Amelioration of the growth and physiological responses of Capsicum annum L. via quantum dot-graphene oxide, cerium oxide, and titanium oxide nanoparticles foliar application under salinity stress

Abstract

Salinity is one of the predominant abiotic stressors that reduce plant growth, yield, and productivity. Ameliorating salt tolerance through nanotechnology is an efficient and reliable methodology for enhancing agricultural crops yield and quality. Nanoparticles enhance plant tolerance to salinity stress by facilitating reactive oxygen species detoxification and by reducing the ionic and osmotic stress effects on plants. This experiment was conducted to study the effects of NaCl salinity stress (0, 100, and 200 mM), and foliar application of quantum dot-graphene oxide, nano-TiO₂, and CeO₂ (zero and 2 g/l) on the growth and physiological responses of Capsicum annum L. The results revealed that the interaction effects of treatments significantly affected plant and fruit fresh weight, chlorophyll a, total soluble solids, phenolics, malondialdehyde, H₂O_2, and proline content. Moreover, catalase activity and sodium, and phosphorus content were responded to the treatments. The highest fresh weight of plants and fruits, fruit diameter, and chlorophyll a content were recorded under no-salinity × quantum dot-graphene oxide foliar use. The highest data for total phenolics content was recorded at NaCl_{100 mM} × quantum dot-graphene oxide. In contrast, the maximum flavonoids content belonged to NaCl_{100 mM} × quantum dot-graphene oxide and NaCl_{100 mM} × TiO₂. The experimental treatments independently affected the number of fruits, chlorophyll b, carotenoids, and vitamin C content, as well as K/Na ratio. The foliar treatment of quantum dot-graphene oxide nanoparticles improved the carotenoids and vitamin C content, stem diameter, and fruit number. The overall results disclosed that, when plants were exposed to high salinity levels; the foliar treatments were unable to effectively mitigate the negative impacts of salt stress on the plant, except for certain traits such as total phenolics, flavonoids, and TSS levels. However, under the low and mild salinity depression, the foliar treatments were enough capable to overcome the salinity defects.

Introduction

Salinity is continuously expanding in Iran due to a decrease in rainfall, a drastic increase in evaporation and transpiration rate, and the mismanagement of water resources¹. The total area of Iran’s rain-fed and irrigated agricultural lands is 18.2 million hectares, with 7.8 million hectares being irrigated. Irrigation of these lands with saline water resources in the arid and semi-arid areas, combined with poor irrigation management and inadequate drainage, can lead to secondary salinization of agricultural soils¹. Salinity occurs due to the accumulation of sodium, calcium, magnesium, potassium, SO₄²⁻, HCO₃⁻, NO₃⁻, and chlorine, or the weathering of rocks containing these ions². Salinity stress affects the physiological, morphological, vegetative, and reproductive attributes of plants through ionic toxicity, ionic imbalance, reduced nutrient absorption, retarded growth rate, and even by the declined photosynthesis potential, and via the secondary osmotic and oxidative stress effects^3–5. As a result of salt stress, ionic toxicity occurs, leading to the replacement of potassium with sodium ions which, in turn, causes structural changes in the cell due to the deteriorative impacts of high concentrations of sodium and chlorine ions within the cell⁶, imposing high electrolyte leakage and eventually cell wall bounded lipids peroxidation⁷A study on pepper showed that, salinity caused a decrease of up to 97% in fruit set potential of plant however, the foliar application of manganese-doped graphene quantum dots enhanced fruit set rates up to 57% in the plant⁸.

The huge limitations in potassium absorption due to stress cause a decrease in turgor pressure and disrupt normal cell metabolism³. The results of one study showed that salinity stress leads to stomata closure, which reduces the consumption of NADPH in the Calvin cycle⁹. This, in turn, decreases the production of oxygen-free radicals and reduces the utilization of NADPH in biochemical reactions^3,10. Furthermore, the effect of salinity stress on the reproductive stage of plants and the development of the fruit is more evident. Salt stress temporarily interferes with the activity of cyclin-dependent kinases, leads to growth restriction, and, consequently, reduces cell division and enlargements in the apical meristems^3,11.

The over-generation of oxygen free radicals is the auxiliary stress effect on plants, which causes damage to biological membranes, proteins, and nucleic acids^3,4,9. Plants are equipped with enzymatic and non-enzymatic defense systems to cope with stressful environments. The production of antioxidant enzymes (superoxide dismutase, catalase, pyruvate oxidase) and low molecular weight solutes (proline, glycine betaine, etc.) plays a pivotal role in reducing the negative effects of stress in plants³. The results of a study showed that salinity significantly reduced the dry weight, relative water content, glycine betaine content, photosynthetic rate, stomatal conductance, and chlorophyll content of the pepper plants⁹. Salinity reduced the pepper plants’ whole growth and yield potential. Using melatonin and arginine helped to improve the morphological characteristics and logically enhanced the content and activity of antioxidant compounds in the plant⁵. In the last decade, agronomic techniques and the application of nanoparticles have played an inevitable role in the advancement of agricultural practices¹². Today, in modern agriculture, nanomaterials are being used as an alternative to chemical fertilizers and pesticides¹². The small size of nanoparticles (1–100 nm) gives rise to various physicochemical properties, such as solubility, surface charge, aggregation, and a high specific surface area. The green synthesis of nanomaterials is an emerging topic in nanotechnology^12,13. Natural products have always fascinated researchers because of their eco-friendliness, economic feasibility, and endless availability. The diverse physicochemical characteristics of nanoparticles allow them to cross biological barriers more efficiently and remediate stressors effects¹⁴. Carbon quantum dots (CQDs) which are a novel class of zero-dimensional carbon nanomaterials with a size range of < 10 nm have gained special attention due to their excellent physical and chemical properties¹³. Activated carbon is a group of carbon materials that are always of interest to researchers and various industries, particularly the agricultural sector. This is because of their high specific surface area, porous structure, high absorption capacity, and low cost. In a research on tarragon, the application of graphene oxide nanoparticles under salt stress increased the activity of SOD enzyme, as well as the zinc and iron content of the plant¹⁵. In Vingna radiataplant under salinity stress, using of sugar-terminated carbon nanoparticles improved seed germination, and strengthened the antioxidant defense system¹⁶. In pepper, the treatment of the Zn-quantum dot improved the growth parameters and plant yield¹⁷. Titanium nanoparticles have been widely used in various industries due to their photocatalytic properties. Titanium as an element is not essential for plants, but in low concentrations, enhances plant stem and root growth by stimulating cell division and improving plant metabolism. Titanium nanoparticles also improve antioxidant activity and soluble sugar content, while reducing hydrogen peroxide levels^18,19. Additionally, titanium nanoparticles promote carbon organization, enhance nutrient absorption, facilitate the biosynthesis of new proteins, and aid in the detoxification of oxygen-free radicals in plants^18–20. In a study on barley, the use of titanium nanoparticles helped to control salinity stress by enhancing photosynthesis capacity in the plants²¹. Cerium is a metal oxide from the group of lanthanides that possesses unique optical, thermal, and electrical properties. These properties have contributed to its extensive utilization in diverse fields such as biology, medicine, and agriculture. The application of 125 mg/liter of cerium oxide in the soil caused an increase in root length, plant growth, and catalase activity in the Coriandrum sativumplant²². Cerium oxide plays an important role in maintaining ionic homeostasis, chlorophyll biosynthesis, regulating the potassium-to-sodium ratio, and scavenging oxygen free radicals produced under stressful conditions^23,24. In general, it should be noted that the impact of nanoparticles on plants under stress conditions depends on various factors, including the growth stage of the plant, the plant species, the concentration of nanoparticles used, and the duration of exposure to stressful conditions^23,24.

