Morphophysiological, biochemical, and nutrient response of spinach (Spinacia oleracea L.) by foliar CeO2 nanoparticles under elevated CO2

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Scientific Reports volume 14, Article number: 25361 (2024 ) Cite this article lanthanum nitride

Nanomaterials offer considerable benefits in improving plant growth and nutritional status owing to their inherent stability, and efficiency in essential nutrient absorption and delivery. Cerium oxide nanoparticles (CeO2 NPs) at optimum concentration could significantly influence plant morpho-physiology and nutritional status. However, it remains unclear how elevated CO2 and CeO2 NPs interactively affect plant growth and quality. Accordingly, the ultimate goal was to reveal whether CeO2 NPs could alter the impact of elevated CO2 on the nutrient composition of spinach. For this purpose, spinach plant morpho-physiological, biochemical traits, and nutritional contents were evaluated. Spinach was exposed to different foliar concentrations of CeO2 NPs (0, 25, 50, 100 mg/L) in open-top chambers (400 and 600 CO2 μmol/mol). Results showed that elevated CO2 enhanced spinach growth by increasing photosynthetic pigments, as evidenced by a higher photosynthetic rate (Pn). However, the maximum growth and photosynthetic pigments were observed at the highest concentration of CeO2 NPs (100 mg/L) under elevated CO2. Elevated CO2 resulted in a decreased stomatal conductance (gs) and transpiration rate (Tr), whereas CeO2 NPs enhanced these parameters. No significant changes were observed in any of the measured biochemical parameters due to increased levels of CO2. However, an increase in antioxidant enzymes, particularly in catalase (CAT; 14.37%) and ascorbate peroxidase (APX; 10.66%) activities, was observed in high CeO2 NPs (100 mg/L) treatment under elevated CO2 levels. Regarding plant nutrient content, elevated CO2 significantly decreases spinach roots and leaves macro and micronutrients as compared to ambient CO2 levels. CeO2 NPs, in a dose-dependent manner, with the highest increase observed in 100 mg/L CeO2 NPs treatment and increased roots and shoots magnesium (211.62–215.49%), iron (256.68–322.77%), zinc (225.89–181.49%), copper (21.99–138.09%), potassium (121.46–138.89%), calcium (118.22–91.32%), manganese (133.15–195.02%) under elevated CO2. Overall, CeO2 NPs improved spinach growth and biomass and reverted the adverse effects of elevated CO2 on its nutritional quality. These findings indicated that CeO2 NPs could be used as an effective approach to increase vegetable growth and nutritional values to ensure food security under future climatic conditions.

Climate change has emerged as a pressing concern in the field of agriculture, posing a significant threat to global food security and sustainability1. The elevated CO2 has been dramatically increased to 407 ppm under anthropogenic activities, which is predicted to reach 550 ppm annually by 20502. Under the current scenario of climate change, the continuous rise in atmospheric CO2 is widely recognized as a significant factor impacting global crop production and there is a growing concern regarding the change in crop nutritional quality threatening food security3. Elevated CO2 increases primary carbon sources which have distinct and significant effects on crop growth and development, including biomass, photosynthesis, and metabolite profiling, regardless of any stress4. In recent years, many studies reported that elevated CO2 levels enhanced dry matter content in root and stem vegetables, leading to greater yield, and this increase is linked to plant physiological mechanisms. However, the response may vary depending on variety and species5,6. Additionally, elevated CO2 reportedly improves photosynthesis and antioxidant defense metabolism by decreasing photorespiration7,8.

Elevated CO2 affects plant nutrient composition by decreasing the content of macro and micronutrients primarily by increasing leaf intracellular concentration and altering plant physiological functions9,10,11,12 exacerbating the current challenges of food security13. Many studies observed that leafy vegetables including spinach, and lettuce, and the grains of major staple food crops including rice and wheat showed a decrease in the content of zinc (Zn) copper (Cu), and Sulphur (S) posing a serious threat on widespread global malnutrition14. A meta-analytic study by Dong et al.15 also reported that elevated CO2 increased the glucose, fructose, and total soluble sugar concentrations in edible portions of vegetables and reduced the iron (Fe), zinc (Zn), and magnesium (Mg) concentrations by 16%, 9.4%, and 9.2% respectively. Wang et al.16 reported that elevated CO2 reduced potassium (K) concentration in wheat root and shoot. Keeping in view the current and future climatic scenarios, it is crucial to devise an approach to enhance crop growth and nutritional quality simultaneously to ensure sustainable food production.

