Seed- and Foliar-Applied Iron Chelate Improves Performance, Physiological, and Biochemical Aspects of Black Cumin (Nigella sativa) under Semi-Arid Conditions

Abstract

The cultivation of medicinal plants plays a crucial role in promoting human health benefits. However, the production of these plants can be affected by drought conditions. This research aimed to investigate the impact of differing water status (non-drought and drought during the flowering to harvest stage) and various iron treatments on the performance of black cumin. The iron treatments included no iron as the control (nFe), no iron with seed hydro-priming (nFe + P), seed iron priming (pFe), seed iron priming with iron foliar spraying (pFe + sFe), and double iron foliar spraying (sFe + sFe). The purpose of these treatments was to assess the effect of iron application methods on plant response under different water conditions. The findings revealed that drought significantly reduced the levels of Chla (15%) and RWC (5.9%), plant height (7%), follicle number (16.7%), seed number (4.6%), 1000-seed weight (3.2%), and seed yield (30.1%). Additionally, drought increased the proline content (90.9%), electrolyte leakage (9.2%), and MDA levels (23.9%). Interestingly, applying iron amendments reduced electrolyte leakage and increased seed yield under both water conditions. The drought-induced increase in proline content was more pronounced in the nFe treatment than in the other treatments. The amount of MDA in the nFe and nFe + P treatments was significantly higher under drought conditions compared to non-drought conditions. In conclusion, the addition of iron amendments helps black cumin plants recover from the effects of drought and reduces damage to seed growth. This means that using both seed iron priming and iron foliar spraying can significantly improve yields. Alternatively, focusing on either seed iron priming or double iron foliar spraying can also boost black cumin production compared to not using iron amendments.

1. Introduction

Black cumin (Nigella sativa L.) is an annual herbaceous plant of the Ranunculaceae family. It is primarily cultivated in certain regions of Iran and finds extensive applications in both the food and pharmaceutical sectors [1]. The plant has a relatively short life cycle and is well suited to semi-arid areas [2]. The composition of black cumin seeds includes 39.02% crude fat, 25.86% carbohydrates, 21.07% crude protein, 6.01% fiber, 5.02% moisture, and 3.02% ash [3]. It has several medicinal properties and is used in the treatment of many conditions, including asthma, bronchitis, rheumatism, and skin disorders. Additionally, black cumin acts as an immune booster, an appetite stimulant, and a liver tonic [4,5]. Black cumin seeds are rich in aromatic and phospholipid compounds, making them valuable in the food, pharmaceutical, and cosmeceutical sectors [6]. In addition, black cumin has antioxidant properties due to the presence of antioxidant substances that contribute to its anti-cancer, anti-diabetic, and anti-inflammatory effects [7].

Water availability for plants is a crucial factor that can significantly impact their structure and performance [6,8]. In agricultural terms, drought refers to an inadequate supply of usable water during the plant’s growth period, leading to a decline in plant productivity [9]. One of the most important and widespread abiotic environmental stressors is drought, which limits crop production and causes significant losses worldwide each year [10]. Drought occurs when cells and tissues are unable to function properly; a prolonged imbalance in water availability affects all metabolic processes in plants, often resulting in reduced crop productivity [11].

In the development of seeds, water and nutrients play a crucial role [2]. The effects of drought on seed characteristics are significant, affecting the germination percentage, seed yield, seed quality, growth, and flowering [12]. Insufficient water availability leads to a reduction in soil water volume, which results in reduced nutrient distribution in the soil. This, in turn, hinders the nutrient uptake by the roots and their subsequent transfer to the aerial parts of the plant [13]. Accordingly, Soltanieh et al. [14] reported that the yield of black cumin decreased by 11.67% under drought stress conditions compared to the control. However, they also found that applying nitrogen and methanol resulted in a yield increase of 5.7% compared to the control. In addition, drought stress and limited water availability have been shown to often result in delayed and uneven plant establishment and suboptimal crop growth and yield [15].