Vegetables are a remarkable part of the human diet, as they are a rich source of minerals, vitamins, and antioxidants¹⁴. Pepper (Capsicum annuumL.) from the Solanaceae family is one of the most widely consumed vegetables in the world. Pepper has long been used as a vegetable, spice, medicine, and as a dyeing natural source^25,26. In traditional medicine, pepper has been used for the treatment of toothache, sore throat, and rheumatism. It is also utilized as an antiseptic, antioxidant, antibacterial, anticancer, and immunomodulatory agent²⁷. Pepper is a good source of natural antioxidant compounds, as well as vitamins C and A.

Climate change and global warming have led to a great decline in rainfall in the arid and semi-arid regions worldwide, particularly in Iran. As a result, a significant portion of Iran is facing the problem of water scarcity and soil salinity. Sustainable agricultural practices in such soils require methodologies to mitigate the effects of stress on plants. This study for the first time aims to evaluate the effect of foliar spraying of different nanoparticles on the growth and physiological responses of pepper plants under salinity stress. We hope that the results will be reliable enough to simulate the same pilot experiments in the field condition and possibly recommend the results to the extension section.

Results

Characterization of CQDs

The Fourier Transform Infrared (FTIR) spectroscopy can be used to determine the functional groups on the surface of CQDs. The result for green-synthesized CQDs is presented in Fig. 1. As shown, the band at 3371 cm⁻¹ region includes a relatively wide peak which is related to stretching bands of either -OH or -NH groups. The peaks at 2898–2976 cm⁻¹ indicate the presence of methylene or methyl (C-H) functional groups due to the presence of the aliphatic hydrocarbons. The peaks at 2340–2360 cm⁻¹ were attributed to the C-N bond. The peak at 1400 cm⁻¹could be identified as C-N, N-H, and -COO groups. The bands at 1000–1100 cm⁻¹ correspond to C-O-C and C-O stretchings, respectively. The narrow band at 883 cm⁻¹ and 669 cm⁻¹ represents the existence of out-of-plane bending of -CH and -OH bonds, respectively. The availability of C-O-C and C-H functional groups on the surface of the CQDs makes it highly hydrophilic^13,28,29.

Fig. 1.

FT-IR spectrum of CQDs.

DLS technique measured the average hydrodynamic size of green-synthesized CQDs as 8.7 nm (Fig. 2).

Fig. 2.

Size distribution of CQDs.

To investigate the optical properties of green-synthesized CQDs; photoluminescence spectra were obtained at different excitation wavelengths. The results revealed that CQDs display the characteristic “excitation-independent emission” behavior and a relatively narrow emission peak at 685 nm (Fig. 3).

Fig. 3.

Emission spectrum of carbon quantum dots at different excitation wavelengths.

Growth traits

Plant height, stem diameter, the average number of fruits per plant, and fruit diameter were influenced by the independent effects of the experimental treatments (Table 1). Salinity negatively affected the mentioned traits. The treatment with no salt stress increased plant height, stem diameter, the average number of fruits per plant, and fruit diameter. Between 100 and 200 mM salinity stress, no significant difference was observed in terms of stem and fruit diameter. However, by adding up the salinity stress from 100 to 200 mM, both the plant height and the average number of fruits per plant were decreased (Table 2). Foliar treatment with quantum dot-graphene oxide enhanced plant height, stem diameter, and the average number of fruits per plant compared to other foliar treatments. Fruit diameter positively responded to the foliar treatment with quantum dot-graphene oxide and titanium nanoparticles (Table 3).

Table 1.

ANOVA for the effects of salinity stress (0, 100, and 200 mM NaCl) and foliar applications (without spraying, TiO₂, CeO_2, and quantum dot-graphene oxide nanoparticles) on some growth and yield-related traits and photosynthetic pigments content of Capsicum annum L. plants. ns, *, ** and C.V. indicate no significance and significant differences at 5 and 1% probability levels, coefficient of variation, respectively.

Source of variation	df	Plant fresh weight	Plant height	Stem diameter	Fruit containing branch number	Average fruit number (each harvest every week)	Fruit fresh weight	Fruit length	Fruit diameter	Chlorophyll a content	Chlorophyll b content	Carotenoids content
Salinity (A)	2	1433.4*	2182*	0.17*	15.4*	73*	2.4*	16.5*	19.0*	27.5*	16.3*	6.98*
Foliar spray (B)	3	538.7*	761.6*	0.05*	5.2**	56.7*	2.3*	10.5*	4.06*	4.5*	1.9*	0.2*
A × B	6	34*	15.1ns	0.01ns	0.33*	31.1ns	0.24*	1.1	0.44ns	0.49*	0.10ns	0.12ns
Error	24	10.3	28.8	0.005	0.03	1.5	0.03	0.42	0.31	0.15	0.19	0.05
C. V. (%)		1.9	8.6	5.9	5.1	10.6	7.2	7.02	5.0	8.9	16.9	10.3

Table 2.

Mean comparisons for the effect of NaCl salinity on plant growth, and physiological traits of Capsicum annuum L.Similar letters show no meaningful difference at a 5% probability level (LSD test).

NaCl salinity (mM)	Plant height (cm)	Stem diameter (cm)	Average fruit number (each harvest every week)	Fruit diameter (mm)	Chlorophyll b (mg/ g FW)	Carotenoids (mg/ g FW)	Vitamin C (mg/ 100 g)
0	74.6 ± 1.05a	1.3 ± 0.07a	14 ± 0.31a	12.4 ± 0.06a	3.8 ± 0.13a	2.9 ± 0.09a	172 ± 0.58a
100	59.1 ± 0.89b	1.1 ± 0.021b	11 ± 0.24b	10.8 ± 0.12b	2.4 ± 0.28b	2.2 ± 0.18b	143 ± 0.29b
200	49.8 ± 0.104c	1.1 ± 0.08b	9.1 ± 0.26c	9.9 ± 0.21b	1.51 ± 0.07c	1.4 ± 0.02c	106 ± 0.98 c
LSD	9.05	0.11	2.06	0.93	0.74	0.38	10.5

Table 3.

