Application of selenium and silicon to alleviate short-term drought stress in French marigold (Tagetes patula L.) as a model plant species

2.1 Plant material and growth conditions

Vegetation experiments were carried out between May and July in unheated greenhouse. Their purpose was to determine the effect of the application of silicon and selenium in relieving drought stress of marigold (T. patula L. ‘Pascal’) as a model plant. Experiment was established in a systematic design with eight replications (a replication was one single plant). The greenhouse was equipped with a modern climate control system. The following ambient conditions were maintained throughout the experiment: temperature of 20–24°C and RH of 65% to 75%.

The seeds were sown individually in rockwool pits made with fingers (17.05). The rockwool was soaked in a standard nutrient solution 48 h before experiments. After 2 weeks, plants were placed in rockwool blocks hydrated with the nutrient solution (Grodan, 100 × 100 × 65 mm), while after another 12 days, they were transferred to a special hydroponic system with recirculation of nutrient solution.

2.2 Plant nutrition

The nutrient solution for plant fertigation is composed of the following (mg dm⁻³): N-NH₄ < 10; N-NO₃, 150; P-PO₄, 50; K, 150; Ca, 150; Mg, 50; Fe, 3.00; Mn, 0.5; Zn, 0.44; Cu, 0.03; and B, 0.011. The pH was 5.50, and the EC was 1.8 mS cm⁻¹. The following fertilizers were used to prepare nutrient solutions: potassium nitrate (13% N-NO₃, 38.2% K), calcium nitrate (14.7% N-NO₃, 18.5% Ca), monopotassium phosphate (22.3% P, 28.2% K), potassium sulfate (44.8% K, 17% S), magnesium sulfate (9.9% Mg, 13% S), Librel FeDP7 (7% Fe), manganese sulfate (32.3% Mn), copper sulfate (25.6% Cu), borax (11.3% B), and sodium molybdate (39.6% Mo). Nitric acid (38%) was used to regulate the pH value. The sources of silicon were as follows: silica sol (200 g SiO₂ dm⁻³, Optysil, Intermag Olkusz) and choline-stabilized orthosilicic acid (ch-OSA; 0.6% Si; Actisil; Yara Poland). ch-OSA was obtained from Bio Minerals N.V., Destelbergen, Belgium. The source of selenium was sodium selenate purum p.a. (Na₂O₄Se₃; Sigma-Aldrich). They were tested at the following concentrations (mg dm⁻³ of NS): silica sol – level I (23.25), level II (31.0); ch-OSA – level I (0.21), level II (0.63); and Se – level I (0.4), level II (0.8). In all combinations, nutrient solution was dosed six times daily per 5 min in each watering cycle. Drought stress was induced in selected combinations for 55 h (from July 17 to July 19) until all the plants tested had wilting symptoms of aerial parts. Then, irrigation was re-enabled for 9 days.

2.3 Measurement of gas exchange parameters

Portable photosynthesis system Ci 340 aa (CID BIOSCIENCE Inc., Camas, USA) was applied to measure the following gas exchange parameters: net photosynthesis activity (P_N), stomatal conductance (g_s), transpiration rate (E), and intercellular CO₂ concentration (C_i). The constant conditions were maintained in the measuring chamber to obtain comparable results. So, the following parameters were sustained at the stable level: CO₂ inflow concentration (400 µmol (CO₂) mol⁻¹), photosynthetic photon flux density (PPFD) 1,000 µmol (photon) m⁻² s⁻¹, chamber temperature 24°C, and relative humidity 50 ± 3%. Investigations were conducted during midday light conditions between 10:00 and 14:00 h. Two gas exchange parameters investigations were performed before and after drought stress.

2.4 Biological measurements

The relative water content (%) [24] was determined for the samples collected before and after drought stress (July 2 and July 25, respectively). On the last day of the experiment (July 28), the weight of plants (individually shoots and inflorescences) was measured.

