Effects of SNP, MgSO4, and MgO-NPs foliar application on Spinacia oleracea L. growth and physio-biochemical responses under cadmium stress | Scientific Reports

Nov 05, 2024

Scientific Reports volume 14, Article number: 26687 (2024) Cite this article

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The effects of foliar application of sodium nitroprusside (SNP), magnesium sulfate (MgSO4) and magnesium oxide nanoparticles (MgO-NPs) on the growth, physiology, and gas exchange parameters of two varieties of spinach (Spinacia oleracea L.) under cadmium (Cd) stress were examined. The experiment was arranged in a completely randomized design with 72 pots. Two varieties of S. oleracea (Desi Palak & Lahori Palak) were used. Two concentrations of Cd (0 µM and 150 µM) in the form of cadmium chloride (CdCl2) were used. Two levels of SNP (0 ppm and 100 ppm) and two levels for each form of Mg i.e. MgSO4 and MgO-NPs (0 and 200 ppm) were foliar sprayed on plants in control and Cd stress. Both varieties behaved similarly under Cd stress and caused reductions in growth, physiology, gas exchange, water content parameters and inorganic ion uptake. However, the biochemical parameters like relative membrane permeability (RMP), malondialdehyde (MDA), and hydrogen peroxide (H2O2) contents were increased. However, all foliar spray treatments increased growth, physiological and gas exchange parameters, water content and inorganic ion uptake. However, this reduced the MDA, RMP, and H2O2 contents. Desi Palak showed the more positive results under foliar application of MgO-NPs. However, Lahori palak showed more positive results under the SNP + MgO-NP treatment. It is concluded that foliar application of SNP, MgSO4 and MgO-NPs could be an innovative approach to alleviated the heavy metals (Cd) toxicity in crop plants.

Rapid industrialization and development over the past century have led to unbearable levels of heavy metal (HM) discharge into agricultural land, causing significant damage to ecological systems1,2. These lands can bear and utilize small amounts of some heavy metals, but extreme amounts can intensely harm sensitive crop yields3. Once such heavy metals penetrate water and soil, their dissipation becomes extremely difficult, and they pose a significant threat to ecosystems and human health via bioaccumulation in the food chain4.

Cadmium (Cd), a primary heavy metal, is common in several environments because of its rapid mobility in soil and water and its ability to form several different forms. Owing to the toxic and mobile nature of Cd, it has gained much attention recently. Even trace amounts of Cd are toxic to plants5. It affects various ecological environments via fertilizers, industrial waste, and sewage discharge6. The World Health Organization (WHO) recognizes Cd as a major source of food contamination and a key carcinogen affecting human beings, plants and animals7. Cd impacts crop growth and development, with high amounts resulting in stunted plant growth, a major decrease in biomass, and various other deficiencies8,9. When plants are exposed to stress induced by Cd, morphological alterations such as growth suppression, leaf yellowing, and curling occur. Physiologically, this stress occurs via enzyme inactivation, metabolic disruption, and crucial accumulation of free radicals such as reactive oxygen species (ROS)10. Furthermore, cadmium ions can replace calcium ions in the reaction centers of photosystem II (PSII), thereby reducing the plants’ photosynthetic capacity and diminishing the light response activity of photosystem II, leading to decreased biomass11. Under heavy metal stress, the levels of the cytotoxic compound methylglyoxal increase. However, to defend against and tolerate Cd stress, plants develop various defense mechanisms12. Although research on heavy metal soil contamination is advancing, conventional soil remediation methods are ineffective in severely polluted areas, necessitating further investigation.

Leafy vegetables grown in soil contaminated with Cd tend to accumulate relatively high levels of this metal13,14. These vegetables are often cultivated on preurban lands polluted with heavy metals, primarily Cd, due to the use of untreated urban wastewater15,16. Consequently, vegetables from these areas have elevated Cd levels in their edible parts, increasing the risk of contamination in the food chain. Spinacia oleracea (spinach) is one of the leafy greens most valued for its essential nutrients17. It is an extensively eaten leafy vegetable that is grown and eaten worldwide, especially in Southeast Asia, throughout the year18. Spinach is scientifically recommended because of its diverse health benefits; it is a rich source of minerals and vitamins, and its leaves are a major iron source17,19. Among different leafy vegetables, HM accumulation has been widely studied. It is a diecious plant and belongs to the Amaranthaceae family20. Different studies have shown that spinach can easily absorb and transport Cd to its shoots, thus increasing the possibility of food chain contamination21,22. In Pakistan, it is widely harvested as a winter crop over a vast field area20.

Nitric oxide (NO) is a tiny, water- and lipid-soluble gas that functions as a major signaling molecule23. Because of its diffusible nature, NO can be obtained from different sources and utilized as an exogenous treatment, such as with sodium nitroprusside (SNP)24. It is linked with many physiological functions in plants, such as stomatal closure, growth and aging phenomena, programmed cell death, and seed germination as a result of biotic and abiotic stresses25,26. In addition to its role in alleviating heavy metal toxicity, NO also decreases metalloid accumulation and might activate ROS and antioxidants in plants facing heavy metal stress27.

Cadmium uptake in plants is retarded by various elements, resulting in the mitigation of Cd stress. The elements that can reduce Cd absorption in plants include Ca, Ni, Cl, P, Cu, Fe, K, Mg, and Mn28,29. Magnesium (Mg) is vital for the proper growth of both animals and plants. A deficiency in this crucial nutrient negatively impacts carbohydrate formation and photosynthetic activity in plants30. In the chloroplast, grana formation is impossible without the addition of Mg. Additionally, Mg activates Rubisco through and facilitates the formation of the Rubisco complex carbamate group31. Magnesium is essential for chelating nucleotidyl phosphate forms and supporting the function of many enzymes32. Plants respond early to magnesium deficiency by enhancing antioxidative mechanisms28,33 and accumulating sugars in leaves, even before any noticeable impact on photosynthetic activity occurs34,35,36,37,38. Recent studies over the last decade have indicated that magnesium ions (Mg2+) may mitigate the adverse effects of heavy metals such as cadmium (Cd2+) and aluminum (Al3+)39,40.

Nanotechnology is increasingly recognized for its promising role in sustainable agriculture, offering various benefits to agroecosystems, such as soil remediation, climate change mitigation, and improved nutrient efficiency41. Currently, there is considerable emphasis on the use of nanoparticles (NPs) to reduce reliance on synthetic fertilizers and enhance plant growth to meet the dietary demands of the rapidly expanding global population42,43,44,45. Owing to their high surface area-to-volume ratio and swift mass transport potential, nanoparticles can be used in agrochemical applications46,47.

Magnesium oxide nanoparticles (MgO-NPs) have drawn significant interest among metallic nanoparticles because of their safe nature and distinctive physical and chemical properties48. Compared with other metal‒oxide nanoparticles, MgO‒NPs are increasingly popular because of their ability to be integrated into biological implants, high recycling potential, strong strength‒to‒weight ratio, functional versatility, hygroscopic nature, low density, low cost and nontoxicity in nature, and powerful functionality. Notably, MgO-NPs can impact plant morphophysiological activities49. They have powerful antibacterial, antioxidative, and anticancer properties50, stimulating plant growth and stress tolerance50. In addition to improving photosynthesis, MgO-NP treatments have been shown to significantly increase the chlorophyll content in plants51. NPs that are inorganic in nature, such as MnO, ZnO, Cu, CaO, SiO2, TiO2, Ag, and MgO, have shown great effectiveness against abiotic stresses along with antimicrobial agents. This provides protection to plants against biotic and abiotic stresses52.

These experiments aimed to increase plant growth, decrease cadmium uptake, and improve food safety. This innovative strategy demands the application of MgO nanoparticles, MgSO4, and nitric oxide (NO) via foliar application, providing potential solutions to decrease cadmium accumulation, increase plant vigor, and improve global food security and safety. The objectives of this study included evaluating the influence of cadmium toxicity on spinach plants; examining various growth, physiological, and biochemical attributes under the influence of MgSO4, MgO-NPs and NO; and analyzing the effectiveness of MgSO4, MgO-NPs and NO in alleviating Cd toxicity in spinach plants.

Seeds of spinach (Spinacia oleracea L.) varieties, Desi Palak and Lahori Palak, were procured from the Ayub Agricultural Research Institute (AARI), Faisalabad. The experiment was conducted at the botanical garden of the University of Education, Township, Lahore. A completely randomized design (CRD) experiment was conducted with three replicates and 72 pots in total. The plastic pots were filled with sand, which was washed several times with distilled water. Healthy seeds of S. oleracea were selected and planted in pots containing prewashed sand. The seeds were sown at equal distances from each other in each pot, with approximately 7–8 seeds per pot. After germination, thinning was done, and 4–5 seedlings were maintained in each pot. To meet nutritional requirements of plants half strength Hoagland’s solution was used for irrigation at regular intervals. In a pilot experiment different concentrations for SNP (0, 25, 50, 100 and 150 ppm) and MgO-NPs (0, 50, 100, 200 and 250 ppm) were used to assess their effects on growth of S. oleracea plants. From this study, 100 ppm for SNP and 200 ppm for MgO-NPs showed optimal improvements in growth of plants. Consequently, these dosages were then used in pot experiment. For cadmium (Cd) stress, cadmium chloride (CdCl2) solution (150 µM) was applied along with Hoagland’s solution. This concentration was selected based on findings of 53. Cd stress was applied at four leaf stage of seedlings. Foliar spray of SNP, MgSO4, and MgO-NPs was done immediately after application of Cd stress. This practice was repeated twice during the whole experiment with intervals of one week. Table 1 represents the layout of treatments employed in this study.