Mean comparisons for the effect of foliar application of TiO₂, CeO_2, and quantum dot-graphene oxide on plant growth, and physiological traits of Capsicum annuum L. Similar letters show no meaningful difference at a 5% probability level (LSD test).

Foliar spray	Plant height (cm)	Stem diameter (cm)	Average fruit number (each harvest every week)	Fruit diameter (mm)	Chlorophyll b (mg/ g FW)	Carotenoid (mg/ g FW)	Vitamin C (mg/ 100 g)
No foliar	52 ± 1.3a	1.1 ± 0.08b	8.4 ± 0.10c	10.3 ± 0.21b	2.04 ± 0.11b	1.5 ± 0.015c	125.2 ± 2.8c
CeO 2	60.2 ± 0.27b	1.17 ± 0.04b	10.8 ± 0.07b	10.9 ± 0.41b	2.5 ± 0.10ab	2.2 ± 0.8b	138.9 ± 1.49b
quantum dot-graphene oxide	74 ± 2.1a	1.3 ± 0.8a	14.4 ± 0.12a	11.9 ± 1.02 a	3.1 ± 0.06a	2.7 ± 0.12a	155.4 ± 2.45a
TiO 2	60.7 ± 0.18b	1.1 ± 0.01b	12.2 ± 0.41b	11.2 ± 0.90ab	2.6 ± 0.17b	2.1 ± 0.04b	143.4 ± 3.1b
LSD	9.0	0.11	2.06	0.93	0.12	0.37	10.2

The combination of no salt stress × foliar spraying with quantum dot-graphene oxide increased plant weight, the number of fruit-bearing branches, fruit weight, and fruit length. Foliar quantum dot-graphene oxide treatment increased the plant’s weight by 15% compared to the control (without salinity and foliar spray). A 95% increase in the fresh weight of the fruit was observed as a result of foliar spraying with quantum dot-graphene oxide compared to the control. In both levels of salinity stress, the least plant and fruit weight was observed under no foliar treatment. Foliar treatments reduced the adverse effects of stress on different plant characteristics (Table 4).

Table 4.

Mean comparisons for the interaction effects of salinity and foliar application of TiO₂, CeO_2, and quantum dot-graphene oxide on the plant fresh weight, fruit containing branch number, fruit fresh weight, and fruit length of Capsicum annuum L. Similar letters show no meaningful difference at a 5% probability level (LSD test).

NaCl salinity	Foliar spray	Plant fresh weight (g)	Fruit containing branch number	Fruit fresh weight (kg/ pot)	Fruit length (mm)
0	0	163 ± 0.42cd	3.2 ± 0.33f	1.5 ± 0.07hi	8.9 ± 0.84de
0	CeO2	166 ± 0.28 cd	4.3 ± 0.21c	2.6 ± 0.084bc	9.6 ± 1.2cd
0	quantum dot-graphene oxide	187 ± 0.07a	5.8 ± 0.02a	3.3 ± 0.42a	12.6 ± 1.2a
0	TiO2	173 ± 2.9b	4.9 ± 0.24b	2.7 ± 0.04b	11.2 ± 1.2b
100	0	153 ± 3.01e	2.5 ± 0.03g	1.4 ± 0.07g-i	7.7 ± 0.04f
100	CeO2	161 ± 1.54cd	3.1 ± 0.2f	2.0 0.10de	8.6 ± 0.09d-f
100	quantum dot-graphene oxide	166 ± 1.14c	4.03 ± 0.41d	2.3 ± 0.13cd	10.4 ± 1.02bc
100	TiO2	161 ± 1.1cd	3.6 ± 0.14e	1.9 ± 0.24ef	9.03 ± 0.25de
200	0	142 ± 2.1f	1.7 ± 0.28i	1.16 ± 0.09i	7.8 ± 0.87f
200	CeO2	149 ± 1.02e	2.1 ± 0.28h	1.5 ± 0.07gh	8.2 ± 0.62ef
200	quantum dot-graphene oxide	160 ± 1.74d	3.1 ± 0.38f	2.0 ± 0.25d-f	9.1 ± 0.29de
200	TiO2	149 ± 3.09e	2.2 ± 0.24h	1.7 ± 0.01fg	8.06 0.38ef
LSD		5.4	0.29	0.33	1.1

Chlorophyll and carotenoids content

Chlorophyll a content was influenced by the interaction effects of experimental treatments, while chlorophyll b and carotenoids content was impacted by the independent effects of experimental treatments (Table 1). By the salinity of 100 and 200 mM, the content of chlorophyll a decreased by 39% and 50%, respectively, compared to the control treatment. A significant increase (p ≤ 0.05) in the content of chlorophyll a was observed under no salt stress × quantum dot-graphene oxide. This treatment showed a 45% increase compared to the control. At both 100 and 200 mM salinity stress levels, foliar spraying with quantum dot-graphene oxide yielded better results compared to titanium and cerium oxide treatments (Table 5). With the salinity of 200 mM, the content of chlorophyll b decreased by 60% and, carotenoids decreased up to 52% compared to the control (Table 2). All foliar treatments enhanced the content of chlorophyll b compared to the conditions without foliar spraying (Table 3). A 74% decline in carotenoids content was observed in the foliar treatment with quantum dot-graphene oxide compared to the control. There was no difference in carotenoids content between foliar treatments with cerium oxide and titanium oxide nanoparticles (Table 3).

Table 5.

Mean comparisons for the interaction effect of salinity and foliar application of TiO₂, CeO_2, and quantum dot-graphene oxide on physiological traits, and catalase activity of Capsicum annuum L. Similar letters show no meaningful difference at a 5% probability level (LSD test).