2.5 Chemical analysis

Chemical analyses were performed for the aboveground parts of plants. Samples (individually shoots and inflorescences) were dried for 48 h at 45–50°C to a stable mass and then ground. Before mineralization, the plant material was dried for 1 h at 105°C. To assay the total forms of N, P, K, Ca, Mg, and Na, the plant material (1 g) was digested in the concentrated (96%, analytically pure) sulfuric acid (20 cm³) with the addition of hydrogen peroxide (30%, analytically pure) [25]. For analyses of total Fe, Mn, Zn, and Cu, the plant material (2,5 g) was digested in a mixture of concentrated nitric (ultra-pure) and perchloric acids (analytically pure) at a 3:1 ratio (30 cm³). To assay the Si, the plant materials (0,5 g) were microwave digested in the mixture of HNO₃, HClO₄, and HF (10, 1, and 0.25 cm³, respectively). HF acid residue was neutralized by adding 3 cm³ 4% H₃BO₃. After mineralization, the following determinations were performed: N-total was determined using the distillation method according to Kjeldahl in a Parnas Wagner apparatus; P was colorimetrically determined with ammonia molybdate; K, Ca, Mg, Na, Fe, Mn, Zn, and Cu were determined using flame atomic absorption spectroscopy (FAAS, on a Carl Zeiss Jena apparatus 5); and Si was determined using the atomic emission spectroscopy method with microwave nitrogen plasma (on an Agilent MP-AES 6200 apparatus). Earlier in the laboratory, the accuracy of the used methods of chemical analyses and the precision of analytical measurements of heavy metals were tested by means of the analysis of the reference material of branched flour (Pseudevernia furfuracea), certified by the IRMM (institute for reference materials and measurements) in Belgium [26].

2.6 Statistical analysis

The data were analyzed using Statistica 13.0 (StatSoft Inc., Tulsa, OK, USA). The results of chemical analyses and plant yielding measurements were processed using ANOVA and the Duncan test (α = 0.05).

Ethical approval: The conducted research is not related to either human or animal use.

3.1 Yield, DM (Dry Matter), and RWC (Relative Water Content)

The experiment showed that the factors under analysis significantly influenced differences in the inflorescence mass (Table 1). When silicon was applied, the mass of inflorescences increased in both the plants grown under the optimal water regime and those exposed to drought stress. The mass also increased along with the intensity of selenium nutrition. There were significant differences in the mass of green parts – it was the lowest in the control plants (I), i.e., 78 g plant⁻¹, and significantly higher in the plants treated with silicon. Selenium mitigated the effects of drought stress, whereas silicon caused the mass of shoots to increase, but the difference was not statistically significant. The total biomass of the aerial parts was the smallest in the control sample with optimal irrigation, but it was significantly greater in other combinations, even those exposed to stress. The factors under analysis significantly differentiated the plants’ characteristics, both the height and width. The values of both parameters improved when silicon was applied to the plants grown under a standard water regime. The height of the plants exposed to the drought stress increased only when Se–I was used. There were similar relationships observed in the plant width. The number of inflorescences was the smallest in the control sample exposed to the drought stress. However, it did not differ significantly from the control sample grown under the standard water regime. Silicon significantly increased the number of inflorescences in the samples grown under the optimal water regime. The source of Si used for the treatment caused differences in the plants exposed to the drought stress.

Selenium also had a positive stimulating effect on the number of inflorescences. The diameter of the stems was the longest in the combinations with the largest mass of aerial parts. Silicon increased the diameter of the shoots. However, the changes were significant only for the plants grown under optimal irrigation. The largest diameter of the shoots was observed in the Se-II combination.

The trace elements used in the experiments caused an increase in the relative water content (RWC) in the leaves of the plants grown under optimal water conditions (Table 2). Both of the tested sources (except silica sol-II) used for the silicon treatment of the plants subjected to drought stress significantly increased the RWC, compared with the control sample. The selenium treatment had no positive effect. Before the drought stress was induced, the Se-II and silicon treatments (except ch-OSA II) had significantly increased the dry matter content. When the plants were exposed to the stress, only Se-I significantly modified this parameter in relation to the control sample.