Engineered nanoparticles NPs are widely used in agriculture due to their small size, unique structure, and large surface area17. Nanoparticle (NPs) supplementation has been demonstrated to prevent nutrient loss and improve crop output sustainably18. Nano fertilizers such as those based on calcium (Ca), zinc (Zn), titanium (Ti), and cerium (Ce) nanoparticles are used in plant nutrition to enhance efficiency and reduce nutrient leakage from fertilizers19. Among these NPs, cerium oxide (CeO2 NPs), a lanthanide metal oxide has shown beneficial functions20,21,22 and possesses excellent reactive oxygen species (ROS) scavenging ability by augmentation of the antioxidant-dense mechanism23 occurs through upregulation of antioxidant activities such as superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), helps to maintain the integrity of chloroplast and cell wall thereby avoiding membrane damage24,25. The unique physiochemical and photoelectric characteristics of CeO2 nanoparticles could be utilized to boost plant photosynthesis through improvements in energy capture and other physiological parameters related to gas exchange attributes, including stomatal conductance and transportation rate26,27,28. Moreover, CeO2 NPs have been known to significantly modify the agronomic traits and nutritional values of plants29. Lower concentrations of CeO2 NPs (10–100 mg/L) improved the growth, biomass, and chlorophyll concentration of maize and peas, and nutrient homeostasis30,31. It has been reported that increased levels of CO2 will alter the behavior of fertilizer and NPs32,33. A recent study by Ayub et al.34 demonstrated that CeO2 NPs enhanced Fe and Zn contents in maize shoots while another study by Rico et al.29 showed that CeO2 NPs increased Mn and S content in wheat.

Currently, few studies examined the impact of nanoparticles in conjunction with elevated CO2 on plant growth and physiology. Du et al.35 reported that elevated CO2 concentrations might alter the impact of nTiO2 on crop nutritional quality. Another study by Saleh et al.36 showed that elevated CO2 mitigated NiO NPs induced phytotoxicity and maintained redox homeostasis. Alsherif et al.32 investigated the synergistic effect of elevated CO2 and selenium nanoparticles on the specified metabolites, antioxidants, and certain photosynthetic parameters and demonstrated a beneficial outcome not observed with individual applications. The interplay between CeO2 nanoparticles and the physiological-biochemical processes of plants is crucial37 under elevated CO2 for long-term application in agriculture and to improve crop productivity and resilience.

Spinach (Spinacia oleracea L.) is a major leafy vegetable with high nutritional value, consumed all over the world due to its rich content of vitamins and nutrients38,39. However, it is well documented that the nutritional quality of leafy vegetables including spinach deteriorated under elevated CO214. The decline in macro and micronutrients poses a significant challenge to food security affecting the nutritional quality of crops and human health. Given the benefits of CeO2 NPs and their ability to enhance nutrient uptake, it is crucial to understand how this interaction plays out under future climatic conditions. Hence, the present study aimed to evaluate the effect of CeO2 NPs on the growth, biochemical, and nutritional quality of spinach, which have not yet been explored under elevated CO2 levels. The main objective of this study was to examine how CeO2 NPs at different concentrations influence nutrient composition and uptake in plants grown under elevated CO2. For this purpose, a pot experiment was conducted under ambient and elevated CO2 levels. We hypothesized that (i) CeO2 NPs and elevated CO2 interactively could improve plant growth (ii) CeO2 NPs would recover and increase spinach nutritional content under elevated CO2 (iii) CeO2 NPs foliar application could regulate stomatal and biochemical traits in spinach in a dose additive manner under elevated CO2. The findings of the present study will provide new insights into plant-NP interaction and advance our understanding of applying agro-nanotechnology under future climatic conditions for better crop quality and sustainable agriculture.

For the present experiment, the soil was collected from Qindong village, Changshu City, Jiangsu province, China (31.6203° N, 120.8493° E) from the upper 0–20 cm of soil layer. The soil was air-dried throughout the week and sieved (0.7 mm) to remove unwanted debris and roots. After this, the sieved soil was characterized for sand, silt, and clay contents according to the standard procedure outlined by Whiting40. The pH of prepared soil was determined with a calibrated pH meter (OHSAUS-ST3100-B), electrical conductivity (EC) with a conductivity meter (FiveEasy Plus FE38), and cation exchange capacity was also calculated41. The Mehlich 3 extraction method was used to measure plant available nutrients in soil42. The metal concentration in the soil was determined by following the method described by Park et al.43. The basic soil physicochemical properties are shown in (Table 1).

The open-top chambers system (OTCs) was located in the School of Environment, Nanjing University, Nanjing, Jiangsu Province, China. The research study conducted by Yin et al.44 provided information on the specifics of OTCs. The experiment took place in two OTCs with a diameter of 1 m and a height of 2 m, in an octagonal shape. Two of the OTCs were randomly selected (ambient; CO2 at 400 ± 10 µmol/mol) and the other (elevated; CO2 at 600 ± 10 µmol/mol). The CO2 flow rate was adjusted, and the CO2 concentration in the elevated chamber was measured using a CO2 analyzer (Li-7000, Li-Cor Inc., Lincoln, Nebraska, USA) until it reached the desired concentration. Potted plants were evenly distributed across the chambers and rotated to ensure adequate sunshine.

