Horn et al., 1946).

2.10 Statistical analysis

The data is given as mean (±SE) of three replicates. The significance of data was tested using ANOVA, and the least significant difference (LSD) was calculated at p <0.05 using SPSS 17.0 for windows. Data followed by the same letter are not significantly different by LSD test at p < 0.05.

3. Results

3.1 Treatment of Toc and Cys mitigates the decline in morphological parameters

Salinity reduced growth parameters, including shoot length (38.88%), root length (49.07%), shoot fresh weight (45.87%), shoot dry weight (49.66%), root fresh weight (33.78%), and root dry weight (33.15%) as compared to control. Application of Toc and/or Cys alleviated the decline and also increased these parameters in unstressed plants. Compared to control, the highest increase in shoot length, root length, shoot fresh weight, shoot dry weight, root fresh weight, and root dry weight was 38.69%, 36.39%, 37.69%, 56.02%, 61.94%, and 62.61%, respectively, in plants treated with both Toc and Cys. However, in NaCl + Toc + Cys, the decline in shoot length was 9.47%, root length was 17.83%, shoot fresh weight was 18.29%, shoot dry weight was 18.70%, root fresh weight was 15.00%, and root dry weight was 12.24% over the control (Table 1).

Table 1. Shoot length, root length, shoot fresh weight, shoot dry weight, root fresh weight, and root dry weight of Trigonella foenum-graecum treated with Toc, Cys, and Toc + Cys under normal and NaCl stress. Mean (±SE) of three replicates has been given, and different letters depict the significant difference at p <0.05.
Shoot length (cm) Root length (cm) Shoot Fresh weight (g) Shoot dry weight (g) Root fresh weight (g) Root dry weight (g)
Control 16.46 ±0.42d 4.66 ±0.28c 3.263 ±0.308c 1.237 ±0.096c 1.080 ±0.054c 0.203 ±0.011d
Toc 18.63 ±0.95c 5.06 ±0.17b 3.700 ±0.200b 1.449 ±0.052b 1.218 ±0.084b 0.263 ±0.020c
Cys 20.16 ±1.24b 5.20 ±0.26b 3.916 ±0.322b 1.477 ±0.065b 1.289 ±0.112b 0.292 ±0.007b
Toc + Cys 22.83 ±1.82a 6.36 ±0.44a 4.493 ±0.408a 1.930 ±0.126a 1.749 ±0.170a 0.330 ±0.020a
NaCl 10.06 ±0.77h 2.37 ±0.08g 1.766 ±0.155f 0.622 ±0.063f 0.715 ±0.057g 0.136 ±0.010h
NaCl + Toc 12.20 ±0.73g 2.98 ±0.31ef 2.026 ±0.115e 0.915 ±0.019e 0.807 ±0.024f 0.149±0.003 g
NaCl + Cys 13.66 ±0.42f 3.30 ±0.26e 2.100 ±0.200e 0.898 ±0.030e 0.873 ±0.020e 0.164 ±0.004f
NaCl + Toc + Cys 14.90 ±0.73e 3.83 ±0.24d 2.666 ±0.155d 1.005 ±0.136d 0.918 ±0.019d 0.178 ±0.003e

It is noted in each table caption that different letters depict the significant difference at p<0.05.

3.2 Toc and Cys application alleviated the decline in chlorophyll pigments

Salinity significantly declined the pigments, including chlorophyll a, chlorophyll b, total chlorophylls, and carotenoids, and the supplementation of Toc and Cys mitigated the decline to some extent. Percent decline observed due to NaCl was 41.14%, 47.09%, 45.04%, and 53.29% for chlorophyll a, chlorophyll b, total chlorophylls, and carotenoids, respectively, over the control. In control-grown plants, applied Toc and Cys increased the pigments, with the highest in plants that received both Toc and Cys. Application of Toc and Cys alleviated the NaCl-induced decline in pigments. In NaCl + Toc + Cys treated plants, the reduction in chlorophyll a was 14.34%, chlorophyll b was 30.83%, total chlorophylls was 23.19%, and carotenoids was 12.56% as compared to control (Table 2).