Two weeks after the treatments were administered, one plant from each replicate was carefully removed, rinsed with tap water, and dried with a soft cloth. The roots and shoots of each plant were then separated via a sharp cutter.

The following growth parameters were measured and recorded via a weighing balance and measuring tape: fresh shoot weight (grams), fresh root weight (grams), shoot length (cm), root length (cm), number of leaves per plant, and leaf area (cm2) of uniform-sized leaves from each plant. After these measurements, the samples were dried at 70 °C in an oven, and the subsequent parameters were documented: dry shoot weight (grams) and dry root weight (grams) (Figs. 1, 2, 3 and 4).

Spinaceae oleraceae L. (Variety- Lahori Palak) plant. Control plant (Cd1S1Mg0), SNP (Cd1S2Mg0), MgSO4 (Cd1S1Mg-I), MgO nanoparticles (Cd1S1Mg-II), SNP + MgSO4 (Cd1S2Mg-I), SNP + MgO nanoparticles (Cd1S2Mg-II).

Spinaceae oleraceae L. (Variety- Desi Palak) plant. Cd plant (Cd1S1Mg0), Cd + SNP (Cd2S2Mg0), Cd + MgSO4 (Cd2S1Mg-I), Cd + MgO nanoparticles (Cd2S1Mg-II), Cd + SNP + MgSO4 (Cd2S2Mg-I), Cd + SNP + MgO nanoparticles (Cd2S2Mg-II).

Spinaceae oleraceae L. (Variety- Lahori Palak) plant. control plant (Cd1S1Mg0), SNP (Cd1S2Mg0), MgSO4 (Cd1S1Mg-I), MgO nanoparticles (Cd1S1Mg-II), SNP + MgSO4 (Cd1S2Mg-I), SNP + MgO nanoparticles (Cd1S2Mg-II).

Spinaceae oleraceae L. (Variety- Lahori Palak) plant. Cd plant (Cd2S1Mg0), Cd + SNP (Cd2S2Mg0), Cd + MgSO4 (Cd2S1Mg-I), Cd + MgO nanoparticles (Cd2S1Mg-II), Cd + SNP + MgSO4 (Cd2S2Mg-I), Cd + SNP + MgO nanoparticles (Cd2S2Mg-II).

The SPAD value was assessed via an Atleaf chlorophyll meter (IC-CHLPLS). A fresh, young leaf was placed into the sensor, and the entry key was pressed. The SPAD value appeared on the screen instantly. Three readings (top, middle, and bottom) were taken from each leaf. This procedure was repeated for each replicate.

An LCpro-SD infrared gas analyzer was used to measure the following growth parameters: photosynthetic rate (Pn), intercellular CO2 (Ci), transpiration rate (Tn), and stomatal conductance (gs). For each replicate, a fully mature, young, fresh leaf was placed in an airtight leaf chamber, and after waiting for two minutes, the values for the aforementioned growth attributes were recorded via an instrument under ambient conditions with specific adjustments. The experimental conditions were as follows: ambient CO2 (Cref) at 415–460 µmol/mol, photosynthetically active radiation (PAR) Qleaf at 914 µmol m− 2 s− 1, leaf chamber temperature (Tch) at 23–27 °C, leaf temperature at 23–27 °C, molar gas flow rate of the leaf chamber (U) at 200.5 µmol s−1, ambient pressure (P) at 997 kPa, and leaf area at 6.25 cm2.

An OS-30p + chlorophyll fluorometer was used to assess the Fv/Fm (maximum quantum yield of PSII) parameter of chlorophyll fluorescence. A fully mature young leaf was selected from each replicate, and a dark period of approximately 15–20 min was provided to each leaf via the fluorometer device’s hairpin-like clips. After 20 min, the measurement was taken and recorded in the device. This process was repeated for each sample.

Arnon54 protocol was used for measuring chlorophyll and carotenoid contents. Fresh, fully mature leaves were taken from each replicate, and 0.5 g was weighed. The leaves were then ground via a prechilled mortar and pestle and mixed with 10 ml of 20% acetic acid solution. The extract was filtered through Whatman filter paper (4). The samples were kept at 4 °C in the refrigerator for 24 h, and the absorbance was measured at 480 nm, 645 nm and 663 nm via a UV/VIS spectrophotometer. The chlorophyll a and b values were measured via the following formulas:

where OD represents the optical density, W represents the weight of the extract and V represents the volume of the extract.

The carotenoid content was measured via the formula described by Lichtenthaler55;

Fresh, fully mature, young and uniform-sized leaves were obtained from every replicate. Leaves were cut into pieces and placed in test tubes containing 20 ml distilled water. Each replicate test tube was vortexed for 5 s, and the electrical conductivity (EC0) of each sample was subsequently determined via an EC meter. The same samples were placed in a refrigerator at 4 °C for 24 h. After 24 h, the samples were removed, and the electrical conductivity (EC1) of each sample was measured. The samples were autoclaved at 120 degrees Celsius for 20 min, and the electrical conductivity (EC2) of each sample was measured. The relative membrane permeability (RMP) of each sample was determined via the formula given by Yang, et al.56.

Fresh fully mature young leaves were collected from each replicate. Any dust present on them with some soft fabric was removed. The fresh weight of each leaf was measured via a weighing balance. Equal-sized Petri plates were taken and filled with distilled water to such a level so that the leaves could be immersed in water. The leaves of each replicate were immersed in water at room temperature (25–27 degrees Celsius) for 3 h. After 3 h, once the leaves became turgid, they were removed from distilled water, and their weight was measured via a weighing balance. After being dried with soft fabric, the same leaves were placed in an oven at 70 degrees Celsius for 24 h, and the dry weight was measured. The RWC was measured via the formula given by Jones and Turner57.

where FW denotes the fresh weight, DW denotes the dry weight, and TW denotes the turgid weight.

A pressure chamber instrument (PMS model 1000) was used to determine the LWP. Fresh, fully mature young leaves from each plant were taken. The leaf was placed in the pressure chamber until bubbles appeared at the tip of the petiole. Note that the reading of the water potential appears on the screen. The same procedure was repeated for each replicate.

Young fully mature leaves from each replicate were taken. Leaves were stored in a refrigerator at 20 °C for seven days. After seven days, the leaves were removed. Leaves were pressed and thawed via a glass rod. Two milliliters of sap from each leaf was transferred to an Eppendorf tube, and the osmotic potential of the leaf was measured via a device osmometer (Loser Messtechnik).

Fresh, fully mature, young leaves were collected from each sample, and 0.5 g was weighed via a weighing balance. Leaves were ground via a prechilled mortar and pestle and mixed with 10 ml of 50 mM phosphate buffer (precooled, pH 7.8). The extracts were stored in centrifuge tubes and centrifuged at 20,000 rpm for approximately 20 min at 4 °C. The supernatant was removed and frozen in a deep freezer. For determination of the catalase activity of 0.1 ml of the enzyme extract, 1.9 ml of 5.9 mM H2O2 and 1 ml of 50 mM phosphate buffer were added to the test tubes, and the absorbance at 240 nm every 30 s for 120 s was measured via a UV/VIS spectrophotometer58.

For APX, a total of 3 ml of APX mixture was made with 0.1 ml of enzyme extract (catalase), 0.1 ml of 7.5 mM ascorbic acid, and 2.7 ml of 50 mM phosphate buffer in test tubes. The absorbance at 290 nm every 30 s for 60 s was measured via a UV/VIS spectrophotometer59.

Similarly, for POD, 0.1 ml of enzyme extract (discussed in catalase), 0.6 ml of 20 mM guaiacol, 0.7 ml of 50 mM phosphate buffer and 0.6 ml of 40 mM H2O2 were added to the test tubes, and the absorbance at 470 nm every 30 s for 150 s was measured via a UV/VIS spectrophotometer58.

The SOD mixture was prepared by adding 0.05 ml of enzyme extract, 0.3 ml of nitro blue tetrazolium (50 µM), 0.3 ml of riboflavin (20 µM), 0.3 ml of methionine (130 mM) and 0.3 ml of 100 µM EDTA-Na2 to the test tube, and the samples were kept in 4000 lx light for approximately 20 min. After 20 min, the absorbance at 560 nm was read via a UV/VIS spectrophotometer60.

Bradford61 protocol was used to measure protein levels in the extract. Coomassie brilliant blue (100 mg) was added to 50 mL of ethanol (95%) to make a Bradford solution. The solution was then added to 100 ml of phosphoric acid (85%). Distilled H2O was added to bring the volume of the solution to 1 L. The optical density (OD) at 595 nm was measured via a UV/VIS spectrophotometer.

