Effects of Water Stress and Mulch Type on Linseed Seed Yield, Physiological Traits, and Oil Compounds

Abstract

This study investigated the effects of three mulch types (straw, vermicompost and “plastic”) plus an untreated control, and three irrigation regimes (RFD: rainfed conditions; SIF: one supplemental irrigation at the flowering stage; SIVF: two supplemental irrigations at the vegetative and flowering stages) on the growth, seed yield, oil composition, and biochemical status of linseed (Linum usitatissimum L.). Linseed plants were best affected by SIVF and straw mulch in terms of seed yield (300 and 222.4 g m⁻², respectively), biomass yield (887.9 and 703 g m⁻², respectively), and concentration of oleic and linoleic acids. Under rainfed conditions, “plastic” mulch application increased stearic acid concentrations, while SIF increased palmitic acid concentrations. Rainfed conditions promoted the accumulation of proline (10.1 μmol g⁻¹ fresh weight), total phenols (6.68 mg g⁻¹ fresh weight), and DPPH radical scavenging capacity (56.5%). Under RFD, plants grown in straw-mulched soil showed the highest total phenol content and DPPH radical scavenging capacity, while control (unmulched) plants displayed the highest proline concentration at this irrigation regime. Enzyme activities, including catalase and superoxide dismutase, were enhanced under straw and “plastic” mulch compared to control plants under rainfed conditions. Our findings suggest that straw mulch represents an effective, sustainable strategy to successfully manage linseed crops, mitigating the adverse effects of water deficit stress on plant performance.

1. Introduction

Oilseed crops are cultivated primarily for their seed oil content, catering to various industries and diets, and, in this respect, soybean, sunflower, rapeseed, cotton, and peanuts are among the key ones [1]. Linseed (Linum usitatissimum L.) is one of the oldest cultivated oilseed species, prized for its versatile applications in foods, paints, varnishes, and linoleum flooring [2].

Linseed is rich in bioactive compounds like unsaturated fatty acids, fiber, proteins, antioxidants, and lignans, offering potential health benefits, including cancer prevention [3], and, in this respect, its α-linolenic acid content is the highest among plant sources [4]. The 18-carbon alpha-linolenic acid is a plant-based source of essential omega-3 polyunsaturated fatty acids that are beneficial for maintaining a healthy diet [4], with positive effects on heart health, nervous system function, and inflammatory regulation [3]. Linseeds are also rich in protein and B-group vitamins [5].

Environmental changes, including decreased rainfall, pose challenges to crop cultivation, with water scarcity significantly impacting crop growth and productivity, especially during critical growth phases [6]. Insufficient moisture disrupts metabolic pathways crucial for water relations, photosynthesis, antioxidant function, and mineral uptake, affecting crop quality and yield [7]. Like other crops, linseed suffers from impaired gas exchange and leaf water relations under water stress [5]. Moisture deficit impedes plant growth by inhibiting cell expansion, cell wall division and differentiation [7], and photosynthesis due to stomata closure [8] as well as destroying chlorophyll due to the excessive release of reactive oxygen species (ROS) [9], leading to decreased productivity [10].

Mulching material protecting soil surface can be organic or inorganic. Organic mulches, derived from natural plant materials, include straw, wood chips, grass clippings, shredded leaves, and compost. The mentioned biodegradable materials enrich the soil with organic matter as they decompose [11], improving soil structure and nutrient content over time. They also allow the retaining of moisture by reducing evaporation and suppress weeds, minimizing competition for nutrients and water while enhancing fertility [12]. During decomposition, organic mulches release essential nutrients [13] and stimulate microbial activity, which is crucial for nutrient cycling and can increase nitrogen availability. Inorganic mulches, made of non-biodegradable materials like “plastic” films, gravel, stones, and synthetic fabrics, offer different benefits. Plastic mulch (especially black and clear types) is widely used for boosting crop yields as it raises soil temperatures, enabling earlier planting and faster growth. The black material absorbs sun rays, thus preventing weed germination and reducing labor costs and resource competition. Unlike organic mulches, they do not decompose, requiring less frequent replacement and lowering long-term costs. Additionally, they preserve soil moisture by limiting evaporation, which is particularly valuable in arid regions [14]. Notably, “plastic” mulch effectively enables the storing of soil moisture, enhances water use efficiency [12], and is particularly valuable for high-value row crops [15]. However, there are drawbacks: under extreme heat, they can overheat the soil, harming plants; since they do not decompose, they fail to enhance soil health or add organic matter; improper disposal can lead to “plastic” waste accumulation in ecosystems. The decision between organic and inorganic mulches depends on climate, budget, and farming objectives [11]. While organic mulches improve long-term soil health through organic matter and microbial activity, inorganic mulches boost short-term yields without sustaining fertility. Although organic mulches may have lower upfront costs (especially if locally sourced), their frequent replenishment raises expenses over time. Conversely, inorganic mulches require a higher initial investment but offer durability, reducing long-term costs [16].

