The fabrication of gold nanoparticles (AuNPs) using spinach leaf extract was carried out following our previously established protocol as detailed in Amir et al. (2024). After synthesis, size and morphology were analyzed using transmission electron microscope (TEM) [Tecnai G2 Spirit BioTwin, FEI].

Spinacia oleracea L. cv. allgreen (Spinach) seeds obtained from the National Seed Corporation, Lucknow, were subjected to surface sterilization using a 0.01% solution of mercuric chloride (HgCl₂). These seeds were then cultivated in 13×11 cm pots filled with a re-formed soil consisting of 70% sand, 20% clay, and 10% peat by dry weight. The cultivation took place in a growth chamber located at the Botany Department, University of Lucknow. Growth chamber was maintained with temperature of 24°C ± 3, at a photoperiod cycle of 16/8 hours light/dark, light intensity of 300 nmol m^-2s^-1 and 60% humidity.

During the experiment, the efficacy of 150 μM S-AuNPs was evaluated for mitigating the adverse effects of salt stress induced by sodium chloride (NaCl) in spinach. The concentration of NaCl was selected based on preliminary screening experiments (data not shown). Four treatment sets were established: the first set served as the control and received no treatment; the second set was subjected to salt stress with 50 mM NaCl; the third set was treated with 150 μM S-AuNPs according to findings from our previously published study (Amir et al., 2024); and the fourth set underwent salt stress (50 mM NaCl) followed by treatment with S-AuNPs (150 μM). The required concentrations of NaCl and S-AuNPs were dissolved in double-distilled water (DDW) and applied to the soil at 15 days after sowing (DAS). Sampling was performed at 30, 45, and 60 DAS to evaluate a range of morphological, physiological, and biochemical attributes. The experimental setup involved all treatments being organized in a completely randomized manner with three replicates (n = 3) for each treatment.

Randomly selected spinach seeds were surface sterilized using a 0.01% HgCl₂ solution for 15 seconds, followed by rinsing four times with DDW to eliminate any residual sterilizing agent. Subsequently, 50 seeds per treatment group were placed in four distinct glass petri plates containing DDW, a 50 mM NaCl solution, a 150 μM S-AuNPs solution, and a NaCl + S-AuNPs solution respectively. These plates were then incubated in a dark chamber (24°C ± 2) for initial 5 days, followed by an additional 2 days under a natural photoperiod at room temperature (25°C ± 3). Three separate replication sets were prepared to assess variability. Germination indices were determined using formulas from previously established protocol (Thomson and El-Kassaby, 1993; Fetouh and Hassan, 2014).

Shoot and root length were measured with a metric scale. The shoot and root fresh weight (fw) of both treated and control plants were assessed using an electronic balance (PGB220, WENSAR). Subsequently, the shoot and root samples were dried out at 48°C for 48 hours and then dehydrated at 70°C for additional 72 hrs to observed their dry mass (Taïbi et al., 2016). Leaf numbers were quantified by manual leaf counting, while leaf area was taken using a leaf area meter (LAM 21, Systronics), as described by Arora et al. (2012).

Fresh leaf weighing 0.5 g were homogenized with 80% (v/v) chilled acetone to extract chlorophyll and carotenoid content followed by centrifugation (REMI Neya-16R, India)) at 10,000 rpm for 10 minutes (min). For chlorophyll, the optical density (O.D.) of the samples was measured at wavelengths of 663 and 645 nm with a UV-Vis spectrophotometer [Shimadzu UV-1601, Tokyo, Japan] following the protocol of Arnon (1949). Further, carotenoid content was calculated by taking the sample O.D. at 480 and 510 nm, as outlined by Maclachlan and Zalik (1963).

Stomatal traits, such as stomatal frequency, were measured according to protocol outlined by Salisbury (1928) and stomatal index was calculated based on the formulation described by Xu and Zhou (2008). Furthermore, the transpiration rate was observed before noontime, using Ganang’s Potometer (Singh et al., 2014). The abscisic acid (ABA) concentration was determined by following the method of Hung and Kao (2003).

Stomata morphology was investigated with a protocol outlined by Faizan et al. (2021). Fresh leaf specimens were immediately preserved with methanol, followed by immersion in a series of diluted ethanol solution. Further, the dehydrated sample was coated with gold and pore length and width was observed with a scanning electron microscope (SEM) [JEOL SM-6510, Tokyo, Japan].