So far studies have shown that silicon affects the water management of plants and improves the efficiency of water consumption. It stimulates enzymatic and nonenzymatic antioxidative defense systems [10]. It affects the production of antioxidants and thus reduces the amount of photo-oxidative damage. It also inhibits the generation of free radicals and helps to maintain the integrity of chloroplast membranes [27]. Silicon may be involved in metabolic or physiological activities of higher plants exposed to drought stress [28]. Silicon treatment decreased the content of thiobarbituric acid reactive substances (TBARS) and the activities of acid phospholipase (AP) and lipoxygenase (LOX) in plants exposed to drought stress [29]. Silicon affects the rate of transpiration by silicating the surface of leaves [30] and reducing the lumen of stomata [31].

It was observed that the silicon treatment of sorghum plants exposed to drought stress improved their cellular hydration [14]; meanwhile in studies conducted on rice (Oryza sativa L.) with varied levels of drought tolerance, it was found that under wet conditions, silicon had no effect on the growth and yielding of plants [4]. Our research showed the opposite tendency. The drought stress reduced the dry weight, root traits, and water potential. In this study, the opposite effect was observed for the DM and RWC. In the previous study, the highest relative water content (RWC) was found in the plants that received foliar silicon treatment [32]. In this study, during the growing season, the RWC also increased in the plants fed with silicon. This increase may have been caused by improved osmoregulation and reduced transpiration [33]. In the study that researched the effect of silicon on the growth and mineral nutrition of maize (Zea mays cv. DK 647 F₁) grown under varied water conditions, it was found that induced water stress reduced the total dry matter content (DM), chlorophyll content, and RWC, but it increased proline accumulation and electrolyte leakage in maize plants [34]. Our study revealed similar tendencies for the DM content at the second term, but the opposite tendencies for the RWC. Silicon treatments improved the physiological parameters, but their levels remained significantly lower than in the control sample, except for the electrolyte leakage and root–shoot ratios, which were higher [34]. In this study, there were similar tendencies observed for the RWC, but the opposite for the DM content. Water-deficient sorghum plants treated with silicon was characterized by the reduced dry matter content [14]. In consequence, the plants’ cellular hydration improved despite the stress. Silicon treatment and water deficit resulted in an increased silicon concentration in potato leaves [35]. The researchers observed higher proline concentrations under water stress and higher silicon availability in the soil, which indicates that silicon may influence osmotic adjustment.

Similar silicon, selenium exhibits antioxidative properties, and it can remove reactive oxygen species under environmental stress, especially water stress [36]. According to Emam et al. [8], plants treated with selenium produce more phenols and flavonoids. Glutathione peroxidase is an important enzyme in the plant’s system of defense from oxidants. Selenium is an essential element in the structure of this enzyme. Selenium may stimulate the plant’s antioxidative system and thus improve tolerance to oxidative stress caused by drought [7]. Selenium ions regulate and ensure the optimal water balance in cells. This is the main mechanism that is considered to provide protection during drought [37]. Earlier research on selenium-fed plants exposed to drought stress showed greater activity of antioxidative enzymes, i.e., catalase (CAT) and oxidoreductase (POX) [7] and an increase in the total carbohydrate content [8]. Our research showed that selenium alleviated short-term drought stress. Selenium supplementation may increase the fresh weight and the water content in broad bean cells [38]. The element regulated the plants’ water management by increasing the efficiency of the uptake of water by the roots and limiting transpiration. In this study, the transpiration level in the selenium-treated plants was higher than in the control combination. Selenium applied to rapeseed leaves could improve the yield quality and the quantity in arid and semi-arid regions [39]. The selenium treatment method significantly affects the effectiveness of nutrition [40]. The authors found that spraying plants with selenium increased their tolerance to drought stress. Our experiment showed that when selenium was applied to the plants’ root zone, it effectively alleviated short-term drought stress. Selenium supply favored the accumulation of biomass of wheat seedlings when the plants were well watered, but it did not significantly affect the biomass accumulation under drought stress although it increased the root activity and some antioxidative indicators [41]. Selenium treatment significantly increased the size of water-deficient wheat plants [42] and increase the yield of plants [43]. As this element is responsible for the activation of the antioxidative system in plant cells, its use has been extended for other stress such as drought, UV radiation, and low and high temperature. Plants treated with selenium exhibit higher tolerance to stress factors, e.g. induced by heavy metals. The use of selenium for nutritional purposes reduces oxidative damage caused by the production of compatible substances. Their compensation prevents cell dehydration. Our study showed that silicon tended to modify changes in the plants’ habit, especially in those that were not exposed to stress. This effect was also observed earlier [44] on pansy plants sprayed with a silicon solution. Silicon nutrition increased the number of lateral shoots in French marigolds [45]. A similar effect was observed in our experiment. Similar effect was found earlier [46] in plants sprayed with a preparation containing silicon produced more buds and flowers or inflorescences with greater diameters than the control plants. Our study showed that selenium nutrition stimulated the plants’ habit. By contrast [6], it was observed that the foliar application of selenium had no effect on the growth of wheat exposed to drought.