A pot experiment was carried out, and each plastic brown cylindrical pot (weight 54 g, diameter 14 cm, height 12 cm) was filled with 1 kg of soil and placed into the open-top chambers. Shubo No. 23 variety of spinach (Spinacia oleracea L.) was used in the study and purchased from Hongshu Seed Industry Technology Company Limited, Beijing, China. Before sowing, spinach seeds underwent disinfection with the solution of H2O2 (2.5%, v/v) and then thoroughly washed with distilled water (DW). Seven seeds of S. oleracea L. have been sowed carefully in each pot. Three seedlings in each pot were left after germination. For foliar spray, cerium oxide nanoparticles (99.9%, < 25 nm) were purchased from Sigma Chemical Company Limited, (Shanghai, China). CeO2 NPs solution was prepared by dissolving CeO2 NPs in DW and sonicated (Ultrasonic Bath XO-4200DT) at 35 °C at 40 kHz for 30 min in water for dispersion. Four different treatments of CeO2 NPs (eCO2 0, 25, 50 and 100 mg/L) and (aCO2 0, 25, 50 and 100 mg/L) with very low and environmentally appropriate doses were applied by hand-held spray bottles to leaves at 72-h intervals. To enhance the adhesion and penetration of CeO2 NPs to leaf surfaces, Tween® 80 was utilized. Plants were watered with tap water having EC (354.4 µS/cm) and pH (8.07). All of the measured parameters were evaluated four weeks after the CeO2 NPs foliar spray was applied while climatic stress persisted until the experiment’s completion. The treatment plan used in this study is shown in (Table 2).

Photosynthetic pigments were measured by following the method proposed by Arnon45. Briefly, the freshly developed leaves were crushed for the extraction of chlorophyll by using 95% ethanol and measured spectrophotometrically (UV2350 UV–Vis spectrophotometer; Unico Shanghai Instrument Co., Ltd). Spinach leaf gas exchange parameters (photosynthesis rate, transpiration rate, stomatal conductance, and intracellular carbon dioxide concentration) were determined by selecting 2nd leaf of each plant from the top side with a LI-6800; Li-Cor Inc., Lincoln, Nebraska, USA.

After harvesting spinach plants, the roots were gently washed with distilled water to eliminate the excessive material and soil. Root length and shoot length were measured by using a meter scale from top to bottom and the total number of leaves was also counted. After being dried in an oven for 48 h at 72 °C, shoot mass and root dry weight were measured. Other plant samples were instantly frozen at − 20 °C for biochemical analyses. For the estimation of superoxide dismutase (SOD) activities, samples were diluted in phosphate buffer (0.5 M at pH of 7.8) and measured as mentioned by Zhang46. Ascorbate peroxidase (APX) activity was estimated by following the method of Nakano and Asada47 and catalase (CAT) activity was measured by following the method of Aebi48. Malondialdehyde (MDA) content was also estimated according to Jaleel49 using a thiobarbituric acid test. The hydrogen peroxide (H2O2) contents were calculated via titanium reagent used by Patterson50. Membrane integrity was determined by following Liu et al.51 with slight modifications (dividing leaves into 4 pieces of 1 cm × 1 cm segments instead of weighing the same number of leaves).

For Ce (cerium) and macro and micronutrient concentration, the dried-powdered (0.1 g) were digested by the mixture of H2O2 and HNO3 (v/v: 4:1) using a hotplate at 160 °C52. Following filtration, the nutrient and Ce concentration was analyzed by ICP-OES (Optima 7000DV ICP-OES, Perkin-Elmer). Samples of spinach leaves standard reference materials GBW10015a (China National Center for Standard Material) were also digested and examined. All element recoveries were 90–99% content.

Leaf tissues have been soaked in glutaraldehyde (4%) in phosphate buffer 25 mM; pH 7 and stored overnight at 4 °C. Following that, the samples were washed thrice for 10 min in a sodium phosphate buffer (25 mM), rinsed with DW, and then dried using a graded series of ethanol (10–90%) and lasting 30 min for each stage53. After 100 s of gold–palladium ion sputtering (HITACHI E-1010), the dehydrated samples were placed on aluminum stubs with carbon double-sided adhesive tabs and examined under SEM; Hitachi Model S-3400N.

The statistical software IBM SPSS (V. 2020) was used for statistical analysis of the collected data and ANOVA was performed followed by Tukey’s HSD post hoc test to analyze significant differences among treatments. The level of significance was (p ≤ 0.05). Lower case alphabetic letters were used to indicate the significant and non-significant differences among treatments54.

For this study seeds of spinach (Spinacia oleracea L. cv Shubo No. 23) were purchased from Hongshu Seed Industry Technology Company Limited, Beijing, China, and this experiment on plants complied with national, and international guidelines and legislation.

Elevated CO2 in general increased growth attributes of spinach plants (Table 3). Specifically, the spinach root length and shoot length increased by 40.51%, and 10.54%, respectively, compared to ambient CO2. The shoot fresh weight increased by 15.53%, the shoot dry weight by 15.95%, the root fresh weight by 13.93%, the root dry weight by 12.47%, the number of leaves by 20.55%, and the leaf area by 16.07% as compared to ambient CO2 without foliar application of CeO2 NPs. CeO2 NPs foliar application further enhanced plant growth attributes at all concentrations. A significant (p ≤ 0.05) and maximum increase in these parameters was observed at the highest concentrations of CeO2 NPs treatments. Specifically, the maximum increase in root length and shoot length was found in 50 and 100 (mg/L) CeO2 NPs treatments by 75.80%, 89.75% and 53.82%, 84.69% respectively, compared to ambient CO2. Similarly, the highest increase in shoot fresh weight and root fresh weight was observed in 50 and 100 (mg/L) CeO2 NPs treatments by 68.51%, 85.86% and 80.73%, 96.72% respectively, as compared to ambient CO2. The shoot and root dry weight yielded the same pattern with an increase of 74.21%, 93.71%, and 78.19%, 88.56% respectively, in 50 and 100 (mg/L) CeO2 NPs. The number of leaves and leaf area per plant also increased significantly (p ≤ 0.05) by 71.61%,45.20%, and 92.22%, 63.45% respectively, in 50 and 100 (mg/L) CeO2 NPs treatments. The results showed that CeO2 NPs significantly promoted spinach plant growth under elevated CO2.

