1 Introduction

Drought stress is one of the greatest abiotic stresses; it inhibits plant growth and productivity and decreases the production of more than 25% of agriculture worldwide. Additionally, drought negatively affects water relationships, absorption, and assimilation of nutrients, photosynthesis, and enzymatic activities (Bano et al. 2021). Prolonged drought might cause significant physiological and developmental alterations in plants (Hasan et al. 2018). Plants that experience drought exhibit a variety of metabolic disorders, including decreased water available in the soil and the necessity for water at evaporation sites, reactions to water status, and the genetic discrepancies of the plant species, thus reducing cell turgor and cell shrinkage (Xiong et al. 2020). Drought causes wilting, stomatal closure, and cell expansion decreases owing to decreases in cell water content, turgidity, and hydrostatic tissue pressure, leading to an inhibition of photosynthesis through alterations in chlorophyll content and damaging photosynthetic equipment, prominent reduction in transpiration, translocation, ion absorption, carbohydrates, nutrient metabolism, and growth promoters (Praba et al. 2009). Additionally, drought stress disrupts the equilibrium of cell homeostasis caused by an excessive production of reactive oxygen species (ROS) including hydrogen peroxide and superoxide radical which induces oxidative stress on proteins, membrane lipids, and interruption of DNA strands (Mittler 2020).

Sunflower (Helianthus annuus L.) is considered one of the most important oilseed crops, ranking third after soybean and peanut, among other oilseed crops in the world. (Thavaprakash et al. 2002). In Egypt, there is a huge shortage of vegetable oil production due to the enormous increase in the annual consumption of vegetable oils, which amounts to 2.66 million tons of oil, including 1820 tons of imported oil, in addition to 3790 tons of locally pressed oil seeds, leaving a gap of 97–98% (Adeleke and Babalola 2020). Approximately half of this amount is produced mainly from cotton seeds, and the other half is extracted from sunflower oilseeds in addition to imported soybeans (Aswaq Financial Co. 2018). Egypt produces about 300–400,000 tons of oil from a variety of crops including sunflowers, canola, soybeans, cotton, flax, olives, oil palm, and some other crops. Nevertheless, while sunflower production per unit area in Egypt has lately increased, productivity remains low, making it critical to increase productivity (FAOSTAT 2022). Only 2% of the consumption demands of edible oil produced by extraction of oilseeds. The remaining 98% was covered by the imported oils, in particular soybean oil, sunflower oil, corn oil, and palm oil (El-Hamidi et al. 2020).

Brassinosteroids (BRs) are a group of steroid growth regulators and signaling molecules and widely distributed in plants; their presence has been confirmed in algae, mosses, and vascular plants (Zullo and Bajguz 2019 and Bajguz 2019). BRs are playing an important role in various physiological processes in developmental processes, such as cell elongation, division, and vascular differentiation (Kour et al. 2021), reproductive development, seed germination, flowering pollen generation, regulation of gene expression, maturation, and plant aging (Nolan et al. 2020). Furthermore, BRs significantly boost transpiration potentiality and raise protein, carbohydrates, and chlorophyll levels (Li and Hi 2020). Additionally, BRs participate in plant’s tolerance to a wide range of biotic and abiotic stresses, including temperature extremes, drought, salinity, and pathogen attacks (Gruszka 2013, Ahanger et al. 2018 and Li et al. 2020). Stigmasterol belongs to the brassinosteroid; it is an integral component of the lipid center of cellular membranes and is the precursor of numerous secondary metabolites involving plant steroid hormones, acting as transporters in acyl, sugar, and protein passage (Krishna 2003).

To the best of our knowledge, little research has looked at the impact of foliar stigmasterol spraying on morphological and physiological traits as well as yield attributes of stressed sunflower plants. Thus, the objective of the current study was to investigate the utility of stigmasterol (with different concentrations) as a promising plant development regulatory substance to improve sunflower tolerance to water deficiency via growth, activities of photosynthetic pigments, osmolytes, anti-oxidative enzyme activities, antioxidant and antioxidant compound contents, cell membrane stability, and H2O2 content, and in addition, to yield characteristics and the nutritional quality of the yielded seeds as well as fatty acid profile of the yielded sunflower seeds.

2 Materials and Methods

2.1 Plant Material and Growth Conditions

A pot test was conducted in two successive seasons of summer 2020 and 2021 (temp. 36–38/17–22 °C, day/night, light intensity; 300 μmol m−2 s−1; and humidity; 60–70%) in the greenhouse of the National Research Center, Dokki, Giza, Egypt (30° 3′0″ N/31° 15′0″E), using sunflower (Helianthus annuus L.) cultivar Sakha 53. Seeds of sunflower were obtained from the Agricultural Research Center, Giza, Egypt. Stigmasterol used was supplied from Sigma–Aldrich. Healthy sunflower seeds were sterilized for 5 min using 5% sodium hypochlorite (NaOCl) and then washed using distilled water. Seeds were sown in pots (50 cm3) containing 7 kg of silty and sandy soil with a ratio of 1:1 to reduce compaction and improve drainage. The pots were divided into two major groups depending on irrigation with different water requirements (80% and 50% WIR), namely D0 (as control) and D1, respectively. Each of the previous groups was divided into four subgroups and foliar treated twice at 30 and 40 days after sowing with different concentrations of stigmasterol (0, 100, 200, and 300 mg L−1), namely Sts 0, Sts 1, Sts 2, and Sts 3, respectively. A preliminary germination experiment using different concentrations of stigmasterol (0.0, 50, 100, 150, 200, 250, 300, 250, and 400 mg L−1) was conducted. Then, the appropriate concentrations of Sts were chosen based on the results of growth characteristics. Each treatment consisted of five replicates distributed in a completely randomized design. Sunflower seeds were planted during the 13th of May of the two summer seasons of 2020 and 2021 and harvested on the 20th of August of the two seasons. Soil was fertilized with the mentioned fertilizers 3 days prior to planting in the following doses: (1) ammonium sulfate (20.5% N) at 10 g per pot; (2) super phosphate (15% P2O5) at a 4 g per pot dose; and (3) potassium sulfate (48% K2O) at a 2 g per pot dose. Soil water irrigation requirement was determined by saturating the soil in each pot with water, drainage for 48 h, and weighing. The soil water irrigation requirement (WIR) or field holding capacity was calculated. The soil water capacity was kept around 80 and 50% of its maximum level by weighing the pots and balancing the daily water loss. Sunflower seedlings were irrigated with the two levels of WIR twice weekly. Thinning was performed 15 days after sowing, and five plants were left in each pot. Pots of each plot are irrigated with one of the following water irrigation levels (80 and 50% WIR). Samples were collected after 50 days from sowing to determine the morphological measurements and chemical analysis. One plant per pot was left for yield determination. The morphological characteristics were plant height (cm), number of leaves and leaves area per plant, and fresh and dry weight of shoot (g per plant). In addition, root length (cm), root fresh and dry weight/plant (g) as well as relative water content (RWC%) of shoot were measured and expressed as percentage, according to the following equation (Alqurainy 2007).