Lipid peroxidation (MDA) content was assessed using the method outlined by Heath and Packer (1968). Fresh leaf specimen weighing 0.5 g was extracted with 0.1% trichloroacetic acid (TCA) and then centrifuged at 12000 rpm at 4°C for 15 min. Resulting supernatant was mixed with 0.5% thiobarbituric acid made in 20% TCA. The mixture was then heated at 95°C for 30 minutes and subsequently cooled in an ice bath. After homogenizing the reaction mixture at 1000×g for 15 min. at 4°C, the absorbance of the supernatant was measured at 532 nm.

The hydrogen peroxide (H₂O₂) content was determined using the method described by Patterson et al (1984). A fresh leaf sample weighing 500 mg was extracted with acetone and then centrifuged at 8000 rpm for 15 min. The resulting supernatant was mixed with a solution containing 20% titanium chloride, concentrated HCl and 17 M ammonia. The obtained precipitate was washed multiple times with acetone, after which 2N H₂SO₄ was added to it. The absorbance was recorded at 410 nm, and H₂O₂ content was calculated from a standard curve prepared using H₂O₂, expressed as μmol g⁻¹ fw.

The content of Superoxide anion (O₂ ^˙−) was determined according to the procedure described by López et al. (2009). The O₂ ^˙− was quantified using a sodium nitrite standard curve and were represented as μmole g^–1 fw.

Electrolyte leakage (EL) was determined using the electrical conductivity method as described in our previous study (Jalil et al., 2017). Activity of lipoxygenase (LOX, EC 1.3.11.12) was assessed following the method outlined by Surrey (1964).

Dichlorodihydrofluorescein diacetate (H₂DCF-DA) was employed for the qualitative assessment of H₂O₂ accumulation (Kumar et al., 2022). For this purpose leaf peels of different treatment groups were immersed in buffer (5 mM MES-KOH, pH 5.8, 5 mM CaCl_2, 0.5 mM sorbitol) containing 10 mM H₂DCF-DA, incubated at RT in dark condition for 30 min, and then washed three times with 5 mM MES buffer (pH 5.8) and were mounted slides using same buffer. Finally, the fluorescence image was observed under a fluorescence microscope (Magnus-MLX1 plus) using FITC filter; excitation and emission of 490 and 515 nm respectively.

The Proline content was estimated following the protocol established by Bates et al. (1973). Fresh leaf sample weighing 0.1 g was crushed with 5 mL of 3% aqueous sulphosalicylic acid and then centrifuged at 10,000 rpm for 15 min. An aliquot of 1 mL was mixed with acidic ninhydrin and glacial acetic acid, incubated at 100°C for 10 min, and subsequently cooled in an ice bath. Further, the mixture was extracted with 4 mL toluene, vortexed for 20 s, and cooled. The absorbance at 520 nm was recorded, and the free proline content was determined from a standard curve, expressed as μmol g^-1 fw.

Ethanol extracts from leaf were prepared to assess non-enzymatic antioxidants and sugar content. Leaf sample weighing 0.5 g was extracted with 10 mL of 80% ethanol and then filtered through Whatman No. 41 filter paper. The residue was re-homogenized with ethanol, and polled together to make a final volume of 20 mL. From the prepared extracts, determination of total sugars were assessed following the method outlined by DuBois et al. (1956), flavonoids were assessed as per Pękal and Pyrzynska (2014) and ascorbate (AsA) was determined from the according to Azuma et al. (1999).

Glutathione (GSH) content was estimated by using protocol as defined by Anderson (1985). A leaf samples weighing 0.5 g were homogenized with an extraction buffer containing 5% (w/v) sulphosalicylic acid and then centrifuged at 10,000×g for 10 min. Subsequently, 0.5 mL of 100 mM PBS and 40 mL of 5, 5′ -dithiobis-2-nitrobenzoic acid were added to 0.5 mL of the supernatant, and the absorbance was recorded at 412 nm.

The antioxidant enzymes extraction procedure was conducted at 4°C following the procedure outlined by Dwivedi et al. (2020). Fresh leaf samples weighing 0.5 g were homogenized with 5 mL of 100 mM PBS (pH 7.6), which included 1 mM EDTA.Na₂, 0.5 mM ascorbate, and 1% Polyvinylpyrrolidone. The homogenates were then centrifuged at 10,000 rpm for 5 min, and the resulting supernatant was utilized as the crude extract for enzymes estimation.

