Silicon dioxide-nanoparticle nutrition mitigates salinity in gerbera by modulating ion accumulation and antioxidants

Gerbera (Gerbera × jamesonii H. Bol cv. ‘Teera Kalina’) plants were planted in 12-L pots that contained 60% perlite and 40% cocopeat. The experiment was conducted at Fadak greenhouse in Maragheh (46°16′ E and 37°23′ N, altitude 1485 m), Iran. During the trial, the photoperiod of greenhouse was 14/10 h (light/dark), 22/18 ± 2°C temperature (day/night) and 75 ± 10% relative humidity. Plants were fed a Hogland nutrient solution containing macro and micro elements (Table 1) in irrigation water for 2 weeks until they were fully grown. The pH of Hogland solution in the tanker was adjusted to 5.5–6 using sodium bicarbonate or sulphuric acid. Then, the salinity level of the nutrient solution was considered as the control (S₀ = 0 mM) with four other concentrations as 5 (S₁), 10 (S₂), 20 (S₃) and 30 (S₄) mM NaCl. Plants were manually irrigated with the salinity treatments at a rate of 400 mL per pot every other day. At the end of each week, pots were irrigated with tap water to prevent leaching and salt accumulation.

After 3 months and just before flowering, the upper surface of leaves of control and salt-treated plants were sprayed until full wetting (ca. 25 mL · plant⁻¹) with solutions containing 0 (distilled water as mentioned C₀), 25 (C₁) and 50 (C₂) mg · L⁻¹ SiO₂-NPs as illustrated in Figure 1. SiO₂-NPs (size ˂50 nm) were prepared from Nanosany Corporation of nanomaterial company, Iran. The SiO₂-NPs properties are illustrated in Table 2. The size and type of nanoparticles used were selected based on the positive results of previous experiments (Zahedi et al., 2020b); indeed, the smaller NPs can enter into plant cells easily (Hossain et al., 2015). In addition, the Transmission Electron Microscopy (TEM) and Scanning Electron Microscopy (SEM) images of mesoporous silica particles (sample NNV-001) synthesised by Nanosany Corporation are illustrated in Figure 2. Then the effect of mentioned SiO₂-NPs spray on vegetative and flowering factors of gerbera was evaluated, and their interactions from morphological, physiological, and nutritional aspects were identified.

Figure 1

Figure 2

Powder of the oven-dried leaf samples (0.5 g) was digested in a solution of nitric acid and perchloric acid (2:1; V/V; Malavolta et al., 1997). The concentration of Na⁺ and K⁺ was quantified using flame photometry (Jeneway, model PFP7) against Na⁺ and K⁺ standards curve of certain concentrations, according to the method of Ren et al. (2005). Ca²⁺ was measured by titration with EDTA and recorded as g · 100 g⁻¹ FW.

Data analyzed by ANOVA software (SAS, version 9.4), and the difference between treatments was determined by the Duncan Multiple Range at p < 0.05. The trial was carried out as a factorial experiment in a completely randomised design (CRD), with three repetitions and each repetition includes two plants.

The number of leaves and flowers on gerbera plants was significantly decreased in salt-treated plants compared to control. Among different salinity levels, the highest salinity (30 mM NaCl) exhibited a profound reduction of 30 and 55% in the number of leaves and flowers on gerbera plants, respectively, versus the control (Table 3).