$$\frac{RWC\left(\%\right)=\left( fresh\ weight- dry\ weight\right)\times 100.}{Fresh\ weight}$$

When signs of full maturity stage, measurements for yield and its components were also recorded (head diameter (cm), head circumference (cm2), seed yield/plant (g), and 100 seeds weights (g).

2.2 Chemical Analysis

Determination of Photosynthetic Pigments. Chlorophyll and carotenoid contents in plant leaves were estimated using the method of Lichtenthaler and Buschmann (2001). Indole acetic acid content was extracted and estimated by the method of Larsen et al. (1962). The level of H2O2 was determined, according to Jana and Choudhuri (1981). According to Hodges et al. (1999), the level of lipid peroxidation was measured by determining the malondialdehyde (MDA) content. The electrolyte leakage was measured, according to the method recorded by Lutts et al. (1996). Lipoxygenase (EC 1.13.11.12) activity was evaluated, according to Doderer et al. (1992).

Assay of Antioxidant Enzyme Activities. Enzymes were extracted following the method of Chen and Wang (2006). Peroxidase (POX) (EC 1.11.1.7) activity was evaluated using the method of Kumar and Khan (1982). Superoxide dismutase (SOD, EC 1.15.1.1) activity was measured according to Chen and Wang (2006). Catalase (CAT) (EC 1.11.1.6) activity was determined, according to the method of Chen and Wang (2006). The level of ascorbate peroxidase (ASP) (EC 1.11.1.11) activity was estimated using Chen and Asada (1992) method. Nitrate reductase (NR) (EC 1.1.1) activity was assayed following the method of Foyer et al. (1998). Glutathione reductase enzyme (GR) (EC 1.6.4.2) activity was evaluated, according to the method of Foyer et al. (1995).

Assay of Non-Enzymatic Antioxidant Enzymes. Non-enzymatic antioxidants were extracted as the method described by Gonzalez et al. (2003). A known weight of the fresh samples (1 g) was homogenized with 85% cold methanol (50 mL v/v) for three times at 90 °C. The free radical scavenging activity by 2,2,-diphenyl-2-picryl-hydrazyl (DPPH) method was estimated, according to Sousa et al. (2007). Total phenol content was described by Gonzalez et al. (2003). Content of glutathione (non-protein SH group) was estimated, according to the method of Paradiso et al. (2008). α-Tocopherol content was assayed, according to the method of Jargar et al. (2012).

Assay of Compatible Solute Accumulation. Proline (Pro) content was determined by Kalsoom et al. (2016). Total soluble sugars (TSS) were estimated by the method of Homme et al. (1992). Total free amino acids (FAA) content was measured by the method of Sorrequieta et al. (2010).

2.2.1 Sunflower Yield and Its Components

The sunflower oil seeds were extracted, according to Das et al. (2002) by using Soxhlet apparatus. Total carbohydrates were evaluated, according to Albalasmeh et al. (2013). The total protein concentration of the supernatant was determined, according to the method described by Bradford (1976) with bovine serum albumin as a standard. Flavonoid compounds were determined, according to the method of Chang et al. (2002).

2.2.2 Determination of Fatty Acid

Fatty acids were extracted, according to Harbone (1984). The methylated samples were analyzed by using GLC apparatus in GVC pye Unicam gas-liquid chromatograph equipped with dual flame ionization detector and dual channel recorder.

2.3 Statistical Analysis

The values were subjected to the analysis of variance (ANOVA) for a factorial in a completely randomized design MSTAT-C (1988). The significant differences between means were compared at p ≤ 0.05 using Tukey’s HSD (honestly significant difference) test.

