Mechanism of Reduction of Drought-Induced Oxidative Stress in Maize Plants by Fertilizer Seed Coating

Abstract

In this study, we investigated the effects of the utilization of seed coating by dedicated fertilizer on the mechanism of oxidative stress reduction in maize growing in simulated drought conditions. A strict pot experiment was conducted for this purpose in a climatic chamber with a phytotron system and controlled temperature and air humidity. Maize seeds were planted and grown in soil with 20% (extreme drought), 40%, and 60% water holding capacity (WHC). The seeds were enhanced using proposed fertilizer and applied at a rate of 2 kg t⁻¹ seeds. The levels of ROS, as well as antioxidant enzymes SOD, CAT, and GPOX, generated by plants enhanced using the seed coating by fertilizer and by control specimens (non-enhanced), were measured 21 days after the seeds were planted. Antioxidant potential and total polyphenol contents in the plants were also determined. The findings show that under drought stress, plants produce high levels of ROS, which is responsible for oxidative stress. However, the latter phenomenon may be reduced using seed coating. Application of seed coating by fertilizer contributed to a 32.7% decrease in ROS in the case of extreme drought (soil with 20% WHC). The treatment also led to increased activity of SOD (61.2%), CAT (45.7%), and GPOX (35.8%), which shows its positive effects on activation of the enzymatic antioxidant system responsible for neutralization of ROS and for reducing the negative effects of drought.

1. Introduction

Maize (Zea mays L.) is one of the most common and the most productive plants grown worldwide [1,2]. Attractiveness of this species for agriculture is linked with the fact that maize can be used in production of food and fodder and can be utilized in industry and energy production [3]. Maize is a high-yield crop, and its production is linked with high water requirements during the growing season. Furthermore, to achieve proper growth, development, and yield, maize plants require optimum amounts of nutrients, including macro- and micro-elements, to be provided with fertilizers [4].

Coating seeds with fertilizers can be an effective way to promote their growth. The use of liquid fertilizer for Stylosanthes seeds coating was investigated and the impact of this technology on seed quality was assessed. Mastermins^® fertilizer was applied to the last layer of the binder material coating on the seeds, and then a Polyseed 76F polymer adhesive was applied onto the fertilizer. This method ensured a significant increase in the concentration of minerals and nutrients supplemented to the seeds. The applied liquid fertilizer had no effect on seed emergence and emergence speed, but influenced germination and germination speed [5]. Coating seeds with fertilizer can also be used in the case of phosphorus deficiency in the early stages of rice seed development. It was concluded that using various phosphorus compounds, it is possible to influence the development of plants in various ways by activating signaling mechanisms. The most promising compound seems to be the direct use of rock phosphate (RP) to coat seeds sown in low-phosphorus soils [6]. The influence of seed coating with micronutrient fertilizer on growth and yield of winter wheat was also tested. The fertilizer was introduced in a polymer coat and contained a mixture of Copper (Cu), Manganese (Mn), and Zinc (Zn) micronutrients. It has been shown that using the proposed fertilizer, plant dry matter yield has increased by up to 20%. The uptake of nitrogen and phosphorus was also improved by growing plant biomass, which significantly influenced the yield [7]. A nutrient seed coating on the emergence of wheat and oats was also tested. It was concluded that applying nutrients directly to seeds can significantly reduce their growth. Further work is needed to examine the mechanisms of injury due to nutrient seed coatings so that safe and effective formulations can be developed [8].

Soil moisture is an abiotic factor significantly affecting maize yields, both at the stage of plant emergence and throughout the entire growth period. Total precipitation and rainfall distribution in the specific months of maize growth affect the size and quality of grain yield. Drought stress, even short-term, is one of the critical environmental stressors adversely impacting growth, development, and yield of plants [9].

Water deficiency during plant growth leads to oxidative stress in plants, which is determined by the production of reactive oxygen species (ROS) in cells [10,11]. ROS are various chemicals containing active oxygen. These include superoxide anion radical (O₂^.−), hydrogen peroxide (H₂O₂), hydroxyl radical (OH·), singlet oxygen (¹O₂), and nitric oxide (NO) [12]. ROS generated in plant cells as a result of drought stress are removed owing to a natural antioxidant mechanism, which involves enzymatic and non-enzymatic antioxidants. The enzymes responsible for decomposition of ROS include, e.g., superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPX) [11,13]. The non-enzymatic antioxidants involved in the decomposition of ROS include low molecular weight compounds easily reacting with free radicals [14].