The method of Velikova, et al.62 was followed for the determination of hydrogen peroxide. Fresh, fully mature leaves were taken from each sample, and 0.5 g was weighed via a weighing system. Prechilled mortar and pestle leaves were ground and mixed with 5 ml (0.1%) trichloroacetic acid, after which the extract was transferred to a conical tube. The same procedure was repeated for every sample. The extracts were subsequently centrifuged at 12,000 rpm (4 °C for 15 min). A 0.5 ml aliquot of the supernatant was removed from the test tubes via pipette. Potassium iodide (1 mL) and phosphate buffer at pH 7 (0.5 mL) were also added to the test tubes. The mixtures were vortexed, and readings at 390 nm were taken via a UV/VIS spectrophotometer.

The Cakmak and Horst63 protocol was used for the measurement of malondialdehyde content. Fully matured fresh leaves were taken from each sample, and 0.5 g was weighed via a weighing balance. Prechilled mortar and pestle leaves were ground and mixed with 3 ml of trichloroacetic acid (1%). The extract was transferred to a conical tube. The same procedure was repeated for every sample. The extracts were subsequently centrifuged at 20,000 rpm (15 min at 4 °C). After centrifugation, 0.5 ml of the supernatant was transferred to test tubes containing 3 ml of a thiobarbituric acid solution (3% thiobarbituric acid produced in 20% trichloroacetic acid solution). The samples were subsequently heated in a water bath at 95 °C for 50 min. The reaction was terminated by placing the samples in an ice-water bath immediately. The samples were centrifuged again (10,000 rpm for 10 min), and the absorbance at 532 nm and 600 nm was determined via a UV/VIS spectrophotometer. The MDA value was calculated via the following formula:

The Bates, et al.64 method was used to analyze the proline content of the leaves. Fresh leaves (0.2 g) were collected from each replicate. The leaves were ground via a mortar and pestle (prechilled) and fixed with 5 mL of sulfo-salicylic acid (SSA- 3%). The mixture was filtered with filter paper (Whatman filter paper). To prepare acid ninhydrin, 2.5 g of ninhydrin was added to O-phosphoric acid (6 M, 40 ml) and glacial acetic acid (60 ml) in a tube. Two mL of filtrate, 2 ml of acid ninhydrin and 2 ml of glacial acetic acid were added to a test tube. The test tubes containing the solution were incubated in an oven for 60 min at 100 °C. The reaction took place in test tubes after the incubation was reversed by placing the incubated tubes in an ice water bath. Then, 4 ml of toluene was added to the same test tube. The test tube was vortexed for a few seconds, and the toluene was mixed thoroughly. The layers were separated from the aqueous solution, and the absorbance at 520 nm was read with a UV/VIS spectrophotometer.

A 0.5 g dried leaf sample was taken from each replicate and mixed in 10 ml of 80% acetone solution. The extract was kept in a conical tube and centrifuged at 4000 rpm (15 min, 4 °C). The supernatant was removed, and the dried residue was transferred to 10 ml of methanol. To measure the amount of total phenolics, the Folin‒Ciocalteu protocol65 was used. Two milliliters of prepared sample, 0.8 ml of sodium carbonate (85%), and 1 ml of Folin-Ciocalteu reagent were mixed gently and left for 30 min. By using a UV/VIS spectrophotometer, the absorbance at 765 nm was measured. The total phenolic content was calculated as gallic acid equivalents per gram of dry material .

A total of 0.1 g of ground dried root and shoot samples from each replicate were collected in test tubes. Two milliliters of H2SO4 was also added to the test tubes. The test tubes were maintained for 24 h. After 24 h, samples were taken and heated on a hot plate. While heating drop by drop, H2O2 was added to the sample until the sample became colorless. The volume of the mixture was increased to 50 ml, and the mixture was filtered. By using a flame photometer (Sherewood model 360), Ca2+ and K+.

The atomic absorption spectrophotometer (AAS) (Hitachi Polarized Zeeman AAS, Z-8200, Japan) method was used for the detection of Cd and Fe under specific conditions (the wavelengths for Cd 228.8 nm and for Fe 248.3 nm, the slit width for Cd 1.3 nm and for Fe 0.2 nm, the lamp current for Cd 7.5 mA and for Fe 10.0 mA, whereas the burner head wasStandard type, C2H2 was used as flame air, the burner height was 7.5 mm, the oxidant gas pressure (flow rate) was 160 kPa, and the fuel gas pressure (flow rate) was 6 kPa) given by AOAC66. The solution used for the detection of Cd and Fe was prepared via the digestion method (discussed in terms of inorganic mineral ions). All glassware used during the analytical process was immersed in 8 N HNO3 overnight and then washed multiple times with deionized water before use.

67 The EDTA titration method was used for the determination of Mg. For the detection of Mg, 10–15 ml of the filtrate containing Mg was placed in a conical flask, and the volume was increased to 20–30 ml with distilled water. Three to five milliliters of buffer solution (NH4Cl-NH4OH) and a few drops of the eriochrome black indicator were added to the same solution. The solution was titrated with 0.01 N EDTA until the color of the solution changed from red to blue (indicating the presence of Mg2+ ions). Initial and final readings were noted. The Mg2+ concentration in the sample was determined via the following formula:

where VEDTA denotes the volume of EDTA used (L), CEDTA denotes the concentration of EDTA (mol/L), MMg denotes the molar mass of Mg2+ (24.305 g/mol), and Vsample denotes the volume of the sample (L).

ANOVA was conducted on all the parameter values via R Studio (R 4.3.3). A significance level of P < 0.05 was employed to perform the means comparison test, utilizing LSD. Pearson’s correlation and principal component analysis were performed on R software using p < 0.05 confidence level.

Results showed that foliar treatments significantly enhanced the growth parameters of both spinach varieties, mitigating the negative effects of Cd stress. Both varieties exhibited significant increases in shoot fresh and dry weight with foliar applications under both control and stress conditions. As shown in Fig. 5A & B, Desi Palak showed the highest increase with SNP (49% and 89% for fresh and dry weight, respectively), while Lahori Palak had the greatest response to MgO-NPs (120% and 112%). Foliar treatments (SNP, MgSO4 and MgO-NPs) also positively influenced root growth (Fig. 5C & D). Desi Palak had the highest root fresh weight increase with SNP (169%), while Lahori Palak showed the greatest increase with MgO-NPs (217%). Root dry weight was also significantly improved, with Desi Palak exhibiting the highest increase (90%) with SNP and Lahori Palak showing the highest increase (120%) with SNP + MgO-NPs. Shoot length, root length, leaf count, and leaf area were all significantly increased by foliar applications (Fig. 6A-D). Desi Palak showed the greatest improvement in shoot length with SNP + MgO-NPs (32%), while Lahori Palak had the highest increase in root length with SNP + MgO-NPs (44%). Leaf count and leaf area were also significantly enhanced by the treatments (Table 2). Foliar applications effectively mitigated the negative effects of Cd stress on spinach growth. Both varieties showed significant reductions in growth parameters under Cd stress, but the treatments helped to alleviate these adverse effects.

Morphological features of two varieties of Spinacia oleraceae under Cadmium (Cd) stress and foliar applied Sodium nitroprusside (SNP), MgSO4 (Mg-I), and MgO nanoparticles (Mg-II). A = Shoot fresh weight, B = Shoot dry weight, C = Root fresh weight and D = Root dry weight. Mean value is represented by data in A-D. Standard error (SE) represented by error bars. Distinct letters (a, b, c, etc.) denote statistically significant variances among the treatment categories, identified by completely randomized four-way ANOVA followed by the LSD test having significance value p < 0.05.

Morphological parameter of two varieties of Spinacia oleraceae under Cadmium (Cd) stress and foliar applied Sodium nitroprusside (SNP), MgSO4 (Mg-I), and MgO nanoparticles (Mg-II). A = Shoot length, B = Root length, C = Leaf count and D = Leaf area. Mean value is represented by data in A–D. Standard error (SE) represented by error bars. Distinct letters (a, b, c, etc.) denote statistically significant variances among the treatment categories, identified by completely randomized four-way ANOVA followed by the LSD test having significance value p < 0.05.

The chlorophyll a contents of both varieties were highly significantly different (Table 3). All the treatments (SNP, MgSO4/MgO-NPs) highly significantly increased the chlorophyll a content. In Desi Palak, MgO-NPs resulted in a maximum increase of 40%, while MgSO4 resulted in a minimum 18% increase in chlorophyll a content, whereas in Lahori Palak, SNP + MgO-NPs resulted in a maximum of 71%, and MgSO4 resulted in a minimum 27% increase in contrast to the control (Fig. 7A). Under 150 µM Cd stress, both Desi Palak and Lahori Palak presented a significant 58% decrease in chlorophyll a content. Under stress conditions, foliar treatments further increased the chlorophyll a content. In the case of Desi Palak, a greater increase of 198% was shown by the MgO-NPs, whereas a smaller increase of 170% was observed in MgSO4. However, in Lahori Palak, SNP and MgO-NPs resulted in a maximum 210% increase, whereas MgSO4 resulted in a minimum 97% increase in the chlorophyll a content compared with that of the control.

Chlorophyll and carotenoides content of two varieties of Spinacia oleraceae under Cadmium (Cd) stress and foliar applied SNP, MgSO4 (Mg-I) and MgO nanoparticles (Mg-II). A = Chlorophyll a, B = Chlorophyll b, C = Carotenoids and D = SPAD value. Mean value is represented by data in A-C. Standard error (SE) represented by error bars. Distinct letters (a, b, c, etc.) denote statistically significant variances among the treatment categories, identified by completely randomized four-way ANOVA followed by the LSD test having significance value p < 0.05.