3 Results

3.1 Impact of Stigmasterol (Sts) and Drought (WIR) on Growth Criteria of Sunflower Plants

The effect of foliar spraying of stigmasterol treatment with different concentrations (0, 100, 200, and 300 mg L−1) on growth indices of sunflower plants grown under drought stress is presented in Table 1. It is obvious that decreasing water irrigation requirement from 80 to 50% WIR (D0 and D1) significantly decreased sunflower growth indices (plant height (cm), leaf number and area/plant, and shoot), while it increased significantly root length (cm), fresh and dry weight (g); and while non-significant decrease was recorded in relative water content (RWC%). The percentage of decreases reached to 14.4%, 10.6%, 18.5%, 20.6%, 15.9%, and 0.78%, in plant height, leaf number and area/plant and RWC%, with 24.1%, 24.2%, and 9.9% percentages of increases in root, length (g), fresh and dry weight (g), respectively Meanwhile, different Sts concentration caused significant gradual increases in the studied growth indices and RWC till 200 mg L−1, and then the increases in response to 300 mg L−1 were less than 200 mg L−1 but still more than the control plants. Moreover, data clearly show superiority of 200 mg L−1 Sts over the control plant and other used Sts treatments (Table 1). Furthermore, 200 mg L−1 could alleviate the negative effect of water stress by 38.6, 16.8, 122.7, 56.5, 30.7, 38.7, 38.7, 41.4, and 2.4 percentages of increases in the studied growth parameters, compared with untreated control (Table 1).

Table 1 Effect of different concentrations of stigmasterol (Sts) application on sunflower plants stressed by drought on growth criteria. D0 (control 80% water irrigation requirement WIR), D1 (50% water irrigation requirement, WIR), Sts 0 (irrigation without stigmasterol treatment), Sts 1 (stigmasterol at 100 mg L−1), Sts 2 (stigmasterol 200 mg L−1), and Sts 3 (stigmasterol 300 mg L−1)
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3.2 Impact of Stigmasterol (Sts) and Drought (WIR) on Photosynthetic Pigments and Endogenous Indole Acetic Acid of Sunflower Plants

The impact of stigmasterol with different concentrations on photosynthetic pigments constituents (Chl. a, Chl. b, carotenoids, and total pigments) Fig. 1, Chl. a/Chl. b, Fig. 2 as well as endogenous indole acetic acid (IAA) (Fig. 3) of sunflower plants grown under drought stress. Drought stress (50% WIR) significantly reduced Chl. a, Chl. b, carotenoids, and consequently total pigments as well as endogenous IAA, compared with control plants (80% WIR). Meanwhile the ratio of Chl. a/Chl. b was increased significantly by 50% WIR. The application of stigmasterol significantly increased those traits gradually to 200 mg L−1, then the increases in 300 mg L−1 were less compared to 200 mg L−1, while decreased Chl. a/Chl. b ratio. Sts treatment at 200 mg L−1 resulted in the highest increments in the above-mentioned traits, compared with other Sts levels, since it gave 96.6 and 79.4 (mg g−1 fresh weight) in Chl. a, 67.4 and 49.1 (mg g−1 fresh weight) in Chl. b, 32.4 and 32.0 (mg g−1 fresh weight) in carotenoids, 196.4 and 160.4 (mg g−1 fresh weight) in total pigments and 50.9 and 39.85 (μg/g fresh weight) in IAA; meanwhile in Chl. a/Chl. b ratio, 200 mg L−1 showed the least decrease 1.43 and 1.62 under normal and water stressed conditions, respectively.

Fig. 1
figure 1

Effect of different concentrations of stigmasterol (Sts) application on sunflower plants stressed by drought on photosynthetic pigment content (μg/100 g fresh weight). D0 (control) (80% water irrigation requirement WIR), D1 (50% water irrigation requirement WIR), Sts 0 (irrigation without stigmasterol treatment), Sts 1 (stigmasterol at 100 mg L−1), Sts 2 (stigmasterol at 200 mg L−1) and Sts 3 (stigmasterol at 300 mg L−1). Mean values within the same column for each trait with the same lower-case letter are not significantly different at p ≤ 0.05. Measurements were done at 50 days after sowing. (Each value represents the mean ± standard error (n = 3)

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Fig. 2
figure 2

Effect of different concentrations of stigmasterol (Sts) application on sunflower plants stressed by drought on chlorophyll a/chlorophyll b ratio. D0 (control) (80% water irrigation requirement WIR), D1 (50 % water irrigation requirement WIR), Sts 0 (irrigation without stigmasterol treatment), Sts 1 (stigmasterol at 100 mg L−1), Sts 2 (stigmasterol at 200 mg L−1) and Sts 3 (stigmasterol at 300 mg L−1). Mean values within the same column for each trait with the same lower-case letter are not significantly different at p ≤ 0.05. Measurements were done at 50 days after sowing. (Each value represents the mean ± standard error (n = 3)

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Fig. 3
figure 3

Effect of different concentrations of stigmasterol (Sts) application on sunflower plants stressed by drought on endogenous indole acetic acid (IAA) (μg/g fresh weight). D0 (control) (80% water irrigation requirement WIR), D1 (50% water irrigation requirement WIR), Sts 0 (irrigation without stigmasterol treatment), Sts 1 (stigmasterol at 100 mg L−1), Sts 2 (stigmasterol at 200 mg L−1) and Sts 3 (stigmasterol at 300 mg L−1). Mean values within the same column for each trait with the same lower-case letter are not significantly different at p ≤ 0.05. Measurements were done at 50 days after sowing. (Each value represents the mean ± standard error (n = 3)

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3.3 Changes in Reactive Oxygen Species and Related Traits

During oxidative stress, reactive oxygen species (ROS) including hydrogen peroxide (H2O2) are generated, while lipid peroxidation is promoted by reactive oxygen species (ROS). The accumulation of ROS in plants damages almost all the cellular components, for example, cell membrane, lipids, pigments, and enzymes. ROS may cause cellular injury if not counteracted by antioxidant enzymes. Figure 4 shows the effect of stigmasterol (Sts) on hydrogen peroxide (H2O2), lipid peroxidation (MDA), and lipoxygenase (LOX) enzyme in sunflower plants grown under drought stress. Drought stress significantly increased oxidative damage parameters including H2O2 which resulted in a significant increase in lipid peroxidation (MDA), and lipoxygenase (LOX) enzyme. Sts application significantly decreased H2O2, which resulted in a significant reduction in MDA. Foliar Sts application at 200 mg L−1 resulted in best results, compared to other Sts levels. Stigmasterol concentration of 200 mg L−1 under normal irrigation resulted in a reduction in oxidative damage by 18.38% in H2O2 and consequently decreased MDA by 15.16%, and LOX by 14.75%, compared to control Sts. However, 200 mg L−1 Sts application under 50% WIR (drought stress) caused a reduction in oxidative damage by 34.19% in H2O2 and consequently decreased MDA by 28.13%, LOX by 31. 15%, compared to control Sts (Fig. 4).

