Evaluation of fenugreek under water stress: agronomic traits, seed oil quality and antioxidant capacity

Abstract

Background

Fenugreek (Trigonella foenum-graecum L.) is rich in seed bioactive compounds, and its yield depends on irrigation. The effects of deficit irrigation treatments on yield, water-use efficiency, and seed quality traits remain insufficiently characterized. This two-year field study (2021–2022) evaluated five irrigation levels. Fenugreek was sown in late March and harvested in late July. In the fully irrigated treatment (FI100), the soil water depleted from field capacity was fully replenished. The FI75, FI50 and FI25 treatments received 75, 50 and 25% of the water applied to FI100, and FI0 was a rainfed control. The aim was to assess the trade-off between yield and quality under water-limited conditions.

Methods

Experiments were conducted in randomized complete-block design with three replications under drip irrigation. Crop water consumptions, irrigation and crop water productivity, and both treatment-specific and seasonal yield-response factors (Ky) were determined. Agronomic traits, seed oil and fatty acid composition, phenolic contents and antioxidant activities were measured and analysed by PCA.

Results

As the average of two years, the highest seed yield occurred under FI100 (1474 kg ha⁻¹) and the lowest under FI0 (533 kg ha⁻¹), a reduction of about 64%. The seasonal Ky exceeded unity in both years (1.12 and 1.18). At the same time, the treatment-specific Ky increased as the deficit became milder (from ≈ 0.97 under FI0 to ≈ 2.8 under FI75), indicating a non-linear response with greatest sensitivity near full irrigation. Water deficit improved seed quality, total phenolics, flavonoids, hydrolysable tannin, and antioxidant activity peaked under FI0, while saturated fatty acids (SFA) increased and polyunsaturated fatty acids (PFA) decreased. PCA explained 89.1% of the variation and revealed a negative relationship between yield/oil-quality traits and phenolic-based antioxidant traits.

Conclusions

Growth and seed yield in fenugreek are highly sensitive to water deficit, whereas seed biochemical quality responds inversely. Irrigation should therefore be planned by objective. Full or near-full irrigation for high yield, and controlled deficit irrigation for phytochemical quality. Where water is the primary limiting resource, FI75 offers a compromise, saving 25% of irrigation water at the cost of about a 41% yield reduction.

Background

Global climate change, rising temperatures, and shifts in precipitation regimes are intensifying the pressure on water resources, threatening sustainable agriculture of arid and semi-arid regions [1, 2]. Because agriculture is the greatest water-consuming sector, enhancing water-use efficiency has become a priority objective [3]. Deficit irrigation, which applies a controlled level of water restriction rather than fully meeting the crop’s water requirements, stands out as an effective water-saving strategy. Indicators such as IWP (Irrigation water productivity), CWP (Crop water productivity) and ky (Yield response factor) which relates the proportional reduction in evapotranspiration to yield loss are fundamental criteria for determining the appropriate irrigation level [4, 5]. The Ky values of greater than 1.0 indicate the species is sensitive to water deficit, reduction in evapotranspiration causes a proportionally greater loss in yield [6]. Modern approaches such as drip, precision, and controlled-deficit irrigation offer important opportunities to enhance water-use efficiency using these indicators [7].

Fenugreek (Trigonella foenum-graecum L.), which exhibits moderate tolerance to water restriction and can be grown across different ecological zones, is a noteworthy species for conditions with limited water resources [8]. This annual legume of southern European and Asian origin offers versatile uses across the agricultural, nutritional, and industrial sectors. Fenugreek seeds contain numerous bioactive compounds including alkaloids, phenolics, flavonoids, saponins, and essential oils [9]. These compounds contribute to nutritional value and health benefits of the plant [10]. Seeds are rich in PUFA (polyunsaturated fatty acids), especially in linoleic and γ-linolenic acids [11]. Furthermore, owing to its high fiber and galactomannan content, fenugreek is largely utilized in food industry as a natural stabilizer, emulsifier, binder, and thickener [9, 12]. Plant seeds and young leaves are used as a spice, food additive, flavoring, and vegetable. Seed phytochemicals have been reported to offer potential benefits against diabetes, hypercholesterolemia, inflammatory diseases, and microbial infections [13, 14]. For this reason, improving not only yield but also quality traits in fenugreek is becoming increasingly important.

Irrigation is a principal factor determining the yield and quality of fenugreek. Irrigation significantly facilitates nutrient uptake and metabolic activity. Water stress may decrease seed yield, plant growth, morphological traits, number of pods per plants and plant development [15, 16], and severe water restriction can lower biological yield by approximately 48% [17]. However, water deficit-induced oxidative stress stimulates antioxidant defense mechanisms by increasing the synthesis of phenolics and flavonoids [18, 19]. Total phenolics and flavonoids, and antioxidant capacity were reported to increase under water stress [11]. Plant response to water stress is characterized by total phenolics, flavonoids and antioxidant activity assays (DPPH, ABTS, FRAP) [20, 21]. Similarly, water stress may influence seed quality traits by altering oil accumulation in seeds and the balance between saturated and unsaturated fatty acids [22, 23].

Although the functional and medicinal importance of fenugreek is well recognized, studies that jointly evaluate the effects of different irrigation levels on plant growth, yield, water-use efficiency, phenolic composition, antioxidant capacity, and fatty acid profile are rather limited. In particular, how the level of deficit irrigation simultaneously affects seed yield, water productivity and the bioactive quality of fenugreek seed remains unclear. Therefore, the present research was designed to determine the effects of five different irrigation levels applied over two growing seasons on the morphological traits, seed yield, irrigation performance indicators, phenolic composition, antioxidant activity, and fatty acid composition of fenugreek, and to identify an appropriate irrigation strategy capable of jointly optimizing yield and quality under water-limited conditions.