The level of ROS generated in plant cells due to drought stress may be reduced by applying appropriate fertilizers. Such treatment, mainly involving application of micronutrients, activates a defense mechanism which protects plants against oxidative stress by increasing the activity of SOD, CAT, and GPX enzymes; as a result, plants are more tolerant to water deficit [15]. Increased resistance of plants to drought, even at the stage of plant emergence, can be achieved by applying seed enhancements. Seed coating by fertilizers are applied to the planting material, usually together with antifungal agents, directly before sowing [16,17].

This study presents the effects of seed coating by complex fertilizer on cells oxidative stress in maize plants growing during simulated drought.

2. Materials and Methods

2.1. Plant Materials and Pots Experiment

A strict pot experiment was performed to assess the effects of seed enhancement on the response of maize seedlings of DKC3787 (FAO: 270) variety to drought-related oxidative stress; the experiment was carried out in a climatic chamber with a phytotron system and controlled temperature and air humidity (MLR-351 H, Sanyo, Japan, Poland). The climate conditions were as a follows: the average day/night temperature was 26/18 °C, day length was 16 h, and relative humidity was around 50%–60%. Maize seeds were planted in soil with pH of 6.0 in H₂O, and with contents of bioavailable forms of P and K amounting to 9.39 mg P₂O₅ 100 g soil⁻¹ and 20.8 mg K₂O 100 g soil⁻¹, respectively. Three levels of soil moisture were applied, i.e., 20, 40 and 60% water holding capacity (WHC), and these conditions were maintained during maize plant germination and growth. Maize seed enhancement was performed by applying Dr Green PRIME fertilizer (Dr Green Sp, z o.o., Chrzanów, Poland) at a rate of 2 kg t seeds⁻¹. The enhancer contains (in 1 kg fertilizer): 250 g P₂O₅, 170 K₂O, 2.5 g B kg, 1.75 g Cu, 35 g Fe, 30 g Mn, 0.25 g Mo, and 32.5 g Zn and a producer special formula with a mixture of amino acids and vitamins. The experiment was performed in 10 replications. One replicate was a pot in which 50 maize seeds were sown. On the 21st day after emergence, successive maize plants from individual pots were separated into the underground plant parts (roots) and above-ground plant parts. After that, all plant parts were weighed and prepared for analysis in order to measure the selected markers of oxidative stress in the above-ground plant parts.

2.2. Reactive Oxygen Species Accumulation

In order to measure the level of reactive oxygen species, 1 g of frozen tissue was homogenized with 4 mL 50 mM of chilled phosphate buffer (pH 7.4). The homogenate was then subjected to centrifugation at 10,000× g for 30 min (4 °C) and the obtained supernatant was designated for analyses. ROS levels in the extracts were determined following a fluorimetric method, using 2′,7′-Dichlorodihydrofluorescein diacetate, in line with the protocol described by Piechowiak and Balawejder [18]. The results were expressed as an increase in fluorescence during 1 min per 1 g of tissue.

2.3. SOD, CAT, and GPOX Activity

In order to measure the activity of superoxide dismutase, catalase, and guaiacol peroxidase, 1g of frozen tissue was homogenized with 4 mL 0.9 NaCl solution containing 2% PVP, 0.05% Triton X-100, and a mixture of protease inhibitors. The homogenates were then subjected to centrifugation at 10,000× g for 30 min (4 °C) and the obtained supernatant was designated for analyses. Activity of superoxide dismutase was assessed using a calorimetric method based on determining the degree of inhibition of adrenalin auto-oxidation by SOD present in the plant extract. A unit of SOD activity was defined as the amount of enzyme which inhibits adrenaline oxidation by 50%. Activity of catalase was assessed in accordance with the methodology presented by Piechowiak et al. [13], which involved colorimetric estimation of H₂O₂ residue in the enzyme mixture comprising catalase from the plant extract. A unit of CAT activity was defined as the amount of the enzyme which results in neutralization of 1 mmol H₂O₂ during 1 min. Activity of guaiacol peroxidase was measured using a colorimetric method in accordance with the protocol presented by Uarrota et al. [19].

2.4. Total Phenolic Content Assay

In order to measure the (total) level of polyphenolic compounds and antioxidant activity using DPPH and CUPRAC assays, 1 g of tissue was homogenized using 75% methanol. The homogenate was then subjected to centrifugation at 7500× g for 30 min, and the obtained supernatant was designated for analyses.