Both varieties presented highly marked differences in terms of chlorophyll b content (Table 3). All the foliar treatments had highly significant effects. In Lahori Palak, MgO-NPs increased the maximum chlorophyll b content by 41%, while MgSO4 increased the minimum chlorophyll b content by 13%, whereas in Desi Palak, MgO-NPs increased the maximum chlorophyll b content by 70%, whereas SNP and MgSO4 in combination increased the minimum chlorophyll b content by 51% in relation to the control. Under 150 µM Cd stress, Desi Palak resulted in a 42% reduction in chlorophyll b content, whereas Lahori Palak resulted in a 43% reduction in chlorophyll b content compared with the control. Foliar treatments increased the Chl b content in stressed plants. Compared with the control, SNP + MgO-NPs increased the maximum chlorophyll b content by 149% and MgO-NPs resulted in a maximum 160% increase in the Chl b content in Lahori Palak and Desi Palak, respectively, whereas MgSO4 resulted in minimum 83% and 112% reductions in the chlorophyll b content in Lahori Palak and Desi Palak, respectively (Fig. 7B).

The carotenoid contents of both varieties were highly significantly different. The foliar application of SNP, MgSO4 or MgO-NPs had highly significant effects (Table 3). In Desi Palak and Lahori Palak, MgO-NPs and Palak SNP + MgO-NPs resulted in maximum increases of 66% and 69%, respectively, whereas MgSO4 resulted in minimum increases of 40% and 29%, respectively, in the carotenoid content compared with the control. There was a 57% and 45% reduction in the carotenoid content under 150 µM Cd stress in Desi Palak and Lahori Palak, respectively, compared with the control (Fig. 7C). The interaction effect of each cadmium treatment was significant. Foliar application increased the carotenoid content in Cd-stressed plants. Compared with those in the control, the maximum carotenoid contents in Desi Palak and Lahori Palak were increased by 198% and 172%, respectively, with SNP + MgSO4 and MgSO4 alone resulting in minimum increases in carotenoid contents of 149% and 107%, respectively. The interaction of SNPxMgSO4/MgO-NPxCd was significant.

Both varieties presented nonsignificant differences in SPAD values. However, all foliar treatments had highly significant effects on the SPAD values (Table 4). In Desi Palak, compared with the control, MgO-NPs led to a maximum increase of 26%, whereas MgSO4 resulted in a minimum increase of 4%. In Lahori palak, SNP + MgO-NPs resulted in the greatest increase of 32%, whereas MgSO4 resulted in the lowest 12% increase. Compared with the control, cadmium stress significantly reduced SPAD values by 39% in Desi Palak and 35% in Lahori Palak. Under stress, foliar treatments increased SPAD values, with significant interactions for each treatment and cadmium. In Desi Palak, MgO-NPs resulted in a maximum 71% increase, whereas MgSO4 resulted in a minimum 46% increase. Lahori palak presented the greatest 86% increase with SNP + MgO-NPs and the lowest 56% increase with MgSO4 (Fig. 7D). The overall interaction among all four factors was nonsignificant.

This study investigated the effects of foliar application of SNP, MgSO4, and MgO-NPs on photosynthetic quantum yield parameters (Fv, Fm, Fv/Fm, Fo, and Fv/Fo) in two spinach varieties (Desi Palak and Lahori Palak) under cadmium (Cd) stress. Results showed that foliar treatments significantly enhanced photosynthetic quantum yield, mitigating the negative effects of Cd stress (Table 5). Cd stress significantly reduced Fv by 50%, Fm by 38%, Fv/Fm by 21%, Fo by 18%, and Fv/Fo by 41% in Desi Palak, while it reduced Fv by 43%, Fm by 28%, Fv/Fm by 19%, Fo by 19%, and Fv/Fo by 33% in Lahori Palak. Foliar treatments significantly increased all parameters in both varieties, with MgO-NPs being particularly effective for Desi Palak. Where it increased Fv by 33%, Fm by 24%, Fv/Fm by 7%, Fo by 17%, and Fv/Fo by 32%. Similarly, in Lahori Palak, SNP + MgO-NPs was particularly more effective, and it increased Fv by 53%, Fm by 42%, Fv/Fm by 7%, Fo by 39%, and Fv/Fo by 34% (Fig. 8A–E).

Chlorophyll fluorescence value of two varieties of Spinacia oleraceae under Cadmium (Cd) stress and foliar applied Sodium nitroprusside (SNP), MgSO4 (Mg-I), and MgO nanoparticles (Mg-II). A = Variable flouresence (Fv), B = Maximal fluorescence (Fm), C = Maximum quantum yield of fluorescence (Fv/Fm), D = Minimal Flourescence (Fo), and E = Fv/Fo of fluorescence (Fv/Fm). Mean value is represented by data in A-C. Standard error (SE) represented by error bars. Distinct letters (a, b, c, etc.) denote statistically significant variances among the treatment categories, identified by completely randomized four-way ANOVA followed by the LSD test having significance value p < 0.05.

Cadmium stress significantly reduced all photosynthetic parameters in both spinach varieties. Desi Palak experienced the most severe effects, with a 63% decrease in net photosynthetic rate, a 44% decrease in intercellular CO2 concentration, a 55% decrease in stomatal conductance, and a 59% decrease in transpiration rate. Lahori Palak, while also affected, showed slightly less pronounced reductions, with a 54% decrease in net photosynthetic rate, a 39% decrease in intercellular CO2 concentration, a 50% decrease in stomatal conductance, and a 42% decrease in transpiration rate. Foliar treatments significantly increased net photosynthetic rate in both varieties. Desi Palak showed the highest increase with MgO-NPs (47%), followed by SNP (35%) and MgSO4 (13%). Lahori Palak exhibited the highest increase with SNP + MgO-NPs (43%), followed by SNP (31%) and MgSO4 (10%), see Fig. 9A. Foliar treatments also positively influenced intercellular CO2 levels. Desi Palak showed the highest increase with MgO-NPs (61%), followed by SNP (47%) and MgSO4 (40%). Lahori Palak exhibited the highest increase with SNP + MgO-NPs (77%), followed by SNP (52%) and MgSO4 (43%), see Fig. 9B. Both varieties exhibited significant increases in stomatal conductance under all treatments. Desi Palak showed the highest increase with MgO-NPs (48%), followed by SNP (32%) and MgSO4 (16%). Lahori Palak exhibited the highest increase with SNP + MgO-NPs (41%), followed by SNP (33%) and MgSO4 (8%), see Fig. 9C. Foliar treatments also positively influenced transpiration rates. Desi Palak showed the highest increase with MgO-NPs (36%), followed by SNP (27%) and MgSO4 (14%). Lahori Palak exhibited the highest increase with SNP + MgO-NPs (51%), followed by SNP (37%) and MgSO4 (31%), see Fig. 9D. Foliar application of MgO-NPs significantly increased net photosynthetic rate, intercellular CO2 concentration, stomatal conductance, and transpiration rates in both spinach varieties under Cd stress (Table 4), effectively mitigating the negative effects of Cd on photosynthesis.

Gas exchange parameters of two varieties of Spinacia oleraceae under Cadmium (Cd) stress and foliar applied Sodium nitroprusside (SNP), MgSO4 (Mg-I), and MgO nanoparticles (Mg-II). A = Net Photosynthetic Rate (Pn), B = Stomatal Conductance (gS), C = Intracellular CO2 (Ci) and D = Transpiration Rate (Tn). Mean value is represented by data in A-D.Standard error (SE) represented by error bars. Distinct letters (a, b, c, etc.) denote statistically significant variances among the treatment categories, identified by completely randomized four-way ANOVA followed by the LSD test having significance value p < 0.05.

All the foliar applied treatments resulted in a highly remarkable reduction in the relative membrane permeability (RMP) content (Table 3). Lahori Palak SNP + MgO-NPs resulted in a maximum 44% reduction, whereas MgSO4 resulted in a minimum 10% reduction. However, in Desi Palak, compared with the control, MgO-NPs resulted in a maximum 40% reduction, whereas MgSO4 resulted in a minimum 21% reduction in RMP content. There was a highly significant increase in RMP content (44% and 34%) in Lahori palak and Desi palak, respectively, under 150 µM Cd stress. When untreated stressed plants were treated with a foliar spray of SNP, MgSO4 or MgO-NPs, the RMP content decreased. Lahori Palak SNP + MgO-NPs resulted in a maximum 37% reduction, whereas MgSO4 resulted in a minimum 12% reduction. However, in Desi Palak, MgO-NPs resulted in a maximum of 38%, whereas MgSO4 resulted in a minimum 14% reduction in the EC value and hence in the RMP content (Fig. 10A).

Relative membrane permeability and Water status of two varieties of Spinacia oleraceae under Cadmium (Cd) stress and foliar applied SNP, MgSO4 (Mg-I) and MgO nanoparticles (Mg-II). A = Relative membrane permeability, B = Relative water content, C = Leaf water potential and D = Leaf osmotic potential. Mean value is represented by data in A-D. Standard error (SE) represented by error bars. Distinct letters (a, b, c, etc.) denote statistically significant variances among the treatment categories, identified by completely randomized four-way ANOVA followed by the LSD test having significance value p < 0.05.