Methods

Field experiments

In this study, the fenugreek (Trigonella foenum-graecum L.) cultivar ‘Güraslan’, a variety widely cultivated in the region and registered in 2004 by the Faculty of Agriculture, Ankara University (Ankara, Türkiye), was used as the plant material. Certified seeds were provided by the same institution, which developed and registered the cultivar and confirmed its identity. Field experiments were carried out at the Research and Application Center of Erciyes University, Kayseri, Türkiye (38°42′N; 35°32′E). All experimental research on plants complied with relevant institutional, national, and international guidelines and legislation. The experiment was conducted between March and July in 2021 and 2022. Experiments were conducted in randomized complete-block design with three replications. Each plot consisted of six 5 m long rows with a 30 cm row spacing. Seeds were hand-sown on 28 March 2021 and 29 March 2022, at a rate of 40 kg ha⁻¹ and a depth of approximately 3 cm. At sowing, a 20–20–0 compose fertilizer was applied as a single basal dose at 250 kg ha⁻¹, providing 50 kg ha⁻¹ of nitrogen (N) and 50 kg ha⁻¹ of phosphorus (P₂O₅). The necessary cultural practices were performed throughout the growing period. The differential irrigation treatments were applied from May until maturity, once pod filling was complete, irrigation was stopped in all treatments to allow harvest. Plants were harvested at physiological maturity after morphological observations had been recorded.

Soil and climate characteristics

Experimental soils were sandy-loam in texture, slightly alkaline (pH 7.70–7.81), low in organic matter (1.12–1.18%), and low in lime content. The electrical conductivity values (0.24–0.79 dS m⁻¹) indicated the absence of any salinity problem. Field capacity (FC) and permanent wilting point (WP) were determined in the laboratory on undisturbed soil samples using a pressure-plate apparatus at 1/3 and 15 atm, respectively. The electrical conductivity of the irrigation water was 0.23 dS m⁻¹, and the pH was 7.60 (Table 1).

Table 1.

Physical and chemical characteristics of the soils of the experimental site

Soil Quality Parameters
Soil Depth (mm)	Texture	ECs (dS m− 1)	pH	FC, Pw (%)	WP, Pw (%)	BD (gr cm− 3)	OM (%)	Lime (%)
0–30	Loam	0.79	7.81	23.55	11.55	1.26	1.12	0.28
30–60	Loam	0.24	7.70	24.49	12.24	1.24	1.18	0.29
Irrigation Water Quality Parameters
pH	ECw (dS m− 1)	Na+ (mg L− 1)	K+ (mg L− 1)	Ca+ 2 (mg L− 1)	Mg+ 2 (mg L− 1)	HCO3 (mg L− 1)	CO3−2 (mg L− 1)	SAR
7.54	0.23	11.51	4.62	27.17	6.27	11.93	˂ 1.00	2.74

EC Electrical conductivity, FC Field capacity, WP Wilting point, BD Bulk density, OM Organic matter

The meteorological data for the experimental site in 2021 and 2022, and for the long-term period (1965–2021), are presented in Table 2. In both years, total rainfall (119.5 and 145.4 mm, respectively) was below the long-term average (185.2 mm), highlighting the need for irrigation during the growing season.

Table 2.

Meteorological data of the experimental site in 2021, 2022, and long-term

Months	Mean Temp. (℃)			Min. Temp. (℃)			Max. Temp. (℃)
	2021	2022	LT*	2021	2022	LT	2021	2022	LT
March	5.2	1.3	4.7	-0.3	-3.3	-1.3	10.8	5.9	10.6
April	10.7	13.6	11.3	4.5	6.4	4.9	16.8	20.8	17.7
May	15.5	14.1	14.7	7.5	7.5	6.9	23.6	20.7	22.5
June	18.9	20.5	18.3	12.1	13.7	9.8	25.8	27.3	26.9
July	23.8	25.1	22.2	14.6	12.7	11.9	31.8	32.5	30.6
Total	-	-	-	-	-	-	-	-	-
Mean	14.8	14.9	14.2	7.7	7.4	6.4	21.8	21.4	21.7

Months	Wind Speed (m s− 1)			Precipitation (mm)			RH (%)
	2021	2022	LT*	2021	2022	LT	2021	2022	LT
March	1.6	1.4	1.3	38.3	32.3	41.8	64.6	70.3	68.3
April	2.2	1.9	1.6	22.7	15.2	52.1	63.5	47.6	62.3
May	1.9	1.5	1.7	21.3	43.4	51.8	56.4	61.8	61.2
June	2.0	1.5	1.4	37.2	54.5	39.5	53.1	59.3	55.8
July	1.8	1.6	1.6	0.0	0.0	10.5	52.7	55.4	49.5
Total	-	-	-	119.5	145.4	185.2	-	-	-
Mean	1.9	1.6	1.5	-	-	-	58.1	58.9	59.4

RH Relative humidity

*LT: Long Term (1965–2021)

Irrigation system and treatments

A drip irrigation system was designed to precisely determine the amount of water to be applied. Polyethylene drip lines with a 16 mm outer diameter and an operating pressure of 4.05 bar were laid out at 30 cm intervals across all six rows, with one lateral per plant row. In-line emitters with a discharge of 2 L h⁻¹ and a spacing of 25 cm were placed to provide a wetting-area ratio of at least 30% [24]. Irrigation water passed through a water meter installed on each plot. The gravimetric method was used to monitor soil moisture every seven days at the 0–30 and 30–60 cm depths. In addition, soil moisture at a depth of 60–90 cm was monitored throughout the experiment, and no deep percolation was detected. To prevent irrigation influence between plots, a 2 m buffer zone was left between them. In observations of the experimental soils, no groundwater was detected to a depth of 2 m, and capillary rise was assumed absent.