Gao et al. [17] reported that white “plastic” film mulching significantly increased the oil flax leaf area, chlorophyll content, and dry matter accumulation; differently, black film mulching with micro-ridge and soil covering, as well as white film mulching with micro-ridge without soil covering, boosted oil flax grain yields by 29%. It was reported that organic mulching significantly improved soil moisture retention and the Camellia oleifera yield, with straw showing the strongest cooling effect and straw+ecological pad providing optimal moisture conservation. Ecological pad led to the highest yield increase and oil content, while gravel yielded peak oil content [18]. Moreover, vermicompost and straw mulch increased soil microorganism activity and water retention, especially in water-deficient conditions [9,19]. Recent studies highlighted the significant benefits of organic mulching in agricultural systems. Research by Youssef et al. [20] revealed that organic mulches, such as rice straw, enhanced soil quality, plant growth, and squash yields. Similarly, Barreda et al. [21] reported that rice straw mulch suppressed weeds and boosted soil fertility and grain production. Other studies also emphasized the impact of straw on water conservation. Paul et al. [22] noted improved water-use efficiency and soil moisture retention, resulting in higher crop yield. Noor et al. [23] linked yield gains to wheat straw regulating soil temperature and moisture. Additionally, Aziz et al. [24] confirmed the dual role of wheat and straw in weed suppression and moisture conservation, further enhancing rainfed wheat yield. Wang et al. [25] found that biodegradable film performance was comparable to that of ordinary “plastic” mulch in terms of oil flax growth duration (≤2 days difference), dry matter accumulation (11–28% lower), and grain filling rate, while all mulching treatments increased yield by ~19%.

A comprehensive understanding of how different irrigation regimes interact with various mulching treatments is essential for optimizing linseed (Linum usitatissimum L.) productivity under diverse moisture conditions. In the present research, the effectiveness of three mulching treatments, i.e., “plastic” mulch, straw mulch, and vermicompost, in improving linseed seed yield, oil quality, and overall crop performance was assessed. Specifically, the effects of the mentioned treatments were determined under three irrigation regimes: rainfed (RFD), one supplemental irrigation at the flowering stage (SIF), and two supplemental irrigations at the vegetative and flowering stages (SIVF). The goal was to identify the most effective strategy for enhancing linseed productivity, quality, and oil content, especially under water-limited conditions.

2. Materials and Methods

2.1. Growing Conditions and Experimental Design

Research was conducted in 2022 at a farm located in Urmia city in Western Azerbaijan province, Iran (37°3′24.82″ N, 44°58′12.42″ E, 1332 m above sea level), characterized by a semi-arid climate, with an average annual temperature of 13.4 °C and annual precipitation of 261 mm, in a loam-clay soil (at 0–0.3 m depth), with 0.12% N, 395 mg kg⁻¹ K, 10 mg kg⁻¹ P, and pH 7.9. The meteorological data (

Table 1. Weather data from October 2021 to September 2022 at the research site in Urmia.

	Oct	Nov	Dec	Jan	Feb	Mar	Apr	May	June	July	Aug	Sep
Monthly average temperature (°C)	3.3	30.6	48.9	19.6	13.4	20.9	18.1	58.4	1.2	1.4	0.1	0.1
Monthly average precipitation (mm)	16.3	9.1	5.4	1.7	0.5	5.3	10.7	16.4	22.4	25.6	28.0	25.3

) were obtained from the Iran Meteorological Organization, and the average annual temperature and precipitation over the last ten years were 12 °C and 390 mm, respectively, at the research site.

Linseed seeds were sourced from the Agriculture and Natural Resources Research and Education Centre of Urmia, Iran. Seeds were sown manually at a 4 cm depth on 17 March 2022. Before sowing, fertilization with 80 kg ha⁻¹ urea and 100 kg ha⁻¹ triple superphosphate was practiced. Plant thinning occurred at the 3–4 leaf stage, manual weeding was performed throughout the vegetation period as required, and no pesticides were used during the growing season.

The experimental protocol was based on the factorial combination of three mulch treatments (straw and vermicompost as organic mulches; “plastic” as synthetic mulch) plus an untreated bare soil control, and three irrigation regimes (RFD: rainfed conditions; SIF: one supplemental irrigation at the flowering stage; SIVF: two supplemental irrigations at the vegetative and flowering stages), using a randomized complete block design with three replicates for the treatment distribution in the field. The mulching materials used in this study consisted of (1) wheat straw, comprising dried and chopped wheat straw (Triticum aestivum) with particle lengths of 5–10 cm, applied uniformly at a rate of 5 kg m⁻²; (2) mature vermicompost exhibiting the following characteristics: pH 6.5–7.5, 60–70% moisture content, >40% organic matter, and a carbon-to-nitrogen (C/N) ratio of ~20; it contained a minimum viable earthworm population of 10 individuals per kg and was applied at a 1 kg m⁻² rate before sowing (3) white “plastic” film (non-biodegradable and not containing starch or other biodegradable additives), made of conventional low-density polyethylene (LDPE) with a thickness of 0.012 mm and a width of 1.2 m. Wheat straw mulch gradually releases carbon and nitrogen compounds into the soil during decomposition, enhancing microbial activity and improving nitrogen cycling through ureolysis and nitrogen fixation processes. The mentioned decomposition also reduces nitrate leaching, leading to higher nitrate accumulation. Additionally, reducing evaporation increases soil moisture retention and stabilizes soil temperature, enhancing plant nitrogen and water use efficiency [26]. Vermicompost releases many essential nutrients, including nitrogen, phosphorus, potassium, and trace elements, without introducing toxic substances. Its balanced C/N ratio (~20) and high microbial load foster microbial diversity and activity, soil structure, moisture retention, and moderate temperature fluctuations [27]. Although white “plastic” mulch does not release nutrients, it modifies the soil microenvironment by preserving moisture and increasing soil temperature. The latter changes indirectly influence the quantity and composition of soil microflora, as elevated temperature and moisture stimulate root exudation, promoting microbial growth and activity [28,29].

