Silicon Induces Salt Stress Amelioration in Sunflower Plants by Improving Photosynthetic Pigments and Mineral Status

Abstract

Silicon (Si) has been extensively studied for its ability to decrease sodium (NaCl) toxicity in various plant species. Nonetheless, the processes that drive these responses are still not well understood. In this study, we investigate the effects of silicon (Si) on the modification of hydrogen peroxide concentration [H₂O₂], photosynthetic pigment content, nutrient accumulation, and the production of root and shoot dry biomass in sunflower (Helianthus annuus L.) plants hydroponically grown with NaCl (0 and 100 mM) in combination with Si (0 and 2.0 mM). Salt stress induced a significant decrease in plant growth due to high [H₂O₂] and a decrease in photosynthetic pigment content and nutritional status, denoting that there is oxidative and ionic stress. Nevertheless, Si addition to the growth medium consistently decreased the [H₂O₂] in sunflower and photosynthetic pigment content, and macro- and micronutrient accumulation, which was associated with an increase in root and shoot dry matter production. These findings indicate that adding Si to the growth medium is crucial for enhancing plant resistance to salt-induced ionic and osmotic stress, making it a promising strategy for improving crop growth and management under salinity conditions.

1. Introduction

Soil salinity has been reported to be increasing due to the effects of climate change, causing a devastating impact on land resources (affecting 7%) and cultivated land (5%) [1]. Salinity is a major environmental factor that restricts crop production in arid and semi-arid regions [2], where it impacts plant growth and yield due to osmotic effects, nutritional imbalances, oxidative damage, and/or toxicity from specific ions [3]. High salt levels in the soil lead to a high osmotic potential, restricting the uptake of water and nutrients by plants [4]. Additionally, ionic stress occurs due to the accumulation of salt reaching toxic levels in mature leaves, which hastens senescence and leads to leaf death [5]. Elevated levels of Na⁺ and Cl⁻ lead to nutritional imbalances, resulting in the increased production of reactive oxygen species (ROS), which negatively impact plant cells, growth, and productivity [6]. High salinity disrupts several essential physiological and biochemical processes, such as transpiration, photosynthesis, protein synthesis, and others [7].

Various strategies are being employed to reduce the adverse effects of salinity on plant growth and productivity. Among these, silicon supplementation is regarded as a significant approach to alleviate the detrimental impacts of salinity on plant growth and yield. Silicon can enhance plant survival under different abiotic stresses, such as mineral deficiency [8], toxicity [9], salinity [10], and drought [11]. All terrestrial plants have Si in their tissues, though the concentration varies significantly between species, ranging from 0.1% to 10% Si on a dry matter basis [12]. Overall, it was shown that both active and passive mechanisms are involved in the uptake and transport of Si in intermediate-type species like sunflower, with their roles varying based on the plant species and external Si concentrations [13].

Previous reports have indicated that Si plays a role in alleviating salt stress by reducing Na⁺ uptake [14,15], ROS accumulation, and transpiration rate [16,17], improving mineral and water uptake [18,19], and regulating antioxidant enzyme activity [19,20], which boosts plant growth. This positive impact of Si on reducing salinity stress, which has been previously confirmed in intermediate Si-accumulating plant species like cucumber [21] and faba bean [18]. Moreover, Si helps improve the upright position of leaf blades, which enhances light penetration and subsequently boosts photosynthesis [22,23]. Also, Si has been shown to prevent chlorophyll degradation and enlarge leaf area, resulting in greater light availability for photosynthesis [23,24]. Furthermore, enhancing plant growth and productivity in salt-affected plants through the use of Si has been documented in various plant species [11,18,23]

The primary aim of this research was to explore the interactive impact of Si on salt-stressed sunflower plants by managing ionic and osmotic stress. We demonstrate that adding silicon (Si) to the growth medium of sunflower (Helianthus annuus L.) plants can alleviate salt-induced ionic and osmotic stress, further improving dry biomass production. These findings indicate that adding Si to the growth medium significantly boosts resistance to salt-induced ionic and osmotic stress, presenting a promising strategy for improving crop growth and management under saline conditions.