In terms of the relative water content (RWC), both varieties presented highly marked differences (Table 3). All the foliar treatments resulted in highly significant increases in RWC. In the case of Desi Palak, MgO-NPs resulted in a maximum 45% increase, whereas MgSO4 resulted in a minimum 16% increase; however, in Lahori Palak, SNP + MgO-NPs increased the maximum 65%, whereas MgSO4 increased the minimum 18% RWC compared with the control. There was a 30% and 28% reduction in RWC in Desi Palak and Lahori Palak, respectively, under 150 µM Cd stress. Under stress conditions, plants treated with SNP, MgSO4 or MgO-NPs presented increased RWC. In Desi Palak, MgO-NPs resulted in a maximum of 78%, whereas MgSO4 resulted in a minimum of 35% increase; however, in Lahori Palak, SNP + MgO-NPs resulted in a maximum of 94%, whereas MgSO4 resulted in a minimum 33% increase in RWC in comparison with the control (Fig. 10B). The overall interaction among all the factors was significant.

Both varieties presented marked differences in terms of leaf water potential (LWP) activity (Table 3). All the foliar treatments had highly significant effects. In the case of Desi Palak, MgO-NPs resulted in a maximum of 64%, whereas SNPs resulted in a minimum 24% increase in LWP activity. However, in Lahori Palak, SNP + MgO-NPs resulted in a maximum of 67%, whereas MgSO4 resulted in a minimum 26% increase in LWP activity. Both varieties resulted in highly significant increases in LWP activity of up to 74% in both Lahori palak and Desi palak under 150 µM Cd stress. The interaction among MgSO4/MgO-NP*Cd was significant. Under stress, when plants were treated with a foliar spray of SNP, MgSO4 or MgO-NPs further upregulated LWP activity. In Desi Palak, MgO-NPs resulted in a maximum of 331%, whereas MgSO4 resulted in a minimum 238% increase in LWP activity. In contrast, in Lahori palak, SNP and MgO-NPs in combination resulted in a maximum of 367%, whereas MgSO4 resulted in a minimum of 233% upregulation of LWP activity in relation to the control (Fig. 10C).

Both varieties presented highly marked differences in terms of leaf osmotic potential (LOP) (Table 3). All the foliar treatments had highly significant effects. In the case of Desi Palak, MgO-NPs resulted in a maximum 20% increase, whereas MgSO4 resulted in a minimum 6% increase in LOP activity. However, in Lahori Palak, SNP + MgO-NPs resulted in a maximum of 36%, whereas MgSO4 resulted in a minimum 11% increase in LOP activity. Both varieties highly significantly upregulated LOP activity by 47% and 56% in Lahori palak and Desi palak, respectively, under 150 µM Cd stress. The interaction among MgSO4/MgO-NP*Cd was highly significant. Under stress, LOP activity was further upregulated in the plants subjected to foliar treatments. In Desi Palak, MgO-NPs resulted in a maximum of 113%, whereas MgSO4 resulted in a minimum of 77% upregulation. In contrast, in Lahori palak, SNP and MgO-NPs in combination resulted in a maximum of 92%, whereas MgSO4 resulted in a minimum of 68% upregulation of LOP activity in relation to the control (Fig. 10D).

The hydrogen peroxide contents of both varieties strongly differed (Table 6). All the treatments resulted in a reduction in the H2O2 content. In the case of Desi Palak, MgO-NPs resulted in a maximum H2O2 content of 37%, whereas MgSO4 resulted in a minimum H2O2 content of 5%. However, in the case of Lahori palak, SNP + MgO-NPs resulted in a maximum reduction of 33%, whereas MgSO4 resulted in a minimum reduction of 13%. Compared with the control, Desi Palak increased H2O2 content by 34%, whereas Lahori Palak increased H2O2 content by 45% under 150 µM Cd stress. The interaction among MgSO4/MgO-NPxCd was highly significant, whereas the interaction among SNPxCd was significant. All the foliar treatments resulted in a reduction in H2O2 content. In Desi Palak, MgO-NPs resulted in a maximum 40% reduction, whereas MgSO4 resulted in a minimum 13% reduction. In contrast, Lahori Palak SNP + MgO-NPs presented a maximum H2O2 content of 30%, whereas MgSO4 presented a minimum H2O2 content of 20% (Fig. 11A).

Effect of foliar application of SNP, MgSO4 (Mg-I) and MgO nanoparticles (Mg-II) on A = Hydrogen peroxide, B = malondialdehyde and C = total soluble proteins in S. oleracea varieties under Cadmium (Cd) stress. Mean value is represented by data in A and B. Standard error (SE) represented by error bars. Distinct letters (a, b, c, etc.) denote statistically significant variances among the treatment categories, identified by completely randomized four-way ANOVA followed by the LSD test having significance value p < 0.05.

All the foliar treatments resulted in highly significant reductions in malondialdehyde (MDA) content (Table 6). In Desi Palak, MgO-NPs resulted in a maximum reduction of 20%, whereas MgSO4 resulted in a minimum 8% reduction. In Lahori palak, SNP + MgO-NPs resulted in a maximum of 32%, whereas MgSO4 resulted in a minimum of 12% reduction in the MDA content relative to that of the control. There was a highly significant 39% and 25% increase in the MDA content in Desi Palak and Lahori Palak, respectively, under 150 µM Cd stress in contrast to the control. The interaction effect of Cd with each treatment was highly significant. The treatment of stressed untreated plants resulted in a reduction in the MDA content. In Desi Palak, MgO-NPs resulted in a maximum 23% reduction, whereas MgSO4 resulted in a minimum 10% reduction. However, in Lahori Palak, SNP + MgO-NPs resulted in a maximum of 36%, whereas MgSO4 resulted in a minimum 8% reduction in the MDA content compared with that of the control (Fig. 11B).

The total soluble protein (TSP) of both varieties highly significantly differed (Table 6). All the treatments resulted in an increase in the amount of TSP. The Desi Palak SNP resulted in a maximum 105% increase, whereas MgSO4 resulted in a minimum 70% increase. However, in Lahori palak, SNP and MgO-NPs resulted in a maximum of 70%, whereas MgSO4 resulted in a minimum of 61% increase in the amount of TSP compared with the control. There was a 25% and 26% increase in the total soluble protein content due to Cd stress in Desi Palak and Lahori Palak, respectively, under 150 µM Cd stress. The interaction of SNPs or MgSO4/MgO-NPs with Cd was highly significant. All the foliar treatments further increased the amount of TSP. The Desi Palak SNP resulted in a maximum 141% increase, whereas MgSO4 resulted in a minimum 102% increase. Similarly, in Lahori Palak, SNP resulted in a maximum of 89%, whereas MgSO4 resulted in a minimum of 75% increase in the amount of TSP compared with the control (Fig. 11C).

Results showed that, in Lahori Palak, Cd stress significantly increased catalase activity by 51%, ascorbate peroxidase (APX) activity by 151%, peroxidase (POD) activity by 43%, and superoxide dismutase (SOD) activity by 32%. Foliar treatments (SNP, MgSO4, and MgO-NPs) further increased these enzyme activities, with SNP + MgO-NPs being particularly effective. SNP + MgO-NPs increased catalase activity by 52%, APX activity by 79%, POD activity by 59%, and SOD activity by 123%. Similarly, in Desi Palak, Cd stress also significantly increased catalase activity by 45%, APX by 158%, POD by 49%, and SOD by 56%. Foliar treatments (SNP, MgSO4, and MgO-NPs) further increased these enzyme activities, with SNP being particularly effective. SNP increased catalase activity by 168%, APX activity by 126%, POD activity by 82%, and SOD activity by 36% (Fig. 12A-D).

Effect of foliar application of SNP, MgSO4 (Mg-I) and MgO nanoparticles (Mg-II) on A = Catalase, B = Ascorbate peroxidase, C = Peroxidase and D = Superoxide dismutase activities in S. oleracea varieties under Cadmium (Cd) stress. Mean value is represented by data in A-D. Standard error (SE) represented by error bars. Distinct letters (a, b, c, etc.) denote statistically significant variances among the treatment categories, identified by completely randomized four-way ANOVA followed by the LSD test having significance value p < 0.05.

Both varieties of spinach presented highly significant differences in terms of leaf proline content (Table 6). All the foliar applied treatments had highly significant effects. SNP resulted in a maximum of 59%, whereas MgSO4 resulted in a minimum 20% increase in proline content in Desi Palak. However, in Lahori palak, SNP + MgO-NPs resulted in a maximum increase of 55%, whereas MgSO4 increased the minimum 10% content in comparison with the control. There was a further 49% and 33% increase in the proline content in Desi Palak and Lahori Palak, respectively, compared with that in the control under 150 µM Cd stress. All the foliar treatments further increased the proline content in the leaves. The interaction of SNPs with Cd was highly significant. In Lahori palak and Lahori palak, the maximum upregulation of 46% and 49%, respectively, was shown by SNP + MgO-NPs and SNP alone, whereas MgSO4 resulted in a minimum of 9% and 14%, respectively, in contrast to the control (Fig. 13A).