The experimental design comprised 36 plots (12 treatments × 3 replicates), each covering a 12 m² surface area (3 × 4 m). Plants were spaced 10 cm along the rows, which were 50 cm apart. Adjacent plots were separated by 1.5 m buffer zones to prevent treatment interference, and the three blocks were 2 m distant from each other to have equipment access (Figure 1).

The agronomic management was uniform across the treatments. Irrigation was applied during the vegetative and flowering stages. RFD only relied on natural precipitation. The amount of water applied (6400 m³ ha⁻¹ per irrigation cycle) was determined based on the soil moisture deficit required to reach field capacity using Walker’s [30] method. Supplemental irrigation accounted for 20% of the average annual long-term rainfall in the area. Supplementary irrigation was carried out at the vegetative stage and at the beginning of flowering, according to the experimental protocol, by a volumetric meter. Irrigation was carried out by installing a volumetric meter in each plot, and each water volume applied to a plot was never removed or transferred from the addressed experimental unit.

Representative leaf samples were randomly collected at the full flowering stage, flash-frozen in liquid nitrogen, and stored at −80 °C for subsequent analysis. Plants were harvested from 5 m² within each plot, excluding the edges, at the end of the growing season on 29 June 2022.

2.2. Growth Parameters

At maturity, samples of 10 linseed plants per plot (30 plants per treatment) were randomly harvested to assess growth parameters, i.e., plant height, branch number, follicle number, seed number per follicle, and 100-seed weight, and on a 2 m² area in each plot center to measure seed and biomass yield. Dry weight as biomass yield was determined after drying plants in a ventilated oven at 72 °C for 48 h. Seeds were separated from follicles and weighed after oven-drying at 72 °C to adjust the moisture content to 14–15%.

2.3. Oil Content and Oilseed Yield

Linseed oil content was extracted from dried seeds using the American Oil Chemists’ Society [31] method. In summary, 5 g of ground seed was subjected to a six-hour extraction using 300 mL n-hexane in a Soxhlet extractor before removing the solvent using a rotavapor (Heidolph, Schwabach, Germany). The resulting oil was collected in a dedicated glass container to facilitate further compound isolation and identification [32].

Seed oil content and yield were determined as follows [33]:

Oil content (%) = (Extracted oil content (g))/(5 g of linseed seed) × 100

(1)

Oil content yield (kg ha⁻¹) = Oil content (%) × Seed yield (g m⁻²)

(2)

2.4. Fatty Acids

Fatty acids were converted into fatty acid methyl esters (FAMEs) to enhance their volatility for GC-FID analysis by mixing 0.1 g of oil with 1.5 mL of hexane and 0.2 mL of 2 N methanolic KOH. The mixture was vortexed for 5 s and then centrifuged at 2500 rpm for 1 min. The upper layer containing the FAMEs was carefully separated and stored at 4 °C for further analysis. GC-FID analysis was conducted using an Agilent 6890 N gas chromatography (GC) instrument (Wilmington, DE, USA) with an FID detector. FAME separations were carried out on an HP-88 capillary column (88% cyanopropyl aryl-polysiloxane, 100 m length, 0.25 mm inner diameter, 0.2 μm film thickness) (Agilent) as follows: initially at 140 °C for 5 min, followed by 4 °C min⁻¹ increase to 240 °C, and finally at 240 °C for 15 min. Nitrogen was the carrier gas at a 1.0 mL min⁻¹ flow rate. The injection port and detector temperatures were set at 260 °C and 280 °C, respectively. The injector operated in split mode with a 1:30 split ratio. Data acquisition and processing were performed using ChemStation (B.03.01) software. A commercially available FAME mixture (Supelco 37 Component FAME Mix, Bellefonte, PA, USA) was used as a reference standard to identify fatty acids, with methyl nonadecanoate (C19:0 ME) serving as the internal standard for quantification.

2.5. Relative Water Content (RWC)

The RWC was determined as follows [34]:

% RWC = [(FW − DW)/(TW − DW)] × 100

(3)

After determining the fresh weight (FW), leaves were soaked in distilled water for 16–18 h. The turgid samples were then quickly blotted dry to remove excess surface water before measuring the turgid weight (TW). The leaves were oven-dried at 70 °C for 24 h to determine dry weight (DW).

2.6. Chlorophyll and Carotenoid Content

Fresh leaf samples (0.5 g) at the full flowering stage were pulverized in liquid nitrogen, mixed with 10 mL of 80% acetone, and homogenized by centrifugation at 4000 rpm for 15 min. Subsequently, the extracted pigments were quantified using a spectrophotometer (N50-Touch, Implen GmbH, Munich, Germany), enabling accurate measurement of chlorophyll a, b, and carotenoid contents at wavelengths of 663 nm, 645 nm, and 470 nm, respectively [35].