Table 2. Chlorophyll a, chlorophyll b, total chlorophyll, and carotenoid content of Trigonella foenum-graecum treated with Toc, Cys, and Toc + Cys under normal and NaCl stress. Mean (±SE) of three replicates has been given, and different letters depict the significant difference at p <0.05.
Chlorophyll a (mg g-1 FW) Chlorophyll b (mg g-1 FW) Total chlorophyll (mg g-1 FW) Carotenoids (mg g-1 FW)
Control 0.522 ±0.022d 0.260 ±0.011d 0.808 ±0.010d 0.244 ±0.015d
Toc 0.612 ±0.011c 0.318 ±0.013c 0.940 ±0.034c 0.297 ±0.009c
Cys 0.718 ±0.029b 0.392 ±0.011b 1.173 ±0.109b 0.347 ±0.006b
Toc + Cys 0.796 ±0.003a 0.441 ±0.006a 1.276 ±0.062a 0.373 ±0.016a
NaCl 0.307 ±0.017h 0.137 ±0.007h 0.444 ±0.015h 0.114 ±0.012h
NaCl + Toc 0.345 ±0.017g 0.155 ±0.004g 0.519 ±0.019g 0.146 ±0.003g
NaCl + Cys 0.402 ±0.023f 0.165 ±0.002f 0.569 ±0.007f 0.194 ±0.011f
NaCl + Toc + Cys 0.447 ±0.017e 0.180 ±0.006e 0.620 ±0.018e 0.213 ±0.009e

It is noted in each table caption that different letters depict the significant difference at p<0.05.

3.3 Influence of applied Toc and Cys application on oxidative stress parameters

Salinity triggered the oxidative stress in Trigonella foenum-graecum by enhancing the concentration of radicals and the rate of lipid peroxidation. The salt-treated plants exhibited an increase of 121.30% in hydrogen peroxide and 159.76% in lipid peroxidation over the control. However, these parameters were reduced due to Toc and Cys application exhibiting a decline of 25.84% and 16.56% due to Toc treatment, 27.94% and 24.01% due to Cys treatment, and 41.18% and 45.54% due to Toc + Cys treatment. Treatment of Toc and/or Cys to salt-stressed plants reduced both hydrogen peroxide and lipid peroxidation, with the highest decline observed in plants that received both Toc and Cys. Contrary to the NaCl-treated plants, a decline of 38.17% and 40.12% in hydrogen peroxide and lipid peroxidation, respectively, was observed in plants treated with NaCl + Toc + Cys (Fig. 1).

(a) hydrogen peroxide and (b) lipid peroxidation in Trigonella foenum-graecum treated with Toc, Cys, and Toc + Cys under normal and NaCl stress conditions. Mean (±SE) of three replicates has been given, and different letters on bars depict the significant difference at p <0.05.
Fig. 1.
(a) hydrogen peroxide and (b) lipid peroxidation in Trigonella foenum-graecum treated with Toc, Cys, and Toc + Cys under normal and NaCl stress conditions. Mean (±SE) of three replicates has been given, and different letters on bars depict the significant difference at p <0.05.

In addition, reduced oxidative stress due to Toc and Cyst application was evident as a significant decline in the activity of LOX and NADPH oxidase. Application of Toc and/or Cys reduced the activity of LOX and NADPH oxidase, with the maximum decline of 46.91% and 48.64% exhibited by plants receiving both Toc and Cys over the control. Plants stressed with NaCl displayed a surge of 109.69% and 126.45% in LOX and NADPH oxidase activity, respectively. Treatment of Toc and Cys to NaCl stressed fenugreek plants imparted a reduction in activity of LOX and NADPH oxidase by 14.75% and 13.39% in NaCl + Toc, by 28.02% and 25.98% in NaCl + Cys, and by 36.69% and 38.57% in NaCl + Toc + Cys as compared to NaCl stressed plants (Fig. 2).

Activity of (a) LOX and (B) NADPH oxidase in Trigonella foenum-graecum treated with Toc, Cys, and Toc + Cys under normal and NaCl stress conditions. Mean (±SE) of three replicates has been given, and different letters on bars depict the significant difference at p <0.05.
Fig. 2.
Activity of (a) LOX and (B) NADPH oxidase in Trigonella foenum-graecum treated with Toc, Cys, and Toc + Cys under normal and NaCl stress conditions. Mean (±SE) of three replicates has been given, and different letters on bars depict the significant difference at p <0.05.

3.4 Treatment of Toc and Cys modulates proline synthesis

The content of proline was augmented by the treatment of Toc and Cys in normal and NaCl-treated plants. Increase in proline was 129.12% due to NaCl, 23.44% due to Toc, 26.92% due to Cys, and 51.76% due to Toc + Cys relative to control. The highest increase of 257.17% was detected in NaCl + Toc + Cys-treated plants, as opposed to the control. The activity of P5CS and γ-GK was increased due to Toc and Cys application, while a decline in the activity of PROX was observed. Salt stress augmented the activity of P5CS and γ-GK by 101.41% and 90.09%, respectively, and a decline of 50.66% was observed in PROX activity over the control plants. Contrary to control, the highest increase of 238.65% and 166.11% in P5CS and γ-GK, respectively, and a decrease of 82.42% in PROX activity was observed in plants treated with NaCl, Toc, and Cys (Fig. 3). In normal grown plants, an increase of 43.94% and 31.43% in P5CS and γ-GK, respectively, was observed by the combined application of Toc and Cys (Fig. 3).