2. Results

2.1. Changes in Root Dry Matter (RDM), Shoot Dry Matter (SDM) and [H₂O₂] After NaCl and Si Treatments

A two-way ANOVA test revealed a significant (p < 0.001) interaction between NaCl and Si for RDM, SDM, and [H₂O₂] (Table 1). Salt stress (100 mM NaCl) evidently decreased the SDM of sunflower plants by ~62% and the RDM by ~47% compared to the non-salt-stressed and Si-untreated plants. However, Si addition to the growth medium significantly (p < 0.0001) mitigated the deleterious effects of salt stress and improved the SDM, RDM, and TDM by ~27%, 68%, and ~31%, respectively, compared to salt-stressed and Si-untreated plants (Table 1). We also found that salt stress (100 mM NaCl) markedly increased [H₂O₂] in leaves, which also indicated adverse effects. Si had no effect on [H₂O₂] under non-salt-stressed sunflower plants; however, [H₂O₂] was decreased by ~47% in salt-stressed sunflower under Si addition compared to salt-stressed and Si-untreated plants and showed significant (p < 0.001) differences (Table 1).

2.2. Changes in Photosynthetic Pigment Concentrations After NaCl and Si Treatments

A two-way analysis revealed that there was a significant (p < 0.001) interaction effect between the NaCl and Si treatments on leaves’ photosynthetic pigments (Table 2 and Table S1). Added NaCl in the absence of Si decreased Chla by 70%, Chlb by ~79%, total Chl by ~74%, and carotenoids (CAs) by ~94% compared to the non-salt-stressed plants without Si addition. However, in salt-stressed plants, Si supplementation significantly increased Chla by ~108%, Chlb by 125%, total Chl by 115%, and CAs by ~41% compared to the salt-stressed and Si-untreated plants (Table 2).

2.3. Changes in Accumulation of Macronutrients and Micronutrients in Shoots and Roots After NaCl and Si Treatments

A two-way ANOVA showed changes in the accumulation of macronutrients and micronutrients and revealed a significant (p < 0.0001) interaction between the NaCl and Si treatments after 30 d in sunflower plants (Figure 1 and Figure 2, Table S1). The accumulation of macronutrients in shoots and roots was lower in sunflower plants exposed to salt treatment (100 mM NaCl) (Figure 1a–f). In addition, Si application had no effect on the accumulation of N, P, K, Ca, Mg, or S in shoots and roots under non-salt-treated plants. Conversely, Si addition increased and showed a significant (p < 0.0002) difference in the accumulation of N, P, K, Ca, Mg, and S in salt-stressed plants compared to the salt-stressed and Si-untreated plants (Figure 1a–f).

The accumulation of Fe, Mn, Zn, and Cu showed a significant (p < 0.0001) interaction between NaCl and Si treatments (Figure 2a–d, Table S1). Si supplementation revealed greater micronutrient accumulation in the shoots and roots of salt-stressed plants compared to the Si-untreated. Meanwhile, the micronutrient accumulation increased in the shoots and roots of salt-stressed sunflower plants under Si treatments compared to the salt-stressed and Si-untreated plants (p < 0.0001) (Figure 2a–d).

3. Discussion

The positive impact of Si on enhancing the growth of various salt-stressed plant species is widely acknowledged [10,18,25]. In summary, the primary function of Si in reducing sodium toxicity in plants is largely due to its positive effects on mineral nutrition, physiology, and biochemical processes, which enhance the plant’s tolerance to abiotic stresses [23,26]. Growth characteristics were improved with Si application, especially dry biomass accumulation, probably by the high photosynthetic pigment content and nutritional status (Table 1, Figure 1 and Figure 2). The positive effects of adding Si, which helps mitigate the harmful impacts of high sodium toxicity on plant growth, have been previously observed in sunflowers [10,20]. Our data on plant growth indicate that sunflower plants exposed to high-salinity conditions with Si supplementation produced approximately 62% more SDM and 47% more RDM compared to those grown in high salinity without Si. These results might also be interpreted as a consequence of reduced [H₂O₂], which likely supports plant growth. This enhanced growth of sunflowers under high salinity can be attributed to Si improving the chlorophyll content in the leaves [23]. Furthermore, the enhancement of plant growth by silicon might be associated with the reduction in damage caused by salt stress [19]. In an initial exploration of this topic, we accepted our hypothesis and discovered that adding silicon to the growth medium serves as a sustainable method to enhance plant growth under salt stress. Silicon has been shown to protect chlorophyll from damage, leading to increased leaf area and higher dry biomass production [27].