2.7. Total Soluble Sugar Content

The phenol-sulfuric acid method was used to estimate the total soluble sugar content of the leaf. Leaf tissue (0.5 g) was powdered in a mortar using liquid nitrogen, mixed with ethanol, and combined with 5% phenol. Subsequently, 5 mL of 98% sulfuric acid was added to the mixture of solutions and incubated for one hour before measuring the solution absorption at 485 nm using a spectrophotometer [36].

2.8. Proline Content

Leaf proline content was determined using the ninhydrin colorimetric method. Briefly, 0.5 g of leaf tissue was ground in a mortar using liquid nitrogen, homogenized in 10 mL of 3% sulfosalicylic acid solution, and then centrifuged at 4000 rpm for 15 min to obtain a clear supernatant. A glacial acetic acid solution of proline ninhydrin acid was prepared in a 1:1:1 ratio for the colorimetric evaluation of proline and equilibrated at 100 °C for one hour to facilitate the reaction between proline and ninhydrin, forming a chromophore. The reaction was terminated by rapidly cooling the solution in an ice bath. To develop the chromophore, 4 mL of toluene was added to the reaction mixture, enabling the extraction of the chromophore into the organic phase. The absorbance of the samples was measured at 515 nm using a spectrophotometer [37].

2.9. Antioxidant Enzyme Extractions and Assays

Antioxidant enzyme activities were quantified by finely pulverizing 100 mg fresh material in 2 mL of 0.1 M KH₂PO₄ buffer, including 5% polyvinylpyrrolidone (PVP) at pH 6. Extracts from the plants were centrifuged for 30 min at 3 °C at 15,000 rpm, with enzyme activity determined from the clear supernatant [38].

Catalase (CAT) activity was measured at 240 nm using hydrogen peroxide (H₂O₂) concentration change. The reaction mixture contained 1.9 mL of 50 mM K₃PO₄, buffered at a pH of 7, 10 mM H₂O₂, and 0.2 mL of enzyme extract. Enzymatic activity was measured in 60 s mg⁻¹ according to variations in protein absorption [39].

Superoxide dismutase (SOD) activity was measured at 560 nm to minimize the photochemical loss of nitroblue tetrazolium (NBT), as described by Beyer and Fridovich [40]. One unit of SOD was defined as the enzyme amount required to inhibit a 50% decrease in NBT.

2.10. Total Polyphenol Content

Total phenolic content (TPC) was measured using the Folin–Ciocalteau method [41]. Briefly, 1600 µL purified water and 10 µL extracts containing methanol were combined and incubated for 5 min at 25 °C with 200 µL Folin–Ciocalteau reagent (10% v/v) prepared in distilled water. After adding 200 µL NaCO₃ (7.5%), the solution mixture was kept at 25 °C in darkness for 30 min. The absorbance of the samples was measured at 760 nm by a UV/visible spectrophotometer (DB-20/DB-20S) for quantitative determination of TPC. The results are presented in milligrams of equivalent gallic acid (3,4,5 trihydroxybenzoic acid) per gram of sample dry weight (mg GAE g⁻¹ dry weight).

2.11. DPPH (2,2-Diphenyl-1-picrylhydrazyl-hydrate) Radical Scavenging Activity

DPPH radical scavenging activity was measured according to Brand-Williams et al. [42]. After combining 2.0 mL DPPH solution with 15 μL methanolic extract, the mixture was left in the dark at 20 °C for 30 min. The absorbance of the resulting solution was determined at 517 nm. Equation (4) was used to calculate DPPH inhibition.

Inhibition (%) = ((Abcontrol − Absample)/(Abcontrol)) × 100

(4)

where Abcontrol and Absample are the absorbances of the control (solution containing 2.0 mL DPPH and 15 μL pure methanol, without extract) and the sample, respectively.

2.12. Data Statistical Processing

To ensure reproducibility, all data represent means ± standard error (SE) derived from three biological replicates (n = 3) per treatment. Data were statistically processed by analysis of variance, and Duncan’s multiple range test was used for mean separation at p ≤ 0.05, using the SAS 9.1 software. Significant differences between treatments are denoted by lowercase letters in the tables and figures.

3. Results

The analysis of variance revealed that both mulch type and irrigation regime significantly influenced flaxseed growth and yield parameters, such as plant height, shoot number, follicle number, seed yield, biomass yield, and oil content (p < 0.05). However, the interaction between mulch and irrigation treatments did not significantly affect the mentioned traits (p > 0.05). In contrast, a significant interactive effect was recorded for three key yield components: follicle seed number, 100-seed weight, and oil yield (

Table 2. Analysis of variance of linseed biometrical and yield traits as influenced by mulch type and irrigation regime.

Source of Variation	Plant Height	Branch Number	Follicle Number	Follicle Seed Number	100-Seed Weight	Seed Yield	Biomass Yield	Oilseed Content	Oilseed Yield
Mulch (M)	**	**	**	**	**	**	**	**	**
Irrigation condition (IC)	**	*	**	*	**	*	*	**	**
M × IC	n.s.	n.s.	n.s.	*	**	n.s.	n.s.	n.s.	*

** and *: significant at p ≤ 0.01 and 0.05, respectively; n.s., not significant.

).