Content of (a) proline and the activity of (b) proline-5-carboxylate synthetase, (c) GK, and (d) PROX in Trigonella foenum-graecum treated with Toc, Cys, and Toc + Cys under normal and NaCl stress conditions. Mean (±SE) of three replicates has been given, and different letters on bars depict the significant difference at p <0.05.
Fig. 3.
Content of (a) proline and the activity of (b) proline-5-carboxylate synthetase, (c) GK, and (d) PROX in Trigonella foenum-graecum treated with Toc, Cys, and Toc + Cys under normal and NaCl stress conditions. Mean (±SE) of three replicates has been given, and different letters on bars depict the significant difference at p <0.05.

3.5 Effect of Toc and Cys on the endogenous concentration of NO and HS

Results showing the effect of NaCl and applied Toc and Cys on the endogenous NO and HS concentrations have been given in Fig. 4. Salinity treatment increased NO by 98.16% and HS by 145.55% over the control plants. In normal grown plants, applied Toc increased NO and HS by 9.30% and 12.60%, Cys increased by 10.32% and 22.34%, while Toc + Cys increased by 25.06% and 41.18% over control. Treatment of Toc and Cys to NaCl-stressed plants resulted in a decline of NO and HS. Compared to NaCl-treated plants, a decline of 20.66% and 23.22% was observed in NO and HS in plants treated with NaCl + Toc + Cys (Fig. 4).

Concentration of (a) NO and (b) HS in Trigonella foenum-graecum treated with Toc, Cys, and Toc + Cys under normal and NaCl stress conditions. Mean (±SE) of three replicates has been given, and different letters on bars depict the significant difference at p <0.05.
Fig. 4.
Concentration of (a) NO and (b) HS in Trigonella foenum-graecum treated with Toc, Cys, and Toc + Cys under normal and NaCl stress conditions. Mean (±SE) of three replicates has been given, and different letters on bars depict the significant difference at p <0.05.

3.6 Influence of Toc and Cys application up-regulates glyoxalase cycle functioning

The activities of glyoxylase I and II were enhanced by 104.94% and 100.25% due to salinity stress, and exogenous Toc and Cys resulted in a further increase in their activities. Relative to control, in unstressed plants, combined Toc and Cys application increased glyoxylase I by 58.69% and glyoxylase II by 58.10%. Contrary to NaCl-treated plants, the activity of glyoxylase I and II increased by 11.57% and 9.039% in NaCl + Toc and by 38.13% and 21.11% in NaCl + Cys. The highest increase observed in the activities of glyoxylase I and II was 221.73% and 163.41%, respectively, in plants treated with NaCl + Toc + Cys over the control (Fig. 5).

Activity of (a) glyoxylase I and (b) glyoxylase II in Trigonella foenum-graecum treated with Toc, Cys, and Toc + Cys under normal and NaCl stress conditions. Mean (±SE) of three replicates has been given, and different letters on bars depict the significant difference at p <0.05.
Fig. 5.
Activity of (a) glyoxylase I and (b) glyoxylase II in Trigonella foenum-graecum treated with Toc, Cys, and Toc + Cys under normal and NaCl stress conditions. Mean (±SE) of three replicates has been given, and different letters on bars depict the significant difference at p <0.05.

3.7 Toc and Cys application alleviated the decline in mineral elements under salt stress

The shoot and root tissue of fenugreek plants treated with NaCl exhibited a substantial reduction in N and K with a considerable increase in Na over the control plants. Contrary to control, NaCl stress resulted in a reduction of 44.79% and 47.71% in leaf N and K, and 30.43% and 46.14% in root N and K. Supplementation of Toc and Cys caused an increase in the N and K of both leaf and root. In plants receiving both Toc and Cys, the content of N and K showed an increase of 35.98% and 31.39% in leaf and 54.12% and 38.62% in root, as opposed to control. Exogenous supplementation of Toc and Cys (individually as well as combinedly) to NaCl alleviated the reduction in N and K. Contrary to NaCl-stressed plants, the leaf N and K improved by 41.20% and 48.11%, respectively. While in root an increase of 27.80% and 60.26% was detected in NaCl + Toc + Cys treated plants (Table 3). Content of sodium increased by 272.99% in root and by 340.84% in leaf as compared to control plants owing to NaCl treatment; however, applied Toc and Cys reduced its accumulation. Contrary to the NaCl-treated plants, Na content showed a decline of 47.69% in root and 28.49% in leaf of plants treated with NaCl + Toc + Cys (Table 3).