Fig. 4
figure 4

a, b, and c Effect of different concentrations of stigmasterol (Sts) application on sunflower plants stressed by drought on hydrogen peroxide H2O2, malondialdehyde MDA, lipoxygenase LOX enzyme (nmole/g fresh weight). D0 (control) (80% water irrigation requirement WIR), D1 (50% water irrigation requirement WIR), Sts 0 (irrigation without stigmasterol treatment), Sts 1 (stigmasterol at 100 mg L−1), Sts 2 (stigmasterol at 200 mg L−1) and Sts 3 (stigmasterol at 300 mg L−1). Mean values within the same column for each trait with the same lower-case letter are not significantly different at p ≤ 0.05. Measurements were done at 28 days after sowing (Each value represents the mean ± standard error (n = 3)

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It is clear from the obtained results in Fig. 5 that drought stress (50% WIR) causes a marked increase in membrane leakage of sunflower plants, compared with those of the reference controls (80% WIR). Meanwhile, application of stigmasterol with different levels caused significant decreases in membrane leakage of treated sunflower plants, compared with those of the reference controls. Sts at 200 mg L−1 under normal irrigation resulted in a reduction in ML by 17.83%, compared to control Sts. However, 200 mg L−1 Sts application under 50% WIR (drought stress) caused a reduction in oxidative damage by 28.13%, ML compared to control Sts (Fig. 5).

Fig. 5
figure 5

Effect of different concentrations of stigmasterol (Sts) application on sunflower plants stressed by drought on membrane leakage (ML%). D0 (control) (80% water irrigation requirement WIR), D1 (50% water irrigation requirement WIR), Sts 0 (irrigation without stigmasterol treatment), Sts 1 (stigmasterol at 100 mg L−1), Sts 2 (stigmasterol at 200 mg L−1) and Sts 3 (stigmasterol at 300 mg L−1). Mean values within the same column for each trait with the same lower-case letter are not significantly different at p ≤ 0.05. Measurements were done at 28 days after sowing (Each value represents the mean ± standard error (n = 3)

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3.4 Changes in Enzymatic Antioxidants, Antioxidant Activities and Non-Enzymatic Antioxidants

3.4.1 Changes in Enzymatic Antioxidants

Drought stress significantly (p ≤ 5%) increased peroxidase (POX), superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (ASP), nitrate reductase (NR), and glutathione reductase (GR) enzyme activities of sunflower plants, compared with well-watered plants (Table 2). Furthermore, distinct levels of stigmasterol (Sts) foliar application caused more significant increases in those studied antioxidant enzymes POX, SOD, CAT, ASP, NR, and GR enzymes, as compared with those untreated sunflower plants. Moreover, exogenous application of 200 mg L−1 Sts gave the highest increases in the above-mentioned enzymes, as compared with other levels of Sts (Table 2).

Table 2 Effect of different concentrations of stigmasterol (Sts) application on sunflower plants stressed by drought on enzymatic antioxidants peroxidase (POX), superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (ASP), nitrate reductase (NR), and glutathione reductase (GR). D0 (control 80% water irrigation requirement WIR), D1 (50% water irrigation requirement WIR), Sts 0 (irrigation without stigmasterol treatment), Sts 1 (stigmasterol at 100 mg L−1), Sts 2 (stigmasterol 200 mg L−1), and Sts 3 (stigmasterol 300 mg L−1)
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3.4.2 Changes in Antioxidant Activities and Non-Enzymatic Antioxidants

Antioxidant activities expressed by DPPH free radical scavenging activity and non-enzymatic antioxidants such as total phenolics (TPh), reduced glutathione GSH, and α-tocopherol. α-Toco were increased in drought-stressed sunflower plants, as compared with well-watered plants (Table 3). Furthermore, different concentrations of Sts caused more significant increases in DPPH, TPh, GSH, and α-Toco. Stigmasterol with concentration of 200 mg L−1 showed superiority in increasing the above-mentioned antioxidants, as compared with all other levels of Sts either under normal irrigation or drought-stress conditions. Thus, it is recommended to apply foliar Sts either under drought or no drought conditions and the best level of Sts application is 200 mg L−1(Table 3).

Table 3 Effect of different concentrations of stigmasterol (Sts) application on sunflower plants stressed by drought on non- enzymatic antioxidants (2,2,-diphenyl-2-picryl-hydrazyl (DPPH%), total phenols (TPh), reduced glutathione (GSH), and α-tocopherol (α–Toco) D0 (control 80% water irrigation requirement WIR), D1 (50% water irrigation requirement WIR), Sts 0 (irrigation without stigmasterol treatment), Sts 1 (stigmasterol at 100 mg L−1), Sts2 (stigmasterol 200 mg L−1), and Sts 3 (stigmasterol 300 mg L−1)
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3.5 Changes in Compatible Solute Accumulation

Accumulation of compatible solutes is known to be a main way for plant defense and survival under abiotic stress, including drought and oxidative stress. Stigmasterol (Sts) impact on compatible solute accumulation (proline, Pro and total soluble sugars, TSS) in addition to free amino acids (FAA) contents of sunflower plants grown under drought stress are presented in Table 4. Decreasing irrigation water requirement from 80 to 50% WIR (drought stress) significantly increased Pro, TSS, and free amino acids over control plants (80%); the percentage of increase in Pro was 28.50%; in TSS, it was 28.48% and in FAA was 26.30%. Furthermore, exogenous application of Sts significantly further increased the accumulation of Pro, TSS, and FAA over untreated control plants. Sts at a rate of 200 mg L−1 gave the highest values, compared to all other Sts rates, so it is recommended to apply foliar Sts either under normal water irrigation or drought stress, and the best level of Sts application is 200 mg L−1 (Table 4).