3.1. Biometrical and Seed Yield Parameters

The mulch type and irrigation regime significantly influenced plant height (Figure S1): Plants that received two irrigations during the vegetative and flowering stages were the tallest (37.0 cm), not significantly different from those irrigated only at the flowering stage, whereas rainfed plants were significantly shorter (−11.1%).

Among mulching treatments, the top plant height (37.4 cm) was recorded under straw mulch, followed by vermicompost, while the control (no mulch) plants were the shortest (−13.4%; Figure S1).

The number of branches and follicles per plant was significantly affected by mulch type and irrigation regime (Figure S1). Plants irrigated during both the vegetative and flowering stages produced the highest number of branches (5.9). In contrast, the lowest branch number (−16.9%) was observed under rainfed conditions, which was not significantly different from plants irrigated only during the flowering stage. As for mulching treatments, straw mulch induced the top number of branches (5.5), which was not significantly different from vermicompost but significantly higher than the control by 12.7% (Figure S1).

Similarly, the highest number of follicles (94.9) was obtained from plants receiving two irrigations, while the lowest (−43.5%) was recorded under rainfed conditions, with no significant difference compared to the single irrigation at the flowering stage. Regarding mulch effects, straw mulch led to the highest follicle number (83.7), not significantly different from vermicompost or “plastic” mulch, while the unmulched control resulted in the lowest number (−31.9%) (Figure S1).

Mulching and irrigation treatments significantly affected the number of seeds per follicle and 100-seed weight (Figure 2). Plants mulched with straw and irrigated twice produced the highest number of seeds per follicle (7.9), which was not significantly different from plants receiving only one irrigation. In contrast, the lowest number of seeds was observed in control plants under rainfed conditions.

The highest 100-seed weight (0.44 g) was also recorded under straw mulch combined with two irrigations, while the control plants under rainfed conditions showed the lowest value (−18.2%), not significantly different from that obtained under one irrigation during the flowering stage (Figure 2).

Mulch and irrigation treatments significantly influenced seed and biomass yield (Figure S2).

Seed yield was significantly higher in irrigated plants compared to rainfed ones. The maximum yield (300.0 g m⁻²) was obtained under two irrigations (vegetative + flowering stages), while the lowest yield (−56.5%) was observed in rainfed control plants. As for mulching treatments, straw mulch had the highest seed yield (222.4 g m⁻²), and the unmulched control had the lowest (−15.8%). No significant differences were recorded between vermicompost and “plastic” mulch treatments. A similar trend regarded the biomass yield: The highest value (887.9 g m⁻²) was recorded under two irrigations and was the lowest (−47.5%) in rainfed control plants. Among mulches, straw produced the highest biomass (703.0 g m⁻²).

3.2. Oilseed Content, Yield, and Fatty Acid Composition

Plants irrigated twice (at vegetative and flowering stages) showed the highest oil content (38.5%), and the rainfed plants had the lowest (33.8%). As for mulching treatments, straw mulch resulted in the highest oil content (37.8%), which was not significantly different from vermicompost or “plastic” mulch, while the control treatment produced the lowest oil content (33.7%) (Figure S3).

The interaction between the mulch type and irrigation regime had a significant effect on oil yield. Under the RFD irrigation, both “plastic” and straw mulch significantly increased oil yield compared to the untreated control under the mentioned irrigation treatment. Corresponding to SIF and SIVF, the application of all three mulch types, vermicompost, “plastic”, and straw, resulted in a significant increase in the oil yield compared to the control. The highest oilseed yield (129.7 g m⁻²) was obtained from plants receiving two irrigations combined with straw mulch, whereas the lowest yield (−69.3%) was recorded in control plants under rainfed conditions, which was not significantly different from those irrigated only once (Figure 3).

The GC-MS and GC-FID analysis identified all major fatty acid components in linseed oil: palmitic acid (4.7–7.4%), stearic acid (5.0–5.8%), oleic acid (21.5–25.9%), linoleic acid (10.7–12.9%), linolenic acid (38.3–52.6%), and arachidic acid (0.21–0.65%) (

Table 3. Composition of linseed oil fatty acids in mulch and stress modifier applications.

No.	Component					Treatment
		RFD	RFD	RFD	RFD	SIF	SIF	SIF	SIF	SIVF	SIVF	SIVF	SIVF
		C	V	PM	SM	C	V	PM	SM	C	V	PM	SM
1	Palmitic acid	6.09	6.18	6.19	6.41	6.3	6.49	7.38	6.96	5.08	5.1	4.49	4.68
2	Stearic acid	5.08	5.29	5.78	5.53	5.61	5.67	5.5	5.66	5.15	5.09	4.97	5.45
3	Oleic acid	21.54	21.93	23.02	23.28	23.65	24.22	24.12	24.78	23.11	25.02	24.96	25.99
4	Linoleic acid	12.06	12.57	12.18	12.28	12.05	12.15	12.32	12.66	10.67	12.85	12.58	12.91
5	Linolenic acid	50.26	52.56	51.99	51.71	50.73	50.86	49.05	51.46	41.09	41.3	38.31	40.46
6	Arachidic acid	0.65	0.21	0.21	0.21	0.21	0.21	0.21	0.21	0.21	0.21	0.21	0.21

C, V, PM, and SM correspond to control, vermicompost, “plastic”, and straw mulch, respectively. RFD, SIF, and SIVF correspond to rainfed, with one supplementary irrigation at the flowering stage and two supplementary irrigations in the vegetative and flowering stages, respectively.