Table 3. Content of sodium, potassium, and nitrogen in root and leaf of Trigonella foenum-graecum treated with Toc, Cys, and Toc + Cys under normal and NaCl stress. Mean (±SE) of three replicates has been given, and different letters depict the significant difference at p <0.05.
Sodium (mg g-1 DW)
 Nitrogen (mg g-1 DW)
 Potassium (mg g-1 DW)
Leaf Root Leaf Root Leaf Root
Control 0.76 ±0.05e 1.20 ±0.07e 24.40 ±1.20d 11.27 ±0.55d 20.83 ±1.75d 11.03 ±0.49d
Toc 0.73 ±0.09f 1.03 ±0.03f 26.25 ±1.10c 13.01 ±0.19c 22.66 ±1.88c 12.15 ±0.70c
Cys 0.74 ±0.03f 1.05 ±0.09f 30.83 ±1.75b 15.15 ±0.51b 25.10 ±1.73b 14.17 ±0.21b
Toc + Cys 0.68 ±0.02g 0.97 ±0.09g 33.18 ±0.84a 17.37 ±0.36a 27.36 ±1.64a 15.29 ±0.54a
NaCl 2.83 ±0.18a 5.32 ±0.22a 13.47 ±0.36h 7.84 ±0.49h 10.89 ±0.34h 5.94 ±0.56h
NaCl + Toc 2.25 ±0.15b 4.74 ±0.17b 14.80 ±1.39g 8.58 ±0.26g 12.41 ±0.42g 6.70 ±0.33g
NaCl + Cys 1.86 ±0.09c 4.36 ±0.23c 17.21 ±0.75f 9.18 ±0.26f 14.20 ±0.26f 8.65 ±0.35f
NaCl + Toc + Cys 1.48 ±0.13d 3.80 ±0.28d 19.02 ±0.46e 10.02 ±0.19e 16.13 ±0.37e 9.52 ±0.33e

It is noted in each table caption that different letters depict the significant difference at p<0.05.

3.8 Toc and Cys application increased the antioxidant activity

Antioxidant enzyme activities assayed showed an increase in the NaCl-stressed plants, and the exogenous Toc and Cys application induced further enhancement, achieving the highest values in plants treated with their combined application. Relative to control, an increase in SOD was 95.51%, APX was 126.21%, MDHAR was 85.68%, DHAR was 99.67%, and GR was 132.06% in NaCl-stressed plants. SOD, APX, MDHAR, DHAR, and GR registered the highest increase of 160.11%, 255.63%, 159.39%, 170.95%, and 219.69%, respectively, in NaCl + Toc + Cys-treated plants over the control. In unstressed plants, the application of Cys and Toc imparted an increase in the activities of these enzymes (Table 4). In addition, the content of Toc, Cys, GSH, and methionine increased by 159.92%, 99.49%, 54.81%, and 96.82% due to NaCl treatment over the control, respectively; however, AsA showed a decline of 21.18%. External treatment of Toc and Cys to unstressed plants caused an increased synthesis of these metabolites, with the highest increase of 107.94%, 50.75%, 28.97%, 77.98%, and 29.26% in Toc, Cys, GSH, methionine, and AsA, respectively, in plants receiving both Toc and Cys. In NaCl-stressed plants, application of Toc and Cys further increased Toc, Cys, GSH, and methionine, attaining the highest increase of 338.08%, 188.14%, 98.26%, and 210.67%, respectively, in plants that received NaCl + Toc + Cys over the control (Table 5).

Table 4. Activity of SOD, APX, DHAR, MDHAR reductase, and GR of Trigonella foenum-graecum treated with Toc, Cys, and Toc + Cys under normal and NaCl stress. Mean (±SE) of three replicates has been given, and different letters depict the significant difference at p <0.05.
SOD (U mg-1 protein) APX (U mg-1 protein) DHAR (U mg-1 protein) MDHAR (U mg-1 protein) GR (U mg-1 protein)
Control 3.76 ±0.35 h 1.84 ±0.10 h 30.44 ±1.25 h 19.70 ±1.21 h 0.94 ±0.03 h
Toc 4.36 ±0.17 g 2.25 ±0.10 g 35.08 ±1.28 g 22.48 ±0.57 g 1.11 ±0.06 g
Cys 4.40 ±0.20 f 2.41 ±0.07 f 38.58 ±2.78 f 26.48 ±1.09 f 1.25 ±0.03 f
Toc + Cys 5.00 ±0.40 e 2.68 ±0.14 e 42.57 ±2.85 e 28.58 ±0.90 e 1.34 ±0.03 e
NaCl 7.36 ±0.33 d 4.17 ±0.27 d 60.78 ±4.02 d 36.58 ±1.16 d 2.19 ±0.17 d
NaCl + Toc 8.04 ±0.18 c 5.02 ±0.24 c 66.97 ±4.21 c 40.84 ±2.41 c 2.58 ±0.02 c
NaCl + Cys 8.71 ±0.12 b 5.85 ±0.19 b 75.18 ±3.98 b 46.61 ±1.66 b 2.76 ±0.09 b
NaCl + Toc + Cys 9.79 ±0.42 a 6.56 ±0.20 a 82.48 ±4.48 a 51.10 ±3.25 a 3.01 ±0.20 a