NaCl salinity	Foliar spray	Chlorophyll a content (mg/g FW)	Total soluble solids content (°Brix)	Total phenolics content (mg/ 100 g)	Flavonoids content (mg/ 100 g)	Catalase activity (µmol H2O2/ mg protein/ min)	Proline content (mg/ g FW)
0	0	5.1 ± 0.35c	2.1 ± 0.08f	7.6 ± 0.41fg	2.4 ± 0.21d	21 ± 1.1g	3.7 ± 0.9i
0	CeO2	6.3 ± 0.98b	2.8 ± 0.24e	6.7 ± 0.87 g	3.8 ± 0.74c	24 ± 1.7f	2.9 ± 0.54j
0	quantum dot-graphene oxide	7.4 ± 0.08a	4.2 ± 0.11c	12.4 ± 0.38b	5.5 ± 0.21b	24.6 ± 1.02f	3.9 ± 0.87i
0	TiO2	5.2 ± 0.51c	3.06 ± 0.24e	10.1 ± 35cd	5.5 ± 0.05b	25 ± 0.87ef	3.8 ± 0.01i
100	0	3.1 ± 0.21fg	3.7 ± 0.41d	8.9 ± 0.37d-f	4.2 ± 0.03c	34 ± 0.28bc	5.4 ± 0.35d
100	CeO2	3.8 ± 0.61de	3.9 ± 0.51cd	9.5 ± 0.09de	4.6 ± 0.08bc	25 ± 0.98ef	4.8 ± 0.24fg
100	quantum dot-graphene oxide	4.8 ± 0.84c	5.9 ± 0.41a	14.5 ± 0.64a	8.8 ± 0.24a	30 ± 0.13d	4.3 ± 0.02gh
100	TiO2	3.9 ± 0.98d	4.1 ± 0.13cd	12.7 ± 0.31b	7.7 ± 0.01a	27 ± 1.1de	5.06 ± 0.01ef
200	0	2.5 ± 0.54g	4.4 ± 0.12bc	8.9 ± 0.31d-f	5.6 ± 0.05b	39 ± 0.37a	8.5 ± 0.12a
200	CeO2	3.2 ± 0.38ef	4.2 ± 0.21c	8.6 ± 0.28d-f	4.4 ± 0.24bc	33 0.41c	5.9 ± 0.34d
200	quantum dot-graphene oxide	3.5 ± 0.27d-f	5.5 ± 0.12a	11.4 ± 0.35bc	4.7 ± 0.21bc	30 ± 0.91d	6.8 ± 0.31c
200	TiO2	2.09 ± 0.11 fg	4.9 ± 0.13b	8.4 ± 0.21ef	3.8 ± 0.02c	36 ± 0.91b	7.4 ± 0.21b
LSD		0.65	0.49	1.6	1.2	2.7	0.47

The content of non-enzymatic antioxidant compounds, including total soluble solids, total phenolics, flavonoids, vitamin C, and proline

The salinity stress of 100 and 200 mM × quantum dot-graphene oxide foliar spray significantly (p ≤ 0.05) increased TSS content. The control (without salinity and foliar spray) had the lowest content of TSS. In conditions without salinity, no difference was observed in terms of TSS between the foliar treatments of cerium oxide and titanium oxide nanoparticles. At 100 mM, both nanoparticles increased the content of TSS. However, at a salinity of 200 mM, titanium oxide nanoparticles enhanced TSS content up to 14% higher than cerium oxide (Table 5).

Total phenolics and flavonoids content was influenced by the interaction effects of the experimental treatments (Table 6). Based on the results, higher phenolics content was obtained in 100 mM salinity × quantum dot-graphene oxide foliar spraying (91% more than the control. A significant (p ≤ 0.05) increase in the flavonoids content was recorded at 100 mM salt stress × quantum dot-graphene oxide and titanium oxide nanoparticles. The control treatment had the least flavonoids content (2.4 mg 100 g⁻¹) (Table 5).

Table 6.

ANOVA for the effects of salinity stress (0, 100, and 200 mM NaCl) and foliar applications (without spraying, TiO₂, CeO_2, and quantum dot-graphene oxide nanoparticles) on some physiological traits of Capsicum annuum L. plants. ns, *, and ** indicate no significance and significant differences at 5 and 1% probability levels, respectively.

Source of variation	df	Total soluble solids content	Vitamin C	Total phenolics content	Flavonoids content	MDA content	H2O2 content	Proline content	Catalase activity
Salinity (A)	2	9.8*	13,150*	18.6*	13.9*	1800*	1043*	39.6*	341*
Foliar spray (B)	3	5.7*	1402*	40.2*	11.02*	89.3*	118.3*	3.02*	25*
A × B	6	0.34*	50.4ns	3.7*	6.54*	31.7*	33.9*	0.94*	37*
Error	24	0.08	37.0	0.92	0.52	3.9	3.6	0.07	2.7
Coefficient of variation (%)		7.1	4.3	9.6	14.3	8.6	10.8	5.3	5.6

The treatment without salt stress affected the vitamin C content in the plant. With the increase of salt stress to 200 mM, vitamin C content decreased by 38% compared to the control (Table 2). Foliar spray of quantum dot-graphene oxide increased (155.4 mg l⁻¹) vitamin C content compared to other foliar treatments. The control treatment had the lowest vitamin C content (125.2 mg L⁻¹) (Table 3).

Proline content was affected by the interaction effects of experimental treatments. The highest (8.5 mg g⁻¹ FW) proline content was observed at 200 mM salinity stress treatment under the condition of without spraying. No difference was observed in the proline content of plants between the control treatment and the treatments without salinity stress when each of the three nanoparticles was sprayed. Under both salinity levels, foliar treatments increased the proline content of the plant (Table 5).

Malondialdehyde and hydrogen peroxide content

A significant (p ≤ 0.05) increase was observed in the malondialdehyde and hydrogen peroxide content under 200 mM salinity in the condition without foliar spraying (Fig. 4A, B). By increasing the salinity stress to 100 mM × without foliar spraying, malondialdehyde and hydrogen peroxide content increased. Under 200 mM salinity, all foliar treatments declined malondialdehyde content. This indicates the positive effect of nanoparticles in reducing the negative effects of salinity stress on the plant (Fig. 4A, B).

Fig. 4.

Mean comparisons for the effect of salinity and foliar application of TiO₂, CeO_2, and quantum dot-graphene oxide on H₂O₂ (A) and MDA (B) content of Capsicum annuum L. Similar letters show no meaningful difference at a 5% probability level by LSD.

Catalase activity

The highest catalase activity was observed at 200 mM salt stress without foliar spraying, which showed up to 85% increase compared to the control. In both levels of salinity, foliar applications increased the activity of catalase enzyme compared to treatments without foliar treatment at the same level of salinity (Table 5).

Elemental content of the leaves

The experimental treatments had independent effects on the nitrogen and potassium content, as well as the potassium-to-sodium ratio of leaves (Table 7). By increasing the salinity to 200 mM, nitrogen and potassium content in the leaves decreased. The treatment without salt stress exhibited the highest nitrogen and potassium content (Fig. 5). Foliar spraying with quantum dot-graphene oxide enhanced the nitrogen content, while foliar treatment of quantum dot-graphene oxide and titanium oxide raised the potassium content of the plant (Fig. 6).

Table 7.

ANOVA for the effects of salinity stress (0, 100, and 200 mM NaCl) and foliar applications (without spraying, TiO₂, CeO2, and quantum dot-graphene oxide nanoparticles) on the elemental content of Capsicum annuum L. plants. ns, *, and ** indicate no significance and significant differences at 5 and 1% probability levels, respectively.

Source of variation	df	N content	P content	K content	Na content	K/Na
Salinity (A)	2	2.75*	0.14*	1.02*	0.24*	67.6*
Foliar spray (B)	3	0.94*	0.016*	0.15*	0.03*	4.1*
A × B	6	0.042ns	0.002*	0.006ns	0.01*	0.83ns
Error	24	0.036	0.001	0.007	0.001	0.42
Coefficient of variation (%)		10.9	4.2	5.6	8.9	15.0

Fig. 5.

Mean comparisons for the effect of salinity on N and K content of Capsicum annuum L. Similar letters show no meaningful differences based on the LSD test.