The level of total phenolic content was analyzed in accordance with the method presented by Matłok et al. [20]. The results were expressed as gallic acid equivalent [mg] per 1 g of dry mass leaves.

2.5. Determination of Antioxidant Activity

The antioxidant activity of maize leaves was assayed with ABTS (2.2′-azynobis-(3-etylobenzotiazolino-6-sulfonian) and CUPRAC (cupric reducing antioxidant capacity) methods presented by Matłok et al. [21].

2.6. Statistical Analysis

The significance of the effect of water holding capacity on the markers of oxidative stress in both fertilizer-coated seed and control plants was assessed using the one-way ANOVA and Tukey’s test at α = 0.05. The significance of differences between the analyzed parameters for fertilized and control plants was determined using the T test for each hydration level separately at α = 0.05. All statistical analyses were performed in Statistica 13.0 software.

3. Results and Discussion

3.1. Maize Plant Mass

The growth and development of young maize plants immediately after their emergence is closely correlated with the water availability immediately after sowing the seeds. Another factor formatting the intensity of plant growth and development is the utilization of seed coating with fertilizer before sowing [17].

The use of the tested fertilizer for maize seed coating influenced the growth plants in the initial stage of development (21 days after sowing) regardless of soil moisture (Figure 1). The application of 2 kg of fertilizer per 1 ton of maize seeds resulted in a significant increase in the weight of the above-ground part of the plants compared to the weight of the above-ground part of the plants without the use of these fertilizer. The highest increase in the weight (50% compared to the control) of above-ground parts of plants as a result of the application of fertilizer was recognized in the case of drought (WHC 20%). Similar results were observed for WHC of 40 and 60% (Figure 1A). The use of dedicated fertilizer also contributed to the development of the plant root system. More intensive growth of maize plants immediately after emergence as a result of the application of fertilizer, especially in drought conditions, resulted in reduction of oxidative stress caused by water deficit. Similarly, phenomena in the case of different plant seeds coated with fertilizer were also confirmed by other researchers. Wiatrak [7] proved that the application of these type of fertilizer on winter wheat seeds resulted in an increase in their biomass yield by 20%.

3.2. Reactive Oxygen Species Accumulation

Germination of maize seeds and plant emergence are significantly impacted by soil moisture. Adverse conditions affecting soil moisture, most importantly droughts, are recognized among abiotic factors which may have a destructive effect on young plants, leading to overproduction of reactive oxygen species (ROS) in their cells and, consequently, to oxidative stress [10].

The current findings show that the ability of young maize plants to produce ROS changed relative to soil water-holding capacity (WHC) and depended on whether or not the fertilizer was applied (Figure 2). It was found that application of fertilizer led to reduced production of ROS in maize seedlings, irrespective of soil WHC. Despite that, the best effects of this fertilizer were observed in extremely dry conditions (20% WHC); applied in this case, the fertilizer contributed to a 32.7% decrease (Figure 1) in the amount of ROS generated by the plants compared to production of ROS in control plants (no fertilizer applied). In the latter case, maize plants were found to produce high levels of ROS, which possibly was an effect of photorespiration linked to drought stress (20% WHC). Photorespiration under drought conditions is responsible for 70% of the total H₂O₂ generated [22]. Consequently, the dry conditions persisting during plant growth contributed to oxidative stress caused by the imbalance between the increased production of and the ability to rapidly detoxify ROS in their cells [23]. As a result, drought stress led to premature ageing and deep degradation of tissues, reflected by visible necroses and growth inhibition in non-enhanced maize plants. Destructive effects of long-term water deficit were also found in plants by other researchers [24,25] who reported that persistent drought stress during soybean plant growth results in oxidative stress, which leads to cell death and a decrease in biomass.

However, higher soil moisture corresponded to lower ROS production in the cells of maize plants (Figure 2). However, at both 40 and 60% soil WHC, cells of plants enhanced with applied fertilizer produced lower amounts of ROS. It seems that coating seeds with fertilizers should be applied only in the case of WHC lower than 60%, but during the emergence period it cannot be ruled out that such conditions will not occur. Reduced production of ROS in cells of maize plants enhanced by fertilizer most likely can be linked to the composition of the fertilizer. It contains many valuable micronutrients and amino acids that can bring down drought-related oxidative stress by reducing production of ROS in cells. Various microelements, such as B, Cu, Fe, Mn, Mo, and Zn, which are contained in Dr Green PRIME fertilizer, have been shown by other researchers to reduce adverse effects produced by stressors and to prevent negative effects of oxidative stress. Nxele et al. [16] demonstrated that treatment of sorghum plants with microelements at low concentrations leads to activation of defense mechanisms against oxidative stress, mainly antioxidant enzymes. As a result, plants are more tolerant to abiotic stresses, including drought. It is likely that amino acids contained in fertilizer contributed to the reduced production of ROS in cells of the enhanced maize plants. Research has shown that by applying amino acids, it is possible to significantly reduce the amounts of ROS produced by plant cells [26]. It has been established that activity of amino acids, such as glutamate, cysteine, phenylalanine, and glycine, may directly or indirectly reduce oxidative stress in plants; therefore, treatments with fertilizers which contain these compounds may mitigate effects induced by oxidative stress [27,28,29].