The irrigation treatments were defined relative to the fully irrigated treatment. At each irrigation, FI100 received the amount of water required to replenish the soil water depleted from field capacity in the 0–60 cm root zone, whereas FI₇₅, FI₅₀, and FI₂₅ received 75, 50, and 25% of the water applied to FI₁₀₀, respectively, and FI₀ was left rainfed. Also, in the FI₁₀₀ treatment, the RAW (Readily available water) fraction was set at 0.5 and irrigation was applied before the soil water dropped below this threshold, so that the crop was kept free of water stress as much as possible. Equation 1 was used to determine the amount of irrigation water to be applied (mm):

1

In this Eq. (1), d denotes the net irrigation depth applied (mm), while Pwfc and Pwm represent the gravimetric soil water content (%) at field capacity and immediately before irrigation, respectively. The term γ is the soil bulk density (g cm⁻³), D is the depth of the soil profile to be wetted (mm), and P is the fraction of the surface area wetted.

Calculation of crop water consumption

Equation 2 was used to determine crop water consumption [25]. Total rainfall recorded at the on-site meteorological station was converted to effective rainfall using the USDA-SCS method [26, 27] and included as the term R in the water-balance calculation. The experiment was conducted under natural rainfall conditions without a rain shelter, with FI0 serving as a rainfed control. Because irrigation amounts were based on measured soil-water depletion before each irrigation, effective rainfall was accounted for.

2

In Eq. 2, ET refers to seasonal crop water consumption (mm), I to the irrigation water supplied, and R to the effective precipitation received over the growing period. Cr accounts for the contribution of capillary rise from the water table, whereas Dp and Rf represent the water lost through deep percolation below the root zone and surface runoff, respectively. The final term, Δs, denotes the change in soil water storage within the profile across the measurement interval.

Water–yield relationship and irrigation efficiency

Equation 3 was used to determine yield-response factor (Ky) indicating proportional yield reduction in response to ET deficit [6].

3

In Eq. 3, Ya and Ym denote the actual and maximum yields (kg ha⁻¹), respectively, while ky is the yield-response factor that relates the relative yield reduction to the relative deficit in water use. ETa represents the actual crop water consumption realized under each treatment (mm), and ETm is the maximum crop water consumption attained under non-limiting water supply.

Equations 4 and 5 were used to determine crop water productivity values [28].

4

5

In these equations, CWP is the crop water productivity (kg ha⁻¹ mm⁻¹), defined as seed yield per unit of water consumed, and IWP is the irrigation water productivity (kg ha⁻¹ mm⁻¹), expressed per unit of irrigation water supplied. Y denotes the seed yield (kg ha⁻¹), I the irrigation water applied over the season (mm), and ET the crop water consumption (mm).

Regression analyses were performed using relative yield (Ya/Ym) and relative water consumption (ETa/ETm) data to establish the relationships between relative yield and water consumption. The seasonal yield response factor (Ky) was obtained for each year from the slope of the linear regression of the proportional yield reduction (1 − Ya/Ym) on the proportional evapotranspiration deficit (1 − ETa/ETm), as described in Eq. 3. In addition, a treatment-specific (point) Ky was calculated for each deficit treatment by rearranging Eq. 3, taking the fully irrigated treatment (FI₁₀₀) as the reference for maximum yield (Ym) and maximum crop water consumption (ETm).

Morphological observations

At physiological maturity, morphological observations (plant height, number of pods per plant, number of branches per plant and first pod height) were made on 10 plants randomly selected from each plot. For seed yield, two side rows and 50 cm sections from the top and bottom of each plot were omitted and remaining area was harvested, plants were threshed and seed yield was expressed in kg ha⁻¹.

Determination of crude oil and fatty acids

Crude oil content was determined by Soxhlet extraction as described in [29]. Briefly, 3 g of ground fenugreek seed was placed in an extraction thimble and extracted with petroleum ether; once extraction was complete, the residue was oven-dried at 95 °C for 1 h, cooled, and weighed, and the crude oil content was calculated gravimetrically. For fatty acid analysis, the oil was first methylated according to AOAC (1990; method 969.33): 100 mg of oil was combined with 3 mL of hexane and 100 µL of 2 N methanolic potassium hydroxide, centrifuged at 5000 rpm for 5 min, and 1 mL of the upper phase was taken. The fatty acid methyl esters were analysed on an Agilent gas chromatograph (model 6890 N) fitted with a Supelco HP-88 capillary column (100 m × 0.25 mm i.d., 0.2 μm) and a flame ionization detector (FID), following Kaplan et al. [30]. The injection volume was 1 µL and the injector temperature was 250 °C. The oven was programmed from 130 °C (held 1 min) to 170 °C at 6.5 °C min⁻¹, then to 215 °C at 2.75 °C min⁻¹ (held 12 min), and finally to 230 °C at 40 °C min⁻¹ (held 5 min), giving a total run time of 40.89 min. The remaining operating parameters were as follows: detector temperature 280 °C; split ratio 50:1; detector H₂ flow 40 mL min⁻¹; dry-air flow 450 mL min⁻¹; column H₂ flow 1.3 mL min⁻¹. The fatty acid methyl esters were identified by comparing their retention times with authentic standards (Supelco 37-component FAME mix; Sigma L8404 and O5632) and quantified as relative percentages of total fatty acids; accordingly, no internal standard was used.

Bioactive analyses of seed extracts

The extraction solvent was selected in a preliminary trial, in which 80% ethanol gave the highest total phenolic recovery and was consequently adopted for all samples. For each extraction, 1 g of ground seed was weighed into a Falcon tube and mixed with 20 mL of solvent. After vortexing for 1 min, the tubes were sealed with parafilm and extracted horizontally in a shaking water bath at 25 °C for 1 h. The extracts were then centrifuged at 7500 g (4 °C, 5 min), and the supernatant was filtered through a 0.45 μm syringe filter prior to analysis. Total phenolic content (TPC) was determined by the Folin–Ciocalteu method of Singleton and Rossi [31] (absorbance at 765 nm; gallic acid standard) and expressed as mg gallic acid equivalent (GAE) 100 g⁻¹ dw. Total flavonoid content (TFC) was measured by the aluminium chloride method of Zhishen et al. [32] (absorbance at 510 nm) and expressed as mg catechin equivalent (CE) g⁻¹ dw. Total hydrolysable tannins (THTC) were quantified with potassium iodate following Willis and Allen [33] (absorbance at 550 nm; tannic acid standard) and expressed as mg kg⁻¹ dw. DPPH radical-scavenging activity was assessed following He et al. [34] (2 mM DPPH; absorbance at 517 nm) and expressed as percentage inhibition. ABTS⁺ radical-scavenging activity was determined following Gong et al. [35] (absorbance at 734 nm) and expressed as µg Trolox equivalent (TE) g⁻¹ dw. Ferric reducing antioxidant power (FRAP) was measured following Malomo et al. [36] (ascorbic acid standard) and expressed as mg ascorbic acid equivalent (AAE) g⁻¹ dw. Iron chelating activity (ICA) was determined with ferrozine following Rival et al. [37] (absorbance at 562 nm) and expressed as percentage chelation relative to the control.