Catalase (CAT, EC 1.11.1.6) activity was determined by measuring the reduction in H₂O₂ and monitoring absorbance difference at 240 nm, following the method described by Aebi (1984).

Superoxide dismutase (SOD, EC 1.15.1.1) activity was assessed using the nitroblue-tetrazolium method, as described by Chen and Pan (1996).

Peroxidase (POD, EC1.11.1.7) activity was measured by monitoring the difference in absorbance at 420 nm, reaction mixture consist of pyrogallol, PBS, H₂O₂, and enzyme extract, following the method outlined by Chance and Maehly (1955).

Ascorbate peroxidase (APX, EC 1.11.1.11) activity was assessed by observing the reduction in ascorbate and observing the difference in absorbance at 290 nm, according to the method described by Nakano and Asada (1981).

Glutathione peroxidase (GPX, EC 1.11.1.9) activity was calculated by recording the decrease in absorbance at 340 nm caused by the NADPH oxidation at 25°C, following the protocol outlined by Smith and Johnson (1988).

Glutathione reductase (GR, EC 1.8.1.7) activity was measured by observing the decline in absorbance at 412 nm resulting from oxidation of NADPH, according to the method described by Halliwell and Foyer (1978).

For nutrient analysis, samples were prepared following the method outlined by Kumar et al. (2014). Leaf samples were washed two times with DDW and immersed in 20 mM EDTA for 5 second. Afterward, the samples washed again with DDW twice to ensure thorough removal of any remaining metal on the plant surface. The washed samples were subsequently dehydrated in an oven at for 105°C for 24 hours. Further, dried samples were digested by using a wet digestion method with a mixture of HNO₃: HClO₄ (7:3, V/V) until clear precipitate were achieved. Each sample was then diluted with DDW to a final volume of 20 mL. The concentrations of Sodium (Na), potassium (K), calcium (Ca), iron (Fe), manganese (Mn), and zinc (Zn) in the leaf samples were analyzed with Atomic Absorption Spectrophotometer (AAS) [Agilent 240FS-AA].

All Measurements were carried out in a randomized manner across three replicates (n=3) for each treatment group. The data presented are mean values accompanied by their corresponding standard deviation (SD). Statistical analyses, including one-way analysis of variance (ANOVA) and Duncan’s multiple range test (DMRT), were performed to identify the significant difference between treatments at 5% level (P ≤ 0.05) by using SPSS 20.0 software (SPSS, Inc., Chicago, IL, USA). Additionally, principal component analysis (PCA) was conducted to assess the correlation between the studied variable and treatment by using OriginPro 2018 software (Origin-Lab Corporation, Northampton, MA, USA).

The synthesis of AuNPs was accomplished using 1 mM chloroaurate (HAuCl₄) as the gold precursor and 0.33 mg/ml spinach leaf extract as reducing agent. The gold reduction process was visually validated by observing the color transformation of the reaction mixture from pale yellow to ruby red. Subsequent UV-Vis spectral scanning verified the formation of AuNPs, showing an absorption peak at 528 nm corresponding to the AuNPs SPR band ( Figure 1A ). Additionally, TEM was employed to assess the core size, shape, and 2-dimensional morphology of synthesized S-AuNPs. The analysis revealed a spherical shape with monodispersed distribution and core size ranging from 14.5 to 20.5 nm ( Figures 1B, C ).

Table 1 illustrated that spinach seeds treated with NaCl exhibited a negative impact on germination-related parameters. However, when supplemented with S-AuNPs + NaCl, increases in GP by 20.4%, GE by 7.6%, and MDG by 6.4% were recorded against NaCl treatment.