3.2 Gas exchange parameters

The intercellular CO₂ concentration increased in the plants supplemented with silicon and selenium (Table 3). The values of all the parameters under analysis decreased in all the combinations, which might indicate a natural physiological process in plants. However, there were differences between the combinations. The P_N of the control plants exposed to the drought stress was lower than in the control plants that were not exposed to the stress. It is particularly important that the P_N level in all the plants treated with silicon or selenium in any combination (except for the plants treated with ch-OSA, which were not exposed to the water stress) was higher than in the control plants, with or without water deficit. The P_N level in the water-deficient plants supplemented with ch-OSA II was significantly higher than in the plants treated with the same supplement but not exposed to the water stress. Measurements of the second gas exchange parameters revealed the highest level in the plants irrigated with silica sol II. However, an elevated level was also noted in the plants with the water deficit. Moreover, the P_N level also increased in the plants supplemented with selenium. Higher P_N levels usually coincided with higher g_s and E levels. However, the E level in the plants treated with silicon (ch-OSA I and silica sol II) under the drought stress was comparable to the E level of the plants with the highest P_N. The C_i level was the highest in the water-deficient plants supplemented with ch-OSA I. The C_i level was also elevated in the well-irrigated plants supplemented with silicon (silica sol II) (Table 3).

The silicon and selenium treatments had positive effect. This particularly applied to an elevated silicon level, which is in agreement with the findings of our previous study [12]. The treatment with selenium at both concentrations positively influenced the photosynthetic process. Our findings are in line with the results of research conducted on other plant species [17]. Proline is an amino acid synthesized by plants as a result of stress. It protects cellular structures (proteins and cytoplasmic membranes) from damage. Silicon has been found to reduce proline levels in plant cells, thus alleviating environmental stress and inhibiting the degradation of the cytoplasmic membrane [32,34]. Our study revealed the protective effect of silicon during drought stress. It was particularly noticeable when higher amounts of silicon were applied. The plants also exhibited the highest transpiration rate and stomatal conductance. Silicon improved the water uptake [13]. However, it increased the hydraulic properties of the roots rather than reducing the water loss. Silicon may enhance water capacity and uptake by the roots [17]. A higher water loss through transpiration was caused by an increase in the relative water content and indicated an enhanced water uptake by the roots. The researchers also noted the elevated stomatal conductance due to improved CO₂ fixation capacity during water stress. This also ensured an optimum CO₂ concentration for carbon reactions and prevented photoinhibition; the transcriptional rate of some photosynthesis-related genes in plants increased and regulated the photochemical process and thus promoted photosynthesis [11]. During drought stress, the intercellular CO₂ concentration (C_i) tended to increase, but silicon reduced it [15]. In this study, this parameter also decreased in all water-stressed plants supplemented with silicon, except ch-OSA I. Higher stomatal conductance was observed in sorghum treated with silicon [14]. In this study, there was a similar effect observed in the plants grown under standard conditions. During the drought stress, stomatal conductance depended on the source and the concentration of silicon. Our investigations confirmed the protective role of selenium during the drought stress. Treatment with higher amounts of selenium caused higher P_N, g_s, and E levels. Similar effects of selenium and silicon on the uptake of water by roots during drought stress were also observed [47]. Selenium prevented drought stress by improving cellular enzymatic and nonenzymatic antioxidative mechanisms [7]. Moreover, selenium improves the cellular water balance [37], which is crucial for the photosynthetic process.