3.3. Antioxidant Enzymes Activity

Simultaneously with production of ROS, the antioxidant system in plant cells is activated. However, the balance between production and accumulation of ROS molecules in the cell is disturbed by various biological and non-biological stressors, possibly causing irreversible damage to the cells [30,31]. The cellular antioxidant system consists of a number of enzymes responsible for decomposition of ROS as well as low molecular weight compounds easily reacting with free radicals [14]. The enzymatic antioxidant system includes enzymes such as superoxide dismutase (SOD) and catalase (CAT). Another enzyme involved in ROS metabolism is guaiacol peroxidase (GPOX) [13].

The ability of young maize plants to activate SOD, CAT, and GPOX enzymes in relation to soil WHC and depending on whether fertilizer was applied is shown in Figure 3. Application of fertilizer, at a rate of 2 kg t⁻¹ seeds, contributed to a significant increase in the activation of enzymes responsible for decomposition of ROS, which are produced in large quantities in drought conditions (20% soil WHC). Application of the fertilizer (at 20% soil WHC) led to increased activity of SOD by 61.2%, CAT by 45.7%, and GPOX by 35.8%, relative to activity of the same enzymes in the control maize plants (no fertilizer applied). High levels of antioxidant enzyme (CAT, SOD, GPOX) activation in maize plants enhanced with fertilizer was reflected by their greater tolerance to drought stress, which was confirmed during observations. The findings showed fewer necroses and less-pronounced growth inhibition in plants enhanced by the application of fertilizer compared to non-enhanced maize plants. Activation of the antioxidant mechanism in cells of maize plants, resulting from the use of the fertilizer, may be linked to its composition, mainly the contents of micronutrients. Effects of micro- or macro-elements on the level of CAT, SOD, and GPOX activation were also reported by other researchers. Rahimizadeh et al. [32] found that application of micronutrients in conditions of severe drought stress increases activity of SOD, GPOX, and CAT by 22%, 79% and 58%, respectively. Similar conclusions were presented by Camak [33], who found that the application of micro-elements (Fe, Zn, Cu, Mn, B, and Mo) results in increased activity of antioxidant enzymes, leading to improved resistance of plants to environmental stressors.

In the case of 40 and 60% soil WHC, the findings show similar activation of antioxidant enzymes, which suggests that, whether or not fertilizer was applied, there was an equilibrium between the amount of ROS generated in maize plant cells and the ability of SOD, CAT, and GPOX to quickly detoxify these. When analyzing changes in GPOX activity, the lowest activity is observed at WHC 40%. This is an interesting observation, indicating that the activity of this enzyme is strongly dependent on WHC.

The fertilization efficiency was most visible in the case of a deep-water deficit, which cannot be ruled out in agricultural production. The fact that no adverse effects of oxidative stress occurred in maize plants growing in soils with 40% and 60% WHC was reflected by the lack of necroses or other damage. Regulation of ROS levels as a result of antioxidant enzyme activation under oxidative stress was also reported by Jubany-Marí et al. [34].

3.4. Antioxidant Activity and Total Phenolic Content

3.4.1. Antioxidant Activity

The presence of oxidative stress, induced by a number of stressors, including drought, results in elevated production of secondary metabolites, mainly low molecular weight antioxidants by plant cells.