From Figure 2 , it is evident that plants exposed to salt stress exhibited reduced growth characteristics, including plant length, leaf number, leaf area and biomass as compared to control plants. However, these parameters were improved by the S-AuNPs application under salt stressed conditions. Shoot lengths were increased by 20.4%, 7.6%, and 6.4%, at 30, 45, and 60 DAS, respectively when supplemented with S-AuNPs + NaCl compared to NaCl treated plants ( Figure 2A ). A similar trend was observed in root length, with increases of 17.4%, 12.4%, and 9.0% at 30, 45, and 60 DAS, respectively ( Figure 2B ). Additionally, leaf number increased by 15.0%, 24.1%, and 11.2%, and leaf area by 23.1%, 7.0%, and 8.0%, respectively, at 30, 45, and 60 DAS against NaCl treated plants ( Figures 2C, D ). Shoot and root fresh mass increased by 36.3%, 31.8%, and 11.9%, and 25.0%, 19.3%, and 10.2%, at 30, 45, and 60 DAS respectively ( Figures 2E, F ). The shoot and root dry mass was also increased in all plants treated with S-AuNPs, with respective increases of 23.3%, 14.0%, and 13.0% for shoot dry mass and 15.3%, 15.0%, and 7.4% for root dry mass, over the salt stressed plants at 30, 45, and 60 DAS ( Figures 2G, H ).

From Figure 3 , it is clear that the chlorophyll contents were decreased by 27.4%, 26.6%, and 22.8% respectively at 30, 45, and 60 DAS under salt stress compared to control plants. However, when supplemented with S-AuNPs with NaCl, chlorophyll content were found to be increased by 19.0%, 20.3%, and 16.9%, respectively compared to NaCl treated plants. In contrast to chlorophyll content, carotenoid content was also improved by 26.1%, 15.8%, and 10.9%, respectively at 30, 45, and 60 DAS when supplemented with S-AuNPs + NaCl in comparison to salt treated plants ( Figure 3B ).

Figure 3 illustrates that plants treated with NaCl experienced reduced stomatal traits (stomatal frequency, index, aperture and transpiration rate) as compared to control plants. However, applying S-AuNPs to salt affected spinach plants led to an increase in stomatal frequency by 12.8%, 9.3%, and 8.7%, and stomatal index by 21.7%, 19.8%, and 14.1%, respectively at 30, 45, and 60 DAS as compared to salt treated plants ( Figures 3C, D ). Similarly, with the combined treatment of AuNPs and NaCl, transpiration rate increased by 12.7%, 10.4%, and 7.4%, respectively at 30, 45, and 60 DAS in comparison to salt-treated plants ( Figure 3E ). In response to the salt stress, the ABA level was increased by 64.0%, 51.7%, and 47.4% against control. However, the combined treatment of S-AuNPs and NaCl reduced the ABA level by 17.1%, 13.7%, and 13.9%, respectively at 30, 45, 60 DAS compared to NaCl treated plants ( Figure 3F ).

In Figure 4 it is shown that plants treated with NaCl had partially closed stomata with reduced aperture size by 34.79% as compared to control plants ( Figures 4A, B ). Conversely, the application of S-AuNPs alone showed a wider aperture compared to control plants ( Figure 4C ). However, when S-AuNPs were applied to salt affected spinach plants, the aperture size increased by 20% as compared to salt-treated plants but remained less than that of control plants ( Figure 4D ).

From Figure 5 it is indicated, that the plants exposed to salt stress exhibited increased production of oxidative stress markers as compared to control plants. However, under combined application of S-AuNP + NaCl, leaf MDA content was decreased by 11.2%, 14.7%, and 5.0%, while H₂O₂ was decreased by 18.5%, 12.9%, and 13.0% respectively at 30, 45, and 60 DAS in comparison to NaCl treated plants ( Figures 5A, B ). A similar trend was observed in superoxide anion content, with decrease of about 14.1%, 13.7%, and 7.0%, respectively with application of S-AuNPs in salt stressed plant over to NaCl treated plants ( Figure 5C ) Beside this, the electrolyte leakage was decreased by 6.6%, 8.4%, and 9.2%, lipoxygenase content was decreased by 4.9%, 10.3%, and 3.6% respectively at 30, 45, and 60 DAS against NaCl treated plants ( Figures 5D, E ).

Figure 6 depicted that, fluorescence microscopy images of H₂DCF-DA fluorescence were used to characterize the formation of H₂O₂ in leaves, where fluorescence intensity is directly proportional to H₂O₂ accumulation. Results illustrate that under salt exposure, H₂DCF fluorescence intensity was increased (brighter green color) against control plants ( Figures 6A, B ). However, the combined treatment of S-AuNPs and NaCl reduced the fluorescence intensity against NaCl treated plants ( Figures 6C, D ), as observed by appearance of diminished green color.