To understand the basis of enhanced growth by elevated CO2 with NPs application, we measured the photosynthetic pigments, gas exchange attributes, and stomatal response. In our results, we observed that elevated CO2 alone without any foliar treatment significantly increased spinach photosynthetic pigment including chlorophyll a, b, and total chlorophyll by 143.39%, 134.52%, and 165.16% as compared to ambient CO2 but a more prominent increase was found when spinach plants were exposed to 50 and 100 (mg/L) CeO2 NPs treatments under elevated CO2 (Fig. 1). The chlorophyll a, b, total chlorophyll, and carotenoids were significantly increased by 300.01%, 313.09%, 351.68%, 85.37%, and 364.15%,383.09%, 398.87%, 114.59% respectively, in 50 and 100 (mg/L) CeO2 NPs treatments as compared to its ambient control (Fig. 1A,D).

The effect of cerium oxide nanoparticles on the chlorophyll contents (A) chlorophyll a, (B) chlorophyll b, (C) total chlorophyll and (D) carotenoids in spinach under ambient and elevated CO2. Different letters among columns indicate statistically significant differences at p ≤ 0.05.

Elevated CO2 significantly influenced spinach leaf gas exchange parameters as compared to ambient CO2 (Fig. 2). The net photosynthetic rate (pn) and intracellular CO2 concentration (Ci) significantly increased by 153.12%and 94.88% in spinach leaves as compared to ambient CO2 (Fig. 2A,B). However, stomatal conductance (gs) and transpiration rate (Tr) reduced significantly (p ≤ 0.05) by 82.21% and 56.02% under elevated CO2 (Fig. 2C,D). In contrast, Spinach plants treated with foliar CeO2 NPs showed increased Tr and gs with increasing foliar CeO2 NPs concentration. The highest increase in transpiration rate (78.95%, 95.65%) and stomatal conductance (8.90%, 39.88%) was observed in 50 and 100 (mg/L) CeO2 NPs as compared to their respective control. Whereas, no significant increase was observed in 25 (mg/L) CeO2 NPs treatment. The SEM image of spinach leaf stomata under both ambient CO2 and elevated CO2 (Figs. 3, 4). Elevated CO2 reduced plant stomata length and width as compared to ambient CO2 (Fig. 4A–E). On the contrary, spinach leaves stomatal length, width, and apertures improved with increasing concentration of NPs elevated CO2 (Fig. 4). Overall, 100 (mg/L) CeO2 NPs were found to be more effective in increasing spinach photosynthetic performance.

The effect of cerium oxide nanoparticles on the gas exchange parameters (A) photosynthetic rate, (B) CO2 concentration intercellular, (C) stomata conductance and (D) transpiration rate in spinach under ambient and elevated CO2. Different letters among columns indicate statistically significant differences at p ≤ 0.05.

Scanning electron microscope (SEM) images of stomata showed the responses of cerium oxide nanoparticles at different treatments (0, 25, 50, and 100 mg/L) on the stomatal aperture of spinach leaves under ambient CO2. (A–D) SEM (scale bar = 100 µm) overview of leaf epidermal surface. The arrow shows the number of stomata. (E–H) SEM (scale bar = 10 µm) showing the structure of guard cells and stomatal aperture.

Scanning electron microscope (SEM) images of stomata showing the responses of cerium oxide nanoparticles at different treatments (0, 25, 50, and 100 mg/L) on the stomatal aperture of spinach leaves under elevated CO2. (A–D) SEM (scale bar = 100 µm) overview of leaf epidermal surface. The arrow shows the number of stomata. (E–H) SEM (scale bar = 10 µm) showing the structure of guard cells and stomatal aperture.

Reactive oxygen species (ROS) are produced in response to oxidative stress in plants. In our study, no obvious increase was found in ion leakage in any of the NPs treatments, demonstrating no damage to the spinach leaf cell membrane under elevated CO2. Furthermore, no significant change in hydrogen peroxides (H2O2) and malonaldehyde (MDA) content was found in all NPs treated plants, which is also a sign of no lipid peroxidation under ambient CO2 and elevated CO2 (Table 4). To assess the NP’s antioxidant capacity under elevated CO2, we investigated spinach plant antioxidant enzyme activities as shown in Table 4. Interestingly, we observed no significant difference in leaf SOD activity in plants among all NPs treatments and under both ambient CO2 and elevated CO2 levels. The catalase (CAT) activity was increased among the treatments under both ambient CO2 and elevated CO2 levels. However, a significant increase was observed in 100 (mg/L) CeO2 NPs under ambient and elevated CO2. Moreover, NPs treatments did not significantly change the ascorbate peroxidase (APX) value except 100 (mg/L) CeO2 NPs. Elevated CO2 with 100 (mg/L) CeO2 NPs significantly (p ≤ 0.05) increased APX by 10.66% compared to ambient CO2.