The highest number of flowers was obtained from S0 (non-saline) which was sprayed with 25 and 50 mg · L⁻¹ of SiO₂-NPs (1.87 and 1.73, respectively) while S5 (30 mM NaCl) without SiO₂-NPs spray had the least number of flowers (0.7). The highest leaf number belonged to 10 mM NaCl along with 25 mg · L⁻¹ SiO₂-NPs spray, while the lowest leaf number was observed in 30 mM NaCl without SiO₂-NPs spray (6.5) (Table 3). Plant shoot weight, especially dry weight, was more affected by salinity. Increased salinity levels resulted in decrease in fresh/dry weight of plant with the maximum and minimum weight ratios of 1.2 and 1.9, respectively. The highest shoot fresh/dry weight was observed in control, and it was not significantly different with S₀C₁ and S₀C₂ treatments. The lowest fresh/dry weight belonged to the S₅C₀ treatment (30 mM NaCl, without SiO₂-NPs). However, in S₅C₂ treatment (30 mM NaCl⁺ 25 mg · L⁻¹ SiO₂-NPs), fresh weight significantly increased in comparison to S₅C₀. Thus, it could be concluded that the modifying effects of SiO₂-NPs reduced the harmful effects of salinity. Results show that stem length was not affected by salinity levels up to 20 mM significantly. However, in 30 mM salinity level without the SiO₂-NPs application, the shortest stem length was measured (6.66 cm vs 19.00 cm in control). Flower diameter was less affected because no significant difference was observed between all treated plants just compared to controls. Also, salinity-treated plants sprayed with 25 mg · L⁻¹ of SiO₂-NP had the most flower diameter. Stem diameter was more affected by salinity as the control sprayed with 25 mg · L⁻¹ SiO₂-NP had the most amount of diameter (0.6 mm) compared with 30 mM of salinity (0.13 mm). Savvas et al. (2002) reported that adding Si to the nutrient solution of gerbera enhanced the stem diameter of the flowers but did not affect the stem length. Increase in salinity levels from 0 up to 30 mM caused a significant reduction in flower number of plant and fresh weight of the flowers approximately by 55 and 18%, respectively, regardless of SiO₂-NPs application. In controls, spraying gerbera plants with SiO₂-NPs (25 mg · L⁻¹) significantly cause to increase in leaf area (14%) compare to un-treated plants. These findings agree with the obtained results on Calendula (Bayat et al., 2013). According to the Munns (2002), inhibition of plant growth and development under salinity may either be because of reduction in water availability or sodium chloride toxicity. Leaf area index is one of the major factors in the growth of plants under salinity stress. As shown in Table 3, leaf areas decreased as the salinity level increased. Control plants sprayed with 25 mg · L⁻¹ SiO₂-NPs had the highest leaf area (260.8 cm²) compared to treated plants with 30 mM salinity (65.7 cm²). Hence, the positive role of SiO₂-NPs treatments in modification of the adverse effects of salinity is undeniable. In other words, silicon increases the stability of cell wall by forming a layer (Marschner, 2011). In addition, SiO₂-NPs particles can better affect xylem humidity and water translocation through their larger surface area through which they can improve the water uptake and cell division and elongation in flowers. Savvas et al. (2002) reported that adding Si to nutrient solution resulted in the most amount of class I flowers and ticker flower stems in gerbera. In addition, Hwang et al. (2005) demonstrated that using potassium silicate enhanced the growth and quality of cut miniature rose ‘Pinocchio’ in a rock wool culture system.

Values of electrolyte leakage and MSI are used indirectly for showing the damage to cell membrane in salinity conditions (Ali et al., 2008). Increasing in salinity level cause to decrease in cell membrane stability (30%) and subsequently increased in EL up to 83% as shown in Table 4 in controls and 30 mM salinity treated ones. However, a beneficial effect SiO₂-NPs in sustainability of cell walls is quite impressive especially at higher ranges of 10 mM salinity. EL is inversely correlated with membrane stability. Using of SiO₂-NPs at 50 mg · L⁻¹ only cause to 11.8 and 45.6% decrease and increase in MSI and EL at 30 mM salinity. The effect of salinity on MSI and electrolyte leakage could be related to damage of plasma membrane, which is caused by reactive oxygen species. Probably, the most suitable factor for monitoring plant status in water deficiency can be the measurement of leaf relative water content as a physiological parameter. Relative water content decreased with increasing in salinity level as difference between the lowest (67.60%; 30 mM) and the highest (90.16%; plants sprayed with 25 mg · L⁻¹ SiO₂-NPs). Presumably, the presence of silicon residues has been found in epidermal cell walls, which are related to water loss of cuticle and extreme transpiration (Mateos-Naranjo et al., 2013). The amount of leaf chlorophyll significantly decreased when gerbera plants were exposed to salt stress. Under several salinity conditions, the severe salinity (30 mM NaCl) cause a high reduction of 40% in the leaf chlorophyll of gerbera plants against controls (Table 4). Reduction in concentration of chlorophyll is likely because of the accumulation of different salt ions and prevention of chlorophyll biosynthesis or membrane deterioration (Ashraf and Bhatti, 2000). It also may be related to the activation of chlorophyllase enzyme and consequently degraded the chlorophyll (Santos, 2004).