It is noted in each table caption that different letters depict the significant difference at p<0.05.

Table 5. Content of GSH, AsA, Cys, Toc, and methionine of Trigonella foenum-graecum treated with Toc, Cys, and Toc + Cys under normal and NaCl stress. Mean (±SE) of three replicates has been given, and different letters depict the significant difference at p <0.05.
Toc (µg g-1 FW) Cys (nmol g-1 FW) GSH (nmol g-1 FW) Methionine (nmol g-1 FW) AsA (nmol g-1 FW)
Control 5.54 ±0.443 h 15.94 ±1.17 h 212.01 ±7.50 h 18.26 ±1.04 h 167.1 ±7.32 c
Toc 9.94 ±0.838 f 18.27 ±1.02 g 230.83 ±9.11 g 21.43 ±1.31 g 181.3 ±9.14 b
Cys 7.35 ±0.418 g 20.56 ±1.57 f 254.01 ±7.31 f 28.40 ±1.86 f 188.6 ±8.22 b
Toc + Cys 11.52 ±1.508 e 24.03 ±1.15 e 273.43 ±8.26 e 32.50 ±2.45 e 216.0 ±10.0 a
NaCl 14.40 ±0.951 d 31.80 ±2.66 d 328.23 ±10.85 d 35.94 ±2.57 d 131.7 ±4.83 f
NaCl + Toc 20.14 ±1.306 b 35.90 ±1.13 c 368.03 ±9.51 c 41.34 ±1.30 c 145.4 ±6.25 e
NaCl + Cys 18.07 ±0.375 c 41.16 ±2.17 b 390.61 ±12.42 b 48.96 ±2.82 b 156.7 ±6.21 d
NaCl + Toc + Cys 24.27 ±0.751 a 45.93 ±1.11 a 420.34 ±9.71 a 56.73 ±3.04 a 170.9 ±5.13 c

It is noted in each table caption that different letters depict the significant difference at p<0.05.

4. Discussion

Increasing salinity has resulted in the shrinking of the productive agricultural land, therefore posing a serious threat to sustainable food production. Implementing or adapting new management techniques can be beneficial in improving crop growth and yield potential significantly. In recent times, exogenous application of phytohormones, mineral ions, and metabolites has been revealed to alleviate the negative effects of stress in plants. In this context, the current study was carried out to assess the role of Toc and Cys, alone and combined, in alleviating the detrimental effects of NaCl stress in fenugreek. Similar to the results of the present study, reduction in plant morphological parameters, including height, fresh and dry weight, has been reported by Qados (2011) in Vicia faba, Ahanger et al. (2019a) in wheat, Abid et al. (2020) in kiwi fruit, and Kumar et al. (2021) in Oenanthe javanica. Salinity stress results in osmotic and ionic stress, causing impediment in root growth, mineral uptake, water uptake, enzyme functioning, etc. thereby significantly altering the plant growth and metabolic functioning (Ahanger et al., 2019a, b; Qin et al., 2020, 2021; Alamer, 2023). In Linum usitatissimum, application of Cys mitigated the decline in growth attributes like length and weight (fresh and dry) of shoot and root (Hussein & Alshammari, 2022). Similarly, Alnusairi (2022) has also reported the alleviation of decline in growth parameters in soybean by the exogenous application of Toc. However, the impact of the joint application of Toc and Cys has not been reported. Reduced shoot and root growth due to NaCl may be attributed to a significant decline in cellular division (Teerarak et al., 2009). In addition, the decline in cellular water content, photosynthesis, and mineral uptake also contributes significantly to NaCl-induced growth restrictions (Ahanger et al., 2019a, b). Appropriate redox maintenance contributes to proper cell cycle progression and tissue proliferation (Chiu & Dawes, 2012), and Cys forms one of the key redox components. In the present study, improved growth in Toc and Cys treated plants may also be attributed to improved nitrogen and potassium uptake with concomitant decline in the accumulation of Na. Nitrogen and potassium are important mineral ions actively regulating several key physiological and metabolic processes, including enzyme functioning, mineral assimilation, photosynthesis, and tolerance to stresses (Ahanger & Agarwal, 2017; Ahanger et al., 2019a; Al-Mushhin, 2022). Decline in nitrogen and potassium was mitigated by the applied Toc and Cys, though individually Cys proved much effective than Toc; however, combined treatments resulted in much evident mitigation. Application of α-Toc to soybean prevented the excess accumulation of Na while increasing K, thereby causing the mitigation of salt stress (Alnusairi, 2022). In Vicia faba, alleviation of the decline in nitrogen (Semida et al., 2014) and potassium (Orabi and Abdelhamid, 2016) due to application of Toc has been reported. Increase in K and decline in the content of Na due to Toc and Cys application may contribute to increased growth and salt tolerance, although the exact mechanisms are largely unknown. Reduction in the accumulation of Na results from the increased functioning of transporters mediating its exclusion and compartmentation from the cell cytoplasm, thereby reducing the negative effects (W, 2018). Applied Toc and Cys may have regulated the functioning of key transport proteins, thereby mediating the efficient salt exclusion to prevent the damaging effects on plant metabolism. Further studies can be interesting to unravel the mechanisms.