To evaluate the potential of CeO2 NPs in strengthening spinach nutritive values, micro and macronutrients in plant different tissues were measured (Table 5). The results of our study depicted that elevated CO2 significantly (p ≤ 0.05) decreased root Mg, Fe, Ca, Zn K, and Mn except Cu content as compared to ambient CO2 level. A statistically significant decrease was observed for shoot Mg, Fe, Cu, K, and Zn except Ca and Mn compared to its ambient control. However, the foliar application of CeO2 NPs increased the concentration of nutrients in spinach under elevated CO2 in a dose-dependent manner. Specifically, the highest Mg content in root and shoot was increased significantly (p ≤ 0.05) by 211.62% and 215.49% respectively, in 100 (mg/L) CeO2 NPs as compared to its elevated CO2 control treatment. The Fe content enhanced in root by 156.68% and shoot 322.77% in 100 (mg/L) CeO2 NPs as compared to the respective control. The content of Zn showed a similar response in root and shoot with a significant increase of 225.89%, and 181.49% respectively, in 100 (mg/L) CeO2 NPs as compared to the respective control. The maximum Cu content found in the root and shoot by 21.99% and 138.09% in 100 (mg/L) CeO2 NPs treatment as compared to the respective control. At 100 (mg/L) CeO2 NPs treatment, the K content in root significantly increased by 121.44% and in shoot by 138.89% as compared to the respective control. Regarding Ca, root Ca content in 100 (mg/L) CeO2-NPs treatment increased significantly by 118.22%, and shoot Ca content increased by 91.32% but this increase was not statically significant. The root Mn also increased significantly by foliar CeO2-NPs application in 100 (mg/L) treatment by 133.15% as compared to elevated CO2 control treatment. However, a statistically non-significant increase of 195.02% was found in shoot Mn content. Overall, CeO2 NPs application reverted the negative effect of elevated CO2 on studied nutrient content. The cerium content in the shoot was significantly higher in CeO2 NPs treatments than in the control, as expected. On the other hand, Ce content in roots was significantly low as compared to CeO2 NPs treated plants under both ambient and elevated CO2 levels (Fig. 5). However, Ce content in root and shoot was significantly (p ≤ 0.05) higher in CeO2 NPs treated plants under elevated CO2 as compared to plants grown under ambient CO2 level.

The effect of cerium oxide nanoparticles on the cerium content (A) Ce in leaves and (B) Ce in roots in spinach under ambient and elevated CO2. Different letters among columns indicate statistically significant differences at p ≤ 0.05.

The present study investigated for the first time, how elevated CO2 and CeO2 NPs interactively affect leafy vegetable growth and nutritional quality. The foliar application of CeO2 NPs from various sources and concentrations has favorable impacts on plants55,56 although many studies reported the negative impact of CeO2 NPs at higher concentrations34,57. There is currently no study on the exposure of CeO2 NPs on plant growth, photosynthetic, and nutritional status under elevated CO2 conditions, previous studies have mostly focused on the effects of elevated CO2 alone. In the current study, we revealed that elevated CO2 improved spinach growth but reduced nutrient content by influencing physiological changes. Additionally, CeO2 NPs interaction further amplified spinach growth and development while increasing spinach nutrient content under elevated CO2.

Elevated enhanced spinach growth and biomass without the interaction of NPs treatments. The increase in plant growth and biomass may be attributed to increased photosynthetic pigments and photosynthetic rate. It is consistent with Alsherif et al.58 who reported that elevated CO2 increased wheat biomass production by stimulating photosynthesis. Similarly, Broberg et al.59 and AbdElgawad et al.60 documented that elevated CO2 improved wheat and rice yield by increasing carbohydrate levels and metabolism. A prominent and significant increase in growth and biomass was found by the foliar application of CeO2 NPs under elevated CO2 in our study (Table 3) and overall, the effect of cerium oxide nanoparticle is diagrammed in (Fig. 6).

Effect of cerium oxide nanoparticles (CeO2 NPs) on spinach crop under elevated CO2.

Previous studies have documented comparable enhancements in both fresh weight (FW) and dry weight (DW) in various plant species following treatment with CeO2 NPs31,61. For instance, Gohari17 reported that CeO2 NPs exposure caused a substantial increase in agronomic traits of grapevine. Another study conducted by Jahani et al.21 also found a positive influence of CeO2 NPs on plants at 50 and 100 (mg/L) application rates under ambient levels. The positive impact of CeO2 NPs at a low concentration in the plant growth medium may be due to the improved efficiency of photosynthesis, higher levels of macronutrients in plant tissues28 increased water uptake, and subsequent enhancements in water balance62, reductions in water loss through leaf transpiration63, and enhancements in the photosynthetic rate, as demonstrated by prior studies. All these factors ultimately result in increased biomass production.