Fig. 6.

Mean comparisons for the effect of foliar application of TiO₂, CeO_2, and quantum dot-graphene oxide on N and K content of Capsicum annuum L. Similar letters show no meaningful differences based on the LSD test.

Phosphorus and sodium content was affected by the interaction effects of experimental treatments (Table 7). The foliar treatment with quantum dot-graphene oxide and titanium oxide nanoparticles under normal conditions and foliar application with quantum dot-graphene oxide under 100 mM sodium chloride salt stress enhanced the phosphorus content of the plant. The lowest phosphorus content was recorded with 200 mM sodium chloride salt stress without foliar spraying. This treatment showed a 40% decrease in phosphorus content compared to the control (Fig. 7). Sodium content of leaves increased under 200 mM salt stress without foliar applications. In both levels of salinity, the foliar treatments reduced the sodium content of leaves compared to the treatment without foliar spraying (Fig. 8).

Fig. 7.

Mean comparisons for the interaction effect of salinity and foliar application of TiO₂, CeO₂, and quantum dot-graphene oxide on P content of Capsicum annuum L. Similar letters show no meaningful differences based on the LSD test.

Fig. 8.

Mean comparisons for the effect of salinity and foliar application of TiO₂, CeO_2, and quantum dot-graphene oxide on Na content of Capsicum annuum L. Similar letters show no meaningful differences based on the LSD test.

The treatment without salt stress exhibited the highest potassium-to-sodium ratio. As the salt stress increased to 200 mM, the ratio of potassium to sodium in the plant decreased up to 68% compared to the control treatment. (Fig. 9). Foliar treatment with all three nanoparticles improved the potassium-to-sodium ratio in the plant (Fig. 10).

Fig. 9.

Mean comparisons for the effect of salinity stress on the K/Na ratio of Capsicum annuum L. Similar letters show no meaningful differences based on the LSD test.

Fig. 10.

Mean comparisons for the effect of foliar application of TiO₂, CeO₂, and quantum dot-graphene oxide on the K/Na ratio of Capsicum annuum L. Similar letters show no meaningful differences based on the LSD test.

.

Pearson’s correlation matrix

Correlations represent the interconnections between diverse attributes and allow the researchers to decide on the right treatment to further progress in the research pass. Moreover, the correlations and clustering allow for relating several treatments and traits to each other to have a realistic idea of the reproducibility of the research theme. The Pearson’s correlations analysis for the assessed attributes of pepper plants under different salinity stress levels and foliar application of CeO₂, TiO₂, and quantum dot graphene oxide are illustrated in Fig 11A. This analysis showed a positive significant correlation among plant height, stem diameter, fruit-containing branches, average fruit number, fruit fresh weight, fruit length, fruit diameter, chlorophyll a, and b, carotenoids, vitamin C content, N, P, K content, and K/Na ratio, while all the mentioned traits revealed a negative significant correlation with Na content, MDA, H₂O₂, proline content, and catalase activity.

Fig. 11.

Pearson’s correlation heat map analysis for the effects of CeO₂, TiO₂, and quantum dot graphene oxide application under salinity stress on pepper plants (A) and heat map pattern of the evaluated traits of the plants under different salinity levels (B). S1, S2, and S3 refer to 0, 100, and 200 mM NaCl treatments, respectively. F1, F2, F3, and F4 refer to 0, CeO₂, TiO₂, and quantum dot graphene oxide foliar application, respectively.

Heat map clusters showed several groups among the traits as presented in Fig 11B. So, the evaluated traits were placed in three clusters; cluster 1 included plant height, stem diameter, fruit-bearing branches, average fruit number, fruit fresh weight, fruit length, fruit diameter, chlorophyll a, and b, carotenoids, vitamin C, N, P, K and K/Na ratio. The cluster 2 comprised of Na content, MDA, H₂O₂, proline, and catalase activity. Also, total soluble solids, flavonoids, and total phenolics content were in cluster 3. Furthermore, cluster pattern and heat map analysis revealed four main clusters in salinity stress levels and foliar application of CeO₂, TiO₂, and quantum dot graphene oxide. Cluster 1 contained the pepper plants foliar sprayed with quantum dot graphene oxide under 0, 100, and 200 mM salinity stress, as well as the plants were grown under 100 mM salinity stress × TiO₂ foliar spraying. Cluster 2 included the pepper plants treated using CeO₂ × 100 mM NaCl treatment. Cluster 3 contained the plants foliar treated with CeO₂ and TiO₂ × no saline conditions. Finally, cluster 4 comprised the treatments of CeO₂ and TiO₂ foliar application under the highest salinity stress, as well as 100 mM NaCl × no foliar application.

Discussion

Globally, salinity stress is a serious threat to sustainable agriculture; as it reduces plant growth and performance by affecting physiological, biochemical, and molecular reactions³⁰. Salinity negatively affects photosynthesis, growth, respiration, and stomatal conductance. The saline conditions trigger osmotic stress by reducing cell water potential³. Plants respond to salinity stress through changes in the anatomical, biochemical, and morphological traits, regulation of ion homeostasis, ion re-organization in the cell, activation of antioxidant machinery, and biosynthesis of super osmoprotectants and phytohormones. The mentioned changes reduce ionic toxicity and salinity stress defects by removing oxygen free radicals, regulating ion distribution, preventing ultrastructural changes on membranes, and enhancing minerals absorption^3,29. The results of the present investigation also showed the detrimental effect of stress on the growth and functional traits of the plant. By increasing the salinity stress to 200 mM, the yield and growth characteristics of the pepper decreased. The selected concentrations of nanoparticles (zero and 2 g/ l) used in the present study were unable to mitigate the negative effects of high salt stress on plant performance. In the present study, the highest plant yield was obtained in the treatment without salt stress × quantum dot-graphene oxide foliar spraying. More possibly, the plant growth and yield are reduced due to ionic imbalance in saline conditions. Furthermore, a possible reason for any decline in plant size after a short or long-term encounter with salinity stress may be due to the stomata closure and the imposed ionic, osmotic, and oxidative stress³¹. This finding is consistent with previous research conducted on tomato³² and Aloe vera³³where it was found that the application of graphene oxide improved the growth and performance of the plant. This improvement was observed through various mechanisms, including enhanced cell division, increased photosynthesis, improved protein-to-amino acid ratio, increased stomatal density, and conductance, as well as enhanced intercellular carbon dioxide concentration³³. In research on wheat, Fe–Mn nanocomposites doped graphene quantum dots increased the photosynthetic pigments content and facilitated the plants’ access to carbon sources, which increased plant growth under salinity stress. The non-toxic nature of this compound and its bio-degradability and ease of operation facilitates the penetration of carbon nanoparticles into the cell which enhances the efficiency of this compound in dealing with stress⁷.