Statistical analysis

Experimental data were subjected to a combined analysis of variance using SAS Statistics software. Differences among irrigation treatments were compared within each year and in the two-year average using the Tukey’s HSD test (p < 0.05). Differences between the two years were evaluated with the use of the LSD test (p < 0.05). Principal Component Analysis (PCA) was performed on the two-year mean data to evaluate the multivariate relationships among agro-morphological, yield, fatty acid, and bioactive traits across the different irrigation regimes. Prior to analysis, all variables were standardized to zero mean and unit variance, and components with eigenvalues greater than 1 (Kaiser criterion) were retained. The first two components accounted for 89.1% of the total variance (PC1 = 81.4%, PC2 = 7.7%). The analysis was carried out in Python.

Results

Applied irrigation water, evapotranspiration, and water-use efficiencies

Amount of irrigation water applied in each treatment and actual crop water consumption values are provided in Table 3. In 2021, the amount of irrigation water applied ranged from 105.5 mm (FI₂₅) to 422.0 mm (FI₁₀₀); the seasonal ETa was highest (568.0 mm) in full irrigation and lowest (185.4 mm) in FI₀ control treatment. In 2022, owing to lower effective rainfall and increased atmospheric water demand, the irrigation water applied to the FI₁₀₀ treatment increased to 443.0 mm, raising the maximum ETa to 623.9 mm, while the FI₀ treatment was limited to 215.9 mm. Water-use efficiencies exhibited nonlinear dynamics under water stress. In 2021, the greatest CWP was obtained from FI₀ treatment (2.73 kg ha⁻¹ mm⁻¹), whereas IWP was highest in the FI₂₅ treatment (6.01 kg ha⁻¹ mm⁻¹). The results showed a completely parallel trend in the 2022 growing season: CWP reached its highest value in the FI₀ treatment (2.59 kg ha⁻¹ mm⁻¹), while IWP was optimized in the FI₂₅ treatment (5.67 kg ha⁻¹ mm⁻¹). These higher values under severe restriction indicate greater yield per unit of water, not higher total yield. In fact, they occurred together with large absolute yield losses. Full irrigation therefore maximized seed yield, whereas the water-productivity indicators peaked under deficit simply because much less water was applied.

Table 3.

Relationships among irrigation levels, evapotranspiration, and water-use efficiency in fenugreek

Treatments	I	P	ETa	CWP	IWP	Ky
2021
FI0	0	119.5	185.4	2.73	-	0.96
FI25	105.5	119.5	279.3	2.27	6.01	1.09
FI50	211.0	119.5	378.8	1.84	3.30	1.54
FI75	316.5	119.5	472.4	1.84	2.74	2.33
FI100	422.0	119.5	568.0	2.51	3.38	-
2022
FI0	0	145.0	215.9	2.59	-	0.97
FI25	110.8	145.0	317.3	1.98	5.67	1.19
FI50	221.5	145.0	421.5	1.72	3.27	1.61
FI75	332.3	145.0	525.8	1.64	2.59	2.76
FI100	443.0	145.0	623.9	2.44	3.43	-

I Irrigation amount (mm), P Rainfall (mm), ETa Actual evapotranspiration (mm/season), CWP Crop water productivity (kg ha⁻¹ mm⁻¹), IWP Irrigation water productivity (kg ha⁻¹ mm⁻¹), Ky Yield response factor

Soil moisture depletion and seasonal water dynamics

Continuous monitoring of root-zone soil moisture throughout the growth period revealed significant consumption and irrigation levels relative to the field capacity (FC) and wilting point (WP) thresholds (Figs. 1 and 2).

Fig. 1.

Dynamic changes of soil water content under various deficit and full irrigation treatments relative to field capacity (FC) and permanent wilting point (WP) thresholds in 2021

Fig. 2.

Dynamic changes of soil water content under various deficit and full irrigation treatments relative to field capacity (FC) and permanent wilting point (WP) thresholds in 2022

In full irrigation and FI₇₅ treatments, soil moisture content remained safely in the upper half of the readily available water (RAW) zone in both seasons. Soil moisture decreased rapidly and consistently under the moderate (FI₅₀), severe (FI₂₅), and rainfed (FI₀) treatments. In both growing seasons, soil moisture in the FI₂₅ and FI₀ treatments fell below the RAW line during the early growth stages. The moisture-depletion curve of the unirrigated FI₀ treatment remained entirely at or below the wilting point during the generative and seed-filling periods. This severe decrease in soil moisture indicates that the plant was exposed to prolonged, high-stress conditions throughout the growing season in both years.

Water–yield relationship and yield response factor (Ky)

Linear regression analysis between proportional yield loss and proportional evapotranspiration deficit produced high coefficients of determination (R² > 0.91) (Fig. 3), reflecting a strong and reliable relationship between the applied water stress and yield losses. The seasonal Ky, calculated from the regression slope of the regression line, was 1.1203 in 2021 and 1.1760 in 2022. Because proportional yield loss progressed faster than the proportional evapotranspiration deficit, fenugreek can be regarded as quite sensitive to water stress. The fact that Ky remained above unity in both consecutive years indicates a stable physiological vulnerability of fenugreek to soil moisture depletion.