As previously mentioned, the hypothesis postulates that providing Si in the growth medium results in approximately 70%~94% more photosynthetic pigments per leaf compared to plants subjected to salt stress without Si treatment (Table 2). This finding suggests that Si can alleviate the harmful effects of salt stress by improving the overall photosynthetic machinery [23]. Another possible mechanism through which silicon (Si) enhances plant productivity under salt-stressed conditions may be its capacity to alter cell wall metabolism. This modification promotes cell enlargement by increasing tissue extensibility, which in turn raises the chlorophyll content in leaves [18,25,28]. Moreover, silicon has been shown to prevent chlorophyll degradation and expand leaf area, thereby enhancing the light available for the photosynthesis apparatus [24]. These results align with our hypothesis and indicate that the enhancement of photosynthesis by silicon represents an economical strategy for plants to manage various salt stress conditions. This might be one reason for the beneficial effects of silicon application in the detoxification of sodium toxicity.

In this study, we found that salt stress led to elevated toxic substance levels in sunflower leaves, promoting [H₂O₂] production (Table 1). This effect was due to the high Na⁺ accumulation [19]. However, supplementing with Si helped the plants maintain lower [H₂O₂] levels [14]. Similar reductions have also been observed in other research studies involving Si supplementation [8,18,29]. This finding suggests that this phenomenon is a common mechanism by which silicon enhances sodium toxicity. Another possible explanation for the decrease in [H₂O₂] could be the increase in antioxidant enzyme activity [18,19,23]. Additionally, the other referenced studies indicate that the addition of Si helps maintain low [H₂O₂] and high membrane stability [30,31]. Additionally, another significant function of Si in alleviating salt stress is the reduction of ROS generation, which consequently improves plant growth [32,33]. This might explain the beneficial impact of silicon application on alleviating salt stress observed in other intermediate Si-accumulating plant species, like cucumbers [34,35] and tomato [36]. Our data directly confirm the initial hypothesis, showing that Si is crucial in boosting resistance to salt-induced osmotic stress.

Our data showed that adding Si during salt stress enhanced the accumulation of macro- and micronutrients in both the leaves and roots (Figure 1 and Figure 2). This might explain why Si treatment is often reported to help reduce Na⁺ toxicity in numerous studies [10,23,26,37]. Our observations may have important implications for the addition of Si to enhance mineral uptake by improving root water uptake in salt-stressed plants [15,38,39,40]. Also, it has been proposed that silicon (Si) enhances salt tolerance in plants by stimulating H⁺-ATPase activity, which leads to increased K⁺ uptake [16,26,34]. Additionally, Si plays a crucial role in maintaining mineral balance in plants under salt stress, due to its ability to enhance nutrient uptake [18,21], which helps to decrease Na⁺ uptake [23,27]. Additionally, silicon significantly enhances the mineral balance under salt stress conditions by promoting ionic [21,41,42] and stoichiometric homeostasis [27]. Furthermore, several studies have reported that silicon (Si) has beneficial effects in enhancing mineral nutrient uptake under high-salinity conditions [26,38,43,44]. The findings of this study suggest that the enhancement of mineral nutrition through Si treatment is a promising method for mitigating the deleterious effects of sodium toxicity. Consistent with the observed results, this can be directly linked to an increase in dry biomass, supporting our hypothesis that Si represents an economical approach for plants to cope with salt stress.