Table 4 Effect of different concentrations of stigmasterol (Sts) application on sunflower plants stressed by drought on osmolyte content Proline (Pro), total soluble sugars (TSS), and total free amino acids (FAA), D0 (control 80% water irrigation requirement WIR), D1 (50% water irrigation requirement WIR), Sts 0 (irrigation without stigmasterol treatment), Sts 1 (stigmasterol at 100 mg L−1), Sts 2 (stigmasterol 200 mg L−1), and Sts 3 (stigmasterol 300 mg L−1)
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3.6 Impact of Stigmasterol (Sts) and Drought (WIR) on Yield Traits of Sunflower Plants

Table 5 shows the effect of Sts on different yield and its components such as head diameter (cm), head circumference (cm), seed yield/plant (g), and 100 seed weight (g) of sunflower plants grown under drought stress. Relative to control, drought stress (D1) (50% WIR) significantly reduced yield components; the percentages of decreases were 26.55%, 26.05%, 36.26% and 29.61% of head diameter, head circumference, seed weight/plant, and 100 seed weight, respectively. While exogenous application of Sts significantly (100, 200, and 300 mg L−1) increased these traits gradually till 200 mg L−1, and then the increases in 300 mg L−1 were less compared to 200 mg L−1. Sts at 200 mg L−1 resulted in the highest values, compared to other treatments under control drought, and drought stress (D0) (80% WIR) gave the highest values (Table 5), as it caused 14.05% and 15.84% of head diameter, 13.79% and 16.58% of head circumference, 19.84% and 25.29% of seed weight and 26.72% and 33.95% of 100 seed weight under 80% and 50% WIR, respectively.

Table 5 Effect of different concentrations of stigmasterol (Sts) application on sunflower plants stressed by drought on yield attributes. D0 (control 80% water irrigation requirement WIR), D1 (50% water irrigation requirement WIR), Sts 0 (irrigation without stigmasterol treatment), Sts 1 (stigmasterol at 100 mg L−1), Sts 2 (stigmasterol 200 mg L−1), and Sts 3 (stigmasterol 300 mg L−1)
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3.7 Changes in Nutritional Values of the Yielded Sunflower Seeds

The impacts of foliar treatment of sunflower plant with stigmasterol Sts different concentrations (0, 100, 200, and 300 mg L−1) on yielded seed quality including the nutritional value of the yielded seeds as oil%, carbohydrates CHO%, protein%, flavonoids%, and DPPH% are presented in Table 6 grown under normal irrigation and drought stress. Data clearly showed that decreasing water irrigation requirement from 80 to 50% WIR (drought stress) caused significant decreases in oil%, carbohydrates CHO%, and protein%, meanwhile increased significantly, as compared with those plants irrigated normally (80% WIR). Sts foliar treatment with different concentrations not only caused promotive effect on the above-mentioned indices of the yielded seeds in plants irrigated with 80% WIR, but also, alleviated the reduced effect of drought stress, compared with untreated controls. Sts with 200 mg L−1 was the most effective treatment, as it gave the highest increases in different studied parameters over the other treatments (Table 6).

Table 6 Effect of different concentrations of stigmasterol (Sts) application on sunflower plants stressed by drought on nutritional values of yielded seeds, D0 (control 80% water irrigation requirement WIR), D1 (50% water irrigation requirement WIR), Sts 0 (irrigation without stigmasterol treatment), Sts 1 (stigmasterol at 100 mg L−1), Sts 2 (stigmasterol 200 mg L−1), and Sts 3 (stigmasterol 300 mg L−1)
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3.8 Changes in Fatty Acid Contents

The constituents of fatty acids of the yielded sunflower seeds are depicted in Table 7. The fatty acids palmitic, stearic, palmitoleic, oleic, linolenic, and linoleic acid comprised most of the fatty acids in control plants; from unsaturated fatty acids, oleic acid was present in higher content followed by linoleic acid. While saturated fatty acid, palmitic was the dominant followed by stearic acid. Drought stress caused great decreases in total fatty acid as well as the contents of oleic, linoleic, and linolenic acids, whereas palmitic and stearic acids increased markedly.

Table 7 Effect of different concentrations of stigmasterol (Sts) application on sunflower plants stressed by drought on fatty acids of yielded seeds, D0 (control 80% water irrigation requirement WIR), D1 (50% water irrigation requirement WIR), Sts 0 (irrigation without stigmasterol treatment), Sts 1 (stigmasterol at 100 mg L−1), Sts 2 (stigmasterol 200 mg L−1), and Sts 3 (stigmasterol 300 mg L−1)
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Moreover, drought stress generated the fatty acid of behenic and lignoceric acids. Nonetheless, stigmasterol treatment with different levels caused marked increments in oleic, linoleic, and linolenic acids, compared with untreated controls either under normal or drought-stressed conditions (Table 7); while, palmitic and stearic acids were decreased relative to untreated control plants. Moreover, drought stress increased markedly total saturated fatty acids while it decreased total unsaturated fatty acids as well as the ratio of total unsaturated/ total saturated fatty acids TUS/TS. On the other hand, Sts treatments highly counterbalanced the effect of drought stress on fatty acid contents, as it improved them. Sts treatment augmented the obtained decrease in total fatty acid under drought stress. Moreover, Sts foliar application could alleviate the drought stress effect by decreasing total saturated fatty acid while increasing total unsaturated fatty acids and the ratio of total unsaturated/total saturated fatty acids TUS/TS. Moreover, the beneficial role of Sts treatment was either in unstressed or drought-stressed plant, the most effective effect recorded with 200 mg L−1 Sts.