Fig. 3.

Relationship between relative yield decrease and relative evapotranspiration deficit for determining the yield response factor (Ky) of fenugreek in the 2021 and 2022 growing seasons

The treatment-specific (point) Ky values, calculated for each deficit treatment relative to the FI₁₀₀ (Table 3), complement this seasonal estimate by revealing a non-linear yield–water relationship. Point Ky increased as the deficit became less severe, rising from 0.96 (FI₀) to 1.09 (FI₂₅), 1.54 (FI₅₀), and 2.33 (FI₇₅) in 2021, with a closely parallel trend in 2022 (0.97, 1.19, 1.61, and 2.76, respectively). Thus, under the most severe deficit (FI₀), the proportional yield loss was almost equal to the proportional evapotranspiration deficit (Ky ≈ 1), whereas under the mildest deficit (FI₇₅), a comparatively small reduction in evapotranspiration caused a proportionally much larger yield loss. This indicates that fenugreek is sensitive to even moderate departures from full irrigation, with a substantial share of the yield loss incurred during the first increment of water withdrawal.

Morphological characteristics and yield components

Different irrigation levels had significant effects on the morphological parameters and seed yields (p < 0.01) both within individual years and in the two-year average (Table 4). The year × irrigation interaction was significant for plant height, first-pod height and pod number, indicating that their response to irrigation level differed between the two seasons, whereas branch number and seed yield showed no significant interaction and responded consistently across years. Plant height (PH) increased with increasing irrigation water volumes in both seasons. As the average of two years, the greatest PH was found in the full irrigation treatment (FI₁₀₀, 59.53 cm), whereas the shortest plant height was recorded in the water-stress treatment FI₀ (32.90 cm). Water restrictions had significant effects on the first-pod height (FPH) and number of branches (NoB). The greatest first-pod height was 26.28 cm in the FI₁₀₀ treatment, and 11.83 cm in the FI₀ treatment. Similarly, the number of branches per plant decreased systematically with increasing water stress and ranged from FI₁₀₀ (1.83 branches) to FI₀ (1.17 branches) across the two-year average. The number of pods per plant (NoP) increased strongly with increasing irrigation levels. The greatest value was found in the FI₁₀₀ treatment (20.80 pods per plant), whereas the lowest value was seen in FI₀ treatment (5.45 pods per plant, representing a reduction of approximately 74%). These morphological and generative losses were directly reflected in seed yield. Seed yield ranged between 507 kg ha⁻¹ (FI₀) and 1428 kg ha⁻¹ (FI₁₀₀) in 2021, and between 559 kg ha⁻¹ (FI₀) and 1520 kg ha⁻¹ (FI₁₀₀) in 2022. Based on the two-year combined data, FI₁₀₀ treatment had the maximum yield of 1474 kg ha⁻¹, followed by FI₇₅ (864 kg ha⁻¹) and FI₅₀ (710 kg ha⁻¹) treatments. The lowest yield was obtained from the FI₀ treatment (533 kg ha⁻¹) under full water constraint.

Table 4.

Morphological characteristics and yield components of fenugreek (Trigonella foenum-graecum L.)

Treatments	PH	FPH	NoB	NoP	Yield
2021
FI0	33.00e	15.00c	1.23c	5.43e	507d
FI25	42.57d	16.23bc	1.57b	10.53d	634c
FI50	49.50c	17.47b	1.57b	14.73c	695c
FI75	55.50b	17.07b	1.73b	18.70b	867b
FI100	63.97a	26.83a	2.03a	23.57a	1428a
2022
FI0	32.80e	8.67c	1.10c	5.47c	559d
FI25	40.67d	21.77b	1.23c	6.10c	628cd
FI50	44.07c	21.13b	1.27bc	8.50b	724c
FI75	48.33b	22.77b	1.47ab	10.23b	861b
FI100	55.10a	25.73a	1.63a	22.90a	1520a
Average of the Years
FI0	32.90e	11.83c	1.17c	5.45e	533d
FI25	41.62d	19.00b	1.40b	8.32d	631c
FI50	46.78c	19.30b	1.42b	11.62c	710c
FI75	51.92b	19.92b	1.60b	16.90b	864b
FI100	59.53a	26.28a	1.83a	20.80a	1474a
Year (Y)	***	**	***	***	ns
Irrigation (I)	***	***	***	***	***
Y × I	*	***	ns	***	ns
CV (%)	5.22	6.41	7.73	10.68	5.64
LSD (Y)	1.85	0.94	0.09	1.03	3.62

Within each year and in two-year average, means followed by different letters differ significantly (Tukey HSD, p < 0.05)

PH Plant height (cm), FPH First pod height (cm), NoP Number of pods, NoB Number of branches, Yield Seed yield (kg ha-1), Y Year, I Irrigation level, ns Not significant, CV Coefficient of variation, LSD Least significant difference

*, ** and *** indicate significance at p < 0.05, 0.01 and 0.001, respectively

Crude oil content and fatty acid composition

The applied irrigation regimes had significant effects on crude oil content (CO) and fatty acid composition of fenugreek seeds (p < 0.01) (Table 5). The year × irrigation interaction was significant for all fatty acids except palmitoleic acid (POA), showing that the effect of irrigation level on fatty acid composition varied between the two growing seasons. Crude oil content decreased significantly in both years with water restriction. As the average of two years, the greatest crude oil content was seen in FI₁₀₀ (5.31%) and FI₇₅ (5.07%) treatments, whereas FI₀ reduced this value to 4.00%. An inverse response was observed in saturated fatty acids and unsaturated fatty acids. The proportions of palmitic acid (PA), a saturated fatty acid, and stearic acid (SA) increased significantly under water stress, with the highest average values of PA (10.77%) and SA (6.08%). With increasing irrigation, these proportions decreased to 9.24% and 4.56%, respectively in the FI₁₀₀ treatment. The proportion of oleic acid (OA), a monounsaturated fatty acid, also increased under water stress, reaching its highest level in the FI₀ treatment in the two-year average (14.84%). The highest LA (47.20%) and α-LA (24.26%) proportions were found in the FI₁₀₀ treatment in the two-year average. Water restriction (FI₀) reduced the LA proportion to 44.04% and the α-LA proportion to 21.98%, attributed to the inhibition of desaturase enzyme activities. The proportions of the minor fatty acids, palmitoleic acid (POA) and behenic acid (BA), did not change significantly across irrigation treatments.