3.3 Chemical composition of plants

When silicon was applied under the standard water regime, it significantly reduced the nitrogen content in the shoots. However, the opposite trend could be observed under the water stress (Table 4). The nitrogen and phosphorus content in the plants treated with selenium was lower than that in the control samples. The phosphorus and calcium content in the plants grown under optimal conditions depended on the carrier. The silicon treatment had positive effect on the phosphorus content in the plants exposed to the water stress. The application of silicon to the plants grown under the optimal water regime significantly reduced the potassium content. In general, there were similar trends observed in the plants exposed to the stress. As the increasing amounts of silicon were applied through both sources to the plants with the water deficit, their calcium content increased significantly compared with the control plants. The silicon treatment of the plants grown without the water deficit had a diversified, source-dependent effect on the calcium content. The sodium content in the shoots of the plants exposed to the drought stress was generally similar to the sodium content in the optimally irrigated control plants. In contrast to the selenium treatment, the silicon treatment had generally positive effect on the sodium content, but it did not affect the magnesium content in the optimally irrigated control plants.

However, the magnesium content tended to increase in the plants exposed to the stress when they were treated with selenium. The treatments also significantly differentiated the content of chemical components in inflorescences. For example, the silicate sol increased the nitrogen content in the plants exposed to the stress, whereas the selenium treatment significantly reduced the phosphorus content. On average, the nitrogen and potassium content in green parts of the plants was about 35–40% greater than that in the inflorescences. The calcium content was as much as about 80% greater. The phosphorus content was similar. Interestingly, the average sodium content in green parts of the plants was about 15% lower, whereas the magnesium content was almost 60% higher than in the inflorescences.

The quantitative relations between the macronutrients and the sodium in the shoots also changed. For example, the N:P ratio in the selenium-treated plants exposed to the stress was higher than that in the plants treated with silicon (Table 5). The N:K ratio in the silicon-treated plants exposed to the stress was noticeably higher than that in the plants treated with selenium. Changes in the quantitative relations between the components were also observed in the inflorescences.

In the silicon-treated plants grown under the optimal water regime and the silicon-treated (except ch-OSA II) and selenium-treated plants exposed to the drought stress, there was a relative increase in the nitrogen content in the inflorescences compared with its content in the shoots (Table 6). There were similar relations concerning the phosphorus content in the plants grown under the optimal water regime and the selenium-treated plants exposed to the stress. In general, the drought stress did not cause changes in the flower-to-shoot potassium content ratio. The silicon-treated plants grown under the optimal water regime were characterized by a smaller flower-to-shoot calcium content ratio (0.15 each) than the other combinations. This trend was not so noticeable in the plants exposed to the stress. In all the combinations exposed to the drought stress, the flower-to-shoot magnesium content ratio was smaller than in the control plants grown under the optimal water regime. In comparison with the control plants, both the selenium and silicon treatment of the plants exposed to the water stress caused noticeable changes in the sodium ratio.

The research showed that in general, the factors tested in the experiment had significant influence on the content of micronutrients and silicon in the shoots. For example, the iron content in the control plants exposed to the drought stress was significantly lower than in the plants treated with silicon, regardless of the carrier and the concentration (Table 7). In general, there were inverse relations concerning the manganese content. The silicon treatment of the plants grown under the optimal water regime significantly increased the manganese content in their shoots. There was a similar upward trend observed in the plants exposed to the drought stress compared with the optimally irrigated control plants.