Photosynthesis is a physiological process that is susceptible to environmental factors and is the indicator of plant health64,65. Elevated CO2 is known to increase photosynthesis9. According to Thompson66 and Khamis67, the plants exhibited enhanced photosynthesis and leaf gas exchange parameters as a result of the higher concentrations of CO2. Here, we found similar trends that elevated CO2 significantly boosted photosynthetic performance in spinach irrespective of NPs application as evidenced by an increase in photosynthetic pigments (Chl a, b and Car) and photosynthetic rate (Figs. 1, 2A). This increase is mainly attributed to the enhancement of Rubisco by increasing the level of CO2 improves carbon fixation, hence increasing greater abundance of non-structural carbohydrates that are essential for the regular growth and metabolic functions of plants68,69. Application of CeO2 NPs under elevated CO2 resulted in a more considerable increment in photosynthetic pigments with increasing concentration. Similar to our results previous studies also reported an increase in chlorophyll a, b, and carotenoids with CeO2 NPs exposure17,68 at optimum concentration under ambient CO2. Ce can form a Rubisco activase super complex and dramatically increase spinach Rubisco carboxylase activity69. These NPs, along with their unique features resembling antioxidative enzymes, may be the reason for the enhancement of photosynthesis and other physiological activities66. CeO2 NPs functioned as a catalyst in the formation of chlorophyll a and b, while also safeguarding chloroplast structure from damage21. A study by Daler,70 found downregulation of the VvCLH1 gene (chlorophyll degradation gene) by CeO2 NP treatment at a moderate concentration (50 mg/L) under drought stress indicates that CeO2 NPs assist in alleviating chlorophyll degradation and sustaining photosynthetic efficiency during abiotic stress, which is associated with diminished oxidative stress. However, some studies indicated that high levels of CeO2 NPs (10,000 μg/L) can hinder photosynthesis71, while 1000 mg/L inhibits it72 as higher CeO2 NPs hinder electron transport in the PSII electronic pathway, leading to a decrease in the efficiency of PSII electron transfer73.

Regarding gas exchange parameters, plants grown under elevated CO2 showed increased photosynthetic rate (Pn) compared to ambient control (Fig. 2A). Higher photosynthetic rates may be attributed to reducing photorespiration by increased levels of CO2 directing more carbohydrates to growth areas, particularly in C4 plants74,75. In line with our results, Alsherif32 revealed that wheat plants showed increased photosynthetic rates under elevated CO2 conditions. Furthermore, elevated levels of CO2 have been observed to reduce stomatal conductance (gs) and transpiration rate (Tr)67,76,77. In coherence with these studies, our results also showed variations regarding Ci and its related parameters including Tr and gs between elevated and ambient CO2 (Fig. 2B–D). Furthermore, the results of SEM from our study depicted stomatal closure associated with decreased gs under elevated CO2 as the closure of stomatal pores in plants is facilitated by increased concentrations of CO2 in the intercellular space (Ci) of leaves compared to ambient CO2 concentrations, triggers the opening of anion channels and causes the plasma membrane of guard cells to depolarize11,78. Reduced stomatal conductance can lead to a slow-down of transpiration rate, ultimately affecting the transport of nutrients and metals from the roots to the shoots79. Foliar application of CeO2 NPs enhanced leaf gas exchange parameters which had been reduced under elevated CO2 levels in dose dose-dependent manner (Fig. 2). In agreement with the current findings, a study reported by Abbas28 demonstrated that foliar application of CeO2 NPs augmented gas exchange parameters i.e. pn and gs in wheat plants at 50 and 100 (mg/L) application rate. The pattern for increasing leaf exchange parameters is also aligned with Djanaguiraman80 who reported that foliar application of CeO2 NPs stimulated photosynthetic rate, stomatal conductance, and transpiration rate simultaneously at optimum concentrations. However, the impact of CeO2 NPs on the photosynthetic function in plants and the overall outcome is contingent upon the physiochemical characteristics of CeO2 NPs, the specific plant species, and the prevailing environmental conditions81. Elevated CO2 by providing carbon for photosynthesis and CeO2 NPs by enhancing pigment accumulation and nutrient content under changing environmental conditions, synergistically improved the photosynthetic capacity of spinach.

MDA and ROS are indicators of oxidative damage in plants under stress. According to biochemical analysis, elevated CO2 did not cause any oxidative stress as evidenced by the effect on MDA and H2O2 (Table 4). Similar to the present study, previous studies also observed no rise in the concentration of reactive oxygen species (ROS) in plants under elevated CO232,67,72,82. Similarly, no oxidative stress was observed in CeO2 NPs treatment at all concentrations under both ambient and elevated CO2 levels (Table 4). CeO2 NPs, when utilized as fertilizers, may enhance root development, activate antioxidant enzyme activity, and inhibit membrane peroxidation and leakage83. Nonetheless, plants encounter oxidative stress due to the excessive accumulation of NPs, which may compromise their defense mechanisms84.

