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Article

Zinc Nanoparticles (ZnNPs): High-Fidelity Amelioration in Turnip (Brassica rapa L.) Production under Drought Stress

by
Jia Li
1,†,
Sara Zafar
2,*,
Ayesha Javaid
2,
Shagufta Perveen
2,
Zuhair Hasnain
3,
Muhammad Ihtisham
1,
Adeel Abbas
4,
Muhammad Usman
5,†,
Ahmed H. El-Sappah
1,6,† and
Manzar Abbas
1,*
1
School of Agriculture, Forestry and Food Engineering, Yibin University, Yibin 644000, China
2
Botany Department, Government College University, Faisalabad 38000, Punjab, Pakistan
3
Department of Agronomy, PMAS Arid Agriculture University, Attock Campus, Attock 43600, Punjab, Pakistan
4
Institute of Environment and Ecology, School of Environment and Safety Engineering, Jiangsu University, Zhenjiang 212013, China
5
Department of Biochemistry and Biotechnology, Faculty of Veterinary and Animal Sciences, MNS University of Agriculture, Multan 66000, Punjab, Pakistan
6
Genetics Department, Faculty of Agriculture, Zagazig University, Zagazig 44519, Egypt
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Sustainability 2023, 15(8), 6512; https://doi.org/10.3390/su15086512
Submission received: 23 January 2023 / Revised: 20 February 2023 / Accepted: 6 April 2023 / Published: 12 April 2023
(This article belongs to the Section Sustainable Agriculture)
Browse Figures
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Abstract

:
The detrimental effects of drought have adverse impacts on the crop yield as global climatic changes put unusual pressure on water resources. The challenge of attaining water security is key for the sustainable development of crops. Zinc (Zn2+) is an important nutrient that helps to alleviate drought stress by modulating the growth and yield of crops. Recently, zinc nanoparticles (ZnNPs) have been used as a novel strategy for the fertilization of crops. This study was specifically developed to observe the comparative effects of ZnNPs and conventional zinc sulfate (ZnSO4) at diverse concentration levels (0.01%, 0.05%, and 0.1%) that could effectively decrease the injurious effect of drought stress on turnip plants. In experiments on the golden turnip variety, drought stress caused a significant reduction in all growth and biochemical attributes, and increased antioxidant enzymatic activity. In a comparison with the conventional fertilizer ZnSO4, the foliar application of 0.1% ZnNPs significantly improved plant height, biomass, root/turnip length, turnip diameter, antioxidant defense system, secondary metabolites, and photosynthetic pigments in the leaves under drought stress. Based on the collected results, it is suggested that the foliar application of ZnNPs, instead of ZnSO4, under drought stress is helpful in increasing the growth and yield of turnip plants.

1. Introduction

Sustainable agriculture is the current global issue, as it includes the efficient use of water and nutrient resources. Sources of good quality irrigation water are being limited, and the extension of new water supplies is not enough to meet the demand of increasing water needs. Optimum water availability is one of the major ecological factors that affect the growth and development of plants [1]. Plant growth is stunted under water-deficit conditions [2], causing considerable impairments in various plants’ physiological and biochemical processes, such as hormonal metabolism, key enzyme activities, reactive oxygen species (ROS) accumulation, and stomatal regulation [2]. Drought stress results in the over-accumulation of ROS, which have drastic effects on proteins, deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and the plasma membrane and, ultimately, affect the proper functioning of organelles [3,4]. To overcome the effects of ROS, plants adopt various counter strategies, i.e., molecular and biochemical mechanisms. These adaptations include the production of antioxidant enzymes, such as peroxidase (POD), catalase (CAT), superoxide dismutase (SOD), and non-enzymatic antioxidants, such as ascorbic acid and phenolics. The drought tolerance phenomenon in plants activates the upregulation of the antioxidant defense system [5,6].
In order to reduce the drastic impacts of stress, the foliar application of micronutrients is useful in overcoming nutrient deficiency and alleviating the adverse effects of water stress on plant growth or, ultimately, on biomass production [7,8]. Foliar application of nutrients is environmentally friendly in comparison to soil applied nutrients, and avoids toxicity on accumulation. It also boosts plant standing crop and its performance, yield, and uniformity under various environmental conditions. The foliar application of micronutrients to enhance crop production, especially Zn, results in high yields [1]. Zn acts as an important micronutrient that helps to maintain plant growth and development. Not only does Zn take part in the biosynthesis of indole acetic acid (IAA), carbohydrate and protein metabolism, but it is also crucial for the regulation of different enzymes, such as dehydrogenases, peptide enzymes, and proteinases, as well as starch formation and seed maturation [9,10,11].
Recently, it has been observed that the application of a controlled-release Zn fertilizer coated with nanomaterials results in an increase in the amount of protein and a decrease in the amount of soluble sugar content in wheat plants [12,13]. The application of nanofertilizers is a significant way to provide nutrients slowly and in a regulated manner, which is vital to alleviate the challenges of fertilizer requirements [1]. It is a fact that when micronutrients or any bulk materials change/transform into the nanoscale range, the chemical, physical, optical, physiochemical, and catalytic properties change drastically [14]. This is because, in the nanoscale range, more surface area is exposed, which ultimately causes a high reactivity with the surrounding cellular and extracellular matrix.
As an essential micronutrient, Zn deficiency causes disturbances in: chlorophyll synthesis; nitrogen metabolism; carbonic anhydrase function; protein synthesis; photosynthesis; cell division; the integrity of the membrane structure; and the functions of and reductions in the root, shoot, and dry matter [15]. Vegetables add good flavors to one’s diet. For the agricultural economy, it is desirable to improve the nutritional quality and micronutrient content of turnip plants under water-limited conditions. Plants readily absorb nanofertilizers. This study was specifically designed to use ZnNPs as nanofertilizer. In the last decade, nanotechnology was mostly used for biomedical applications. Nowadays, nanofertilizers are being widely used by the agriculture sector. It has been previously reported that nanofertilizers not only boost agricultural output but also protect crops from abiotic stresses. Therefore, nanofertilizers have sparked a lot of interest in increasing agricultural yields. Growers’ profit margin ratios are increased when they employ nanofertilizers to increase yield and product quality [16].
Turnips are highly valued for their nutritional content, which includes vitamins, carbohydrates, folate, and minerals, such as iron, copper, manganese, calcium, potassium, phosphorus, and magnesium. However, under drought conditions, the seed germination rate, yield, and development of turnip plants are significantly reduced, as reported previously [17,18]. The purpose of this study was to investigate the comparative effect of exogenously applied Zn as ZnNPs and ZnSO4 in order to determine whether either of these materials can overcome the negative effects of water scarcity on the morphological, physiological, and biochemical characteristics of turnip plants, and to observe the potential of conventional and nano-zinc fertilizer to improve yield of turnip plants.