Under salinity stress, molecular oxygen acts as an electron acceptor and triggers the production of reactive oxygen species (ROS) in the chloroplasts, mitochondria, and peroxisomes. An increase in the concentration of free radicals causes damage to the cell membrane, enzyme inactivation, and harm to the cell components³⁴. The balance between the production and elimination of intracellular ROS must be tightly regulated and/or they have to be efficiently scavenged by an antioxidant pool¹⁴. Hydrogen peroxide is a regulator of various processes, including growth, biochemical responses, and oxidative reactions. The effect of hydrogen peroxide on these processes depends on its concentration. An increase in hydrogen peroxide concentration has been reported in various environmental stresses^15,35. By increasing the salinity stress from 100 to 200 mM in the present study, the levels of hydrogen peroxide and malondialdehyde increased in the plant. In line, the highest H₂O₂ and MDA content was recorded under 150 mM NaCl + TiO₂spray in eggplant¹⁴. Plants under salt stress require an additional source of energy to develop salt tolerance mechanisms and promote growth. To cope with salt stress, plants possess enzymatic and non-enzymatic defense systems. SOD is the most important enzyme for destroying superoxide radicals in plants. Hydrogen peroxide produced as a result of stress is converted into H₂O₂ by catalase, guaiacol peroxidase, and ascorbate peroxidase. Catalase reduces the harmful effects of free radicals by converting H₂O₂into oxygen and water³. Perhaps one of the reasons for the increase in catalase content observed in the present study, under the salinity stress of 200 mM sodium chloride is the plant’s response to neutralize oxygen free radicals. In the research conducted on rice³⁶, tarragon¹⁵, beans¹⁸, and eggplant¹⁴, it was found that the use of nanoparticles such as cerium oxide, graphene oxide, and titanium oxide reduced the negative effects of stress, including hydrogen peroxide content and the production of oxygen free radicals. In the present study, the use of nanoparticles at different salinity levels resulted in a decrease in hydrogen peroxide levels. These compounds were reliably decomposed into water and molecular oxygen through the use of various nanoparticles. It appears that the use of nanoparticles can lead to an increase in the concentration of compounds such as proteins, soluble sugars, and phenolics. This increase can help mitigate the negative effects of hydrogen peroxide on plants. An increase in the concentration of proteins and soluble sugars has been reported in studies conducted on Trachyspermum ammi³⁷ and Abelmoschus esculentus³⁸. In the present study, the foliar application of quantum dot-graphene oxide enhanced the total soluble solids content under salt stress conditions, thus supporting the aforementioned findings. Phenolic compounds are water-soluble, non-enzymatic antioxidant compounds that protect cells from environmental stresses by eliminating oxygen free radicals. These compounds prevent the production and accumulation of free radicals within the cell. An increase in the content of phenolic compounds has been reported in mung bean plants due to the application of quantum dots³⁹, which aligns with the findings of the present study.

Accumulation of soluble compounds such as proline, glycine betaine, and sugars under stress conditions plays an important role in the osmotic regulation of the cell. This is achieved through the establishment of water flow, osmotic protection, carbon storage, and elimination of oxygen free radicals³. In the present study, the highest proline content was obtained under salt stress of 200 mM sodium chloride, without foliar spraying. It seems that under stress conditions, proline acts as a source of nitrogen and energy, and even acts as a scavenger of free radicals in the cell. It also helps to maintain the integrity of the cell membrane³. An increase in proline content was reported in beans under salinity stress following the application of TiO₂¹⁸. Similar results were reported by Hareem, et al.¹⁷in chili emphasizing the enhanced proline content under stress conditions. Increased expression of the D1-pyrroline-5-carboxylate synthetase genes, PvP5CS1 and PvP5CS2 plays an eminent role in proline biosynthesis under stress conditions in plants^40,41. P5CS is a crucial enzyme in the biosynthesis of proline. Increasing the activity of P5CS under conditions of environmental stress causes the accumulation of proline and improves osmotic regulation in plants, which helps protect the plant against stress⁴¹.

Quantum dot nanoparticles are known as carriers in plants. The impact of nanoparticles on mitigating the effects of environmental stress has been documented in various studies^32,36. Our results showed that non-enzymatic antioxidant compounds, such as phenolics, flavonoids, and soluble solids content increased when plants were subjected to 100 mM salinity stress and treated with foliar spraying of quantum dot-graphene oxide nanoparticles. In the current study, the application of titanium oxide nanoparticles and quantum dots-graphene oxide enhanced the flavonoids content of pepper. Similar results were reported regarding the positive effect of cerium oxide and titanium nanoparticles under saline conditions^18,36. Cerium oxide, as a catalytic inhibitor, plays a crucial role in eliminating free radicals and mitigating the adverse effects of stress on plants. In general, salinity stress stimulates the uptake of sodium and greatly declines potassium content in plants. This occurs due to the elevated concentration of sodium ions in the soil or nutrient solution, which interferes with the nutrient absorption balance and dynamics. Under stressful conditions, nanoparticle treatment declines the absorption of sodium ions by plants. The results of the present study demonstrated that the use of nanoparticles reduced sodium absorption under all salinity levels. Furthermore, the application of quantum dot-graphene oxide improved the absorption of nitrogen, phosphorus, and potassium in plants. Under stress conditions, a decrease in membrane-bound H⁺-ATPase activity is one of the causes of increased sodium levels in the cell. Foliar spraying with nanoparticles enhances the activity of this channel and inhibits the entry of sodium into the cell⁴². In the present study, the utilization of quantum dots reduced the absorption of sodium ions by pepper. Quantum dot reduces the oxidative stress in plants through the expression of genes related to the salicylic acid biosynthesis pathway⁴³. Similar results were reported regarding the reduction of sodium absorption under stress conditions due to the application of quantum dots in tarragon plants¹⁵. As already known, nanoparticles easily penetrate plant cells and alleviate the adverse effects of salinity in the main part due to their small size and large surface area¹⁴.

Chlorophyll is the prominent pigment that plays a vital role in the process of photosynthesis. A decrease in chlorophyll and carotenoids biosynthesis has been observed in most plants under salt stress. This decrease is attributed to a remarkable reduction in chlorophyll biosynthesis and an increase in the ratio of decomposition to conversion of chlorophyll⁴⁴. The accumulation of sodium ions in chloroplasts increases the production of oxygen free radicals, which eventually destroy the cell wall⁴⁵. Under salinity stress, the stomata closure results in reduced CO₂ uptake, limiting carboxylation by the lessened internal CO₂levels which go to increased photorespiration. The production of reactive oxygen species is also enhanced in plants when exposed to stress which leads to oxidative damage to cellular organelles and genetic material^14,31. Inhibition of photochemical activities and downstream regulation of chloroplast-encoded genes due to salt stress caused a decrease in chlorophyll content⁴². A decrease in chlorophyll content due to salinity stress has been reported in several studies^15,44,46. These are consistent with the results of the present study showing a reduced pattern in chlorophyll and carotenoid content in the plant when subjected to increased salinity levels. Gai et al. identified bZIP (basic leucine zipper) genes in pepper plants and found that the CabZIP25 gene increases the plant’s tolerance to salt stress⁴⁷. The results showed that the chlorophyll content decreased in the peppers in which the CabZIP25 gene was switched off. In the present study, cerium oxide nanoparticles and quantum dot-graphene oxide foliar application increased the chlorophyll content of the plant. This finding aligns with previous studies conducted on the use of cerium oxide in rice to mitigate the negative effects of salinity stress³⁶, as well as the use of graphene oxide in rice and corn plants⁴⁸. Perhaps one of the reasons for the higher pigment content is related to the enhanced absorption of carbon dioxide. This increase is attributed to the heightened activity of the RuBisco enzyme, which plays a crucial role in the process of photosynthesis⁴⁶. In our study, quantum dot graphene oxide foliar use increased the chlorophyll content of the plant, which is in line with the above-mentioned studies.