Effect of foliar application of SNP, MgSO4 (Mg-I) and MgO nanoparticles (Mg-II) on A = Leaf proline, and B = Total phenolics content in S. oleracea varieties under Cadmium (Cd) stress. Mean value is represented by data in A and B. Standard error (SE) represented by error bars. Distinct letters (a, b, c, etc.) denote statistically significant variances among the treatment categories, identified by completely randomized four-way ANOVA followed by the LSD test having significance value p < 0.05.

Both varieties presented highly marked differences in total phenolic content (Table 6). All the foliar applied treatments resulted in highly significant effects. The Desi Palak SNP presented a maximum elevation of 64%, whereas MgSO4 presented a minimum elevation of 26%. However, in Lahori Palak SNP + MgO-NPs, the maximum phenolic content increased by 64%, whereas MgSO4 increased the total phenolic content by a minimum of 13% compared with that of the control. Compared with that of the control, the total phenolic content of Desi palak further increased by 50%, whereas the total phenolic content of Lahori palak increased by 36%. All the foliar spray treatments further increased the total phenolic content. The interaction of MgSO4/MgO-NPs with Cd was highly valuable. In Lahori Palak and Desi Palak, SNP + MgO-NPs and SNP alone resulted in maximum increases of 51% and 38%, respectively, in the phenolic content, whereas MgSO4 resulted in minimum increases of 15% and 11%, respectively, in contrast to the control (Fig. 13B).

Cd stress significantly reduced K+ and Ca2+ ion content in both varieties. In shoot, Desi Palak showed more pronounced reductions (44% and 46%, respectively) compared to Lahori Palak (34% and 44%, respectively). Results showed that foliar treatments significantly increased the levels of both K+ and Ca2+ ions in both spinach varieties, mitigating the negative effects of Cd stress. In the shoots, both varieties exhibited significant increases in K+ and Ca2+ ion content under all treatments. Desi Palak showed the highest increase in K+ ions with MgO-NPs (45%), while Lahori Palak had the greatest response to SNP + MgO-NPs (40%). For Ca2+ ions, Desi Palak showed the highest increase with MgO-NPs (57%), while Lahori Palak had the greatest response to SNP + MgO-NPs (37%). Similarly, in the roots Desi Palak again showed more pronounced reductions (48% and 25%, respectively) compared to Lahori Palak (50% and 35%, respectively). Both varieties also exhibited significant increases in K+ and Ca2+ ion content with foliar spray of SNP, MgSO4 and MgO-NPs. Desi Palak showed the highest increase in K+ ions with MgO-NPs (93%), while Lahori Palak had the greatest response to SNP + MgO-NPs (79%). For Ca2+ ions, Desi Palak showed the highest increase with MgO-NPs (55%), while Lahori Palak had the greatest response to SNP + MgO-NPs (33%). Cd stress significantly reduced K+ and Ca2+ ion content in both varieties, with Desi Palak showing more pronounced reductions (48% and 25%, respectively) compared to Lahori Palak (50% and 35%, respectively), see Fig. 14A–D.

Effect of foliar application of SNP, MgSO4 (Mg-I) and MgO nanoparticles (Mg-II) on A = K+ ion shoots, B = Ca+ ion shoots, C = K+ ion roots and D = Ca+ ion roots of S. oleracea varieties under Cadmium (Cd) stress. Mean value is represented by data in A–D. Standard error (SE) represented by error bars. Distinct letters (a, b, c, etc.) denote statistically significant variances among the treatment categories, identified by completely randomized four-way ANOVA followed by the LSD test having significance value p < 0.05.

Both varieties presented highly significant differences in shoot Cd content (Table 7). When 150 µM Cd stress was applied, 0.004 ppm shoot Cd was detected in Desi Palak, whereas 0.005 ppm shoot Cd was detected in Lahori Palak. The interaction among SNPs and MgSO4/MgO-NPs was highly significant. Similarly, the interaction among MgSO4/MgO-NP*Cd was highly significant, whereas the interaction among SNP and Cd was significant. All the foliar treatments (SNP, MgSO4, and MgO-NPs) resulted in a decrease in shoot Cd content. In Desi Palak, MgO-NPs reduce the maximum Cd content by 53%, whereas MgSO4 reduces the minimum Cd content by 21%, whereas in Lahori Palak, SNP and MgO-NPs decrease the maximum Cd content by 40%, whereas MgSO4 decreases the minimum Cd content by 14% in relation to the control (Fig. 15A).

Effect of foliar application of SNP, MgSO4 (Mg-I) and MgO nanoparticles (Mg-II) on Cadmium (Cd), Iron (Fe) and Magnesium (Mg) content in S. oleracea varieties under Cadmium (Cd) stress. A = Shoot Cd content, B = Shoot Fe contentb, and C = Shoot Mg content. Mean value is represented by data in A–C. Standard error (SE) represented by error bars. Distinct letters (a, b, c, etc.) denote statistically significant variances among the treatment categories, identified by completely randomized four-way ANOVA followed by the LSD test having significance value p < 0.05.

In terms of shoot Fe content, both varieties presented highly significant differences (Table 7). All the foliar spraying treatments had highly significant effects. In Desi Palak, the maximum increase in the Fe content was caused by 23% MgO-NPs, whereas the minimum increase of 9% was caused by MgSO4. However, in Lahori Palak, SNP + MgO-NPs resulted in a maximum of 48%, whereas MgSO4 resulted in a minimum 17% increase in the Ca+ ion level. Under 150 µM Cd stress, there was a 52% and 47% reduction in the Ca+ ion content in Desi Palak and Lahori Palak, respectively, compared with that in the control. All the foliar treatments resulted in an increase in the Fe content. In Desi Palak, MgO-NPs increased the maximum Fe content by 53%, whereas SNP and MgO-NPs increased the minimum Fe content by 109%, whereas in Lahori Palak, SNP and MgO-NPs increased the maximum Fe content by 82%, whereas MgSO4 increased the minimum Fe content by 81% in relation to the control (Fig. 15B).

Both varieties presented highly significant differences in terms of shoot Mg content (Table 7). All the foliar spraying treatments had highly significant effects. In Lahori palak, the maximum increase in Mg content was given by SNP + MgO-NP, with a value of 52%, whereas the minimum increase of 28% was given by SNP. However, in Desi Palak, the maximum Mg content of the MgO-NPs was 56%, whereas the minimum Mg content of the SNP was 11%. Under 150 µM Cd stress, there was a 39% and 43% reduction in the Mg content in Lahori palak and Desi palak, respectively, compared with that in the control. All the foliar treatments resulted in an increase in the Mg content. In Lahori Palak, SNP + MgO-NPs increased the maximum Mg content by 111%, whereas SNP increased the minimum Mg content by 72%, whereas in Desi Palak, both SNP + MgO-NPs and MgO-NPs alone increased the maximum Mg content by 98%, whereas SNP increased the minimum Mg content by 46%, in contrast to the control (Fig. 15C).

Pearson’s correlation in Fig. 16 showed that Cd in shoot of both S. oleracea varieties is in positive correlation with stress markers i.e. lipid peroxidation in the for MDA, H2O2 and membrane permeability. Whereas, these parameters are negatively correlated with growth, water relation and gas exchange and photosynthesis related parameters. This showed that Cd entry in the shoot caused oxidative stress in S. oleracea plants. It distorted photosynthetic pigments, reduced water uptake and inferred gas exchange attributes. Ultimately, Cd stress reduced growth and biomass of S. oleracea plants. Furthermore, principal component analysis for variety Desi Palak and Lahori Palak shown in Figs. 17 and 18, respectively, validates results of Pearson’s correlation. Cd stress (7) in PCA biplot of both varieties is separated well from control (1) and all remaining treatments applied in this work. This revealed clear cut effects of applied Cd stress on all studied parameters in this work. Further, PCA results showed that first two components PC1 and PC2 had maximum contribution of 93.74% for variety Desi Palak and 93.38% for variety Lahori Palak. For both varieties, parameters aligned with PC2 included stress markers and Cd contents in shoot of plants. While parameters aligned with PC1 included growth, biomass, water relation, photosynthetic pigments, gas exchange and biochemical parameters. Parameters of these two PCs are in negative correlation which showed adverse effects of Cd stress on growth and physiology of S. oleracea plants.

Pearson’s correlation for all studied attributes of S. oleracea plants under the effect of Cd toxicity with or without SNP, MgSO4 and MgO-NPs foliar spray. Upper triangle is for variety Desi Palak and lower triangle is for variety Lahori Palak.

Principal components analysis of all studied attributes of S. oleracea variety Desi Palak under the effect of Cd toxicity with or without SNP, MgSO4 and MgO-NPs foliar spray. The arrow length showed the contribution of each attribute. Numbers 1, 2, 3…, 12 represents different treatments of SNP, MgSO4 and MgO-NPs applied in control (1–6) and Cd stress (7–12) conditions.

Principal components analysis of all studied attributes of S. oleracea variety Lahori Palak under the effect of Cd toxicity with or without SNP, MgSO4 and MgO-NPs foliar spray. The arrow length showed the contribution of each attribute. Numbers 1, 2, 3…, 12 represents different treatments of SNP, MgSO4 and MgO-NPs applied in control (1–6) and Cd stress (7–12) conditions.