4 Discussion

Drought stress is one of the environmental stressors that have a negative impact on sunflower plant growth, different metabolic activities, seed production, and nutritional quality. Stigmasterol exogenous treatments were evaluated to diminish the negative effects of drought stress on sunflower plants. Low water irrigation caused variations in a variety of biochemical, physiological, and molecular attributes that reduced nutrient availability and decreased plant growth and productivity (Tariq et al. 2019 and Kapoor et al. 2020).

According to the current study, sunflower plants under drought stress exhibited less growth features and yield components. These findings supported earlier studies on quinoa, wheat, flax, and moringa plants (Elewa et al. 2017, Bakry et al. 2019, Sadak et al. 2019 and Abd Elhamid et al. 2021).These reductions might be resulted from inhibiting the production of protein kinases, restricted cellular division, and expansion resulting in a reduction in apical growth (Kapoor et al. 2020 and Jing et al. 2022). Furthermore, water stress restricts the Calvin cycle’s ability to fix CO2 resulting in low fresh and dry matter production (Ozturk et al. 2021). These decreases might potentially be the result of pigment and protein alterations, oxidation of chloroplast, and lipids (Marcinska et al. 2013). The equilibrium among both leaves’ water supply and the rate of transpiration is indicated by the relative water content (RWC) of leaves. Decreased relative water content means less pressure, leading to reduced water available for regulator cell enlargement (Avramova et al. 2015). The lower leaf RWC may be due to reduced water availability, or the roots may become unable to reabsorb water lost through transpiration (Soltys-Kalinaet al. 2016).

Impaired photosynthesis caused via chloroplast degradation due to severe effects of drought stress from the significant signs of drought stress impacts (Sardans and Peñuelas 2012). These decreases are substantiated by previous studies on Moringa oleifera (Ezzo et al. 2018) and chickpea plant (Bakhoum et al. 2022). These reductions might be caused by oxidation of chloroplast lipids, modifications to the structures of pigment and protein molecule, or chlorophyll degradation by proteolytic enzymes like chlorophyllase (Marcinska et al. 2013) deterioration in chloroplast molecule and finally stomatal closure (Jomo et al. 2016).

Moreover, water deficiency decreased endogenous IAA of sunflower seedlings; these findings are consistent with those obtained by Abd Elhamid et al. (2021) on Moringa oleifera and Sadak and Ramadan (2021) on white lupine plants. Bano and Yasmeen (2010) reflected these decreases to the improvement in IAA oxidase activity, which would boost IAA degradation. Reduced IAA contents led to cell division and/or cell expansion decreases which in turn reduced growth rate.

Drought stress increased oxidative stress damage in terms of free radical accumulation such as H2O2 and lipoxygenase activity (LOX), leading to lipid peroxidation and membrane leakage. These free radicals react with lipid, protein, and DNA causing oxidative damage and disturb normal cellular functions (Gill and Tuteja 2010). These obtained data are supported with those obtained by Elkelish et al. (2019), Sadak and Bakhoum (2022), and Sadak (2022). ROS can permeate from sites which produce them to other sites of cells, harming delicate cell structures (Yin et al. 2015). A correlation between the increased H2O2 in leaves and the decreased chlorophyll content in wheat plants under salinity stress is also stated by Angelika et al. (2013). Membrane leakage ML is a crucial indicator of damaged cell membranes. Additionally, the loss of membrane integrity which resulted in increased ion leakiness and, as a consequence, an increase in membrane leakage that led to decreased membrane stability (Prabha and Negi 2014).

Under stress conditions, plants have internal mechanisms which reduce stress triggered by oxidative impacts by eliminating overproduced ROS (Kapoor et al. 2020). Antioxidants, both enzymatic and non-enzymatic, have a strong ability to scavenge excess ROS. It was intriguing to see how stigmasterol treatment increased the antioxidant system of the sunflower plant by raising enzyme activity and non-enzymatic component concentration (Sadak and Bakry 2020). O2•− radical is removed from numerous cellular locations, including the photosynthetic system, by superoxide dismutase (SOD), while H2O2 is scavenged by either CAT in the cytoplasm or by the ascorbate-glutathione cycle in chloroplast and mitochondria (Elkeilsh et al. 2019). Drought-stressed sunflower plants exhibited improved CAT and POX activities which involved quenching ROS, decreasing cellular damage and enhancing oxidative capacity to defend against stress (Sofy et al. 2021); while increases in SOD, POX, and CAT activities improve the elimination of excess ROS molecules, conferring higher stress resistance on sunflower plants. Increased antioxidant functioning prevents the vulnerability of photosynthetic electron transport to O2•− in the PSII reaction center, which otherwise results in irreversible oxidation of D1 protein (Borella et al. 2019).

Total phenols are an example of secondary metabolites that positively affect defense mechanisms by efficiently scavenging different ROS (Hasan et al. 2018). Total phenol contents of sunflower plant increased due to drought; these increases could be due to increased activity of enzymes known to be involved in their production (Bhattacharya et al. 2010). The same effect was obtained Abd Elhamid et al. (2021) who observed that drought increased phenol levels of Moringa olefera.