The amount of proline in leaves gerbera plant under salinity increased by 32, 384, 1,046 and 1231% under 5, 10, 20 and 30 mM NaCl treatments, respectively; but incorporation of SiO₂-NPs sprays on plants limited the proline accumulation. Results were in agreement with Moussa (2006) and Lee et al. (2010) in maize and soybean, respectively. Sever salinity (30 mM) cause to decrease in protein by 54% compared to controls (Table 4). It has been demonstrated that proline is a possible source of carbon and nitrogen for rapid recovery of plant after exposure to salt stress. In addition, it is a membrane and some macromolecules stabiliser as well as scavenger for reactive oxygen species. Some articles have concluded that SiO₂-NPs have harmful effects, but it has also been concluded that the toxic effect of SiO₂-NP could be due of an alteration in the pH of the growing media after SiO₂-NP addition (Slomberg and Schoenfisch, 2012). In any case, the amount of proline in the nutrient solution increased from 2.74 to 32.41 mmol · g⁻¹ FW as the salinity level increased, but adding SiO₂-NPs to the nutrient solution prevented proline accumulation. Similar results were obtained in strawberry (Avestan et al., 2019). Since lipid peroxidation was significantly lower in gerbera plants treated with Si under salinity than in the same treated plants without Si application, SiO₂-NPs have beneficial effect in preventing lipid peroxidation induced by salinity. This effect of Si was more considerable at 20 and 30 mM NaCl. Salinity caused a 54% decrease in protein compared to controls (Table 4).

Antioxidant enzyme activities play an important role as reactive oxygen species scavengers, which can improve the ability of plant tolerance under stress conditions. Following the increase in salinity, the changes in activities of SOD, GPX, APX, H₂O₂ and MDA had similar tendency as their activities were simulated by salt stress (Figures 3 and 4). However, the increase was higher in controls than in plants treated with SiO₂-NPs. Under salt stress, the activity of APX and GPX was significantly increased after the application of SiO₂-NPs (Figure 3A and B) but not in SOD, although the difference between treated and untreated plants was not significant (Figure 3C). According to Figure 3C, spray of 50 mg · L⁻¹ SiO₂ nanoparticles could suppress the increase of SOD activity in plants under 10 mM salinity. This probably indicates that plants are not affected by these stress conditions. The increase in the activity of antioxidant enzymes by silicone spray under salinity is the protective way for inhibition of oxidative stress in plants which is the first defense mechanism of salinity reduction induced upon Silicone application (Soundararajan et al., 2014). Improving in growth characteristics and nutrition uptake by supplementation of SiO₂-NPs might be result of a reduction in oxidative stress as by activation of APX and GPX although the activity of SOD was unchanged. Results were in agreement with Abdul Qados (2015) in faba bean sprayed with nano silicon under salinity stress.

Figure 3

Figure 4

The decrease in the amount of malondialdehyde and electrolyte leakage followed by SiO₂-NPs application might be due to activation of antioxidant enzymes and consequently protect the plants from oxidative stress, increase in the stability of membrane and protect plant from harmful effects of reactive oxygen species (Rubinowska et al., 2014). In addition, it seems that increase in the Ca²⁺ uptake can cause to protect of plant from oxidative stress.