In contrast to our results, some studies reported increased MDA content and ion membrane leakage with CeO2 NPs57,85 at 100 (mg/L) higher concentration. Salehi et al.53 reported that foliar CeO2 NPs caused oxidative stress and induced membrane damage in plants in a dose-dependent manner. However, the observed variations are likely to be influenced by factors such as the plant growth conditions, the specific plant species, the application method, and the concentration and duration of exposure17,85. Regarding antioxidant enzyme activity, we found variability among results activities under elevated CO2 and CeO2 NPs (Table 3). In the present study no, significant change in antioxidant enzyme activities including SOD, CAT, and APX under elevated CO2 was observed in control treatments, which is aligned with some previous studies9,58. However, with the foliar application of CeO2 NPs changes in antioxidant enzyme activities were observed (Table 4). Previous research has reported that the presence of low levels of CeO2 NPs has a positive effect on the overall antioxidant capacity of plants86. The CeO2 NPs possess enzyme-like properties and are regarded as nano enzymes for enhancing plant tolerance to abiotic stress. This effect is attributed to the ability of CeO2 NPs to mimic enzymes actions, acting both as oxidant and antioxidant plants, with significant protective function linked to their ability to mimic the activity of superoxide dismutase (SOD), as described by Korsvik87 and Hussain88. In addition, CeO2 NPs possess a distinct and exceptional surface redox chemistry that significantly influences the biochemical and physiological processes in plants89. Considering SOD activity, we found no significant increase in all CeO2 NPs treatments which is aligned with Ma85 and Gohari17 who reported no change in SOD activity under CeO2 NPs exposure indicating that elevated SOD activity correlates with higher levels of H2O2. Prior research has shown that CeO2 NPs can act as ROS scavengers at extremely low levels, but can also stimulate the generation of H2O2 in large quantities25,85. Similar to current results, the previous studies also reported an increase in CAT activity under CeO2 NPs treatments and antioxidant gene expression analysis demonstrated the upregulation of CAT and APX activities in spinach exposed to CeO2 NPs24. The increase in these antioxidant enzyme activities suggests the direct or indirect involvement of elevated CO2 and CeO2 which is reported previously58,90,91.

Crops are one of the main sources of vital nutrients and modification in plant’s nutritive values could lead to significant health risks81. Elevated CO2 alters and degrades plant quality, even though it can increase the yield and productivity of agricultural products15. Our results also demonstrated a significant reduction in spinach root and shoot nutrient content mainly Mg, Fe, Cu, Zn, and K as compared to ambient CO2 (Table 5). Like the present study, Giri92 also found detrimental effects of increased CO2 on leaf nutritional quality. The study reported a decrease in protein content as well as lower levels of macronutrients (N, P, K) and micronutrients (Cu, Zn, Mg, S) in the edible portion of lettuce and spinach. The levels of nutrient elements like Fe, Cu, and Zinc in rice and wheat were reportedly reduced under elevated CO276. One possible cause of the reduction in nutritional content in leaves is linked to the dilution effect observed in plants cultivated under increased CO2 levels7,93. Another possible reason reported is that increased levels of carbon dioxide can decrease transpiration-driven mass flow, potentially resulting in lower nutrient absorption from the soil. Soares94 examined the substantial influence of increased CO2 levels on the nutritional content of key consumable crops such as wheat, rice, legumes, and vegetables, revealing a significant reduction in both macro and micronutrients across all these crops.

According to the literature, adding NPs to a growth medium or its foliar application can substantially impact the balance of metals within plants and alter their nutritional composition95,96. In this study, the use of CeO2 NPs counteracted the adverse effects of elevated CO2 on the nutrient content of plants. CeO2 NPs significantly improved the nutrient content of spinach with increasing concentration (Table 5). The potential benefits of CeO2 NPs in increasing nutritional values have been reported by many studies at optimum concentrations29,53,97. Similar to the present results, Liu98 reported that CeO2 NPs treatment dramatically raised the content of macronutrients (P and S) and micronutrients (Mn, Fe, Cu, and Zn) by 32.9%, 24.5%, 93.1%, 27.6%, 54.3%, and 69.7% respectively, in Cucumber. A recent study by Gui99 reported a significant increase in Zn content in Zea Mays under CeO2 NPs (75 and 225 mg/kg) treatments but a decrease in Mn and Cu which is inconsistent with our results. The relative expression of the nutrient (Na and K) genes was upregulated under stress and remained elevated following CeO2 application, indicating that CeO2 nanoparticles might sustain nutrient equilibrium by controlling the ion transporter gene30. Another study revealed that Go and KEEG enrichment analysis of proteomic data indicated that CeO2 NPs primarily affected pathways associated with proteins and amino acid functions, suggesting an improvement in fruit nutritional quality by CeO2 NPs100.

A proposed mechanism by which CeO2 NPs increase photosynthesis and nutrient uptake under elevated CO2 is outlined based on current findings (Fig. 6) These might adsorb nutrients to their surface and facilitate the uptake of these nutrients (Fig. 6). Another factor that contributes might be the improved photosynthetic efficiency and gas exchange attributes i.e. stomatal conductance and transpiration rate, especially under elevated CO2 and regulate nutrient uptake and absorption. Studies have demonstrated that foliar applied was transported to the roots via the phloem or moved upwards to different portions of the aboveground tissue through the xylem62,83. In plants, NPs predominantly accumulate within vacuoles and the cell wall, while vascular tissues enable their movement both upward and downward to various plant parts101. Overall, our findings revealed that CeO2 NPs have the potential to increase crop nutrient content and growth, and this approach may be implemented on a wide scale in the field; however, optimal CeO2 NPs concentrations for various crops must be determined before application. Assessing the impact of CeO2 NPs on soil microbial communities and ecosystem stability in the field under elevated CO2 presents intriguing future research opportunities (Fig. 7).