An earlier work by Sadak et al. (2019) discovered a rise in Pro and TSS contents of flax varieties under drought stress. Osmolytes found in the cytoplasm of plants have a basic effect on lowering cell osmotic potential and stabilizing cell turgor (Serraj and Sinclair 2002) without reducing actual water contents (Pathan et al. 2004). While proline (Pro) is one of the most significant osmolytes linked to plants experiencing oxidative stress and helps to stabilize subcellular structures (such as proteins and membranes) as well as scavenge free radicals, proline accumulation may be a way for plants to escape water stress (Huang et al. 2014). Herein, the increased production of proline and TSS might be the result of different biosynthetic and catabolic pathways being regulated. Furthermore, major cellular structures and their functionality could be safeguarded by adequate production of proline, TSS, and free amino acids. For example, proline protects the carboxylase activity of RuBisCO (Sivakumar et al. 2000). Proline is also a crucial component for controlling water absorption, preserving ROS homeostasis, and modulating communication (Mattioli et al. 2009).

Yield and its components showed significant losses in sunflower plants subjected to drought stress, while Sts treatment led to significant increases in yield components. Drought stress decreases plant growth, hence decreasing yield components. Therefore, the plant yield might result from integrating various metabolic processes. Thus, yield is affected by factors that disrupt these processes. In this study, the decreases in yield traits might be resulted from growth cessation, so plants are suffering from stress. The reduction in yield is associated with delayed flowering and decreasing flower number and pod set (Khan et al. 2016). The nutritional values of sunflower plants yielded seeds such as oil, total carbohydrates (CHO), and protein contents and were significantly reduced by drought, whereas their flavonoids contents and antioxidant activity increased. Similar findings were obtained in flax plant species by Sadak and Bakry (2020). Variations in a seed’s nutritional values such as oil, total carbohydrate, protein, and flavonoid contents are important since they are closely related to various biochemical and physiological metabolic processes (Anjum et al. 2003). Water stress lowered chlorophyll content of leaves, which in turn reduced photosynthetic activity.

Consequently, lowering carbohydrate deposit in mature leaves and, as a result, lowering the transit of carbohydrates from leaves to developing seeds (Elewa et al. 2017). To preserve energy and defend against stress, plants’ metabolic systems generally or the seeds they produce undergo changes that slow down the growth and development of seeds (Ragaey et al. 2022). In addition, seed oil and protein affect plant responses to environmental signals, particularly osmotic adjustment, either directly or indirectly (Singh and Sinha 2005). Meanwhile, the increased amount of flavonoid and antioxidant activity under drought conditions may reflect defense against stress conditions (Shimoi et al. 1996 and Geetha et al. 2003), since drought stress was accompanied by increased production of reactive oxygen species (Rezazadeh et al. 2012).

Sunflower oil’s fatty acid profile under drought stress was significantly countered by stigmasterol foliar treatments. The obtained decreases in the unsaturated fatty acids which are accompanied with increments in saturated fatty acid contents in sunflower oilseeds, due to decreasing water irrigation requirements (50%), are in accordance with the results of Hassan et al. (2021) on flax plant. According to Hajlaoui et al. (2009), the ability to adapt to drought stress may manifest as a potential decrease in desaturase activity of Schizochytrium limacinum. Malkit et al. (2002) also stated various plants might be shielded from oxidative stress via rebuilding membranes with less polyunsaturated fatty acids. Additionally, this less unsaturation degree hindered the elasticity of membrane (Zhao and Qin 2005; Upchurch 2008) and restricted permeability (Konova et al. 2009). Furthermore, Petcu et al. (2001) and Baldini et al. (2002) stated that drought significantly lowered oleic acid amount in sunflower oil, and they reflected this result to the enzyme ∆-9 desaturase that began to be active on the 8th day following flowering along with a rise in the biosynthesis of the oil. By desaturating stearic acid (C18:0), it has been proposed that this enzyme is responsible for the accumulation of oleic acid (C18:1) (Mckeon and Stumpf 1982). The second desaturation of oleic acid in linoleic acid is catalyzed by ∆-12 desaturase, another enzyme that contributes to the formation of oleic acid (Stymne and Appelqvist 1980). Palmitic (C16:0) and stearic acid (C18:0) saturated fatty acids were increased significantly in response to water stress. This indicates that palmitic acid and stearic acid may play an important role in drought tolerance mechanism of plants (Hashem et al. 2011).