Furthermore, drought conditions lead to a decrease in photosynthesis, an increase in respiration, and a decrease in assimilates within the plant, resulting in plant starvation [16]. Enzyme systems are also disrupted by drought, reducing the activity of active oxygen and increasing the peroxidation of membrane lipids, resulting in cell membrane damage and increased electrolyte leakage [6,16]. Plant nutrition is extremely important in improving overall plant performance [17]. It not only enhances the quality of plants, but also helps them to withstand different types of stress. When plants are well nourished and have all the nutrients that they need, they are better able to withstand drought and produce a higher quantity and quality of crops [6]. However, continuous farming practices, high yield demands, and excessive use of NPK fertilizers are leading to deficiencies in essential micronutrients such as iron. This deficiency adversely affects plant performance [18,19]. Iron is essential in plants because it is involved in several reactions and processes, including nitrogen fixation, enzyme activity, and photosynthesis. It is also important in the structure of cytochromes and acts as a transporter in photosynthetic systems. Iron is also required for metabolic processes such as chlorophyll synthesis and electron transfer. Increasing iron levels in leaves can lead to a higher chlorophyll content, improved photosynthetic activity, and, ultimately, increased crop yield. Excessive soil calcium (Ca) and carbonate levels from over-liming can lead to iron deficiency due to a high soil pH and competition for calcium at the surface of roots [20]. Additionally, iron deficiency can also be a concern in organic farming, particularly due to soil conditions and management practices that limit the use of synthetic inputs. Although synthetic chemicals are restricted in organic systems, certain natural inputs are permitted. Iron chelates, for example, are permitted under EU organic regulations (Regulation No. 2018/848), making them a viable option for addressing iron deficiency and potentially boosting yields in organic systems.

Waqas Mazhar et al. [21] demonstrated that after seed priming, hydrogen peroxide and malondialdehyde levels were reduced by 71% and 66%, respectively. In addition, an increase in the activity of the antioxidant enzymes peroxidase, superoxide dismutase, and catalase of 56%, 28%, and 39%, respectively, was observed, highlighting the potential of iron oxide particles to mitigate water stress. In a study conducted by Ali et al. [22], it was found that Trachyspermum ammi (L.) showed a notable enhancement in performance under both water stress and normal irrigation conditions. This improvement was observed when Fe-chelated glutamate was used for fertigation compared to the use of FeSO₄. Ali et al. [23] demonstrated that the foliar application of iron-chelated aspartate was more effective than FeSO₄ in enhancing plant–water relations, increasing essential amino acids, and improving nutrient acquisition, especially Fe accumulation with improved translocation. They concluded that the foliar application of iron-chelated aspartate was superior to FeSO₄ in inducing drought tolerance in sunflower plants.

The growth of black cumin primarily occurs during summer, relying on irrigation in semi-arid regions where the water supply is often limited. Therefore, implementing agricultural techniques to enhance plant resilience to drought is crucial. This study hypothesizes that iron supplementation methods will enhance the drought tolerance of black cumin by improving physiological processes such as photosynthesis, antioxidant defense mechanisms, and nutrient uptake. In this way, this study aimed to enhance the drought tolerance of black cumin through iron supplementation methods.

2. Materials and Methods

2.1. Experimental Location

The research farm of Shahrekord University, located at latitude 32 degrees and 21 min North and longitude 50 degrees and 49 min East, with an altitude of 2050 m above sea level, was selected as the site to conduct this experiment, in the year 2021.

2.2. Climate and Soil Characteristics of the Region

According to the Köppen classification, the Shahrekord region has a cold temperate climate with hot and dry summers. The annual rainfall in this area occurs mainly during the winter season, which does not align well with the growing season of the black cumin plant. Figure 1 illustrates the temperature and rainfall patterns in the Shahrekord region for the year 2021 and the previous three-year period (2018 to 2020). It is worth noting that the average monthly maximum air temperature in May during the study period was higher compared to the corresponding month in the previous three years.

Table 1. The physical and chemical properties of the soil used in the experiment.

Texture	pH	Electrical Conductivity	Organic Carbon	Field Capacity	Total Nitrogen	Available Phosphorus	Available Potassium	Copper	Iron	Manganese	Zinc
		(dS/m)	(%)	(%)	(%)	(mg/kg)	(mg/kg)	(mg/kg)	(mg/kg)	(mg/kg)	(mg/kg)
Clay loam	8.0	1.5	0.76	23.1	0.01	11.6	83	0.77	1.9	6.2	0.63

provides an overview of the physical and chemical characteristics of the soil in the experimental area. Electrical conductivity (EC) was measured using an electrical conductivity meter from a saturated paste extract [24], and pH was measured using a pH meter. Macronutrients, including total nitrogen, were measured using the Kjeldahl method [25], available phosphorus using a spectrophotometer [26], and available potassium using the ammonium acetate method with a flame photometer [27]. Micronutrients, including the available zinc, copper, iron, and manganese, were also measured using atomic absorption spectroscopy with the DTPA method [28].