Gerbera plants treated with SiO₂-NPs had higher Ca²⁺ and K⁺ content in leaves especially at 25 mg · L⁻¹ level of SiO₂ nano particles, in comparison with other treatments, regardless of the salinity level (Table 5). Supplementation of SiO₂-NPs also led to the decrease in Na⁺ content as compared with controls and treatments with salinity lower than 30 mM both sprayed with 25 mg · L⁻¹ SiO₂-NP were in the range of 0.64 up to 2.97 g · g⁻¹ FW. The highest Na⁺ content (3.17 g · g⁻¹ FW) was related to 30 mM salinity, whereas the least was for control plants treated with 25 mg · L⁻¹ SiO₂-NPs (0.64 g · g⁻¹ FW). Leaf Na⁺ content increased from 0.89 to 3.17 g · g⁻¹ FW and in the opposite trend K⁺ content decreased from 1.77 to 0.85 g · g⁻¹ FW following the increase in salinity levels up to 30 mM. Salinity not only can disrupt K⁺ uptake but also might disrupt the cell membrane, thus affecting its power of ion selection (Perez-Alfocea et al., 1996). Niu et al. (2012) showed that zinnia was sensitive to salinity as plant height became shorter and more compact as well as increase in electrolyte conductivity of irrigation water. Also, dry weight of shoot in EC values of 4.2 dS · m⁻¹ reduced by 50% and Na⁺ and Cl⁻ accumulated excessively, whereas Ca²⁺, Mg²⁺ and K⁺ did not change substantially. One of the effects of salinity is the elimination of K⁺ by plant roots and consequently imbalance in plant physiology since K⁺ is necessary to the synthesis of protein. Losses of K⁺ cause to reduce of plant growth (Chen et al., 2007). As shown in Table 4, SiO₂-NPs can prevent protein degradation at high NaCl concentrations by up to 17 and 45% at 25 and 30 mM NaCl, respectively. The incorporation of SiO₂-NPs improved the absorption of K⁺ and likely prevent protein degradation. The application of SiO₂-NPs improved leaf potassium level under salt stress. It also significantly reduced the level of leaf Na⁺ and caused to improve in the K⁺/Na⁺ and Ca²⁺/Na⁺ ratios in leaves. These results are in agreement with Kafi and Rahimi (2011) on purslane and Xu and Liu (2015) an aloe. However, the highest Ca²⁺/Na⁺ and K⁺/Na⁺ ratios were related to the control plants sprayed with 25 and 50 mg · L⁻¹ SiO₂-NPs and the lower values are related to 20 and 30 mM level of salinity regardless of the SiO₂-NPs treatment (Table 5).

This finding means that the improving effects of SiO₂-NPs were so evident in 10 mM salinity. On the other hand, salinity caused to decrease in Ca²⁺ content by 61%. Therefore, under salinity stress, the calcium requirement of plant is higher than those in non-saline conditions. Also, salt stress effect on leaf Ca²⁺/K⁺ ratio negatively, as decreased by 21% under 30 mM salinity, whereas in the same situation application of 25 and 50 mg · L⁻¹ SiO₂-NPs, it was reduced only by 7 and 10%, respectively.

Increased resistance to salinity levels in gerbera plant under the application of SiO₂-NPs most likely was because of the reduction in Na⁺ uptake and detoxification of plant from Na⁺ by increasing in Na⁺ binding on cell wall (Kafi and Rahimi, 2011). Because of the same mechanisms of both Na⁺ and K⁺ uptake (Niu et al., 1995), SiO₂-NPs can increase K⁺ uptake by suppressing Na⁺ uptake. It seems that silicon acts as a competitive inhibitor for Na⁺ therefore, using 25 mg · L⁻¹ SiO₂-NPs led to the decrease in Na⁺ content by 28% (control), 2% (5 mM), 14% (10 mM), 15% (20 mM) and 6% (30 mM) of salinity levels. The auxiliary effect of 25 mg · L⁻¹ SiO₂-NPs in K⁺ uptake in control was 31%, whereas in 30 mM salinity it was 27%. This indicates the higher effect of SiO₂-NPs under stress conditions and ion homeostasis of gerbera plants was kept well. The improvement of salt stress by using SiO₂-NPs treatments was accompanied with improved membrane stability, enhancing the activity of enzymes and nutrition uptake. It has been known that Si can be beneficial for some crop species. Therefore, it has been used increasingly as a supplement in hydroponic nutrient solutions (Savvas et al., 2002). Under salinity, it has been demonstrated that the beneficial effects of silicone are because of the decreased level of Na⁺ (Matoh et al., 1986; Bradbury and Ahmad, 1990; Liang et al., 2003), increased level of K⁺ (Liang et al., 1996) and enhaced photosynthesis rate in some plants (Liang, 1998; Al-Aghabary et al., 2004).

Pearson correlation analysis showed that Na⁺ concentration was cor related with EL and MSI, positively. Similarly, a positive correlation was detected between Na⁺ and antioxidative enzyme activities (SOD, APX and GPX), oxidative markers (MDA and H₂O₂) and proline. In contrast, Na⁺ concentration displayed a negative correlation with morphological parameters (leaf and flower number, plant FW and DW, stem length and diameter, flower diameter and LA) (Figure 5).

Figure 5