Figure 5F illustrate that upon salt exposure proline content was increased by 20.0%, 27.16% and 30.59% at 30, 45, and 60 DAS, respectively as compared to control. However, combined treatment of S-AuNPs and NaCl reduced the proline content by 5.7%, 12.6% and 8.11% respectively at 30, 45, and 60 DAS compared to salt treated spinach plant.

From Figure 7 it is marked that the level of soluble sugar, including reducing and non-reducing sugars were decreased under salinity as compared to control plants. Total soluble sugar was found to be increased by 14.5%, 10.4% and 8.4% at 30, 45, and 60 DAS, respectively, when treated with S-AuNPs with NaCl as compared to salt treated plants ( Figure 7A ). A similar trend to that of total soluble sugar was observed in reducing and non-reducing sugar ( Figures 7B, C ). Non enzymatic antioxidant flavonoid, AsA and GSH content were also affected under salt stressed plant. Flavonoid content were found to be decreased by 6.1%, 5.7% and 9.8% respectively at 30, 45, and 60 DAS, when supplemented with S-AuNPs + NaCl against NaCl treated plants ( Figure 7D ). Ascorbate content were increased by 14.8%, 6.3% and 9.8%, reduced glutathione content increased enormously under salt stress but were respective increase of 13.0%, 17.2% and 3.4% respectively under co-application of S-AuNPs and NaCl as compared to NaCl treated spinach plants ( Figures 7E, F ).

Figure 8 results illustrate that increased antioxidant enzyme activities were observed in salt-stressed spinach plants compared to the control. However, the combined treatment of S-AuNPs with NaCl, further improved these enzymes activities as compared to salt-stressed plants. Specifically, CAT activity increased by 16.0%, 41.3%, and 26.7%, POD activity by 4.2%, 7.0%, and 16.4%, and APX activity by 61.0%, 23.0%, and 14.3%, respectively, at 30, 45, and 60 DAS, compared to respective salt-treated plants ( Figures 8B–D ). Conversely, under the combined treatment of S-AuNPs and NaCl, SOD activity decreased by 20.3%, 38.8%, and 38.3%, respectively, at 30, 45, and 60 DAS, compared to salt-stressed plants ( Figure 8A ). Furthermore, the activity of GR and GPX increased by 6.5%, 6.7%, and 12.5%, and 6.31%, 11.7%, and 11.5%, respectively, at 30, 45, and 60 DAS, under S-AuNPs and NaCl treatment compared to salt-stressed spinach plants ( Figures 8E, F ).

It is marked from Table 2 that the plants treated with NaCl affected the cationic mineral profiles of spinach. The Na⁺ content and Na⁺/K⁺ ratio was increased by 58.6% and 122% respectively, under NaCl treatment as compared to control. However, they decreased by 23.9% and 28.8% respectively, under S-AuNPs + NaCl supplementation as compared to NaCl stressed plants. Similarly, Na⁺/K⁺ were also surge under salt stress. In contrast, the contents of K⁺, Fe⁺², Ca⁺² and Zn⁺² decreased under salt stress against control. Conversely, combined treatment of S-AuNPs and NaCl increased the K⁺, Ca⁺², Fe⁺², Mn⁺², and Zn⁺² content by 24.3%, 9.9%, 18.3%, 20.6%, and 29.8% respectively at 45 DAS as compared to salt treated plant.

PCA was utilized to investigate the inter-relationships among the variables under study ( Figure 9 ). The loading and score plot revealed significant variations among the variables and treatment groups; with PC1 contributing 80.8% of the variation and PC2 contributes 15.7% variation. Notably, treatments involving S-AuNPs were clustered within the first two components, indicating a substantial impact of S-AuNPs in alleviating the adverse effects on NaCl-treated plants. PC1 predominantly influenced most of the spinach response variables, excluding Na⁺, MDA, LOX, H₂O₂, EL and proline, which were primarily represented by PC2. This suggests a correlation and co-variation among the spinach response variables, excluding the mentioned ones. Additionally, in the score plot, S-AuNPs exhibited a significant contribution to PC1 and showed a strong negative correlation with PC2. Furthermore, the positive correlation of S-AuNPs + NaCl with PC1 confirms the beneficial regulatory effect of S-AuNPs in ameliorating the detrimental impact of NaCl stress on spinach plants.