In this context, one of the main limitations for sunflower production is its sensitivity to abiotic stress like salinity and drought, which decreases the grain yield and oil production and quality. In addition, future research should pay attention to the root microlevel due to sunflower crop expansion. We also highlight the need to link field studies through land use impacts and the zoning policy that has been implemented in Brazil. Finally, exogenous application of various organic or inorganic sources of Si and their possible ameliorative effects on biotic and abiotic stress in plants signify a promising future.

4. Materials and Methods

4.1. Localization and Growth Conditions

The research was conducted at the School of Agricultural and Veterinary Sciences (FCAV), São Paulo State University (UNESP) located on the Jaboticabal Campus (21°15′22″ S and 48°18′58″ W) in Brazil. We used a glasshouse with natural lighting. Daytime temperatures peaked at around 33 °C, while nighttime temperatures dropped to 20 °C. The relative humidity ranged from 60% to 75%, and the photoperiod was set to 14 h of light and 10 h of darkness.

4.2. Plant Material and Nutrient Solution

The yellow dwarf sunflower (H. annuus L. ‘Double Sungold’) was used. Five seeds were evenly placed in pots (4 dm⁻³ polyethylene containers) filled with washed sand (3.7 dm⁻³) and watered daily with demineralized water until emergence. Five days after plant emergence a quarter-strength modified Hoagland nutrient solution (NS) [45] was first applied with the pH adjusted to approximately 5.8 (±0.2) for one week. Then, the NS was increased to half-strength for two weeks, followed by an increase to three-quarters strength until the experiment concluded. Two seven-day-old seedlings were removed, leaving three uniform seedlings.

The pot experiment utilized a factorial arrangement (2 × 2) in a randomized block design with five replicates. A total of 40 pots were divided into 4 groups: non-salt-stressed and salt-stressed (100 mM), both combined with the absence or presence of Si (2 mM) in the growth medium. NaCl was used to induce salt stress treatment and to stabilize the sodium and potassium silicate (SINAKE) in order to add Si into the growth medium.

4.3. Salt Stress and Si Treatments

After a week of acclimating the plants, salt treatment was initiated by adding 100 mM NaCl to the nutrient solution (NS) and continued for 30 experimental days. Si treatment was administered through the NS throughout the entire experimental period, following the method outlined by Calero et al. [10]. For treatments without Si addition, K concentration was equilibrated by supplying potassium chloride (KCl).

4.4. Experimental Methods

4.4.1. Plant Growth Determination

After 30 days, sunflower seedlings were collected and washed with distilled water, neutral detergent solution (0.2%), HCl solution (0.1%), and twice-deionized water [10]. Then, sunflower plants were separated into roots and shoots, collocated in paper bag, and dried at 60 °C in a forced-ventilation oven (TE 394-3, Tecnal, Piracicaba, São Paulo, Brazil) until achieving a constant dry mass (DM). RDM and SDM were promptly determined on a digital balance (model ATX224, Shimadzu, Nakagyo-ku, Japan).

4.4.2. Experimental Methods

Thirty days after beginning the salinity treatments, the levels of photosynthetic pigments such as chlorophyll a (Chla), chlorophyll b (Chlb), total chlorophyll (Chlt), and carotenoids (CAs) were measured as previously described by Lichtenthale et al. [46,47]. Leaf discs were collected from the first fully expanded leaf blade (from the top) and placed in 10 mL of 80% acetone (v/v). The mixture was then centrifuged at 4000× g at 4 °C for 10 min. The supernatant was separated and utilized for the chlorophyll assay. Individual samples were analyzed in triplicate.

4.4.3. Hydrogen Peroxide Determination

Leaves of sunflower seedlings were collected to determine the H₂O₂ concentration. Then, the Alexieva et al. [48] methodology was used to estimate the H₂O₂ concentration. Briefly, 0.2 g of fresh leaves was homogenized in trichloroacetic acid (TCA) at 0.1% and centrifuged at 11,000× g for 15 min. The supernatant was added to the 100 mM potassium phosphate buffer (pH 7.50) and the 1.0 M potassium iodide solution. Then, the material was incubated in an ice bath for 1 h, the absorbance was read at 390 nm, and the H₂O₂ concentration was determined using an H₂O₂ concentration curve, known as the standard curve. The H₂O₂ results were expressed in μMol g⁻¹ of fresh matter (FM).