2.3. Experimental Design and Treatments

The field experiment, conducted under conventional conditions, followed an RCBD factorial design with three replications. The field experiment consisted of 30 separate plots, each measuring 4 m². These plots were placed one meter apart, with a two-meter distance between blocks. The study investigated the impact of differing water status (non-drought and drought) and five levels of iron amendments. The iron treatments included no iron as a control (nFe), no iron with seed hydro-priming (nFe + P), seed iron priming (pFe), seed iron priming with iron foliar spraying (pFe + sFe), and double iron foliar spraying (sFe + sFe). Iron chelate (DTPA (Diethylene Triamine Pentaacetic Acid)) at a concentration of 4000 mg/L was utilized to supplement the seeds with iron amendments and for foliar spraying. Seeds were soaked in either water or an iron solution for 12 h, and foliar spraying (4000 mg/L) was carried out after the vegetative growth stage of the black cumin plant and before flowering. The drought test was applied from flowering to harvest, starting on 21 July 2021 and ending on 9 October 2021. Flowering was identified as the most sensitive stage to water deficit in order to provide effective treatments under stress-prone conditions. During this period, the non-drought treatments were irrigated every three days, while the drought treatments were irrigated once a week. After the appearance of wilting symptoms in plants (23 October), the irrigation of these plots was continued until the harvest stage, similar to the non-drought treatment.

2.4. Crop Management

To prepare the field, the land was initially plowed, followed by ground leveling and plot work. The land was then prepared for planting in July 2021. On 22 June 2021, five rows of seeds, each measuring 2.5 m long, were planted in each plot. The plant density was 50 plants/m². NPK (20-20-20) fertilizer was applied at two stages, 30 and 44 days after planting, at a rate of 120 kg/ha. The seeds were sown in rows to achieve a final density of 100 plants per square meter. Irrigation was carried out throughout the growth period, adjusted to the water requirements of the plants and the prevailing environmental conditions. Tape irrigation was employed as the irrigation method in this experiment. During the growth period, necessary measures, such as weeding and pest and disease control, were implemented, and relevant observations were recorded. Finally, the plants were harvested on 23 October, when the leaves had turned yellow and the follicles had browned. At an early stage of flowering, specifically, 87 days after planting (17 September 2021), samples of developed young leaves were collected for further investigation into the following parameters. For the biochemical analyses, 10 plants from each experimental unit (plot) were harvested.

2.5. Measurements

2.5.1. Determination of Photosynthetic Pigments

The measurement of photosynthetic pigments was carried out according to the method reported by Lichtenthaler and Buschman [29]. The quantity of photosynthetic pigments was determined in milligrams per gram of fresh weight of plant tissue, using the following equations:

$$\mathrm{C}\mathrm{h}\mathrm{a}\hspace{0.17em}({\displaystyle \frac{\mathrm{m}\mathrm{g}}{\mathrm{m}\mathrm{L}}})=12.25\times {\mathrm{A}}_{663.2}-{2.79\times \mathrm{A}}_{646.8}$$

(1)

$$\mathrm{C}\mathrm{h}\mathrm{b}\hspace{0.17em}({\displaystyle \frac{\mathrm{m}\mathrm{g}}{\mathrm{m}\mathrm{L}}})={21.51\times \mathrm{A}}_{646.8}-{5.10\times \mathrm{A}}_{663.2}$$

(2)

$$\mathrm{C}\mathrm{h}\mathrm{l}\mathrm{t}\hspace{0.17em}\left({\displaystyle \frac{\mathrm{m}\mathrm{g}}{\mathrm{m}\mathrm{L}}}\right)=\mathrm{C}\mathrm{h}\mathrm{l}\mathrm{a}+\mathrm{C}\mathrm{h}\mathrm{l}\mathrm{b}$$

(3)

$$\mathrm{C}\mathrm{a}\mathrm{r}\mathrm{o}\mathrm{t}\mathrm{e}\mathrm{n}\mathrm{o}\mathrm{i}\mathrm{d}\mathrm{s}\hspace{0.17em}({\displaystyle \frac{\mathrm{m}\mathrm{g}}{\mathrm{m}\mathrm{L}}})={\displaystyle \frac{{1000\times \mathrm{A}}_{470}-\left(1.82\times \mathrm{C}\mathrm{h}\mathrm{a}\right)-(85.02\times \mathrm{C}\mathrm{h}\mathrm{b})}{198}}$$

(4)

A = the quantity of light absorbed by the extract at specific wavelengths.

Chla, Chlb, and Chlt = chlorophyll a, chlorophyll b, and chlorophyll a + b, respectively.