). Notably, linolenic acid was the predominant component, accounting for 38–53% of the total fatty acids, significantly higher than all other acids. Under straw mulch, plants showed the highest oleic acid content (25.99%) with SIVF and the lowest (21.5%) under rainfed conditions. The maximum linoleic acid percentage (12.9%) was elicited by straw mulch and two irrigations, while the minimum (10.7%) was recorded in bare soil. Palmitic acid peaked (7.4%) with “plastic” mulch and single irrigation and was the lowest (4.7%) under straw mulch and two irrigations. Linolenic acid showed its highest levels (50–53%) in rainfed treatments and the lowest (38.3%) with “plastic” mulch and two irrigations, suggesting that irrigation may reduce omega-3 content. Stearic acid ranged from 5.0% to 5.8% under “plastic” mulch and rainfed conditions. The exceptional concentration of linolenic acid, representing over 50% of the total fatty acids in some treatments, confirms its status as the most nutritionally and industrially significant component of linseed oil.

3.3. Physiological Traits

Relative Water Content

Linseed relative water content (RWC) was significantly influenced by mulch type and irrigation regime (

Table 4. Analysis of variance of linseed physiological traits as influenced by mulch type and irrigation regime.

Source of Variation	Relative Water Content	Chlorophyll a	Chlorophyll b	Carotenoids	Total Soluble Sugars	Proline Content	CAT	SOD	Total Phenol Content	DPPH Radical Scavenging Capacity
Mulch (M)	**	**	**	*	**	**	**	**	**	**
Irrigation regime (IR)	*	*	n.s.	**	**	**	**	*	**	**
M × IR	n.s.	**	*	**	**	**	**	**	**	**

** and *: significant at p ≤ 0.01 and 0.05, respectively; n.s., not significant.

, Figure S4). Plants receiving two irrigations during the vegetative and flowering stages exhibited the highest RWC (54.8%), which was not significantly different from that recorded under one irrigation during flowering; in contrast, rainfed plants showed the lowest RWC (39.5%). With respect to mulching treatments, straw mulch resulted in the highest RWC (52.6%), not significantly different from “plastic” mulch, and the bare soil-grown plants (control) showed the lowest value (44.7%).

3.4. Photosynthetic Pigments

The interaction between the mulch type and irrigation regime significantly affected the levels of chlorophyll a, chlorophyll b, and carotenoids (Table 4), which were the highest in plants grown in straw-mulched soil under two irrigations (1.5, 0.6, and 1.37 mg g⁻¹ fresh weight, respectively).

However, the interaction between the mulch type and irrigation was significant on chlorophyll b and carotenoids, with no significant differences between the organic and synthetic mulches under two irrigations; in contrast, plants grown in bare soil under rainfed conditions showed the lowest levels of chlorophyll a, b, and carotenoids (0.8, 0.3, and 0.7 mg g⁻¹ fresh weight, respectively) (Figure 4).

Moreover, no significant differences in chlorophyll b were recorded between the unmulched control and organic or synthetic mulches under rainfed conditions. Similarly, no carotenoid differences were observed between vermicompost-treated plants and the control under rainfed conditions (Figure 4).

3.5. Total Soluble Sugars and Proline Content

The interaction between the mulch type and irrigation regime was significant in terms of the total soluble sugar and proline content (Table 4, Figure 5).

Straw mulch combined with two irrigations resulted in the highest total soluble sugar content (18.8 mg g⁻¹ fresh weight), while the plants grown in bare soil under rainfed conditions showed the lowest sugar content (−20.2%), not significantly different from that elicited by mulch with vermicompost under rainfed conditions. As for proline content, plants grown in unmulched soil under rainfed conditions had the highest level (10.1 μmol g⁻¹ fresh weight), while no significant differences were observed between vermicompost and “plastic” mulch treatments. Interestingly, two irrigations at the vegetative and flowering stages resulted in the lowest proline content under straw mulch (5.0 μmol g⁻¹ fresh weight), and additionally, one irrigation at the flowering stage did not lead to significant differences between vermicompost and bare soil.

3.6. Antioxidant Enzyme Activity

The interaction between the mulch type and irrigation regime significantly influenced the activities of antioxidant enzymes (Table 4, Figure 6).

Under rainfed conditions, plants treated with straw mulch showed the highest CAT and SOD activities (6.9 and 16.2 μmol min⁻¹ g⁻¹ fresh weight, respectively). No significant differences in CAT activity were recorded between straw and “plastic” mulch treatments under rainfed conditions (Figure 6). Similarly, there were no significant differences in SOD activity between vermicompost and “plastic” mulch treatments under rainfed conditions.

Unmulched soil-grown plants irrigated twice during the vegetative and flowering stages showed the lowest antioxidant enzyme activities (0.7 and 4.8 μmol min⁻¹ g⁻¹ fresh weight for CAT and SOD, respectively) (Figure 6).

3.7. Total Phenol Content and DPPH Radical Scavenging Capacity

The interaction between the mulch type and irrigation regime significantly influenced both total phenol content and DPPH radical scavenging capacity (Table 4, Figure 7).