4.4.4. Macro- and Micronutrient Determination

The oven-dried root and shoot samples were then ground into a fine powder. Wet digestion of the dried sunflower plant material was carried out following the method of Bataglia et al. [49]. Initially, 0.1 g of dried, ground plant material was digested using 7 mL of concentrated H₂SO₄ and 1.5 mL of a diacid mixture composed of nitric and perchloric acids in a 3:1 ratio. The extract was then diluted to a total volume of 30 mL in volumetric flasks to determine the concentrations of macro- (g kg⁻¹) and micronutrients (mg kg⁻¹). The accumulation of each nutrient was calculated by multiplying its concentration by the corresponding dry biomass [10].

4.5. Statistical Analysis

Data on RDM, SDM, Chla, Chlb, total Chla + Chlb, CAs, [H₂O₂], N, P, K, Ca, Mg, S, Fe, Mn, and Cu were subjected to normality distribution checks using the Kolmogorov–Smirnov test and Levene test (p < 0.05) for unequal variance. After confirming normality and unequal variance, a two-way ANOVA was performed to examine the main effects of two levels of NaCl (Na) and two levels of Si (Si), and their interaction (Na × Si). When the F test indicated significant differences among treatments, mean comparisons were carried out using the Scott–Knott test (p < 0.05). All analyses were conducted using the statistical software R v. 4.4. [50].

5. Conclusions

In conclusion, our study assumes the hypothesis that Si supplementation is an economic and efficient alternative to ameliorate the growth of salt-stressed sunflower plants. The decreasing H₂O₂ concentration and increasing photosynthetic pigments and nutritional status is an important mechanism of Si for improving the growth of salt-stressed sunflower seedlings. Our findings suggest that Si supplementation plays an important role in detoxifying sodium toxicity by regulating ionic and osmotic stress, which leads to increased growth production.

Author Contributions

Conceptualization, A.C.H. and D.A.C.; methodology, R.d.M.P.; validation, A.C.H., D.A.C. and R.d.M.P.; formal analysis, A.C.H.; investigation, A.C.H., D.A.C., K.P.C., D.O.V. and G.d.S.S.J.; resources, R.d.M.P.; data curation, A.C.H.; writing—original draft preparation, A.C.H.; writing—review and editing, A.C.H., R.d.M.P., K.P.C. and D.O.V.; visualization, R.d.M.P.; supervision, A.C.H.; project administration, R.d.M.P.; funding acquisition, A.C.H. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

All relevant data are within the paper.

Acknowledgments

The authors are grateful to the São Paulo State University (UNESP), School of Agricultural and Veterinarian Sciences (FCAV), for providing the necessary facilities for this study. We would also like to thank GENPLANT research for their technical support of the project. We wish to thank to CAPES for providing the fellowship for the first author. The authors want to acknowledge the helpful comments on earlier versions of this manuscript of Marcelo Lattarulo Campos.

Conflicts of Interest

The authors declare no conflicts of interest.

Footnotes

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Figures and Tables

Figure 1. Shoot and root N accumulation (a), shoot and root P accumulation (b), shoot and root K accumulation (c), shoot and root Ca accumulation (d), shoot and root Mg accumulation (e), and shoot and root S accumulation (f), grown under NaCl (0 and 100 mM NaCl) treatments combined with the absence or presence (2 mM) of Si. Data are means ± standard deviation (SD) (n = 5). Different normal small letters (e.g., a, b) or italic small letters (e.g., a, b, c) indicate significant differences between Si treatments under non-salt stress and salt stress conditions, respectively, and different uppercase letters (e.g., A, B) show significant differences between non-salt and salt treatments for the same Si treatment (e.g., 0 mM or 2 mM) according to Scott–Knott test (p [less than] 0.05).