2. Materials and Methods

2.1. Acquisition of ZnNPs and Experimental Design

The effects of varying levels of foliar applications of Zn at concentrations of 0.01%, 0.05%, and 0.1%, selected from previous studies, and a control (a negative or a positive control) were evaluated in a field experiment with 30-day-old golden turnip plants under drought stress [19]. Zinc nanoparticles (ZnNPs, 40 nm) and ZnSO4 were purchased from Sigma-Aldrich and used as fertilizers. The Randomized Complete Block Design (RCBD) was employed as an experimental design.

2.2. Seed Selection and Germination

We obtained turnip seeds from Ayub Agricultural Research Institute (AARI), Faisalabad. For the individual foliar application of ZnNPs and ZnSO4, the seeds were sown in soil (Table 1) in a field following the RCBD design. Drought stress was induced by watering the control plants with an interval of one week and by watering the stressed plants with an interval of two weeks. A foliar spray of micronutrients was applied after 28 days of germination [20]. The plants were harvested after two weeks of the application of the Zn micronutrient to investigate the effects of the ZnNPs and ZnSO4 on the activation of various biochemical, growth, and yield attributes under water-limited conditions.

2.3. Measurement of Plant Height, Shoot Length, Diameter, and Weight

When the turnip plants reached full maturity, the plant heights of five randomly selected plants from the experimental area were recorded. After the measurement of the plant heights of the randomly selected plants, the average height was measured in centimeters (cm). Similarly, the shoot length was also measured in centimeters (cm) one week after the foliar application of the ZnNPs and ZnSO4. After the harvesting of the crops, five turnip plants were randomly selected from each subplot to measure the turnip length (cm), diameter (cm), and weight (g). For the measurement of the turnip fresh weight, five randomly selected turnips were weighted, and the average weight was calculated. The selected turnips were dried in an oven at 75 °C, and the dry weight was also calculated.

2.4. Analysis of Chlorophyll and Carotenoid Contents

The chlorophyll (Chl a and Chl b) contents were determined by following the recommendations of Arnon [21] and calculated as described by Davies [22]. We excised 0.5 g samples of the leaves of the plants treated with the ZnNPs and ZnSO4, and we soaked them in 5 mL of 80% acetone. Subsequently, centrifugation was performed at 1000× g for 5 min. The wavelength (λ) of the supernatant was measured by using a spectrophotometer at λ = 645 nm, λ = 663 nm, and λ = 480 nm. The chlorophyll contents were determined by using the following formulae:
C h l o r o p h y l l . a = [ 12.7 O D 663 − 2.69 O D 645 ] × V / 1000 × W
C h l o r o p h y l l . b = [ 22.9 O D 645 − 4.68 ( O D 663 ) ] × V / 1000 × W
Carotenoid (g mL−1) = Acar/Em × 100
Note: Acar = OD 480 + 0.114(OD 663) − 0.68 (OD 645), Em × 100 = 2500, OD = optical density, V = sample extract volume, and W = sample weight.

2.5. Analysis of Activity of Antioxidant Enzymes

In order to calculate the contents of antioxidant enzymes, 0.5 g samples of the leaves of the turnip plants treated with the ZnNPs and ZnSO4 were homogenized in 50 mM phosphate buffer at pH 7.8, and they were centrifuged at 15,000× g for 20 min at 4 °C. The supernatant was carefully moved to a new tube and used for the determination of enzyme activity [23].