Under rainfed conditions, linseed plants grown with straw mulch exhibited the highest total phenol content (6.7 mg g⁻¹ fresh weight) and DPPH radical scavenging capacity (56.5%), significantly outperforming other treatments (p ≤ 0.05). In contrast, plants grown in bare soil with SIVF showed the lowest values (4.3 mg g⁻¹ fresh weight phenols; 21.3% DPPH activity).

No significant differences were recorded between the mulch types in terms of DPPH activity, under RFD and one irrigation at flowering (SIF), and of phenol content, with two irrigations at the vegetative and flowering phenological phases (Figure 7).

4. Discussion

Water shortage stress impairs plant growth by reducing cell viability, development, division, and elongation, which diminishes vegetative and reproductive growth [43]. Also, it decreases stomatal conductance due to inhibited Rubisco activity, raising crop water potential [44]. Stress-induced decline in agronomic traits and seed yield is linked to prioritized root resource allocation under low soil moisture or reduced chlorophyll content, limiting photosynthesis [45]. Stomatal closure during moisture stress reduces gas exchange, CO₂ intake, and Rubisco activity, decreasing dry matter production [46].

The results indicated that irrigation regime and mulch type had independent effects on several morphological and yield-related traits in linseed. Plant height, number of branches per plant, number of follicles per plant, seed yield, oil seed content, and biomass yield were significantly affected by irrigation and mulch separately. The highest values for these traits were observed under two irrigations and straw mulch, while the lowest values were recorded under rainfed and no mulch conditions.

In contrast, oil yield, seed number per follicle, and 100-seed weight showed a significant interaction between the mulch type and irrigation regime. These traits reached their maximum levels under two irrigations combined with straw mulch and the minimum in the rainfed control.

The mentioned findings suggest that most agronomic traits responded independently to irrigation and mulching, but some reproductive traits were enhanced only through their combined application. This underlines the importance of optimizing both water availability and mulch type to improve linseed productivity under semi-arid conditions.

Mulching has been widely reported to support crop growth and yield under water-limited conditions by improving nutrient availability, enhancing soil moisture retention, and regulating root zone temperature [47]. Oil yield in linseed is a function of both seed yield and oil content, which may be influenced by mulching and irrigation management. Previous studies have shown that water stress tends to reduce oil content in linseed due to impaired physiological processes such as reduced chlorophyll content, enzyme activity, and assimilate translocation [48]. Conversely, the application of organic or synthetic mulches has been associated with improvements in oil content and yield, likely due to enhanced leaf water potential, CO₂ assimilation, hormonal balance, and nutrient uptake [49].

In other oilseed crops such as sunflower, mulching under moisture stress has been reported to improve nutrient absorption and carbohydrate availability, thereby enhancing oil accumulation in seeds [50,51]. In this study, straw mulch combined with two irrigations showed the highest total soluble sugar content. Since carbohydrates play a central role in fatty acid biosynthesis, their availability and translocation may contribute to improved oil composition under optimal water and mulch management [52]. In the current study, although most yield-related traits responded independently to irrigation and mulch, significant interactions were recorded on oil yield, seed number per follicle, and 100-seed weight. The latter outcomes suggest that, similarly to previous findings, combined mulching and irrigation may synergistically improve certain reproductive traits and oil productivity in linseed. Organic and synthetic mulches may also modulate soil microbial activity, enzymatic processes, and osmotic adjustment [47,53], which could partially explain the enhanced oil yield recorded under specific treatment combinations. However, further physiological and biochemical analyses are needed to confirm the mentioned mechanisms.

Our results demonstrated that linseed oil composition is highly responsive to mulching strategies and irrigation regimes, with linolenic acid being the predominant fatty acid across all treatments. Interestingly, the highest linolenic acid content (up to 52.6%) was observed under rainfed conditions, while its lowest content (38.3%) was recorded under “plastic” mulch with two irrigations. The latter trend suggests that higher water availability may reduce the synthesis of omega-3 fatty acids, likely due to altered enzymatic or metabolic pathways under non-stress conditions. Similar findings were reported by Zare et al. [48], who observed increased linolenic acid accumulation under water-deficit conditions in linseed, indicating a potential adaptive mechanism to oxidative stress [48]. In contrast, oleic and linoleic acids showed an increasing trend with improved water availability and mulching, particularly with straw mulch and two irrigations. In this study, the application of organic and inorganic mulches, as well as irrigation levels, increased the amount of RWC. It was reported that organic mulching using rice husk significantly enhanced RWC and leaf water potential compared to non-mulched treatments under limited irrigation. The latter improvement in plant water status contributed to stable yield levels while reducing overall water use, thereby increasing water use efficiency [54]. Similarly, in a two-year field study on sorghum grown in newly reclaimed soils, the combined application of straw mulch and organic compost significantly encouraged RWC and plant physiological traits under deficit irrigation [55].