Heavy metals (HMs), especially cadmium (Cd), interfere with a number of physiological processes in plants. This interference results in stunted growth, reduced photosynthesis, and insufficient chlorophyll synthesis. In the present study, highly significant reductions in plant growth parameters, such as shoot and root fresh weights, shoot and root dry weights, and shoot and root lengths, were detected under Cd stress. The leaf area and number of leaves per plant also noticeably decreased under Cd stress. A similar decrease was detected in menthol mint plants exposed to Cd stress68. The stunted growth under Cd stress might be due to minimal uptake of water and essential mineral nutrients via roots69, an altered rate of cell division, alterations in cellular expansion and structures70, and the modification of proteins through the disruption of hydrogen–sulfur (H–S) bonds. Consistent with previous research, it was concluded that the elevated uptake and transport of Cd via the phloem interferes with water absorption, thereby decreasing plant biomass. This is a key factor leading to reduced plant growth under cadmium stress71. Figure 19 showed adverse effects of Cd stress on growth of S. oleracea plants.

Growth of S. oleracea plants under Cd stress.

There was a significant improvement in all the morphological factors under the foliar-applied SNP, MgSO4, and MgO-NP treatments. Our findings are supported by previous research showing that NO supplementation increases various plant morphological parameters, such as shoot and root dry weights, as well as shoot length, in plants exposed to heavy metal toxicity from Pb and Cd72. The main causes of such enhancements might include NO and phospholipid bilayer interactions, increased membrane fluidity, and softening of the cell wall, thus stimulating plant growth and cell expansion73. Another reason could be that NO acts as a phytohormone, altering various plants’ physiological processes74. Possible mechanism of SNP, MgSO4 and MgO-NPs entry, target sites and response by plants is shown in Fig. 20.

NO, MgSO4 and MgO-NPs entry mechanism, target sites and responses in plants.

Similarly, MgO-NPs were found to increase both shoot and root length, as well as fresh biomass, in horse gram-bearing plants75. It has been reported that Fe3O4 nanoparticles (NPs) increase the length of both shoots and roots in barley seedlings76. These results were also supported by research by Raliya, et al.51, who reported an increase in the shoot length of cluster bean after foliar application of biologically produced MgO-NPs compared with the control. Similarly, Jayarambabu, et al.77 reported that maize treated with MgO-NPs (biologically synthesized), with an average particle size of 40 nm, presented greater shoot length than did the control when the seed soaking method was used. The increase in plant height due to Mg could be due to its key role in loading phloem and transporting photoassimilates to plant growing Sect78. The increase in leaf number caused by MgO-NPs may result from the activation of the photosynthesis-related enzyme ribulose-1,5-bisphosphate (RuBP) carboxylase, cell division, and osmoregulation by Mg, thus encouraging new sprouts and the production of fresh leaves78.

In the present study, there was a significant reduction in the SPAD value under Cd stress. A similar reduction in SPAD value was observed in tomato plants under Cu stress79. An enzyme, Chlorophyllase regulates chlorophyll molecule turnover. Cd, which is known to increase chlorophyllase activity, thus assisted in the degradation of chlorophyll molecules instead of their synthesis. The enzymes Protochlorophyllide reductase and δ-aminolavulinic acid dehydratase take part in chlorophyll biosynthesis, and chlorophyllase activity stimulation is also decreased by Cd. The combined repressive impact of these reactions resulted in a decrease in the SPAD value80. Hayat, et al.81 reported that Cd-stressed plants subjected to brassinosteroid treatment counteract the above detrimental impacts on Solanum lycopersicum. Cd-induced stressed plants treated with SNP, MgSO4, or Mg-NPs presented increased SPAD values. A similar increase was previously reported in Oryza sativa plants via the application of zinc oxide nanoparticles82 and in faba bean, soya bean and oregano via foliar application of Mg83. Mg is not only a central atom in the chlorophyll molecule but also mandatory for chlorophyll biosynthesis and Mg-chelatase activation84. NO is directly internalized by proteins (low-molecular-weight), along with transcription factors, repressors and activators, via sulfhydryl groups. This interconnection impacts transcription as well as translation by affecting the expression of genes capable of the production of enzymes essential for chlorophyll biosynthesis, and the NO-induced impact on transcription and/or translation may involve the expression of specific genes responsible for the synthesis of enzymes involved in chlorophyll biosynthesis85.

The gas exchange parameters and the maximum efficiency of photosystem II (PSII), represented by Fv/Fm, a stress indicator for plants86, were significantly affected by Cd stress, as indicated by a notable reduction in their values. Our results are consistent with the findings of Zouari, et al.87, who examined the impact of CdCl2 on gas exchange characteristics in olive plants and reported significant decreases. Similarly, Farooq, et al.88 reported comparable results in cotton seedlings. Additionally, fenugreek trees grown in Cd-contaminated soil presented reduced stomatal conductance and transpiration rates, as noted by Rehman, et al.89. Furthermore, a decrease in water loss via transpiration and reduced stomatal gas conductance were observed in barley grown in Cd-contaminated environments90. Similarly, increased Cd levels led to decreased stomatal conductance and transpiration rates in carrots91, maize92, and spinach93. Moreover, other plant species, such as corn92, mustard greens94, and field mustard95,96, manifested lower carbon dioxide (CO2) concentrations in their cells.

Numerous studies have shown that metals such as Cd impact gas exchange in plants through stomatal closure, lessening CO2 uptake, and changing water relationships97,98. Furthermore, cadmium-induced suppression of photosynthesis might be due to disturbances in the electron transport chain (ETC), obstruction of vital chlorophyll synthesis enzymes, intervention with the PS-II reaction center, substitution of central Mg ions in chlorophyll, and decreased phosphorus absorption, which are essential for pigment production. Cadmium can harm the microscopic structure of chloroplasts by disrupting the organization of granum stacks86. The ability of chloroplasts to absorb light energy significantly decreases in the presence of Cd, which impacts several photosynthesis-related functions99.

An increase in the fluorometric parameter Fo indicates photo damage, whereas a decrease in the Fm parameter indicates increased nonradiative energy. Fv/Fo specifies the number and size of active photosynthetic centers in chloroplasts and thus the photosynthetic strength of plants100. In the present study, the Fv and Fm parameters decreased under Cd stress. A similar reduction in the Fm value was observed in three species of mangrove plants grown under saline conditions101. A reduction in the Fm value might be linked to depletion of water splitting enzyme complex activity and possibly related cyclic electron transport in and throughout the PS II system101. The decrease in Fv/Fm and PSII efficiency suggests that Cd stress impaired PSII activation. This phenomenon might be due to harm to the antennae pigment and quinone (QA) reoxidation disturbance, which evolves from a decrease in or partial obstruction of electron transport from PSII to PSI86. However, foliar application of SNP, MgSO4, or MgO-NPs mitigated the effects of Cd stress. NO might stop photosynthetic pigment degradation by preserving the photosynthetic membrane, D1, D2 proteins, rubisco and cytochrome b6/f. The observed increases in the photosynthesis rate, transpiration rate, stomatal conductance, and water use efficiency with SNP treatment might be due to the impact of NO on stomatal closure86.

The increased absorption of iron (Fe) and magnesium (Mg) due to NO resulted in higher chlorophyll production, which, in turn, improved the rates of transpiration and photosynthesis102. When magnesium is deficient, reduced chlorophyll levels lead to decreased light energy absorption. Therefore, the elevated magnesium levels resulting from foliar application of MgO-NPs increased photosynthetic activity and light absorption, as indicated by the improved Fv/Fm ratio and increased yield of ΦPSII103. In line with this,104 reported that nanoparticles (NPs) enhanced light-dependent photosynthetic reactions in Lemna minor. Similarly, Krishnaraj, et al.105 reported that silver NPs might interact with proteins associated with photosystems. Our results support the idea that MgO-NPs improve light utilization efficiency, thereby benefiting both growth and photosynthesis. Magnesium has been shown to positively impact the net assimilation of carbon dioxide in various plant species and reduce the negative effects of heavy metals or metalloids on plants103.

Under Mg deficiency, the Fv/Fm ratio is the lowest. This reduction in Fv/Fm might be due to lower total chlorophyll contents in the Mg-deficient state106. Mg acts as a cofactor for numerous enzymes involved in carbon fixation and metabolism during photosynthesis. Deficiency of Mg results in sucrose accumulation on leaf surfaces prior to any notable depletion in chlorophyll content or photosynthetic activity107.

In the present study, physiological parameters, including Chl a, Chl b, and Carotenoids, were highly significantly reduced under Cd stress. An analogous decrease was previously noted in pea (Pisum sativum)86. Chlorophyll levels in rice108, barley90, tomato109,110,111, corn112 and cabbage113,114 exposed to Cd. This decline may be attributed to cadmium-induced increases in chlorophyllase activity, the suppression of enzymes crucial for photosynthetic pigment synthesis, and the oxidative stress-induced degradation of photosynthetic pigments115.

When plants were subjected to foliar application of SNP, MgSO4 or MgO-NPs, physiological parameters, including Chl a, Chl b, and Carotenoids values, increased. A comparable rise in chlorophyll levels resulting from NO supplementation was observed in Pisum sativum86, a finding supported by studies in Zea mays116, Lactuca sativa117,118, fava bean119 and Brassica juncea120 under Cd stress. Additionally, Kanjana78 reported a similar chlorophyll increase in cotton plants due to magnesium.