2.6. Analysis of Superoxide Dismutase (SOD) Activity

SOD activity was analyzed by following the method of Giannopolitis and Ries [23], which is based on the inhibition of the photochemical reduction of nitrobluetetrazolium (NBT) at λ = 560 nm. SOD activity was calculated by the addition of 50 µL enzymatic extract, 50 μM NBT, 1.3 μM methionine, and 50 mM phosphate buffer at pH 7.8. In the chamber, the solution was placed under a 30 W fluorescent light source. SOD activity was determined based on the inhibition of the reduction of NBT by xanthine oxidase. The absorbance was measured using a UV-visible spectrophotometer at λ = 560 nm. One SOD unit was characterized as the amount of enzyme necessary for a 50% inhibition rate of NBT reduction, in contrast to the tubes with the enzyme extract.

2.7. Analysis of Catalase (CAT) Activity

The reaction mixture for CAT consisted of 50 mM phosphate buffer at pH 7, 5.9 mM H2O2, and 0.1 mL enzyme extract. After every 20 s, the absorbance was recorded at λ = 240 nm. Each unit of activity is a change in the absorbance of 0.01 unit min−1 [24].

2.8. Analysis of Peroxidase (POD) Activity

The peroxidase activities of the turnip plants treated with the ZnNPs and ZnSO4 were recorded against the peroxidation of guaicol and H2O2. We added 50 mM phosphate buffer at pH 5, 20 mM guaiacol, 40 mM H2O2, and 0.1 mL enzyme extract. The absorbance was measured after every 20 s at λ = 470 nm with a spectrophotometer [24].

2.9. Analysis of Malondialdehyde (MDA) Contents

In order to measure the MDA contents, 0.5 g samples of ground leaf tissue were added to 6% trichloroacetic acid and centrifuged at 12,000× g for 10 min. In the next step, 1 mL of the supernatant was added to 3 mL of thiobarbituric acid (0.5% prepared in TCA). The mixture was placed at 95 °C in a water bath for 30 min while shaking. The sample was centrifuged for 10 min. The absorbance of the samples was measured with a spectrophotometer at λ = 532 to 600 nm [25].
M D A l e v e l n m o l = ∆ ( A 532 n m − A 660 n m ) / 1.56 × 105

2.10. Measurement of Contents of Ascorbic Acid (AA)

In order to measure the AA contents, 0.5 g samples of the leaves detached from the ZnNP- and ZnSO4-treated turnip plants were homogenized in 6% TCA and centrifuged at 14,000 rpm for 10 min. Subsequently, 2 mL of 2% dinitrophenyl hydrazine and two drops of 10% thiourea were added to 0.5 mL of the reaction mixture. The solution was boiled for 20 min and cooled at room temperature, and 5 mL of 80% H2SO4 was added [26]. Subsequently, the absorbance was measured at λ = 530 nm with a spectrophotometer. Finally, the AA contents were calculated using a standard curve and a standard solution series, ranging from 20 to 300 ppm of ascorbic acid contents.

2.11. Measurement of Total Phenolic Contents

In order to measure the total phenolic contents, the fresh leaves of the turnip plants treated with the ZnNPs and ZnSO4 were homogenized in 80% acetone and centrifuged at 14,000 rpm for 10 min. The supernatant was carefully decanted into a new protease-free tube. Subsequently, 100 µL of the plant extract was added, followed by the addition of 2.5 mL of 20% Na2CO3 and 500 µL of Folin–Ciocalteu reagent. Using a UV-visible spectrophotometer, the absorbance was read at λ = 750 nm [27].

2.12. Analysis of Total Soluble Proteins (TSPs)

In order to analyze the total protein contents, 0.25 g samples of the fresh leaves of the turnip plants treated with the ZnNPs and ZnSO4 were detached and homogenized in 5 mL of phosphate buffer at pH 7, and centrifuged at 10,000 rpm for 10 min. The supernatant was moved to a new protease-free tube and used to measure the absorbance at λ = 595 nm with a spectrophotometer, as described by Bradford [28].

2.13. Analysis of Total Soluble Sugar (TSS) Contents

Around 0.25 g samples of the fresh leaves of the turnip plants treated with the ZnNPs and ZnSO4 were homogenized in 5 mL of 1% acidic methanol and centrifuged at 14,000 rpm for 10 min. Subsequently, 200 µL of the supernatant was moved to a new tube, and 3 mL of anthrone was added. The reaction mixture was heated to 95 °C for 10 min, followed by cooling at room temperature. Finally, the absorbance was measured at λ = 595 nm with a UV-visible spectrophotometer [29].

2.14. Analysis of Total Free Amino Acid (TFA) Contents

In order to analyze the effect of the ZnNPs on proteins, the total free amino acid contents were measured by following the already established method of Crampton et al. [30]. To 1 mL of turnip plant leaf extract, after the treatment with the ZnNPs and ZnSO4, 1 mL of 10% pyridine and 1 mL of 2% nin-hydrin were added. Subsequently, the mixture was heated for 30 min and placed at room temperature to cool down. Finally, the absorbance was measured at λ = 570 nm by employing a spectrophotometer.