Figure 2. Accumulation of micronutrients in shoots and roots of sunflower plants. Shoot and root Fe accumulation (a), shoot and root Mn accumulation (b), shoot and root Zn accumulation (c), and shoot and root Cu accumulation (d), grown under non-salt and salt stress (100 mM NaCl) treatments combined with the absence or presence (2 mM) of Si. Data are means ± standard deviation (SD) (n = 5). Different normal small letters (e.g., a, b) in the same column indicate significant differences among Si treatments under non-salt stress; different italic small letters (e.g., a, b) shown significant differences among Si treatments under salt stress conditions and different uppercase letters (e.g., A, B) indicate significant differences between non-salt and salt treatments at the same level of Si according to the Scott–Knott test (p [less than] 0.05).

Table 1

Effect of absence or presence (2 mM) of Si on H₂O₂ concentration, root dry matter (RDM), and shoot dry matter (SDM) of sunflower plants grown under unstressed and salt stress (100 mM NaCl) conditions.

Treatments		H2O2 Concentration	RDM	SDM
NaCl	Si
mM		µMol g−1 FM	g plant−1
0	0	6.97 ± 0.27 aB	0.95 ± 0.01 aA	2.46 ± 0.07 bA
	2	6.79 ± 0.14 aB	0.97 ± 0.03 aA	2.48 ± 0.06 bA
100	0	22.15 ± 0.51 aA	0.29 ± 0.03 bB	1.35 ± 0.05 bB
	2	11.78 ± 0.34 bA	0.46 ± 0.03 aB	1.73 ± 0.07 aB

Data are means ± standard deviation (SD) (n = 5). NaCl (0 mM); NaCl (100 mM); Si (0 mM); Si (2 mM). Different normal small letters (e.g., a, b) or italic small letters (e.g., a, b, c) in the same column indicate significant differences between Si treatments under non-salt stress and salt stress conditions, respectively, and different uppercase letters (e.g., A, B) indicate significant differences between non-salt and salt treatments for the same Si treatment (e.g., 0 or 2 mM) according to Scott–Knott test (p < 0.05). FM, fresh matter.

Table 2

Effect of absence or presence (2 mM) of Si on photosynthetic pigment (Chla, Chlb, Chlt, and CAs) concentrations in sunflower plants grown under unstressed and salt stress (100 mM NaCl) conditions.

Treatments		Photosynthetic Pigments
NaCl	Si	Chla	Chlb	Total Chl	CAs
mM		µg cm−2
0	0	0.40 ± 0.01 aA	0.38 ± 0.01 aA	0.78 ± 0.01 aA	3.04 ± 0.01 aA
	2	0.41 ± 0.01 aA	0.39 ± 0.01 aA	0.80 ± 0.02 aA	3.06 ± 0.02 aA
100	0	0.12 ± 0.01 bB	0.08 ± 0.01 bB	0.20 ± 0.02 bB	1.57 ± 0.03 bB
	2	0.25 ± 0.01 aB	0.18 ± 0.01 aB	0.43 ± 0.01 aB	2.21 ± 0.02 aB

Data are means ± standard deviation (SD) (n = 5). Chla, chlorophyll a; Chlb, chlorophyll b; Chlt, chlorophyll (a + b); CAs, carotenoids. Different normal small letters (e.g., a, b) or italic small letters (e.g., a, b, c) in the same column indicate significant differences between Si treatments under non-salt stress and salt stress conditions, respectively, and different uppercase letters (e.g., A, B) indicate significant differences between non-salt and salt treatments for the same Si treatment (e.g., 0 mM or 2 mM) according to Scott–Knott test (p < 0.05).

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/stresses4040057/s1, Table S1: Results of the two-way ANOVA of the effects of NaCl, Si, and their interaction on shoot and root dry biomass, H₂O₂ concentration, photosynthetic pigments, and shoot and root macro- and micronutrient accumulations (n = 5).

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