Table 5.

Fatty acid composition of fenugreek (Trigonella foenum-graecum L.)

Treatments	CO	PA	POA	SA	OA	LA	ɣ-LA	α-LA	BA
2021
FI0	3.71d	11.21a	0.33	5.35a	14.57a	44.39d	1.63a	22.22d	0.28a
FI25	4.39c	9.53b	0.34	5.07b	13.55b	46.38c	1.56a	23.31c	0.26b
FI50	4.75b	9.33c	0.33	4.72c	13.07c	46.98b	1.47b	23.84b	0.26b
FI75	5.25a	9.29c	0.33	4.64d	12.79d	47.07b	1.35c	24.24a	0.29a
FI100	5.44a	9.23c	0.33	4.49e	12.55e	47.47a	1.26c	24.38a	0.29a
2022
FI0	4.29b	10.33a	0.29	6.80a	15.11a	43.69d	1.74a	21.74d	0.29
FI25	4.31b	9.80b	0.30	5.79b	14.42b	45.28c	1.59b	22.53c	0.30
FI50	4.53ab	9.41c	0.29	5.20c	14.27b	45.51bc	1.57b	23.46b	0.30
FI75	4.90ab	9.37c	0.30	4.97c	14.22b	45.95b	1.34c	23.56b	0.29
FI100	5.17a	9.24c	0.30	4.63d	13.18c	46.93a	1.28c	24.15a	0.29
Average of the Years
FI0	4.00c	10.77a	0.31	6.08a	14.84a	44.04d	1.69a	21.98e	0.29
FI25	4.35b	9.66b	0.32	5.43b	13.99b	45.83c	1.58b	22.92d	0.28
FI50	4.64b	9.37c	0.31	4.96c	13.67c	46.25b	1.52c	23.65c	0.28
FI75	5.07a	9.33cd	0.32	4.81d	13.50c	46.51b	1.34d	23.90b	0.29
FI100	5.31a	9.24d	0.31	4.56e	12.86d	47.20a	1.27e	24.26a	0.29
Year (Y)	ns	*	***	***	***	***	***	***	***
Irrigation (I)	***	***	ns	***	***	***	***	***	ns
Y × I	**	***	ns	***	***	**	**	***	***
CV (%)	4.17	0.91	5.22	1.39	1.12	0.38	1.91	0.35	2.89
LSD (Y)	0.15	0.07	0.01	0.05	0.12	0.13	0.02	0.06	0.01

Within each year and in two-year average, means followed by different letters differ significantly (Tukey HSD, p < 0.05)

CO Crude oil (%), PA Palmitic acid (%), POA Palmitoleic acid (%), SA Stearic acid (%), OA Oleic acid (%), LA Linoleic acid (%), ɣ-LA ɣ-Linolenic acid (%), α -LA α -Linolenic acid (%), BA Behenic acid (%), Y Year, I Irrigation level, ns Not significant, CV Coefficient of variation, LSD Least significant difference

*, ** and *** indicate significance at p < 0.05, 0.01 and 0.001, respectively

Phenolic profiles, total bioactive contents, and antioxidant activities

The total bioactive content, phenolic profiles, and antioxidant capacities of the fenugreek extracts showed a very strong (p < 0.01) secondary-metabolite accumulation response to the applied water-restriction strategies (Table 6). The year × irrigation interaction was significant for all phenolic and antioxidant variables except FRAP, indicating that their response to irrigation level was modulated by seasonal conditions.Total phenolic content (TPC), total flavonoid content (TFC), and total hydrolysable tannin content (THTC) increased significantly with increasing water stress. As the average of two years, the greatest TPC (159.4 mg GAE 100 g⁻¹), TFC (3.26 mg CE g⁻¹), and THTC (3205 mg kg⁻¹) values were seen under fully unirrigated conditions (FI₀). Under fully irrigated (FI₁₀₀) conditions, these parameters respectively decreased to 119.1 mg GAE 100 g⁻¹, 2.58 mg CE g⁻¹, and 1005 mg kg⁻¹. This indicates that the plant synthesizes polyphenolic compounds intensively, a response commonly associated with water deficit, although defense mechanisms were not directly measured in this study. In parallel with the bioactive accumulation, the antioxidant activities (ABTS, DPPH, and FRAP) of the extracts also peaked under water stress conditions. As the average of two years, the greatest free-radical-scavenging activities, ABTS (44.01 µg TE g⁻¹) and DPPH (58.73%), were seen in the FI₀ treatment, whereas the lowest values were recorded in the FI₁₀₀ treatment (37.44 µg TE g⁻¹ and 38.64%, respectively). This parallel increase indicates that the antioxidant capacity of the seed is largely driven by its phenolic compounds. Iron-chelating activity (ICA), however, behaved differently and varied between years, pointing to a distinct basis. The highest FRAP was recorded in the FI₀ treatment (0.20 mg AAE g⁻¹). ICA was significant in 2021, reaching its highest level in the FI₀ treatment (96.83%); however, it did not differ significantly among treatments in 2022. It was determined that increased water stress strongly activates the antioxidant defense system in fenugreek seeds.

Table 6.