2.15. Analysis of Flavonoid Contents

To measure the flavonoid contents, 0.5 g samples of the leaves of the turnip plants treated with both the ZnNPs and ZnSO4 were homogenized in 5 mL of 80% acetone, and, subsequently, filtration was performed to remove coarse contents. About 0.5 mL of the filtrate was placed into a new tube and diluted by adding 2 mL of ddH2O, followed by the addition of 0.6 mL of 5% NaNO2, and the tube was kept at room temperature. After five minutes, 0.5 mL of 10% AlCl3 was added to the solution. As the last step, 2 mL of 1 M NaOH was added to the reaction mixture, and the absorbance was measured at λ = 510 nm by following an already published methodology [31].

2.16. Statistical Analysis

Statistix v8.1 was employed to perform a statistical analysis of the data. The mean and standard errors were determined using Microsoft Excel 2007, and the difference in the means was calculated using the least significant difference (LSD) test at the 5% probability level.

3. Results

3.1. ZnNPs Enhanced Leaf Growth Attributes

3.1.1. ZnNPs Enhanced Shoot Length of Turnip Plants

We observed significant, drastic effects of drought stress on the shoot length and plant height of the turnip plants (Figure 1 and Table 2). Around 23% of the total decrease in shoot length was observed in the turnip plants grown under drought stress. However, the foliar application of 0.05% ZnSO4 and 0.1% ZnNPs enhanced the shoot and root lengths of the turnip plants under drought stress (Figure 1a). Notably, plant height decreased by 44% under drought stress. The foliar application of 0.1% ZnNPs enhanced plant height by 89%, while 0.1% ZnSO4 enhanced plant height by 62% under drought stress (Figure 1b). In comparison with ZnSO4, the foliar spray of 0.1% ZnNPs significantly increased the plant height and fresh and dry weights of the turnip plants.

3.1.2. ZnNPs Ameliorate Chlorophyll and Carotenoid Contents in Leaves

A significant decrease in the Chl a, Chl b, and total chlorophyll contents was observed in the leaves of the turnip plants under drought stress (Figure 2). The Chl a and Chl b contents in the leaves of the turnip plants reduced by 59% and 52%, respectively. The exogenous foliar applications of 0.1% ZnNPs and ZnSO4 enhanced the Chl a, Chl b, and total chlorophyll contents. Notably, the foliar spray of 0.1% ZnNPs under normal irrigation enhanced the contents of Chl a and Chl b significantly in comparison to those under drought. There was no significant decrease in the carotenoid contents in the leaves of the turnip plants under drought stress (Figure 2d). Despite this, 0.1% ZnNPs and ZnSO4 significantly enhanced the carotenoid contents under drought stress.

3.1.3. ZnNPs Enhanced Antioxidant Enzyme Activity in Leaves

Under drought stress conditions, the leaves of the golden turnip variety displayed a significant increase in antioxidant enzyme activities, including MDA, SOD, POD, and catalase activities (Figure 3). Among these, the highest increase in MDA contents was observed in the turnip leaves upon the foliar application of 0.01% ZnSO4, followed by 0.05% ZnNPs under drought stress (Figure 3a). The highest SOD activities were observed upon the foliar application of 0.01% ZnNPs and ZnSO4 under drought stress, which were 46.4 unit min−1 and 51.9 unit min−1, respectively (Figure 3b). By contrast, the highest POD activity was observed in the leaves of the control turnip plants, followed by 0.01% ZnNPs (Figure 3c), indicating the need to further investigate the interaction between the contents of POD and ZnNPs. The foliar application of 0.01% ZnNPs and ZnSO4 improved catalase enzyme activities to 6.85 unit min−1 and 6.65 unit min−1, respectively (Figure 3d). In conclusion, it was only under drought stress that the antioxidant enzymes in the turnip plants displayed the highest activity and assisted in stress tolerance, upon the foliar application of 0.01% ZnNPs followed by 0.01% ZnSO4. However, the exogenous spray of the ZnNPs and ZnSO4 significantly decreased the devastating effects of the drought on the plants.

3.1.4. ZnNPs Enhanced Sugars, Proteins, and Secondary Metabolites in Leaves

The total soluble sugar (TSS), total soluble protein (TSP), and total free amino acid (TAA) contents remained unchanged, while the total phenolics, flavonoids, and ascorbic acid (AA) contents decreased significantly in the turnip leaves under drought stress (Table 2 and Figure 4). The application of 0.1% ZnNPs enhanced the TSS, TSP, TAA, and total phenolics in the turnip leaves under drought stress (Figure 4a–d), while no significant changes in the flavonoid or AA (Figure 4e,f) contents were observed. The most significant increase was observed in the TFA (6.40 mg g−1 f.wt., 6.07 mg g−1 f.wt.) and TSP (1.94 mg g−1 f.wt., 1.93 mg g−1 f.wt.) under the treatment of 0.1% ZnNPs. Similarly, the foliar application of the ZnNPs and ZnSO4 enhanced the ascorbic acid contents in the turnip leaves by 1.69 mg g−1 and 3.87 mg g−1 under drought stress, respectively.