Salt stress disrupts cytoplasmic homeostasis and alters the potassium-to-sodium ratio in the cell, leading to an accumulation of sodium in the cytosol³. In the present study, subjecting the plants to salinity stress of 200 mM enhanced the sodium content while decreasing the levels of potassium, nitrogen, phosphorus, and potassium-to-sodium ratio in pepper plants. Foliar spraying with all three nanoparticles added up the potassium-to-sodium ratio compared to conditions without foliar spraying at the same salinity level. This indicates the effectiveness of nanoparticles in reducing the negative effects of stress. The mechanism of salinity tolerance in plants not only requires adaptation to sodium toxicity but also necessitates an increase in potassium absorption by the plant. Plants also develop ionic tolerance by activating various signaling cascades triggered by the entry of salt into the root system. The plant reduced the net influx of Na⁺ in the root and translocation of Na ⁺toward the shoots⁴⁹. The response of sensitive and resistant plants to salt stress differs in terms of the potassium-to-sodium ratio. Sensitive cultivars have a high sodium-to-potassium ratio⁵⁰. Reducing the sodium-to-potassium ratio is the main mechanism that induces salt tolerance in the plant. Stimulating the activity of highly salt-sensitive genes, such as SOS1: Na⁺/H⁺antiporter, triggers the exit of sodium ions from the root cells and hence enhances stress tolerance⁵¹. Along with SOS1: Na⁺/H⁺ antiporter activity, HKT family proteins also protect plants against salt stress. The cellular response of plants to salinity stress appears much earlier than the physiological responses. A decrease in the potassium-to-sodium ratio occurs due to the accumulation of sodium in the plant. Replacing sodium with potassium in plant tissues leads to an increase in potassium leakage through the cell, which is caused by the stimulation of K⁺efflux channel activity⁵². A former study on pepper showed that the foliar use of Zn-quantum dot biochar increased the N, P, and K content in plants under drought stress¹⁷. In our study, salinity reduced N, P, and K content in plants. In contrast, quantum dot graphene oxide treatment increased elemental content in the plants. Carbon-based nanomaterials have been shown to minimize the adverse effects of abiotic stress on plants by promoting the nutrition potential and strengthening the defense system, scavenging reactive oxygen species, or improving calcium signaling pathways and overall by the improved environmental adaptability^8,10,22.

Materials and methods

The present study was conducted in the research greenhouse of Azerbaijan Shahid Madani University during the years 2021 and 2022. The average daily temperature was 27^oC, with a night-time temperature of 22^oC. The relative humidity ranged from 60 to 70%. The seeds were obtained from Pakan Bazar Company in Isfahan, Iran, and were planted according to the relevant institutional and national guidelines and legislation. The seeds were first planted in a tray filled with cocopeat. At the three-leaves stage (10 weeks after seed germination), the plants were transferred to 7-liter pots. The planting substrate used included a homogenous mixture of agricultural soil, sand, and animal manure in a ratio of 1:1:1. The characteristics of soil and fertilizer are mentioned in Table 8. To fully establish the plants, they were watered for two weeks. In the 4-leaf stage, salt stress was applied with concentrations of 0 mM (without salt stress), 100 mM, and 200 mM sodium chloride. The salt stress began from 50 mM and then gradually increased every 3 days until reaching the treatments concentration (100 mM, and 200 mM). In the early growing stages in the greenhouse, the plants were irrigated with water containing the desired salt concentrations every two days. 300 ml of water was used for any pot during each watering cycle until the water was drained from the bottom of the pot. To prevent salt buildup, the plants were rinsed with fresh water every 10 days. Foliar treatment was conducted using two concentrations: zero (distilled water) and 2 g per liter (freshly prepared) of carbon quantum dot nanoparticles, cerium oxide, and titanium oxide. Cerium oxide and titanium oxide nanoparticles were purchased from the US-NANO company. The initial foliar spraying was conducted 48 h before applying the salt stress, and the second foliar spraying was repeated two weeks thereafter. Necessary crop care was performed throughout the entire 14th -week period of plant maintenance. At the end of the 14th week, the plant samples were taken to study the desired traits. The present study had 12 treatments, each with three replications. Each experimental unit consisted of two pots. To measure the weight of the plant, the plants were removed from the soil, and the weight of the aboveground part of the plant was measured using a digital scale. The length and diameter of the fruit and stem were recorded using a digital caliper. The height of the plant was measured in centimeters.

Table 8.

Physico-chemical characteristics of soil and organic manure.

Soil texture	pH	EC (ds/m)	N (%)	P (mg/kg)	K (mg/kg)
Sandy loam	6.8	1.2	0.3	47	201
Animal manure	8.1	1.9	1.0	64	301

Synthesis of activated carbon nanoparticles (CQDs)

The green synthesis of CQDs was done by Mulberry leaves via a green route^12,13. To this end, after collecting the mulberry leaves, the leaves were washed with water to remove any dust and impurities. Then, they were washed with distilled water several times. Next, the chopped leaves were mashed and extracted with ethanol. Later, the ethanol extract was heated in autoclaves at 150 °C for 5 h to carbonize the extract. After cooling down to room temperature, the resultant was centrifuged for 30 min at the speed of 6000 rpm to obtain CQDs suspension.

Characterization of quantum dot –graphene oxide nanoparticle

The Fourier Transform Infrared (FTIR) spectrum of the carbon quantum dots (CQDs) was recorded on a Vector 22 (Bruker, Ettlingen, Germany) Fourier transform infrared spectrometer using KBr as the mulling agent. The dynamic light scattering (DLS) measurements were taken on the Zetasiser instrument ZEN3600 (Malvern, UK MAL 1001767) with a He-Ne laser beam at 511 nm and 25 °C. A spectrofluorimeter with a xenon arc lamp of 150 watts and a scanning speed of 4000 rpm (Jasco, model FP-6200, Japan) was applied to record the fluorescence spectra of different solutions. An electric muffle furnace (Fan Azma Gostar, model FM8P, Iran) was used for heating purposes. An electronic analytical balance (PFB300-3, Kern, Germany) was used to weigh the solid materials.