Salinity significantly declined the chlorophyll pigments and the carotenoids in fenugreek, and application of Toc and Cys alleviated the decline considerably, with the highest alleviation observed in plants that received both Toc and Cys. Salinity stress triggers excessive degradation of chlorophyll and also alters the biosynthesis by mediating a significant decline in the activities of enzymes catalyzing chlorophyll biosynthesis (Turan & Tripathy, 2015; Alamer, 2023). Earlier, exposure to salt stress has been observed to reduce the content of chlorophylls and carotenoids in different crop plants like wheat (Ahanger & Agarwal, 2017; Alamer, 2023), soybean (Soliman et al., 2020), Oenanthe javanica (Kumar et al., 2021), and Azolla spp. (Narayan et al., 2024). In fenugreek, Lamsaadi et al. (2023) have also confirmed reduced chlorophyll, photosynthesis, and PSII functioning due to NaCl stress, causing a significant decline in growth. Salinity alters chlorophyll synthesis and the chloroplast structure, thereby affecting the growth and productivity (Wang et al., 2024). Exogenous treatment of Cys to Linum usitatissimum (Hussein & Alshammari, 2022) and Toc to soybean (Alnusairi, 2022) has been reported to mitigate the decline in pigments caused by NaCl stress; however, their combined effect has not been reported. Increased chlorophyll and carotenoid synthesis in Toc and Cys treated plants depicts their advantageous role in preventing impairment to the photosynthetic performance of fenugreek plants. Greater pigment synthesis results by up-regulation of the chlorophyll biosynthesis pathway, improved uptake of minerals like nitrogen, magnesium, and others. (Qin et al., 2024b; Li et al., 2024). Increased chlorophyll synthesis depicts better metabolic functioning, which can also contribute to fine-tune the overall plant performance.