3.2. ZnNPs Enhanced Growth Attributes of Root/Turnip

3.2.1. ZnNPs Enhanced Root/Turnip Length

Drought stress significantly decreased the turnip fresh weight (F.W), dry weight (D.W), length, and diameter (Figure 5). The root/turnip length grown under drought stress decreased by 21%. We observed a significant increase in the F.W of the turnips upon the foliar application of 0.1% ZnNPs (Figure 5a). In contrast to the ZnNPs, the foliar application of 0.05% ZnSO4 enhanced the turnip D.W (Figure 5b). The turnip length and diameter were significantly enhanced upon the foliar application of 0.1% ZnNPs. In conclusion, we observed a significant effect of 0.1% ZnNPs on fruit growth under drought stress.

3.2.2. ZnNPs Enhanced Sugars, Proteins, and Secondary Metabolites in Root/Turnip

We observed a significant decrease in the TSP but an increase in the TSS and TAA in the roots/turnips under drought stress (Figure 6). The highest sugar contents were observed upon the foliar application of 0.01% ZnNPs, followed by 0.01% ZnSO4 (Figure 6b). Similarly, the foliar application of 0.1% ZnNPs followed by ZnSO4 enhanced the TAA and TSP in the roots/turnips under drought stress. In conclusion, the foliar spray of 0.1% ZnNPs enhanced the protein and sugar contents of the turnips under drought stress.

3.2.3. ZnNPs Enhanced Antioxidant Enzyme Activity in Root/Turnip

Under drought stress, the golden turnip genotype displayed a variable antioxidant enzyme activity in the roots/turnips (Figure 6). We observed a slight increase in the contents of H2O2 and MDA and a slight decrease in the contents of ascorbic acid in the turnips under drought stress (Figure 6a,e,f). The foliar application of only 0.01% ZnNPs significantly enhanced the contents of H2O2 when compared to the application of 0.1% ZnSO4 (Figure 6a). By contrast, the foliar application of the ZnNPs and ZnSO4 decreased the MDA contents in comparison to those of the control, but it still enhanced the MDA contents in comparison to those of the normally irrigated plants (Figure 6e). The foliar application of 0.1% ZnNPs and ZnSO4 significantly enhanced the ascorbic acid contents in the turnip roots under drought stress to 1.74 mg g−1 and 1.62 mg g−1, respectively (Figure 6f). However, the exogenous spray of the ZnNPs and ZnSO4 significantly ameliorated the negative effects of the oxidants on the turnip roots.

3.2.4. Correlation among Growth Attributes

Pearson’s correlations revealed significant positive correlations under control conditions of: root fresh and dry weights with the root length; total chlorophyll contents in the leaves; shoot length; total soluble sugars in the leaves; total soluble proteins in the roots; and ascorbic acid, phenolics, and flavonoids in the leaves (Figure 7). However, a significantly negative correlation was evident for SOD, POD, and CAT in the leaves under control conditions. Under water stress conditions, a negative correlation was evident between the root fresh weight and the total soluble proteins and ascorbic acid in the roots/turnips. For further validation of Pearson’s correlation results and an evaluation of the responses of the turnips to different ZnNP and ZnSO4 applications, a principal component analysis was executed (Figure 8). The results of Pearson’s correlation were confirmed by a PCA biplot. We imaged the direction of the cases on a factor plane in PCA with a biplot. A clear separation of all of the different parameters of the leaves and roots of the turnip in response to the different levels of ZnNPs and ZnSO4 was exhibited by Dim1 and Dim2. The contribution of Dim1 to the total variance was 51.5% in comparison to the contribution of 9.6% of Dim2. A clear separation of the leaf and root attributes with respect to the treatments was observed in the PC analysis (Figure 8).