2.5.2. Determination of Proline Content

The extraction of proline from the samples was conducted according to the method described by Bates et al. [30]. Initially, 0.5 g of the plant sample was ground in 10 mL of a 3% sulfosalicylic acid solution to create a homogeneous mixture. The resulting extracts were then transferred to a Falcon tube and placed in an ice bath. The samples were centrifuged at 15,000 rpm for 10 to 15 min at 4 degrees Celsius to separate any additional materials from the solution. Alternatively, a glass funnel and filter paper could be used to filter the samples instead of a centrifuge. Subsequently, 2 mL of the centrifuged plant extract was transferred to a new Falcon tube, and 2 mL of ninhydrin acid and 2 mL of glacial acetic acid were added and mixed thoroughly. The samples were then heated in a hot water bath for 1 h and then placed in an ice bath. Finally, 4 mL of toluene was added to the resulting solutions and vortexed for 20 s. The Falcon tubes were then left undisturbed for 15–20 min to allow the complete separation of the two phases. The top layer containing toluene and proline was used to measure the concentration of proline. Toluene was also used as a control for the spectrophotometer.

2.5.3. Determination of Relative Water Content (RWC)

The leaf relative water content (RWC) of the leaves was assessed following the protocol outlined by Martinez et al. [31]. The youngest fully developed leaf from each experimental group was carefully isolated and immediately transported from the field to the laboratory. A standardized sample was then taken from the central region of the leaf blade and immediately weighed. The samples were then immersed in distilled water for 24 h, protected from light, and kept at a temperature of 4 °C. At the end of the soaking period, the samples were removed from the distilled water, excess surface moisture was removed, and the saturated weight of the leaves was recorded. The leaves were then oven-dried at 70 °C for 24 h, after which the dry weight was determined. Finally, the RWC of the leaf was calculated using the formula:

$$\mathrm{R}\mathrm{W}\mathrm{C}\left(\%\right)={\displaystyle \frac{\mathrm{F}\mathrm{w}-\mathrm{D}\mathrm{w}}{\mathrm{S}\mathrm{w}-\mathrm{D}\mathrm{w}}}\times 100$$

(5)

• FW = leaf fresh weight immediately after collection;
• DW = leaf dry weight after oven drying;
• SW = leaf-saturated weight following exposure to distilled water.

2.5.4. Determination of Membrane Lipid Peroxidation

The technique employed by Heath and Packer [32] was utilized for the assessment of membrane lipid peroxidation. The quantification of lipid peroxidation in the air organ was assessed by measurements of the amount of malondialdehyde (MDA), a by-product of lipid peroxidation, using the thiobarbituric acid (TBA) reaction. A spectrophotometer was employed to measure the extent of lipid peroxidation in the leaf membrane at wavelengths of 532 and 600 nm. The concentration of malondialdehyde was determined using an extinction coefficient of 155,000 µM⁻¹ cm⁻¹, and the results were expressed in micromoles per gram fresh weight according to Narwal et al. [33].

$$\mathrm{M}\mathrm{D}\mathrm{A}\left(\mathrm{m}\mathrm{m}\mathrm{o}\mathrm{l}/\mathrm{g}\mathrm{F}\mathrm{W}\right)={\displaystyle \frac{\mathrm{A}532-\mathrm{A}600}{\mathrm{Ɛ}\mathrm{d}\mathrm{F}\mathrm{W}}}\times \mathrm{V}$$

(6)

• A = absorption rate;
• Ɛ = extinction coefficient of malondialdehyde at 532 nm (155 m/M/cm);
• V = sample volume (L);
• FW = weight of fresh leaf tissue in the sample (0.1 g);
• d = width of cut (cm).

2.5.5. Membrane Electrolyte Leakage (EL)

To assess the extent of electrolyte leakage from leaf cells, the leaf samples were rapidly transported from the field to the laboratory. Leaf fragments weighing 0.1 g were extracted from the central part of the leaf blade and placed in lidded glass containers containing 10 mL of deionized water. The samples were then immersed in a hot water bath maintained at a constant temperature of 32 °C for 2 h. The electrical conductivity of the samples, indicating electrolyte leakage, was determined using an electrical conductivity meter (EC meter) (C1). The samples were then autoclaved at 121 °C for 20 min, and their electrical conductivity was measured after cooling to 25 °C (C2). The percentage of electrical leakage across the membrane was then calculated using the following equation [34]:

$$\mathrm{E}\mathrm{L}\left(\mathrm{\%}\right)={\displaystyle \frac{\mathrm{C}1}{\mathrm{C}2}}\times 100$$

(7)

• C1 = primary electrical conductivity;
• C2 = final electrical conductivity.