Pigments content

Chlorophylls and carotenoids contents were quantified by Prochazkova et al. (2001) methods at 645, 665, and 470 nm. 0.5 g leaf samples were extracted using dimethyl sulfoxide (DMSO, Sigma Aldrich, Germany) for 4 h at 65 °C in the dark⁵³.

Total soluble solids (TSS) content

One-gram leaf sample was used for the determination of total soluble solids content. The total soluble solids were quantified with a hand refractometer (Erma, Tokyo, Japan), and the data are presented as °Brix.

Elemental composition

The leaves were dried at 80 °C for 48 h. The dried plants were ground in a Wiley mill to particles less than 0.42 nm. 0.3 g of samples were acid-digested (2 N HCl) and analyzed for nutrient content⁵⁴. Na⁺ and K⁺content was quantified with the flame photometric method (Corning, 410, England). N content was assessed with the Kjeldahl method. Phosphorous was determined with the Vanadat Molybdate method⁵⁵.

Preparation of fruit extract

To extract, 5 g of the fruits were ground with the help of 80% ethyl alcohol in a mortar and dried in an incubator at 37 °C for one day. The resulting solution was centrifuged at 32,869 × g for 10 min. The top layer of solution was passed through a Minisart filter (0.45 μm pore size, regenerated cellulose). Then, it was concentrated to 20 ml using a rotary evaporator. The sample was stored in a freezer at −80 °C until the measurement of antioxidant compounds.

Measurement of total phenolics content

The Folin-Ciocalteu reagent was used to measure the total phenolics content of the samples^56,57. 0.2 g of Folin-Ciocalteu reagent was added to a 15 ml falcon containing 0.2 ml of the extract, then 0.2 ml of distilled water was added. The resulting mixture was kept at room temperature for 6 min. Then, 0.2 ml of 7% sodium carbonate was added and kept at room temperature for 90 min. The absorbance of the sample was recorded at 750 nm based on the gallic acid standard.

Flavonoids content

The colorimetric method was used to measure flavonoids content in the fruit extract^56,57. In a 15 ml falcon, 4 ml of distilled water and extract were poured, then 0.3 ml of 5% NaNO₂ was added to the mixture and it was placed at room temperature for 5 min. 0.3 ml of 10% aluminum chloride was added to the solution and rested for 6 min. In the next step, 2 ml of normal sodium was added to the solution. In the last step, its volume was increased to 10 ml by adding distilled water. The absorbance of the sample was read at 510 nm based on the catechin standard using a spectrophotometer.

Hydrogen peroxide content

0.2 g of leaf sample was powdered in liquid N₂ and then ground in ice-cold 0.1% trichloroacetic acid (TCA). The sample was centrifuged at 12,000 × g for 15 min. 0.5 mL of the supernatant was mixed with 0.5 mL of 10 mM potassium phosphate buffer (pH = 7.5) and 1 mL of 1 M potassium iodide. The H₂O₂ content was evaluated using standards of 5 to 1000 µM of H₂O₂and the absorbance of samples and standards was measured at 390 nm⁵⁸.

Malondialdehyde (MDA) content

For determination of malondialdehyde content, 0.2 g of leaf sample was homogenized in 0.1% TCA, and the extract was centrifuged for 15 min at 12,000 × g. 0.5 mL of the extract and 1.5 mL of 0.5% thiobarbituric acid (TBA) in 20% TCA was incubated at 95^°C for 30 min and then cooled in an ice bath. The absorbance was determined at 520 nm and corrected for non-specific absorbance at 600 nm. The MDA content was determined using the extinction coefficient of 155 mM cm⁻¹59.

Proline content

5 mL of 3% homogenized sulfosalicylic acid was added to 0.2 g of pepper leaf sample. The extract was centrifuged for 7 min at 6037 × g. 1 mL of the supernatant was mixed with the same volume of ninhydrin acid and 1 mL of glacial acetic acid, then, the samples were incubated in a water bath (100^°C) and then for 30 s in an ice bath. After 30 min, a red phase formed above the samples. The red phase supernatant was used for the proline content measurements at 520 nm⁶⁰. The proline content was computed using a standard curve of proline.

Catalase (CAT) activity

0.5 g of pepper leaf samples were homogenized with 0.1 M cold potassium phosphate buffer at pH: 7.5, with 0.5 mM EDTA. 0.05 mL from the resulting supernatant was added to 1.5 mL of 0.1 mM phosphate buffer at pH: 7, and 1.45 mL of double-distilled water. By adding 0.5 mL of 75 mM hydrogen peroxide reaction was started, and a decrease in absorption was recorded at 240 nm for 1 min⁶¹.

Vitamin C content

Vitamin C content was determined by titrating the aqueous extract with a solution of 2.6-dichlorophenol dye to a faint pink color⁶².

Experimental design and data analysis

The experiment was conducted with three replications as a factorial based on CRD. Analysis of variance (ANOVA) was performed by MSTAT-C ver. 2.1. Moreover, the significant differences among the means were evaluated with the Least Significance Difference(LSD) test at p < 0.05. Standard deviations (n = 3) were evaluated for the traits. Pearson’s correlation and cluster dendrogram heat maps were depicted in R software (R Foundation for Statistical Computing, version 4.1.2).

Conclusion

Salinity stress negatively affected the growth traits of the plant. Foliar spraying with quantum dot-graphene oxide increased the content of non-enzymatic antioxidant compounds (phenolics, flavonoids, and TSS) under salinity stress of 100 mM. Salinity reduced the elemental content in plants. Overall, pepper plants can be considered salt-sensitive crops capable of production under mild saline conditions. In our experiment, all foliar treatments were partially effective in alleviating the salinity effects, especially under low salinity levels. However, for the majority of traits, quantum dot graphene oxide gave promising results. Exposing the plants to 200 mM salinity drastically affected the growth traits and concomitantly increased the stress-responsive attributes such as the content of malondialdehyde, H₂O₂, proline, and sodium. Otherwise, foliar treatments were unable to overcome the stress effects under high salinity conditions. However, as mentioned, the quantum dot graphene oxide induced reliable outcomes and is worthy of further evaluation with more detailed studies in the greenhouse and even under field conditions. Thereafter, we will be able to conduct parallel studies with some other analogous compounds to possibly extend the cultivation of this valuable vegetable under the marginal lands.

Author contributions

Conceptualization M.B.H. and L.V.M.; data curation, L.V.M., and F.R.; formal analysis and methodology, M.B.H., L.V.M., and F.R.; project administration, M.B.H.; visualization, L.V.M., M.B.H., L.K., and F.R.; writing original draft, L.V.M.; writing-review and editing. M.B.H. All authors have read and agreed to the published version of the manuscript.

Funding

The authors wish to thank the Azarbaijan Shahid Madani University, Iran, for the financial support of the study.

Data availability

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

Declarations

Ethics approval and consent to participate

All procedures were conducted following the relevant institutional, national, and international guidelines and legislations.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.