Figure 4 presents anti-radical activity of maize plants identified using CUPRAC (cupric reducing antioxidant capacity) and DPPH (2,2-difenylo-1-pikrylohydrazyl) assays, relative to soil WHC and depending on whether fertilizer was applied. The findings show the highest antioxidant potential in maize plants growing under drought stress (20% WHC). However, in the non-enhanced plants, antioxidant potential was 11.0% (CUPRAC) and 41.3% (DPPH) higher compared to the respective values observed in plants enhanced with fertilizer. The significantly higher potential in control plants most possibly was an effect of oxidative burst in the apoplastic space of cells, which led to increased activation of the mechanisms involved in biosynthesis of low molecular weight antioxidants, easily reacting with free radicals [35]. In the case of plants enhanced by the application of Dr Green PRIME fertilizer, improving tolerance of plants to drought oxidative stress depends on WHC level by activation SOD, CAT, and GXOP, which leads to reduction of ROS generation in most cases. Similar conclusions were presented by Zarabi et al. [36], who found that negative effects of water stress may be reduced by adequate nutrient management. On the other hand, an increase in antiradical activity observed in plants growing under drought stress was also reported by other researchers. Adebayo and Menkir [37] demonstrated that resistance and yield of maize plants growing in dry soil correlated with better antioxidant properties of the plants. Likewise, Farooq et al. [38] found significantly higher antiradical activity in plants growing in dry conditions. Furthermore, the researchers established that higher level of enzymatic and non-enzymatic antioxidants may also improve tolerance of plants to drought by scavenging ROS.

Antioxidant potential (CUPRAC and DPPH) of maize plants growing in soil with 40% WHC was significantly lower than antioxidant potential measured in plants growing in extremely dry soil (20% WHC), which reflects lower oxidative stress. Antioxidant potential identified in plants growing in soil with 40% WHC using a PPDH assay was in the range from 106.6 mg of trolox 100 g⁻¹ (plants enhanced with fertilizer) to 128.4 mg of trolox 100 g⁻¹ (control plants). Antiradical activity of maize plants measured using a CUPRAC test was between 663.2 mg of trolox 100 g⁻¹ (enhanced plants) and 736.3 mg of trolox 100 g⁻¹ (non-enhanced plants). The significantly lower antioxidant potential (by 20% in DPPH and 9.9% in CUPRAC assay) identified in maize plants enhanced with fertilizer reflects the positive effects of nutrients contained in the fertilizer on the level of oxidative stress. Other researchers also observed decreases in oxidative stress induced by specific stressors when plants were supplied with certain nutrients, mainly potassium, phosphorus, and microelements, which are also contained in fertilizer. Dehnavi et al. [39] demonstrated that by applying micronutrients, it is possible to improve resistance of plants to environmental stressors, such as drought. Tittal et al. [40] showed that activity of antioxidant enzymes (SOD, CAT, and APX) responsible for neutralization of ROS increased after potassium fertilizer was applied to sorghum seedlings.

Enhanced and non-enhanced maize plants growing in soil with 60% WHC were found with similar antioxidant potential, which reflects the fact that there was no water-related stress inducing oxidative stress.

3.4.2. Total Phenolic Content

Soil moisture at the time of plant emergence and during the initial growth stages, as well as application of fertilizer, affected the total contents of polyphenols in the leaves (Figure 5).

As in the case of antioxidant potential, the highest contents of polyphenols were found in plants growing in extremely dry conditions (20% soil WHC). Polyphenolic contents were closely related to the applied fertilizer, which contributed to a reduction in oxidative stress and consequently to 15.9% lower contents of polyphenols, compared to control plants (no fertilizer applied), where the highest production of ROS was observed. Increased levels of polyphenols due to abiotic stressors were shown by other researchers in several crops. Gharibi et al. [41] demonstrated that drought stress leads to higher contents of polyphenols in Achillea pachycephala.

Polyphenolic content in leaves of maize plants growing in soils with 40 and 60% WHC and enhanced with fertilizer were significantly lower than in plants growing in soil with 20% WHC, as in the case of antioxidant potential.

4. Conclusions

Drought occurring at the time of maize plant emergence and during initial growth stages is a significant abiotic factor inducing oxidative stress, which leads to growth inhibition, necroses, and eventually death of the plants. Negative effects of drought stress may be reduced by supplementation of required macro- and micronutrients applied at the stage of planting in the form of fertilizer. It was found that application of Dr Green PRIME fertilizer at a rate of 2 kg t⁻¹ maize seeds results in a significant decrease in production of ROS (32.7% lower value compared to non-enhanced plants) in plant cells, in extremely dry conditions (20% soil WHC). Furthermore, application of macro- and micronutrients contained in the fertilizer led to a significant increase in activation of the enzymes SOD (increase by 61.2%), CAT (increase by 45.7%), and GPOX (increase by 35.8%), responsible for decomposition of ROS. The comprehensive activity of fertilizer ingredients on coated seeds allows to keep maize plants subjected to drought stress in better condition in the early stage of emergence.