2.5.6. Morphological Parameters

Plant height was measured at the early seed stage (87 days after planting) using a metal meter. Additionally, the number of lateral branches was counted at the late flowering stage (80 days after planting). Towards the end of the growing season (125 days after planting), when the plants had turned yellow but the follicles had not yet split, 10 plants were randomly selected from each plot. Yield components such as the number of follicles per plant, the number of seeds per follicle, and the thousand seed weight were measured. Seed yield in kg/ha was determined by removing one square meter of plants from the center of each plot on 25 October 2021. The seeds from each plot were dried for 48 h in an oven at 72 °C and then weighed using a digital balance.

2.6. Data Analysis

Statistical analysis of the data for the parameters assessed was performed in a factorial manner using a randomized complete block design using SAS software (version 9.4). The preliminary tests, including tests for normality and homogeneity of variances, were conducted before performing the factorial analysis. The significant interaction effects of the experimental treatments were also compared. Comparisons of means were carried out using the LSD test at the 5% probability level.

3. Results

3.1. Photosynthetic Pigments

Statistical analysis (

Table 2. Analysis of variance (mean square) for the effect of iron amendments and drought status on biochemical parameters in black cumin.

S.o.V a	Df	Chlorophyll a	Chlorophyll b	Chlorophyll a + b	Carotenoids	Proline Content	Relative Water Content	Malondialdehyde	Electrolyte Leakage
R	2	2.07 ns	0.38 ns	3.31 ns	0.07 ns	0.05 ns	21.6 ns	0.34 ns	0.001 ns
W	1	60.7 **	2.72 ns	89.1 **	0.16 ns	3352	316 **	9.8 **	0.40 **
Fe	4	1.0 ns	0.54 ns	2.2 ns	0.31 ns	283 **	31.2 ns	16.2 **	0.22 **
W × Fe	4	3.65 ns	0.42 ns	5.4 ns	1.8 *	261 **	35.1 ns	7.9 **	0.61 ns
Error	18	3.3	0.69	6.2	0.49	3.5	33.4	0.78	0.01
C.V (%)		10.4	11.7	10.1	9.7	9.7	7.3	16.4	4.3

^a S.o.V, source of variance; R, replication; W, water status; Fe, Fe-chelated. ^ns, *, and ** indicate non-significant and significant at p < 0.05 and p < 0.01, respectively.

) revealed that the levels of Chla and Chl a + b were affected by drought conditions (p < 0.01). Specifically, the concentration of Chla decreased by 15% under drought stress (16.0 mg/mL) compared to non-drought conditions (18.9 mg/mL). On the other hand, the amount of Chlt decreased by 13% under drought stress (22.8 mg/mL) compared to non-drought conditions (26.3 mg/mL) (Figure 2). The interaction between drought and iron amendments had a significant effect on the carotenoid content (Table 2; p < 0.05). Under drought conditions, the sFe + sFe treatment exhibited lower carotenoid levels compared to the pFe + sFe treatment, while the opposite trend was observed under non-stressed conditions (Figure 3a).

3.2. Proline Content

Proline content showed a significant response to drought, iron amendments, and their interaction (p < 0.01, Table 2). Under non-drought conditions, there was no significant difference in proline levels between plants treated with iron and those without iron. However, under drought conditions, plants treated with iron had significantly lower proline levels compared to the nFe treatment. The lowest proline content was observed in the pFe treatment (0.02 μmol/g) under non-stress conditions, while the highest content was recorded in the nFe treatment (0.08 μmol/g) under drought conditions (Figure 3b).

3.3. Relative Water Content (RWC)

The analysis of variance indicated that the leaf RWC was affected by drought stress (p < 0.01; Table 2), while the effect of iron amendments and the interaction between drought and iron amendments were not statistically significant. The leaf RWC was 75.5% under drought conditions and 82% under non-drought conditions. Drought stress led to a 7% reduction in leaf RWC compared to non-drought conditions (Figure 4a).

3.4. Membrane Lipid Peroxidation

Membrane lipid peroxidation was assessed by the malondialdehyde (MDA) assay. The results of the analysis of variance indicated that the amount of MDA was significantly affected by drought, iron amendments, and their interaction (p < 0.01; Table 2). In the absence of drought, the MDA content of the nFe + P and pFe + sFe treatments was lower than that of nFe, while the pFe and sFe + sFe treatments did not show a significant difference compared to nFe. Under stress conditions, plants receiving iron amendments had lower MDA levels than those treated with nFe and pFe. The highest MDA levels were observed in the nFe and pFe treatments under drought conditions (8.8 and 8.8 μmol/g, respectively), while the lowest levels were found in the pFe + sFe treatment under both drought and non-stress conditions (1.3 and 3.4 μmol/g, respectively) (Figure 3c).