Phenolic profiles, total bioactive contents, and antioxidant activities of fenugreek (Trigonella foenum-graecum L.) extracts

Treatments	ABTS	TPC	DPPH	ICA	TFC	FRAP	THTC
2021
FI0	44.34a	158.9a	57.44a	96.83a	3.26a	0.19a	2958a
FI25	42.88a	149.5b	43.02b	94.37ab	2.78b	0.18ab	1127b
FI50	39.88b	142.1c	41.96bc	93.37abc	2.73b	0.17ab	1066b
FI75	37.54c	137.6c	40.57c	91.00bc	2.56c	0.16bc	947c
FI100	37.01c	117.0d	33.31d	89.25c	2.50c	0.15c	914c
2022
FI0	43.67a	160.1a	60.01a	90.18	3.25a	0.21a	3452a
FI25	42.04b	158.6a	56.96b	92.04	3.02b	0.19b	3138b
FI50	41.99b	154.0a	57.76ab	91.90	2.88bc	0.19b	2964c
FI75	39.31c	125.3b	45.74c	93.53	2.78cd	0.18b	1871d
FI100	37.88c	121.2b	43.97c	92.78	2.65d	0.18b	1095e
Average of the Years
FI0	44.01a	159.4a	58.73a	93.50	3.26a	0.20a	3205a
FI25	42.46b	154.0b	49.99b	93.21	2.90b	0.18b	2133b
FI50	40.94c	148.1c	49.86b	92.64	2.81c	0.18b	2015c
FI75	38.43d	131.4d	43.16c	92.27	2.67d	0.17bc	1409d
FI100	37.44d	119.1e	38.64d	91.02	2.58e	0.16c	1005e
Year (Y)	*	**	***	ns	***	***	***
Irrigation (I)	***	***	***	ns	***	***	***
Y × I	**	***	***	***	**	ns	***
CV (%)	1.60	1.65	1.74	1.59	1.64	4.49	1.86
LSD (Y)	0.05	1.8	0.06	1.12	0.04	0.01	28

Within each year and in two-year average, means followed by different letters differ significantly (Tukey HSD, p < 0.05)

TPC Total phenolic content (mg GAE 100 g⁻¹), TFC Total flavonoid content (mg CE g⁻¹), THTC Total hydrolysable tannin content (mg kg⁻¹), FRAP Ferric reducing antioxidant power (mg AAE g⁻¹), ICA Iron chelating activity (%), ABTS Radical scavenging activity (µg TE g⁻¹), DPPH Radical scavenging activity (%), Y Year, I Irrigation level, ns Not significant, CV Coefficient of variation, LSD Least significant difference

*, ** and *** indicate significance at p < 0.05, 0.01 and 0.001, respectively

Principal Component Analysis (PCA) biplot integration

PCA was applied to combined data of two years to assess the effects of different irrigation levels on morphological, yield, fatty acid, and phenolic traits of fenugreek and to clarify their relationships. The first two components explained 89.1% of the total variance (PC1: 81.4%; PC2: 7.7%), so the treatments were clearly separated along PC1.

The FI₁₀₀ and FI₇₅ treatments were positioned on the negative side of PC1 and were closely associated, at narrow vector angles, with seed yield, plant height, number of pods, number of branches, first-pod height, crude oil (CO), and the LA and α-LA. This indicates that adequate irrigation promotes vegetative and generative growth, oil accumulation, and the synthesis of these unsaturated fatty acids. In contrast, the FI₀ and FI₂₅ treatments were positioned on the positive side of PC1, overlapping consistently with the TPC, TFC, THTC and with the antioxidant indicators ABTS, DPPH, FRAP, and—to a lesser extent—ICA. The saturated fatty acids PA and SA, together with OA and γ-LA, accumulated in the same region, suggesting that under water deficit the plant shifts its membrane lipids toward a more saturated profile while channeling metabolic carbon toward the phenolic-based antioxidant defense system. The FI₅₀ treatment, subjected to moderate water restriction, occupied an intermediate position between these two opposing poles (Fig. 4).

Fig. 4.

Principal Component Analysis (PCA) biplot illustrating the multivariate relationships among agro-morphological traits, phenolic profiles, and fatty acid compositions of fenugreek under different irrigation regimes

Discussion

The different irrigation levels markedly affected crop water consumptions, water-use efficiency, and yield performance of fenugreek. As irrigation decreased, seasonal ETa declined (Table 3), and in the FI₀ and FI₂₅ treatments the root-zone moisture fell below the RAW and WP thresholds, indicating that the plants were exposed to prolonged water stress during the generative period (Figs. 1 and 2). Plant height, first-pod height, number of branches and pods, and seed yield decreased with increasing water restriction levels, with the highest yield obtained under FI₁₀₀ and the lowest under FI₀. This demonstrates that vegetative and generative development in fenugreek depends largely on access to soil moisture [38]. The seasonal Ky exceeded unity in both years, confirming that fenugreek is sensitive to water deficit, with proportional yield loss outpacing the proportional reduction in evapotranspiration [39]. Moreover, the treatment-specific Ky values increased as the deficit became milder approaching unity under the most severe restriction (FI₀) but peaking under the mildest restriction (FI₇₅) indicating a non-linear response in which sensitivity is greatest near full irrigation and a large share of the yield penalty is incurred during the first increment of water withdrawal. Indeed, under FI₀, seed yield decreased by approximately 64% relative to FI₁₀₀, and the number of pods per plant declined from 20.8 to 5.45. It is known that water stress during the reproductive period causes yield loss by limiting cell expansion, stomatal conductance, photosynthesis, and assimilate translocation [40]. Furthermore, exposure of reproductive growth to heat and drought stresses suppresses the seed filling rate, thereby decreasing seed yield [41]. This physiological limitation particularly impaired pod and seed formation. The findings comply with the results of Kaplan et al. [38] and Badawy et al. [42], reporting supported plant growth and yield levels with adequate irrigations.