4. Discussion

In recent years, drought stress has adversely affected crop production across the globe. In this study, we observed a significant decrease in the shoot and root lengths of turnip plants under drought stress, similar to that observed in bean [32], rice [33], and soybean [34]. Drought stress causes a severe halt in the growth of turnip plants by limiting the rate of supply of metabolites to growing tissues, leading to the improper regulation of biochemical and physiological processes [35]. The foliar application of micronutrients is one of the most effective ways for improving stress tolerance in many plants [36], to mitigate the adverse effects of drought. In this study, the foliar application of 0.1% ZnNPs resulted in considerable growth in comparison to the plants sprayed with ZnSO4. Similarly to our findings, Zafar et al. [37] reported that the foliar application of ZnNPs improved the growth of okra plants under salt stress by enhancing the total soluble sugar and total free amino acid contents. Similarly, Mahajan et al. [38] stated that zinc oxide nanoparticles (ZnONPs) increased root and shoot lengths.
Upon the aerial application of nanoparticles, the nanoparticles are mechanistically absorbed through the stomata, enhance oxygen transport and the absorbance of sunlight in photosystems I and II, and result in a higher biomass production [39]. In the present study, the foliar application of ZnNPs enhanced photosynthetic pigments, including the Chl a, Chl b, and total chlorophyll contents, under both stress and normal conditions (Figure 2). However, the carotenoid contents slightly increased under drought stress. These results are somewhat similar to findings in spinach [37]. According to Wang et al. [40], the decline is attributable to a stress-induced decrease in damage to the photosynthetic system, which decreases photosynthesis and chloroplast disruption and increases the chlorophyllase enzyme in plants.
Zn is a micronutrient found in plants, and it aids in the regulation of metabolism and the biosynthesis of photosynthetic pigments. The beneficial effect of zinc (ZnSO4 and ZnNPs) on chlorophyll contents could be due to its role in strengthening the chloroplast ultrastructure or in enhancing antioxidant enzyme activity, delaying senescence and decreasing reactive oxygen species generation [41]. Chlorophyll degradation and reduction and improved photosynthetic efficiency are linked to the protective effect of Zn fertilizer [42]. In this study, the exogenous application of 0.1% ZnNPs reduced the stress-related effects and increased the chlorophyll content.
The plants under water stress showed an increase in antioxidant enzyme activity, as also reported by Sachdev et al. [43]. It has been reported that the upregulation of antioxidants is helpful in drought tolerance in different plants, such as maize [44] and chickpea [45]. In this study, under water stress, the foliar application of ZnNPs and ZnSO4 helped to maintain SOD, POD, and CAT activities. The antioxidant defensive mechanism is activated by Zn applied as ZnNPs and ZnSO4, and it reduces oxidative stress damage [37]. The H2O2 and MDA contents were observed to be the highest under stress conditions as also reported in linseed [2]. However, the exogenous foliar application of Zn as ZnNPs and ZnSO4 improved the activity of antioxidants under water stress. Zinc plays an important role in the osmotic adjustment of plants and prevents membrane damage from stress-induced ROS. The flavonoid, total phenolic, and ascorbic acid contents increased with the application of Zn as ZnNPs and ZnSO4. Similar observations were reported by García-López et al. [46] in peppers. It was reported by Ain et al. [47] and Itroutwar et al. [48] that an effective source of Zn fertilizer for the growth of plants is the foliar application of ZnNPs.
The total soluble sugars and total free amino acids in the leaves and roots of the turnip plants were recorded to be the highest under stress conditions, which is consistent with the observations of Živanović et al. [49]. According to Noreen et al. [50], Zn supplementation aids in the maintenance of increased ascorbic acid levels. Increased levels of osmo-protectants, such as soluble sugars, play an important role in cellular stress defense and act as modulators of plant sensitivity [36,51]. In this study, zinc significantly enhanced all growth parameters of the non-stressed and stressed plants. It seemed that Zn counteracted the negative effects of drought on tuberous roots and turnip growth. The highest values of RL, RD, and RFW were observed in the ZnNP-treated plants, followed by ZnSO4 when compared with the untreated plants. Growth parameters, such as plant height, root length and diameter, and fresh and dry weights, significantly decreased under water stress [52].

5. Conclusions

In this study, ZnNPs foliar application significantly increased the physical parameters of turnips in comparison to those of control plants under water stress. The application of ZnNPs (0.1%) was the most promising foliar application in improving the growth, primary and secondary metabolites, and yield of the turnip plants. The application of ZnNPs had more prominent effects than a conventional fertilizer on the turnip plants. Therefore, under limited water availability in agricultural systems, the foliar application of ZnNPs is an economical option for increasing fertilizer output and sustainable production of crops.

Author Contributions

Conceptualization, J.L., M.A., Z.H. and S.Z.; Methodology, J.L., M.A., S.Z., Z.H. and M.U.; Experiments, J.L., A.J., S.P., Z.H., M.I., S.Z., M.U., A.A. and A.H.E.-S.; Formal Analysis, M.A., J.L., S.Z., Z.H., M.I. and A.H.E.-S.; Writing—Review and Editing, J.L., M.A., M.U. and S.Z.; Supervision, M.A. and Z.H.; Project Administration, J.L.; Funding Acquisition, J.L.; Writing—Original Draft, M.A., S.Z. and J.L.; Writing—Review and Editing, M.A., S.Z. and Z.H. All authors reviewed the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This work was funded by Yibin University’s High-Level Talent Project (No. 2018RC07), Yibin University, Yibin, China.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data used for the analysis in this study are available within the article, and the datasets used or analyzed during the current study are available from the corresponding author upon reasonable request.