3.5. Membrane Electrolyte Leakage

Membrane electrolyte leakage in black cumin leaves showed a significant response to drought and iron amendments (p < 0.01), although the interaction between these two factors was not significant (Table 2). Drought increased leaf membrane electrical leakage. The leaf membrane electrical leakage was 63.1% and 58.5% in drought and non-drought conditions, respectively (Figure 4b). Plants that received iron amendments exhibited lower rates of electrolyte leakage compared to those that were iron-deficient. The pFe + sFe and pFe + Sfe treatments showed the lowest levels of electrolyte leakage (Figure 4c).

3.6. Plant Height

The growth of black cumin was significantly influenced by drought, iron amendments, and their combined effect (p < 0.01,

Table 3. Analysis of variance (mean square) for the effect of iron amendments and drought status on morphological parameters in black cumin.

S.o.V a	Df	Plant Height	Branch/Plant	Follicle/Plant	Seed/Follicle	1000-Seed Weight	Seed Yield
R	2	0.01 ns	0.05 ns	4.34 ns	0.83 ns	0.04 ns	12,883 ns
W	1	44.7 **	0.02 ns	90.8 **	52.7 **	0.07 *	143,558 **
Fe	4	11.6 **	0.92 *	47.9 **	49.7 **	0.12 **	87,822 **
W × Fe	4	4.61 **	0.40 ns	3.44 **	12.2 **	0.02 ns	5394 ns
Error	18	0.10	0.24	0.49	0.82	0.01	2204
C.V (%)		0.67	0.55	3.1	0.79	3.6	7.0

^a S.o.V, source of variance; R, replication; W, water status; Fe, Fe-chelated. ^ns, *, and ** indicate non-significant and significant at p < 0.050 and p < 0.01, respectively.

). Under normal irrigation conditions, the application of iron amendments did not result in increased plant height. In fact, the sFe + sFe treatment significantly reduced plant height compared to the control treatment. However, when exposed to drought conditions, plants that received iron amendments, except for the treatment with sFe + sFe, exhibited a greater plant height compared to the control treatment (Figure 5a).

3.7. Branch Number

Based on the analysis of variance table (Table 3), the effect of iron amendments on the number of branches per plant was found to be significant (p < 0.05), while no significant effects of drought or the interaction between drought and iron amendments were observed. There was no significant effect of drought or the interaction between drought and iron amendments on the number of branches per plant. The treatment without iron amendments resulted in an average of 8.79 branches per plant, whereas the treatment with iron amendments resulted in a 5% increase in the number of branches per plant (Figure 6a).

3.8. Follicle Number

The analysis of variance revealed that the effect of drought (p < 0.01), iron amendments (p < 0.01), and their interaction (p < 0.05) on the number of follicles per plant was statistically significant (Table 3). Both seed priming and iron fertilization significantly increased the number of follicles per plant compared to the treatment without iron amendments, regardless of drought conditions (Figure 5b).

3.9. Seed Number

According to the results presented in Table 3, the number of seeds per follicle showed a significant response to drought, iron amendments, and their interaction (p < 0.01). Under both drought and non-drought conditions, the application of iron amendments significantly increased the number of seeds per follicle compared to those without iron treatments. However, the rate of increase in the number of seeds per follicle was higher under drought conditions than under non-drought conditions (Figure 5b).

3.10. Thousand-Seed Weight

Table 3 shows the results for the weight of a thousand seeds, indicating a significant response to both drought and iron amendments. The effect of drought resulted in a 3% reduction in thousand-seed weight compared to non-drought conditions, as illustrated in Figure 7a. On the other hand, pFe and pFe + sFe treatments showed an increase in thousand-seed weight compared to nFe and nFe + P treatments. Notably, the highest weight per thousand seeds was observed in the pFe and pFe + sFe treatments, with an average of 2.8 and 2.7 g, respectively, as shown in Figure 6b.

3.11. Seed Yield

There was a significant effect of drought and iron amendments on the yield of black cumin (p < 0.01), with the interaction between these two factors not being significant (Table 3). Seed yield under non-drought and drought conditions was 751 and 524 kg/ha, respectively, and the variance between them was statistically significant at the 5% confidence level (Figure 7b). The application of iron to the plants resulted in a significant increase in seed yield compared to nFe (28.5–43.3%). The significant advantage of plants treated with iron over nFe + P suggests that seed priming with iron is not dependent on hydropriming (Figure 6c).