A different trend emerged for water-use efficiency. The yield per unit of irrigation water (IWP) was highest in the FI₂₅ treatment and decreased with subsequent irrigation increments. This indicates that the initial irrigation water provided the highest marginal return, whereas additional water provided progressively lower returns. However, because the high efficiency under FI₂₅ was accompanied by severe yield losses, focusing solely on water-use efficiency is not sufficient from an agronomic and economic standpoint [43]. Although FI₇₅ still entailed a marked yield reduction relative to full irrigation, it gave the highest yield among the deficit treatments and thus represents a compromise between water saving and yield among the deficit treatments, and may therefore be preferred where water—rather than yield—is the primary limiting resource. It has also been reported for various field and vegetable crops that applications at about 70–80% of full irrigation increase water-use efficiency while keeping yield loss at an acceptable level [44–46]. However, yield increased substantially as irrigation approached the full level. Therefore, the superiority of full irrigation should not be overlooked when maximum yield is the target. Within this framework, FI₇₅ may be considered a reasonable option for fenugreek production in regions where water is limited, provided that the associated yield reduction is acceptable.

In contrast to the yield traits, water restriction enriched bioactive compounds of fenugreek seeds. Total phenolics, flavonoids and hydrolysable tannin levels increased as water stress intensified and reached their highest values under the non-irrigated FI₀ condition. Full irrigation markedly reduced this accumulation. This shows that water stress stimulates secondary-metabolite biosynthesis in fenugreek as an adaptive defense mechanism developed against oxidative damage [47]. Phenolic compounds have also been reported to support drought tolerance by stabilizing cell membranes, regulating hormonal signaling, and facilitating osmotic adjustment [48, 49]. In parallel, the ABTS and DPPH radical-scavenging activities and the FRAP values also increased under conditions of high water stress. This suggests that antioxidant capacity is largely driven by phenolic constituents [50]. Flavonoids, an important subgroup of phenolics, may help maintain cellular redox balance by participating in the detoxification of hydrogen peroxide produced under drought [51]. These findings indicate that water restriction strengthens the antioxidant defense system of fenugreek, consistent with the physiological responses observed in Borago officinalis [42, 52] and Ehsanzadeh, 2025). Iron-chelating activity, by contrast, showed a more irregular response: high ICA was observed only under FI₀ conditions in 2021, whereas no significant differences among treatments were observed in 2022. This suggests that the compounds responsible for chelation are regulated differently from the phenolic antioxidants and are more strongly influenced by annual environmental conditions.

Fatty acid composition of fenugreek seeds was remarkably altered by water stress. Under water restriction, SFA (palmitic and stearic acid) levels increased, whereas PUFA (linoleic, α-linolenic and γ-linolenic acid) levels decreased. This decrease points to a drought-related reorganization of the membrane lipid structure and can be regarded as an adaptive response that preserves membrane stability and limits lipid peroxidation [53]. The decline in unsaturated fatty acids may be associated with the suppression of fatty acid desaturase enzymes under water stress; reduced activity of these enzymes limits double-bond formation, thereby decreasing the synthesis of LA and α-LA acids while increasing the proportion of saturated fatty acids [54, 55]. Similarly, in drought-tolerant and drought-sensitive bermudagrass genotypes, LA and α-LA acids have been reported to decrease and palmitic and stearic acids to increase under water deficit [56]. Such changes can be attributed to reorganization of the membrane lipid structure in response to drought stress [54, 57].

Overall, this study shows that fenugreek is highly sensitive to water deficit in terms of growth and seed yield, but its biochemical quality traits respond in the opposite direction to water stress. While severe water restriction caused significant yield losses, the FI₇₅ treatment offered an intermediate trade-off between water saving and yield, though still at a substantial yield cost; in contrast, water restriction increased phenolic compound accumulation, antioxidant activity, and stress-related fatty acid changes. Therefore, irrigation management in fenugreek cultivation should be planned according to the production objective. Full or near-full irrigation appears more appropriate when high seed yield is targeted, whereas controlled deficit irrigation appears more suitable when phytochemical quality and functional value are targeted [58, 59].

Biplot analysis allows the user to input two-way data [58]. It also allows differences among treatments to be screened across the examined traits and to identify the most prominent treatments [60, 61]. The present biplot separated the traits into two contrasting groups. Yield, plant height, number of pods and branches, crude oil, and the PFA (linoleic and α-linolenic acid) were positioned at narrow angles, indicating positive relationships among themselves, and were associated with the full and near-full irrigation treatments (FI₁₀₀, FI₇₅). In contrast, total phenolics, flavonoids, and hydrolyzable tannins and antioxidant activities (ABTS, DPPH, FRAP) clustered in the opposite direction and were associated with the water-deficit treatments (FI₀, FI₂₅). The location of these two groups at opposite ends of the PC1 axis indicates a negative relationship between the yield/oil-quality traits and the phenolic-based antioxidant traits; that is, as irrigation was associated with higher yield and unsaturated fatty acids with lower, phenolic accumulation and antioxidant capacity.

Conclusions

This study demonstrated that growth and seed yield in fenugreek are highly sensitive to water deficit, whereas the biochemical quality of the seed responds in the opposite direction. However, deficit irrigation inevitably reduced yield: relative to full irrigation, seed yield declined by about 41, 52 and 57% under FI₇₅, FI₅₀ and FI₂₅, respectively. FI₇₅ therefore represents a compromise that saves water at the cost of a substantial yield reduction, rather than an option that maintains yield. Increasing water stress raised the functional value of the seed by enhancing phenolic accumulation, antioxidant activity, and stress-related fatty acid changes. Therefore, irrigation should be planned according to the production objective: full or near-full irrigation when maximum seed yield is the priority, and controlled deficit irrigation when phytochemical quality is targeted. Because the findings are limited to 2 years and a single location, and because the underlying mechanisms were not directly measured but inferred, multi-year, multi-location studies supported by physiological and molecular analyses are recommended to confirm and extend these results.

Authors’ contributions

I.S.V. : Conceptualization, Data curation, Formal analysis, Methodology, Field Experiment, Writing-original draft, Writing-review and editing.

Funding

This research received no external fundindg.

Data availability

Data will be made available on request.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.