Acknowledgments

Authors would like to extend their sincere appreciation to kind administration of Yibin University, Yibin, China, and Government College University, Faisalabad, Pakistan, for providing them with such a prestigious and well-equipped platform for research and development. They are also grateful to the kind administration of the department of Science and Technology of Sichuan Province, China.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Effect of foliar application of ZnNPs and ZnSO4 on (a) shoot length and (b) plant height of the golden turnip variety under drought stress. Error bars specify the standard error, and columns with different letters exhibit variations among treatments. LSD, least significant difference at 0.05.
Figure 1. Effect of foliar application of ZnNPs and ZnSO4 on (a) shoot length and (b) plant height of the golden turnip variety under drought stress. Error bars specify the standard error, and columns with different letters exhibit variations among treatments. LSD, least significant difference at 0.05.
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Figure 2. Effect of foliar application of ZnNPs and ZnSO4 on (a) Chl a, (b) Chl b, (c) total chlorophyll, and (d) carotenoid contents of turnip leaves under drought stress. Error bars specify the standard error, and columns with different letters exhibit variations among treatments. LSD, least significant difference at 0.05.
Figure 2. Effect of foliar application of ZnNPs and ZnSO4 on (a) Chl a, (b) Chl b, (c) total chlorophyll, and (d) carotenoid contents of turnip leaves under drought stress. Error bars specify the standard error, and columns with different letters exhibit variations among treatments. LSD, least significant difference at 0.05.
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Figure 3. Effect of foliar application of ZnNPs and ZnSO4 on (a) MDA, (b) SOD, (c) POD, and (d) CAT activity of the golden turnip variety under water-deficit conditions. Error bars specify the standard error, and columns with different letters exhibit variations among treatments. LSD, least significant difference at 0.05.
Figure 3. Effect of foliar application of ZnNPs and ZnSO4 on (a) MDA, (b) SOD, (c) POD, and (d) CAT activity of the golden turnip variety under water-deficit conditions. Error bars specify the standard error, and columns with different letters exhibit variations among treatments. LSD, least significant difference at 0.05.
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Figure 4. Effect of foliar application of ZnNPs and ZnSO4 on leaf biochemical attributes, including (a) total soluble sugar, (b) total soluble protein, (c) total free amino acids, (d) total phenolics, (e) flavonoids, and (f) ascorbic acid, of the golden turnip variety under drought stress. Error bars specify the standard error, and columns with different letters exhibit variations among treatments. LSD, least significant difference at 0.05.
Figure 4. Effect of foliar application of ZnNPs and ZnSO4 on leaf biochemical attributes, including (a) total soluble sugar, (b) total soluble protein, (c) total free amino acids, (d) total phenolics, (e) flavonoids, and (f) ascorbic acid, of the golden turnip variety under drought stress. Error bars specify the standard error, and columns with different letters exhibit variations among treatments. LSD, least significant difference at 0.05.
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Figure 5. Effect of foliar application of ZnNPs and ZnSO4 on (a) fresh weight, (b) dry weight, (c) length, and (d) diameter of the golden turnip variety under drought stress. Error bars specify the standard error, and columns with different letters exhibit variations among treatments. LSD, least significant difference at 0.05.
Figure 5. Effect of foliar application of ZnNPs and ZnSO4 on (a) fresh weight, (b) dry weight, (c) length, and (d) diameter of the golden turnip variety under drought stress. Error bars specify the standard error, and columns with different letters exhibit variations among treatments. LSD, least significant difference at 0.05.
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Figure 6. Effect of foliar application of ZnNPs and ZnSO4 on (a) H2O2, (b) total soluble sugars, (c) total soluble proteins, (d) total free amino acids, (e) MDA, and (f) ascorbic acid of the golden turnip variety under drought stress. Error bars specify the standard error, and columns with different letters exhibit variations among treatments. LSD, least significant difference at 0.05.
Figure 6. Effect of foliar application of ZnNPs and ZnSO4 on (a) H2O2, (b) total soluble sugars, (c) total soluble proteins, (d) total free amino acids, (e) MDA, and (f) ascorbic acid of the golden turnip variety under drought stress. Error bars specify the standard error, and columns with different letters exhibit variations among treatments. LSD, least significant difference at 0.05.