4. Discussion

The findings of this study indicate that drought conditions lead to a decrease in Chla levels, resulting in a decrease in the photosynthetic activity of the plants. This decrease in photosynthetic activity is attributed to a reduction in the efficiency of carbon utilization and an increase in the production of ethanol and lactate, which, in turn, leads to a decrease in the synthesis of chlorophylls [35]. Additionally, when drought occurs during the reproductive stage of the plant, it accelerates leaf senescence and reduces photosynthetic pigments, as noted in [36]. The changes in carotenoid levels in plants subjected to different water conditions, particularly the FeS + FeS treatment, demonstrate that carotenoids are relatively increased under drought conditions and play a crucial role in maintaining the photosynthetic system. Under conditions of oxidative stress, carotenoids can convert singlet oxygen into triplet oxygen and act as antioxidants by scavenging the generated radicals, as highlighted in [37]. Kakulvand et al. [36] also reported that Trigonella foenum-graceum exhibited the highest amount of carotenoid under drought conditions. Proline, a compound that mitigates the inhibitory effects of ions on enzymes, plays an important role in regulating osmotic pressure in plants [6]. In addition, proline may help stabilize antioxidant enzymes and promote the production of reactive oxygen species (ROS) in mitochondria by influencing respiratory electron transport processes. The degree to which proline synthesis and/or accumulation increases in response to drought can serve as an indicator of the severity of drought-related stress in plants [38]. In our research, plants treated with iron exhibited significantly lower levels of proline under drought conditions compared to those without iron treatment, highlighting the effectiveness of iron supplementation in mitigating drought stress in plants. This can be attributed to the effect of seed soaking on the enhancement of the root system, thereby reducing the susceptibility to drought. Furthermore, this study observed that iron nutrition increased plant tolerance to drought and reduced the need for proline production. In our study, we observed a 7% decrease in leaf relative water content (RWC) under drought conditions compared to non-drought conditions. Similarly, Mirjahanmardi and Ehsanzadeh [38] reported significant reductions in leaf RWC in fennel plants exposed to drought stress. RWC is directly influenced by soil moisture, with a decrease in RWC corresponding to a decrease in soil moisture and tension. Plants experiencing moisture stress minimize intercellular space and water content by increasing osmotic substances in their tissues, allowing them to absorb water from the soil with greater force. Consequently, the relative amount of water decreases under drought conditions, as explained in [39]. Under drought conditions, stomatal closure reduces the fixation of carbon dioxide, while photoreactions and electron transfer proceed normally [40]. During these conditions, the limited supply of NADP to accept electrons leads to oxygen serving as an alternative electron acceptor. This leads to the accumulation of harmful species such as superoxide radicals, hydrogen peroxide, and hydroxyl radicals [16]. The accumulation of reactive oxygen species during stress damages various cellular components, including lipids, proteins, carbohydrates, and nucleic acids, causing lipid peroxides to penetrate the cell membrane [6]. By improving the efficiency of plant antioxidant systems, iron plays a crucial role in regulating free radicals and mitigating their detrimental effects on membrane systems. Consequently, the foliar application of iron appears to increase plant tolerance to moisture stress by increasing the production of free radical scavenging enzymes, thereby reducing the plant’s exposure to stressful conditions. According to Waqas Mazhar et al. [21], iron plays a key role in managing free radicals and their adverse effects on membrane systems by elevating the activity of plant antioxidant systems. Consequently, the inclusion of iron in black cumin may protect the membrane by increasing the production of free radical scavenging enzymes, resulting in reduced leakage. Similarly, Saeidi Aboueshaghi et al. [39] observed a decrease in electrolyte leakage in red bean leaves following the foliar application of iron element.

As soil moisture diminishes, protoplasm degradation occurs along with a decrease in cell mass, resulting in a significant reduction in cell size and cell division rate. This ultimately leads to a decrease in the growth rate and photosynthetic levels of the plant [41]. Priming black cumin seed appears to stimulate root development and improve the plant’s access to nutrients, consequently promoting plant growth. In line with these findings, Waqas Mazhar et al. [21] also demonstrated that seed priming with iron oxide nanoparticles enhances various plant parameters, including yield attributes. Their study showed that this treatment reduces oxidative stress markers and increases the activity of antioxidant enzymes. These results underscore the potential of iron supplementation in mitigating water stress and improving the overall agronomic profile of plants. The involvement of iron compounds in cell division and growth-related responses contributes to the increased plant height compared to the control [42]. Additionally, Hansen et al. [43] found that iron-deficient plants exhibited slower growth and reduced height compared to optimal conditions. In particular, iron increases the leaf surface area and photosynthetic material, which, when transported to growing parts such as the stem, increases the number of nodes and subsequently boosts the number of branches [39].