In our research, water deficit during plant growth reduced chlorophyll a, b, and carotenoids, and a significant interaction between the mulch type and irrigation regime was observed on the mentioned pigment contents, consistent with reports by Gholami and Zahedi [43]. The highest pigment concentrations were recorded in plants grown with straw mulch under SIVF and the lowest in the unmulched RFD control. Abd El-Mageed et al. [56] reported similar findings to ours, indicating that water stress significantly decreased the content of chlorophyll a, chlorophyll b, and total carotenoids in sorghum leaves. They also observed that the application of mulching and organic compost enhanced the concentration of leaf photosynthetic pigments and the plant’s ability to withstand drought stress. Drought-induced chlorophyll reduction is linked to water deficiency, impacting chlorophyll decomposition and oxidative damage, causing lipid, protein, and pigment breakdown [57].

Lahmod et al. [7] reported that straw mulch increased chlorophyll content and photosynthesis by fostering soil properties like water content, energy, bulk density, and nutrients, supporting crop growth.

In our study, the total soluble sugar content decreased under rainfed conditions, but the use of “plastic” and straw mulch under these conditions elicited sugar accumulation, likely by alleviating drought-induced limitations. In contrast, proline content significantly increased under rainfed and unmulched soil conditions, reflecting heightened osmotic stress, while the application of mulches (especially straw) lowered proline levels by enhancing moisture retention and reducing plant stress. Soluble sugars, derived from carbon reserve hydrolysis, accumulate under drought stress and function both as carbon sources and as signaling molecules that mitigate oxidative damage by reducing reactive oxygen species (ROS) levels [57,58]. Moreover, they help regulate cellular osmotic balance, maintain turgor pressure, and stabilize membranes, thereby enhancing drought tolerance [9,55]. In our study, mulch application (both straw and “plastic”) under water-limited conditions contributed to higher sugar content, suggesting improved osmotic regulation and carbon balance. Additionally, proline, as a primary organic osmolyte, accumulated in drought-stressed plants, thus supporting osmotic adjustment, protecting cytosolic enzymes, and stabilizing proteins and membranes [59]. However, under mulched treatments, proline content was significantly lower, indicating a reduced perception of stress due to improved water availability. The mentioned pattern suggests the role of mulch in mitigating physiological drought and lessening the need for osmoprotectants like proline. Antioxidant enzymes play a crucial role in protecting plants against oxidative damage by scavenging reactive oxygen species (ROS) under drought-stress conditions. SOD acts as the first line of defense by dismutating superoxide radicals (O₂⁻) into hydrogen peroxide (H₂O₂), which is subsequently detoxified by CAT into water and oxygen, preventing oxidative damage to cellular components such as lipids, proteins, and nucleic acids [6,8,60,61]. In this research, CAT and SOD enzyme activities in linseed significantly increased under water stress, particularly in plants treated with straw mulch, while under two irrigations, control (bare soil) plants showed the lowest levels of these enzymes. The latter outcome aligns with previous findings suggesting that water deficit conditions trigger the activation of antioxidant enzymes, which are strongly associated with enhanced stress tolerance [62,63]. In a field study on sesame (Sesamum indicum L.), the application of wheat straw mulch led to increased activity of antioxidant enzymes CAT and SOD under water-deficit conditions. This enzymatic enhancement was reported as a defense mechanism to mitigate oxidative stress caused by drought, highlighting the beneficial role of mulching in improving drought tolerance in sesame [6]. Our findings demonstrate that both organic and synthetic mulches significantly enhanced total phenolic content and DPPH radical scavenging capacity across irrigation regimes, suggesting that these treatments improve the plant’s overall antioxidant defense system. The latter aligns with previous research indicating that mulches can boost phenolic synthesis under water deficit conditions, thereby enhancing plant tolerance to oxidative stress through increased ROS scavenging activity [64,65]. However, while phenolic compounds contribute substantially to the mentioned activity [32], the DPPH assay reflects the combined antioxidant effects of multiple metabolites, including thiols, ascorbic acid, and soluble sugars. Overall, the present findings emphasize the role of mulching, particularly straw mulch, in enhancing linseed physiological and biochemical responses under water-limited conditions. By improving soil moisture retention and reducing evaporation losses, mulching effectively supported higher relative water content, photosynthetic pigment stability, and antioxidant enzyme activities while also modulating osmoprotectants such as soluble sugars and proline. The mentioned integrated responses either reflect improved stress tolerance or align with previous studies in various crops, confirming mulching as a strategy to mitigate drought-induced damages and maintain plant productivity under a sustainable management, safety, and health perspective [66].

5. Conclusions

Water deficit stress adversely affected various agronomic and physiological traits in linseed plants, including seed and biomass yield, RWC, chlorophyll a, chlorophyll b, and carotenoid content; however, it increased proline and total phenol concentrations, antioxidant capacity, and CAT and SOD activity. Straw mulch effectively mitigated the adverse impacts of moisture shortage stress on linseed plants by increasing seed and biomass yield, RWC, chlorophyll a, chlorophyll b, and carotenoid contents, osmotic adjustment, as well as antioxidant activity, and stimulating oilseed production, though also synthetic mulch had positive effects. The positive responses observed under organic and synthetic mulches suggest their potential to help linseed plants withstand water stress. The “plastic” and straw mulch applications under rainfed conditions or with one irrigation at the flowering stage improved linseed fixed oil quality and enzymatic and non-enzymatic activities. Within the examined mulch treatments, straw is recommended as a valuable source of beneficial substances for successful L. usitatissimum management, also reducing the harmful consequences of moisture shortage stress and providing a sustainable solution for linseed production, even under rainfed conditions.