Proposed mechanism by which CeO2-NPs recover nutrient content and improve growth under elevated CO2. Plants grown under elevated CO2 increase plant growth and biomass but have low nutrient content due to altered plant physiology. CeO2-NPs adsorb nutrients and translocate to the aerial parts of plants, and increase plant stomatal conductance and transpiration rate thereby improving micro and macronutrients within the spinach under elevated CO2.

The rise in atmospheric CO2 positively stimulates plant growth but negatively affects crop nutritional quality. The use of CeO2 NPs in our study presents a promising strategy and demonstrates that spinach growth and photosynthetic pigments were remarkably improved by the foliar application of CeO2 NPs in a dose-additive manner. Elevated CO2 alone showed an increased photosynthetic rate, while significantly decreased stomatal conductance and transpiration rate. The application of CeO2 NPs treatment improved the overall gas exchange parameters of the spinach plant. Regarding spinach plant biochemical attributes, no alteration in oxidants and antioxidant enzyme activities was observed under elevated CO2 levels. However, an increase in antioxidant enzymes i.e. CAT and APX were observed at higher concentrations of NPs (100 mg/L). Elevated CO2 decreased roots and shoots macro and micronutrient of spinach as compared to ambient CO2 level. Interestingly, foliar CeO2 NPs significantly and dramatically increased the macro and micronutrient content of the roots and shoots of spinach and recovered the nutrient deficits caused by elevated CO2. In aggregate, CeO2 NPs have the potential to increase crop productivity and quality by modulating plant photosynthesis mechanisms under elevated CO2 leading to better agriculture productivity under future climatic conditions.

These findings offer insight into the key mechanism by which CeO2 NPs affect spinach growth and quality under changing environmental conditions. Our future study will focus on elucidating the underlying mechanisms by investigating how CeO2 NPs affect molecular pathways involved in photosynthesis and crop nutrient metabolism. Specifically, we will focus on analyzing the expression of key nutrient transporter genes such as ZIP, YSL, and MTP and the regulation of signaling pathways associated with nutrient transport and photosynthesis. Furthermore, future research will include studying microbial community structure to study plant-soil-microbe interaction under CeO2 NPs and elevated CO2 levels. Understanding this molecular and microbial interaction will provide mechanistic insight to fully grasp their broader agricultural implications.

All data generated or analyzed during the study are included in this article.

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The authors wish to acknowledge the financial support provided by the Science and Technology Innovation Program of Jiangsu Province (No. BK20220036). We also gratefully acknowledge the financial assistance by China Scholarship Council (CSC).

This work was financial supported provided by the Science and Technology Innovation Program of Jiangsu Province (No. BK20220036).

State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Nanjing, 210023, Jiangsu, China

Shoaib Ahmad, Adiba Khan Sehrish, Fuxun Ai, Xueying Zong & Hongyan Guo

Department of Biology, College of Science and Arts, Najran University, 66252, Najran, Saudi Arabia

Department of Zoology, College of Science, King Saud University, 11451, Riyadh, Saudi Arabia

Department of Biology, Faculty of Science, University of Tabuk, 71491, Tabuk, Saudi Arabia

Department of Environmental Sciences, Government College University, Faisalabad, 38000, Pakistan

Joint International Research Centre for Critical Zone Science, University of Leeds and Nanjing University, Nanjing University, Nanjing, 210023, China

Quanzhou Institute for Environment Protection Industry, Nanjing University, Beifeng Road, Quanzhou, 362000, China

Department of Biological Sciences and Technology, China Medical University, Taichung 40402 , Taiwan

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S.A.: Formal analysis, methodology, writing—original draft, writing—review and editing. A.K.S.: Writing—review and editing the final draft. F.A.: Data curation, writing—review and editing. X.Z.: Investigation. S.O.A.: Methodology, software. M.A.A. Writing—review and editing the final draft. K.A.A.-G.: Formal analysis, writing—review and editing. S.A.: Resources, funding acquisition. H.G.: Supervision, project administration, resources, funding acquisition.

Correspondence to Shafaqat Ali or Hongyan Guo.

The authors declare no competing interests.

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Ahmad, S., Sehrish, A.K., Ai, F. et al. Morphophysiological, biochemical, and nutrient response of spinach (Spinacia oleracea L.) by foliar CeO2 nanoparticles under elevated CO2. Sci Rep 14, 25361 (2024). https://doi.org/10.1038/s41598-024-76875-z

DOI: https://doi.org/10.1038/s41598-024-76875-z

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Morphophysiological, biochemical, and nutrient response of spinach (Spinacia oleracea L.) by foliar CeO2 nanoparticles under elevated CO2 | Scientific Reports