Iron supplementation under drought conditions resulted in a higher increase in the number of follicles per plant compared to non-drought conditions. The presence of iron had a greater effect on the changes in follicle number than soaking without iron. Iron plays a crucial role in strengthening the plant’s antioxidant system, particularly under drought conditions, which helps maintain the plant’s photosynthetic capacity. This, in turn, ensures an adequate supply of assimilates for the transformation of flowers into follicles. Mahmoud et al. [42] concluded that foliar spray with Fe-chelated greatly improved all yield and component parameters compared to a control in Vicia faba (L.). Drought stress negatively affects the fertility of flowers and reduces seed production in the follicles. Additionally, research indicates that under high-temperature stress during flowering, maintaining optimal spikelet water status through increased transpiration rates and reduced internal spikelet temperatures can improve spikelet fertility, pollen fertility, and anther dehiscence, and ultimately increase overall fertility, especially in heat-tolerant rice genotypes [44]. Drought conditions before and during flowering disrupt pollination and result in sterility in pollen grains, leading to a decrease in seed production in the plant [41]. The reduction in seed numbers in iron-deficient plants highlights the negative impact of not utilizing these materials to prepare reproductive organs for seed production. The availability of micronutrient fertilizers can alleviate resource limitations and contribute to a higher seed number in plants [22].

Black cumin is a limited-growth plant, so increased drought conditions accelerate the transition from the vegetative to reproductive stage. This transition results in a decrease in the number of branches, the number of follicles produced per plant, and the weight of 1000 seeds. The photosynthetic period of black cumin is shortened under drought stress, resulting in a reduced transfer of photosynthetic material from leaves to seeds and, ultimately, a reduction in 1000-seed weight under low-irrigation conditions. The addition of iron, which is crucial in photosynthetic processes and carbohydrate accumulation, enhances the 1000-seed weight, as observed in soybean plants by Jalil Sheshbahreh et al. [13]. Under drought conditions, the seed yield of black cumin can be reduced due to the effects of the oxidants generated, resulting in lipid peroxidation and cell membrane damage. This process, together with the increased cost of proline production, leads to the impairment of photosynthetic pigments and reduced performance in Nigella components. The decrease in photosynthetic leaves is attributed to a shortened seed-filling period and premature leaf senescence. Iron is essential in chlorophyll production and electron transport during photosynthesis, with ferredoxin serving as an iron-carrying protein involved in electron transport. Consequently, an increase in leaf iron content enhances chlorophyll content, boosts photosynthetic activity, and, ultimately, increases black cumin yield, aligning with the findings of Jalil Sheshbahreh et al. [45] in soybean. Under stressful conditions, increased leaf iron levels lead to increased chlorophyll content, enhanced photosynthetic activity, and, ultimately, expanded leaf surface area, as noted by Ali et al. [22]. Another study has demonstrated that the foliar application of iron and zinc increased wheat seed yield by 19.5% under drought stress conditions. The researchers concluded that these micronutrients enhance radiation absorption and improve the plant’s efficiency in light utilization and photosynthetic activity. This is achieved by promoting healthy vegetative growth, which includes an increase in the leaf area and number of leaves [46].

5. Conclusions

A short drought in the flowering stage significantly reduced the photosynthetic pigments, vegetative growth, and yield components, and its effects were also observed in changes in proline and membrane damage, all of which indicate the plant’s sensitivity to water limitation. The application of iron chelate amendments reduced drought-induced damage and improved the number of follicles and seeds under drought conditions, resulting in a higher yield of black cumin. In general, it can be concluded that the addition of iron moderates the physiological effects of drought stress on black cumin and reduces the damage caused by this stress. Therefore, Fe seed priming plus Fe spraying significantly increases seed yield in semi-arid conditions. As a second priority, the addition of Fe seed priming or double Fe spraying can also increase the yield of black cumin compared to no iron amendments. The experimental results of the study could depend on the climatic conditions; as a result, future studies could include soil water retention curves to precisely adjust water stress levels and gain a more holistic understanding of plant–soil–water dynamics.