An evident decline in the oxidative stress attributes was detected in Toc and Cys treated fenugreek plants under unstressed and stressed conditions. Salinity triggered the excess generation of hydrogen peroxide, increased the activity of LOX, and lipid peroxidation. Similar results of increased hydrogen peroxide and lipid peroxidation under salt stress have been observed by Ahanger et al. (2019a) in wheat, Soliman et al. (2020) in soybean, Kumar et al. (2021) in Oenanthe javanica, and Azeem et al. (2023) in Moringa oleifera. Salinity-mediated enhancement in oxidative damage significantly affects the membrane functioning and the key metabolic pathways like photosynthesis (Taibi et al., 2016; Azeem et al., 2023). Application of Toc has been observed to decline the accumulation of hydrogen peroxide, therefore causing a decline in lipid peroxidation (Alnusairi, 2022). Orabi and Abdelhamid (2016) has also reported reduced lipid peroxidation due to exogenous Toc application in Vicia faba. In Carex leucochlora, Toc application reduced radical accumulation and lipid peroxidation, leading to greater protection of photosynthetic pigments and improved growth (Ye et al., 2017). Similarly, in Linum usitatissimum, treatment of Cys resulted in a significant decline in the lipid peroxidation under salt stress (Hussein & Alshammari, 2022). Plants accumulating greater Cys content exhibit a considerable decline in lipid peroxidation (Van Nguyen et al., 2021). Reduced hydrogen peroxide concentration in Toc and Cys treated plants reflects their potential to prevent the oxidative effects of hydrogen peroxide in fenugreek. Toc hold the phospholipid bilayer of polyunsaturated fatty acyl chain, preventing any damage to it (Sattler et al., 2003). At lower concentrations, hydrogen peroxide proves beneficial for crop stress tolerance and also contributes to strengthening the tolerance mechanisms (Smirnoff & Arnaud, 2018). Therefore, it can be said that the interactive Toc and Cys application can be helpful in avoiding the damaging influence of hydrogen peroxide. This was evident in terms of reduced LOX and NADPH oxidase activity. In salt-stressed plants, the LOX and NADPH oxidase activity were triggered significantly, reflecting in greater peroxidation of lipids and the generation of radicals, respectively. LOX converts polyunsaturated fatty acids into the fatty acid hydroperoxides through oxidation (Singh et al., 2022). Further decline in the activities of LOX and NADPH oxidase due to Toc and Cys depicts the beneficial impact on growth by reducing the production of toxic radicals. Stresses like nickel (Sirhindi et al., 2016) and arsenic (Qin et al., 2024a) increased the activity of NADPH oxidase in different plants. NADPH oxidase is a key player in ROS signalling and belongs to the NOX family encoded by the RBOH gene. NADPH oxidases produce O2- and are activated by salinity at transcriptional and translational levels (Liu et al., 2020). Salt stress resulted in increased NADPH oxidase activity and the expression levels of CsRboh, causing an increase in the accumulation of ROS in cucumber (Kabala et al., 2022). Increased activity of the LOX due to salt stress being observed earlier by Ahanger et al. (2019a, b). Declined LOX and NADPH oxidase activity due to application of exogenous protectants reflects in improved growth and the stress mitigation potential of the applied molecules (Roychoudhary et al., 2011; Sirhindi et al., 2016; Qin et al., 2024a, b).

Application of Toc and Cys proved effective in increasing proline accumulation under both growth conditions. In response to salinity stress, plants accumulate increased concentrations of proline, which has been observed in Solanum tuberosum (Jaarsma et al., 2011), soybean (Soliman et al., 2020), and Panicum miliaceum (Mushtaq et al., 2023). Applied Toc increased proline in Vicia faba (Semida et al., 2014; Orabi & Abdelhamid, 2016) and soybean (Alnusairi, 2022). In Linum usitatissimum, treatment of Cys reduced the accumulation of proline under salt stress (Hussein & Alshammari, 2022). In the present study, increased proline accumulation was related to the differential regulation of the enzymes catalyzing proline metabolism. Treatment of Toc and Cys increased the activity of P5CS and γ-GK, while reducing the PROX activity reflecting in increased proline reserves. In response to salinity, increased P5CS and γ-GK activity with a decline in PROX subsequently resulting in increased proline accumulation has also been observed by Iqbal et al. (2015) and Mushtaq et al. (2023). Similarly, this differential influence on proline metabolizing enzymes has been reported under chromium stress also (Qin et al., 2024b). Alnusairi (2022) has also observed that Toc application increased the activity of γ-GK in salt-stressed soybean. In the present study, increased proline accumulation in Toc and Cys treated plants was due to their impact on the proline metabolizing enzymes. Proline is a multifunctional amino acid acting as an osmolyte, radical scavenger, and helps in quick recovery from stress (Szepesi & Szollosi, 2018). Increased activity of proline-synthesizing enzymes with declined activity of catabolizing enzymes due to Toc and Cys treatments depicts their beneficial role in avoiding the harmful impact of salt stress in fenugreek. Plant genotypes accumulating greater proline exhibit declined hydrogen peroxide accumulation and can better counter the oxidative effects of stresses (Jaarsma et al., 2011). Besides this, salinity caused a significant enhancement in the endogenous concentration of NO and HS. However, treatment of Toc and Cys reduced their concentrations in salt-stressed plants, while in unstressed plants, a slight increase in their concentrations was observed due to their treatments. Increase in the endogenous concentration of NO (Ahanger et al., 2019b; Alamer, 2023) and HS (Mostofa et al., 2015; Alamer, 2023) has been reported due to salt stress. Maintaining optimal concentrations of NO and HS have a beneficial impact on growth and metabolism by mediating germination, photosynthesis, stress signalling, and assisting in strengthening the tolerance mechanisms (Saini et al., 2024).