The iron and zinc content in the inflorescences was more stable than in the shoots. In general, there were no differences in the content of these nutrients between the combinations. The silicon treatment of the plants exposed to the drought stress reduced the copper content in their inflorescences. There was a similar tendency concerning the manganese content. Both silicon carriers caused generally significant differences. In all the combinations, the silicon content in the plants exposed to the drought stress was significantly higher than in the optimally irrigated control plants.

There were noticeable differences in the content of microelements and silicon in the inflorescences in relation to the shoots (Table 8). For example, when silicon was applied to the plants grown under the optimal water regime as well as those exposed to the water stress (except ch-OSA I), the relative iron content increased. Both silicon carriers modified the inflorescence-to-shoot iron content ratio.

The factors analyzed in this study caused changes in the quantitative relations between micronutrients in both the shoots and inflorescences (Table 9). For example, the Fe:Zn ratio in the silicon-treated plants exposed to the drought stress was noticeably higher than in the control plants, regardless of the sources. There was a similar relation observed for the copper and manganese content. The silicon treatment narrowed the Fe:Zn ratio in both the plants grown under the optimal water regime and those exposed to the short-term drought stress. However, the selenium treatment resulted in different Fe:Zn and Fe:Cu ratios.

The average iron content in the shoots was 8% higher than in the inflorescences. Most of the combinations also had higher zinc and copper content in the green parts than in the inflorescences. There was a similar relation observed for the manganese content – in all the combinations, it was higher in the green parts than in the inflorescences.

Changes in the uptake of components and consequently changes in the chemical composition of plants should be treated as their adaptation to specific environmental factors. For example, the uptake and the translocation of nutrients in plants can be modified by silicon [48]. Also drought stress significantly increased the content of K, Na, Ca, Mg, and Fe in plants, whereas silicon treatment reduced the content of these elements [4]. In this study, the opposite tendency was observed for the N, P, K, and Ca content in the shoots, but a similar tendency for the Na content. Similar to this study, it was observed that silicon affected the chemical composition of water-stressed plants [34]. The silicon concentration in the plants was correlated with its content in the nutrient solution. In this study, the silicon content in the shoots of the plants grown under the optimal watering regime was significantly higher than in the control plants. There were increasing but insignificant tendencies observed in the plants exposed to the water stress. Water stress decreased the calcium and potassium concentrations in plants, but silicon treatment increased their levels [34]. In this study, a similar effect was observed only for the calcium content (at higher silicon concentrations in the nutrient solution). It was found that the silicon treatment may improve the growth of plants and increase its yield during drought, but it could not be a full substitute for an adequate water supply. The silicon treatment caused an upward trend (without significant differences) in the yield of plants exposed to the short-term drought stress. However, the selenium treatment caused significant differences. Silicon treatment reduced the sodium content in rice plants exposed to salinity stress [10]. In contrast to the findings of the other scientist [49], in this study, the simultaneous silicon treatment of the plants exposed to the drought stress caused an upward trend in the sodium content (without statistically significant differences). Selenium supplementation disorders the metabolism of amino acids and thus increases the content of soluble proteins and the nitrate reductase activity in plants exposed to drought stress [50]. When wheat plants were supplemented with selenium in an exogenous form, their uptake of potassium, iron, and zinc improved [18]. Selenium supplementation also improves turgor and regulates transpiration and the accumulation of soluble sugars and free amino acids. Selenium supplementation also improved the wheat grain yield by 24% and simultaneously increased the straw calcium content [18]. In this study, the selenium treatment had positive effect on the calcium content in the shoots. The effect of selenium on the accumulation of potassium and phosphorus was found in the studies on rapeseed [39] and rice [8]. The studies conducted on spinach showed that selenium significantly affected the calcium content [19]. In this study, the nitrogen and phosphorus content in the shoots of the plants exposed to the short-term drought stress exhibited a downward trend, whereas the calcium and magnesium content exhibited an upward trend. Selenium may limit the uptake of metals such as Mn, Zn, Cu, Cd, and Pb [20,21,51]. In this study, when the plants exposed to the drought stress were treated with selenium, the manganese content tended to decrease (insignificant differences), whereas the zinc content tended to increase.