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Figure 7. Pearson correlation between different attributes of the golden turnip variety reveals the impact of treatments under control and drought stress on turnip leaves and roots. * Highly correlated either positive or negative with colors. TSP_L = total soluble proteins in leaves; TSP_R = total soluble proteins in roots; TFA_L = total free amino acids in leaves; TFA_R = total free amino acids in roots; TSS_L = total soluble sugars in leaves; TSS_R = total soluble sugars in roots; SOD_L = superoxide dismutase activity in leaves; POD_L = peroxidase activity in leaves; CAT_L = catalase activity in leaves.
Figure 7. Pearson correlation between different attributes of the golden turnip variety reveals the impact of treatments under control and drought stress on turnip leaves and roots. * Highly correlated either positive or negative with colors. TSP_L = total soluble proteins in leaves; TSP_R = total soluble proteins in roots; TFA_L = total free amino acids in leaves; TFA_R = total free amino acids in roots; TSS_L = total soluble sugars in leaves; TSS_R = total soluble sugars in roots; SOD_L = superoxide dismutase activity in leaves; POD_L = peroxidase activity in leaves; CAT_L = catalase activity in leaves.
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Figure 8. Principal component analysis (PCA) showing the relationship among different measured variables of turnip leaves and roots under control and drought stress. (A) Comparison of non-stressed and stressed plants (B) Comparison among treatments. T0 = control; T1 = 0.01% ZnNPs; T2 = 0.05% ZnNPs; T3 = 0.1% ZnNPs; T4 = 0.01% ZnSO4; T5 = 0.05% ZnSO4; T6 = 0.1% ZnSO4.
Figure 8. Principal component analysis (PCA) showing the relationship among different measured variables of turnip leaves and roots under control and drought stress. (A) Comparison of non-stressed and stressed plants (B) Comparison among treatments. T0 = control; T1 = 0.01% ZnNPs; T2 = 0.05% ZnNPs; T3 = 0.1% ZnNPs; T4 = 0.01% ZnSO4; T5 = 0.05% ZnSO4; T6 = 0.1% ZnSO4.
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Table
Table 1. Physicochemical properties of soil.
Table 1. Physicochemical properties of soil.
Soil TextureLoamCO32− (meq/L)Nil
ECe (dS/m)2.04HCO3− (meq/L)2.75
pH8.3Zn (ppm)2
Organic Matter (%)0.76Available P (ppm)3.1
Saturation35Available K+ (ppm)80
Table
Table 2. Mean square values of plant growth and leaf biochemical attributes of turnip (Brassica rapa L.) subjected to foliar application of ZnNPs and ZnSO4 under drought stress.
Table 2. Mean square values of plant growth and leaf biochemical attributes of turnip (Brassica rapa L.) subjected to foliar application of ZnNPs and ZnSO4 under drought stress.
TreatmentDfPlant HeightShoot LengthRoot LengthRoot F.wt.Root Dry.wt.
Treatment (T)656.769 ***10.706 ***102.039 ***278.404 ***17.229 ***
Drought (D)1712.595 ***94.5 ***288.095 ***1069.086 ***313.786 ***
T × D618.595 ***3.388 ns7.373 ns169.156 ***15.288 ***
Error262.9341.55.7311.5470.825
TreatmentDfRoot DiameterChl aChl bTotal ChlCarotenoids
Treatment (T)64.769 ***0.098 ns0.057 ns0.275 ns0.023 ns
Drought (D)121.715 ***0.922 ***0.417 ***2.577 ***3.020 ***
T × D61.562 *0.017 ns0.013 ns0.027 ns0.224 ns
Error260.5940.0500.0270.1120.102
TreatmentDfTSSTotal Soluble ProteinsTotal Free Amino AcidsPhenolicsFlavonoids
Treatment (T)60.138 ns4.095 ***4.916 ns5087.534 ***80.230 ***
Drought (D)112.565 ***25.582 ***56.619 *41979.97 ***728.29 ***
T × D60.359 ns0.699 ns0.695 ns1900.97 ***26.758 ***
Error260.4170.3477.336297.3542.251
TreatmentDfAscorbic AcidMDASODPODCAT
Treatment (T)63.959 ***42.619 ns273.537 ***13.307 **1.029 *
Drought (D)194.109 ***1004.729 ***4234.264 ***131.109 ***17.379 ***
T × D63.063 ***61.158 ns5.520 ns6.245 ns0.135 ns
Error260.18832.56748.8532.6860.388
TreatmentDfTSSTotal Soluble ProteinsTotal Free Amino AcidsMDAH2O2
Treatment (T)60.526 ns1.949 ***5.329 ns22.424 *0.993 ns
Drought (D)110.327 ***15.113 ***63.385 ***827.807 ***85.347 ***
T × D60.906 *0.090 ns1.512 ns30.321 **3.085 *
Error260.3520.2812.747.0960.875
***, **, and * = significant at 0.001, 0.01, and 0.05 levels, respectively; Df = degrees of freedom; ns = non-significant; T × D = interaction between treatment and drought; Chl a = chlorophyll a; Chl b = chlorophyll b; TSS = total soluble sugar; MDA = malondialdehyde; SOD = superoxide dismutase; POD = peroxidase; CAT = catalase.
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Li, J.; Zafar, S.; Javaid, A.; Perveen, S.; Hasnain, Z.; Ihtisham, M.; Abbas, A.; Usman, M.; El-Sappah, A.H.; Abbas, M. Zinc Nanoparticles (ZnNPs): High-Fidelity Amelioration in Turnip (Brassica rapa L.) Production under Drought Stress. Sustainability 2023, 15, 6512. https://doi.org/10.3390/su15086512

AMA Style

Li J, Zafar S, Javaid A, Perveen S, Hasnain Z, Ihtisham M, Abbas A, Usman M, El-Sappah AH, Abbas M. Zinc Nanoparticles (ZnNPs): High-Fidelity Amelioration in Turnip (Brassica rapa L.) Production under Drought Stress. Sustainability. 2023; 15(8):6512. https://doi.org/10.3390/su15086512

Chicago/Turabian Style

Li, Jia, Sara Zafar, Ayesha Javaid, Shagufta Perveen, Zuhair Hasnain, Muhammad Ihtisham, Adeel Abbas, Muhammad Usman, Ahmed H. El-Sappah, and Manzar Abbas. 2023. "Zinc Nanoparticles (ZnNPs): High-Fidelity Amelioration in Turnip (Brassica rapa L.) Production under Drought Stress" Sustainability 15, no. 8: 6512. https://doi.org/10.3390/su15086512

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