Exogenous treatment of Toc and Cys increased the activity of antioxidant enzymes, and the highest activity was observed in plants treated with both. Salinity has been reported to increase the activity of antioxidant enzymes in Brassica juncea (Iqbal et al., 2015), wheat (Ahanger et al., 2019a), soybean (Soliman et al., 2020), Moringa oleifera (Azeem et al., 2023), and Lonicera japonica (Song et al., 2024). The increased functioning of SOD results in the elimination of superoxide and preventing its harmful impact on the functioning of delicate organelles, including chloroplasts (Ahanger et al., 2017; Azeem et al., 2023). In addition, APX, MDHAR, DHAR, GR, AsA, and GSH are the vital components of the intriguing radical neutralizing mechanism. The ascorbate-glutathione cycle, which not only eliminates hydrogen peroxide but also contributes to redox maintenance and the protection of electron transport (Ahanger et al., 2017; Hasanuzzaman et al., 2019). Treatment of Toc and Cys significantly increased the functioning of this cycle by increasing the activity of the enzymes and concentration of AsA and GSH, thereby providing extra protection to fenugreek to withstand the salt stress. Supplementation of Toc imparted an increase in the activity of antioxidant enzymes, resulting in mitigation of the oxidative impact of salt stress (Orabi & Abdelhamid, 2016). Alnusairi (2022) has also demonstrated increased activity of SOD, APX, and GR as well as the accumulation of AsA and GSH due to Toc treatment. Exogenous supplementation of Cys up-regulated the functioning of antioxidant enzymes in Arabidopsis, subsequently contributing to the mitigation of oxidative effects of mercury stress (Kim et al., 2022). Similarly, the applied Cys increased the content of endogenous AsA, GSH, GSSG, Cys, and phytochelatins, resulting in increased growth and photosynthesis in cadmium-stressed Brassica juncea (Xue et al., 2024). Reports about the combined influence of Toc and Cys are not available; therefore, a crosstalk mechanism is suggested between Toc and Cys that regulates the tolerance mechanisms against NaCl stress. In the present study, exogenously applied Toc and Cys increased the endogenous levels of the nonenzymatic antioxidants, thereby improving the potential to counter the negative effects of NaCl. Toc in chloroplasts protects its envelope, thylakoid membranes, and plastoglobuli from the toxic effects of free radicals, mainly 1O2 and OH (Munne-Bosch, 2005). Plants increase Toc accumulation in response to stresses (Mishra et al., 2014; Ahanger et al., 2019a). Cys, glutathione, AsA, and Toc protect plant growth from stress adversaries by maintaining redox homeostasis and mediating radical scavenging (Ahanger et al., 2017; Hasanuzzaman et al., 2019; Faizan et al., 2023). The impact of applied Toc and Cys on these key metabolites is less reported; therefore, further research studies are needed to understand the actual mechanisms. In addition, treatment of Toc and Cys was effective in increasing the activities of glyoxylase cycle enzymes. Improved activity of the glyoxylases leads to quick elimination of toxic methylglyoxal from cells (Kaur et al., 2014). Increased activity of the glyoxylase cycle due to NaCl stress has earlier been demonstrated in tobacco (Hoque et al., 2008), rice (Mostofa et al., 2015), and wheat (Alamer, 2023). Further improvement in their activities due to Toc and Cys treatments confers quick removal of toxic methylglyoxal and hence protecting the plant metabolism. It has been reported that carrizo citrange plants overexpressing the glyoxylase cycle enzymes showed improved salt tolerance (Alvarez-Gerding et al., 2015).

5. Conclusions

Exogenous Toc and Cys proved beneficial in ameliorating the harmful effects of salt stress in fenugreek. The highest increase in growth and pigment synthesis due to combined Toc and Cys treatment was well correlated with a significant decline in the oxidative stress parameters in them. In addition, the differential regulation of proline metabolism, improved antioxidant system, and the glyoxylase cycle functioning due to Toc and/or Cys treatments confer their beneficial role in mitigating the NaCl stress. In addition, reduced accumulation of Na ions and the increase in nonenzymatic metabolites support the efficiency of applied Toc and Cys in mitigating the negative effects of salinity in fenugreek and can significantly contribute to improved growth and yield.

CRediT authorship contribution statement

Houneida Attia and Khalid H. Alamer: Collaboratively designed and wrote the manuscript, contributed to the literature search and approved the manuscript.

Declaration of competing interest

The authors declare that they have no competing financial interests or personal relationships that could have influenced the work presented in this paper.

Data availability

All the raw data in this research can be obtained from the corresponding author upon reasonable request.

Declaration of Generative AI and AI-assisted technologies in the writing process

The authors confirm that there was no use of artificial intelligence (AI)-assisted technology for assisting in the writing or editing of the manuscript and no images were manipulated using AI.

Funding

This project was funded by the Deanship of Scientific Research (DSR) at King Abdulaziz University, Jeddah, Saudi Arabia under grant no. (IPP: 582-662-2025). The authors, therefore, acknowledge with thanks DSR for technical and financial support.

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