The increase in chlorophyll content induced by NO supplementation might stem from decreased oxidative stress and protection of cadmium-exposed plants from chlorophyll pigments. Carotenoids, which function as light-absorbing pigments, have the ability to safeguard chlorophyll and membranes against harm by extinguishing triplet chlorophyll and eliminating oxygen from excited chlorophyll86. Moreover, the increase attributed to Mg could be linked to its unique presence within the porphyrin ring framework, which plays a pivotal role in chlorophyll synthesis facilitated by Mg chelatase activity. As a fundamental element in all chlorophyll types, Mg is indispensable for plant photosynthesis. Without Mg, chlorophyll is incapable of harnessing the solar energy essential for photosynthesis. Additionally, Mg significantly influences partitioning, assimilate utilization, photophosphorylation (including ATP production), and photooxidation processes within leaf tissues121,122.

In the present study, all the antioxidant levels as well as the proline and total phenolic contents were upregulated under Cd stress. In combination with SNP, MgSO4/MgO-NPs further increased the levels of all the above parameters. However, the MDA, RMP and H2O2 values decreased under Cd stress and increased in response to foliar application. Under stress conditions, the accumulation of H2O2 in plants is attributed to various adverse factors, such as lipid membrane harm, stomatal closure, reduced leaf moisture, oligosaccharide production, osmotic balance adjustments and increased osmolyte levels25. In response to unfavorable environmental conditions, plants increase the generation of ROS. To counteract this, plants deploy a defense strategy through the synthesis of numerous enzymatic antioxidant proteins, including POD, CAT, and SOD, aimed at mitigating the generation of ROS in harsh environmental conditions123. Similarly, in response to elevated levels of ROS, the total phenolic content of the plants increased under all the treatments. This result was supported by124. However, a similar increase in phenolic and proline contents under abiotic stress was reported by125. Previous findings have also revealed a decrease in MDA levels due to the application of proline126.

Numerous research findings indicate that the application of nitric oxide (NO) serves to deter the buildup of ROS amid stressful conditions, altering gene expression as a signaling agent. Consequently, this safeguard mechanism aids in shielding plant cells from the oxidative harm incurred during stress127. In times of stress, SOD facilitates the transformation of O2 and H2O2, diminishing their accumulation, whereas POD guarantees the elimination of ROS by eradicating H2O2 within the extracellular environment. Conversely, CAT detoxifies ROS compounds by converting H2O2 to O2128. The improved antioxidant capabilities exhibited by MgO NPs suggest their substantial capacity to combat oxidative stress, maintain the integrity of chloroplast membranes, minimize the production of free radicals, and preserve the cellular balance of ROS129. The high proline content under stress might be due to the following reasons: as a compatible entity within cells, proline is sproutable in facilitating osmotic adaptation, stabilizing subcellular frameworks, and eliminating free radicals. Additionally, the accumulation of proline might mitigate stress-induced cellular acidification or stimulate oxidative respiration to fuel the recuperative process. Furthermore, increased proline synthesis during stress periods could sustain NAD(P)/NAD(P)H ratios within ranges conducive to normal metabolic functions130.

There was a significant reduction in the RWC, leaf water content and osmotic potential under Cd stress. Similar results were reported by131. Osmotic adaptation in plants could stem from the buildup of inorganic or organic solutes, yet the proportionate input of these substances fluctuates among different species, cultivars, and even compartments within a single plant132.

An overabundance of cadmium (Cd) resulted in a reduction in manganese (Mn), magnesium (Mg), calcium (Ca), and potassium (K) levels in the present study, potentially resulting in a deficiency of ions in plants. These findings indicate that the stress from Cd impedes root-to-shoot ion transfer in plants. Cadmium impacts nutrients by competing for identical transport systems133. The stability of ions within a cell is closely linked to how plants adapt to the toxicity of heavy metals73. The K+ ion content increased due to the foliar application of SNP or MgSO4/MgO-NPs. The application of Mg via roots induces competition for K+ uptake from the soil. However, when applied foliarily, Mg does not compete for K+ uptake, as it is absent as a competitor at the root level, and the decreased Mg content at the root surface increases the uptake of K+ ions by roots78. The elevation in Ca and K concentrations resulting from NO application can be rationalized through (i) the capacity of Ca and K to displace Na, arising from the mutual rivalry between ions for transport sites on carrier proteins, or (ii) a reduction in the plasma membrane pH gradient size, leading to a diminished net intake of Na and the suppression of membrane-bound carrier proteins, hence preserving the equilibrium of Ca and K134. Our findings revealed a highly significant reduction in the Fe and Mg contents of the shoots but a marked increase in the accumulation of Cd in the shoots of both varieties under stressed conditions. However, as foliar treatment with SNP, MgSO4 or MgO-NPs, either alone or in combination, alleviated the toxic effects of Cd and not only decreased Cd accumulation in shoots but also increased the Fe and Mg contents. A similar reduction in Fe and Mg was observed in Catharanthus roseus under Cd stress, where increasing CAT and POD activity could play a role in mitigating Cd-induced oxidative stress in plants135. An improvement in Cd toxicity and nutrient uptake due to foliar application of SNPs was also previously reported in Amaranthus tricolor by24, who reported that SNPs act as signaling molecules that mediate the relationships among tolerance-related genes and their expression through different regulatory genes and their pathways. Hence, oxidative stress induced by Cd can be alleviated by reducing the H2O2 and MDA contents, which are increased under stress conditions. The other reason for this mitigation may be the formation of a Cd‒NO complex73. An improvement in nutrient uptake due to nanoparticles was reported previously in Oryza sativa45 and Brassica juncea120. Hence, all the above findings showed that foliar application of SNP, MgSO4 and MgO-NPs not only mitigated cadmium toxicity in spinach but also improved morphophysiological and biochemical processes in plants. Additionally, it improved inorganic ion uptake and the uptake of other necessary nutrients in plants.

The effects of foliar application of sodium nitroprusside (SNP), magnesium sulfate (MgSO4) and magnesium oxide nanoparticles (MgO-NPs) on the growth, physiology, biochemistry, and gas exchange parameters of two varieties of spinach (Spinacia oleracea) under cadmium (Cd) stress were examined. Two varieties of Spinacia oleracea L. (spinach plant Desi Palak/Lahori Palak) were used. Two concentrations of cadmium (0 µM and 150 µM) in the form of cadmium chloride (CdCl2) were used. Two strains of SNP (0 ppm and 100 ppm), two strains of each form of Mg (0 ppm and 200 ppm), were foliar sprayed on specific plants alone or in combination. Both varieties behaved similarly under Cd stress and caused reductions in growth, physiology, gas exchange, water content parameters and inorganic ion activity. However, the biochemical parameters and RMP, MDA, and H2O2 contents were increased. However, all foliar spray treatments increased growth, physiological and gas exchange parameters, water content and inorganic ion activity. However, it reduced the MDA, RMP, and H2O2 contents. The foliar treatments further increased the biochemical parameters. Desi Palak showed the greatest effect under foliar application of MgO-NPs. However, Lahori palak showed the greatest effect under the SNP + MgO-NP treatment. Thus, foliar application of SNP, MgSO4 and MgO-NPs alleviated the toxicity caused by Cd stress in spinach. Thus, we concluded that foliar treatments with NO and MgSO4/MgO-NPs, both alone and in combination, improved spinach plant growth and all physiological and biochemical parameters. Further field trials should be performed on different vegetative plants using both NO and MgSO4/MgO-NPs as novel techniques to alleviate the toxicity of Cd to Spinacia oleracea plants.

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

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The authors would like to extend their sincere appreciation to the Researchers Supporting Project Number (RSP2025R182) King Saud University, Riyadh, Saudi Arabia.

Researchers Supporting Project Number (RSP2025R182) King Saud University, Riyadh, Saudi Arabia.

Department of Botany, Division of Science and Technology, University of Education, Lahore, Pakistan

Hafsa Taj, Zahra Noreen, Sheeraz Usman, Anis Ali Shah & Maham Rafique

Department of Chemistry, Division of Science and Technology, University of Education, Lahore, Pakistan

Muhammad Aslam

University Centre for Research and Development, Chandigarh University, Gharuan, Mohali, Punjab, 140413, India

Vaseem Raja

Botany and Microbiology Department, College of Science, King Saud University, Riyadh, Saudi Arabia

Mohamed A. El-Sheikh

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HT; Experimentation and Methodology, ZN; Supervision and Validation, MA; writing-revised draft preparation, SU & MR; writing-original draft preparation and Statistical analysis, AAS; Conceptualization, Data curation and Formal analysis, VR & MAE; Conceptualization and Investigation. All authors read and approved the final manuscript.

Correspondence to Zahra Noreen or Anis Ali Shah.

The authors declare no competing interests.

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Taj, H., Noreen, Z., Aslam, M. et al. Effects of SNP, MgSO4, and MgO-NPs foliar application on Spinacia oleracea L. growth and physio-biochemical responses under cadmium stress. Sci Rep 14, 26687 (2024). https://doi.org/10.1038/s41598-024-77221-z

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Received: 14 September 2024

Accepted: 21 October 2024

Published: 04 November 2024

DOI: https://doi.org/10.1038/s41598-024-77221-z

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