Nevertheless, treatment of sunflower seedlings with Sts could lessen the impact of drought stress on growth, RWC%, and yield related traits. The obtained data are comparable to those obtained in flax and wheat plants by Hassan et al. (2021) and Hussein et al. (2022). Concurrently, Sts play a regulatory role in plant development (He et al. 2003). Among these functions are cell expansion and vascular differentiation (Rao et al. 2002). Hartmann (1998) stated that phytosterols are biogenic precursors for many components important for plant growth, including membrane permeability and modulation of enzyme activity. Additionally, Rogowska and Szakiel (2020) stated that these improvements might be due to improving water uptake and utilization efficiency, improving cell division, and/or cell enlargement. Moreover, Sts foliar spraying improved the impaired effects of water stress on photosynthetic pigment components. These findings reinforce earlier research in which exogenously applied Sts increased photosynthetic pigment levels in chickpea and Arabidopsis under salt and drought stress (Li et al. 2012). Refereeing to the earlier investigations BRs caused increases in different photosynthetic pigments of wheat (Dong et al. 2017), flax (Bakry et al. (2019), and duckweed (Chmur and Bajguz 2021). This improving effect could be related to its ability to boost oxide assimilation in the Calvin cycle by enhancing RuBisCO’s initial activity. Stigmasterols (Sts) are phytohormones that regulate plant defense responses (Sharma et al. 2014), in addition to enhanced photosystem II efficiency (Filova et al. 2013). Chmur and Bajguz (2021) reflected those increases in pigment contents to the improvement of plant ability to absorb light and the stimulation of the activity of enzymes involved in chlorophyll production. Additionally, the increases in chlorophyll a/b ratios resulted by stigmasterol treatment may be caused via more rapid degradation of chlorophyll b than chlorophyll a. This may be clarified by the fact that the first step in the degradation of chlorophyll b includes conversion to chlorophyll a despite that indole acetic acid sustains the protective role of many crops against biotic and abiotic stress through antagonistic or synergistic interaction with other phytohormones such as gibberellic acid, cytokinin, and abscisic acid. The enhanced IAA levels obtained in response to stigmasterol treatments may lead to an enhancement of enzyme activity and as a result, increased growth parameters in stigmasterol-treated plants (Bakry et al. 2019). Hussein et al. (2022) stated that stigmasterol treatment improved numerous endogenous phytohormones (including IAA) which play an important role in plant development as signals and regulators. Exogenous Sts also improved membrane leakage and at the same time decreased lipid peroxidation and lipoxygenase activity by lowering ROS levels. Lowered ROS production maintains the structural and functional integrity of membranes, maintaining cellular function (Nahar et al. 2016). The positive impact of Sts in defending membranes and stabilizing the key cellular processes is demonstrated by the decreased ROS overproduction and lipoxygenase activity in seedling treated with Sts. Recent research on soybean plant showed that Sts application lowered lipoxygenase activity and ROS production, which led to a decline in lipid peroxidation under water stress (Hasan et al. 2020).

Similar results are obtained earlier by Bassuany et al. (2014) on flax plants. The possible mechanism for improved membrane leakage in response to stigmasterol treatment was the obtained decrease in lipid peroxidation (as indicated by MDA content) in Sts-treated plants. Furthermore, the induction of SOD upregulation, which has aided in the regulation of photosynthetic activity and the structural and functional maintenance of PSII, may be the cause of STs’ treatment effects on antioxidant enzymes in sunflower plants. According to Hassan et al. (2021), stigmasterol treatment resulted in enhanced activity and transcript levels of certain antioxidant enzymes, which improve drought tolerance. Additionally, plants treated with Sts showed an increase in non-enzymatic antioxidants; at the same time, H2O2 and MDA levels dropped, proving that Sts had repaired the antioxidant systems. Sts may therefore lead to an enhancement in the antioxidant system, enhancing ROS scavenging and resulting in increased drought tolerance (Hassan et al. 2021). Improved DPPH%, GSH and α-toco accumulation due to Sts treatment might have significantly contributed to drought tolerance through (a) stabilizing redox homeostasis, (b) scavenging ROS, and (c) preserving the activity of ascorbate-glutathione cycle enzymes including APX and GR. Additionally, GSH plays a crucial signaling role in shielding photosynthetic pigments from stress brought on by oxidative conditions (Foyer and Shigeoka 2011). Increased GSH production in conjunction with GR upregulation is important for maintaining NADP levels for steady photosynthetic electron transport. The same results were obtained in soybean plants under oxidative stress (Hasan et al. 2020). Sts improved yield as well as growth, ROS homeostasis, TPh, antioxidant enzymes, antioxidant, and Rubisco as found by Bassuany et al. (2014) and Hassan et al. (2021).

Stigmasterol treatments markedly enhanced sunflower seed output and its constituents, including oil, total carbohydrate, protein, and flavonoid concentrations, under both normal and drought-stress conditions. Additionally, these increments in carbohydrate levels may be attributed to improved photosynthetic activity, which would accelerate the transfer of carbohydrates from leaves to growing grains (Bassuany et al. 2014). Furthermore, Sts application increased antioxidant activity comparing with control plants. The increase in scavenging activity can be considered an advantage of treatment used that could be attributed to the increases in total phenols and total flavonoids (Yu et al. 2002). The obtained results of increased unsaturated fatty acid content as well as TU/TS (Table 7) in response to Sts treatments might be explained by cell defense against oxidative stress via suppression of lipid peroxidation and free radical scavenging (Velikova et al. 2000). However, the quantity and quality of oil output were greatly enhanced by treatment with Sts. Generally, the concentration of essential fatty acid of an oil determines its quality (Johnson and Bradford 2014), but certain oil quality features suitable for certain industrial uses can also be obtained by water stress.

5 Conclusion

According to our study’s findings, drought stress (50% of water irrigation requirement) reduced sunflower growth attributes, photosynthetic pigments, indole acetic acid, and phenolic. Meanwhile, it increased antioxidant enzymes and antioxidant compounds as well as some osmo-protectants. Furthermore, yield analysis revealed declines in several yield attributes, yielded oil, carbohydrates, and protein percentages, while it increased flavonoid and antioxidant activity percentages as well as altered fatty acid components. These findings supported the negative impact of drought via demonstrating how it stresses sunflower plants. In contrary, exogenous stigmasterol treatment of sunflower plant reduced these negative effects and enhanced quantity and quality of the yielded seeds and oil output. Stigmasterol modulated photosynthetic pigments, ameliorated oxidative stress disorders, accumulated osmo-protectants, enhanced endogenous indole acetic acid contents and phenolic biosynthesis, and triggered superoxide radical- and hydrogen-peroxide-scavenging enzymes. All of these alterations had a positive correlation with the growth, and hence the yield of drought stressed sunflower plants. These were concomitant with improved oil, carbohydrate, protein, and flavonoid contents of seeds as well as fatty acid composition of oil.