Brackish water (BW) was prepared by adding different concentrations of sodium chloride (NaCl) salt into distilled water. For the incubation experiment, there were four different rates of BW prepared as 10, 20, 30, and 40 dSm⁻¹ by adding NaCl to 100 mL of distilled water. Similarly, for the pot experiment, there were three different rates of BW prepared as 5, 10, and 15 dSm⁻¹ by adding NaCl at the rate of 0.32%, 0.8%, and 1.2%, respectively, into 1 L of distilled water separately.

Simple biochar (SBc) was prepared from rice husk at a pyrolysis temperature of 500°C for 3 h (hrs) in a furnace (TF-1200X). Later, that biochar (Bc) was ground and passed through a 2 mm sieve. Zinc enriched biochar (ZnBc) was prepared by mixing the 10 g Bc with distilled water (1:5 w/v) in a mechanical vessel. The mixture was heated to raise the temperature (100°C); then, 2.97 g of Zn(NO₃)₂.6H₂O was added to the mixture till the homogenous mixture was attained after continuous mixing and heating for 3 h. The Bc was dried in the oven at 60°C to remove the excess water from the prepared Bc (Li et al., 2018). Manganese enriched biochar (MnBc) was prepared by mixing the Bc with distilled water (1:5 w/v) in a mechanical vessel. The mixture was heated to raise the temperature (100°C); then, MnCl₂ 4.H₂O was added to the mixture till the homogenous mixture was attained after continuous stirring and heating for 3 h. The Bc was dried in the oven at 60°C to remove the excess water from the prepared Bc (Li et al., 2018). Iron enriched biochar (FeBc) was prepared by mixing the Bc with distilled water (1:5 w/v) in a mechanical vessel. The mixture was heated to raise the temperature (100°C); then, the iron chloride (FeCl₃) and iron sulfate (FeSO₄) were added (2:10 w/v) to the mixture. The homogenous mixture was attained after continuous stirring and heating for 3 h. The Bc was dried in the oven at 60°C to remove the excess water from the prepared Bc (Khan et al., 2022).

Fourier-Transform Infrared (FTIR) characterization of all sorts of BCs was performed at the Center for Advanced Studies in Physics (CASP), Lahore.

An incubation study was conducted in the research laboratory of the Sustainable Development Study Centre at Government College University, Lahore. The main purpose of this study was to investigate the most efficient applied rate of four different BCs to tackle the severity of BW stress. The temperature of the laboratory was 25°C with a humidity level (of 40%) during the incubation period. The bhal soil (200 g) was used in that experiment (Rabiya et al., 2022). There were four rates of BW (10, 20, 30, and 40 dSm⁻¹) and two rates of SBc, ZnBc, MnBc, and FeBc (0.1 and 0.5%) used in the experiment with triplicates. The purpose of this experiment was to evaluate that how higher concentration of salts affect the soil properties and how addition of simple and enriched Bc strengthen the soil physicochemical properties. The details of 32 treatments were provided in Table 1.

One replicate of each treatment was harvested after the 8^th, 16^th, and 24^th day of incubation (DAI), and the post-harvested soil was analyzed for different parameters such as pH, EC, Na^+, and Chloride (Cl⁻). The pH of the soil was measured by using a standardized pH meter (YSI pH100) and EC by using a standardized electrical conductivity meter (Extech EC300). Soil solutions were prepared with a 1:2 soil/distilled water ratio and samples were shaken for 30 min using orbital flask shaker (SK-O330-Pro Biobase). Finally, readings were examined after 1 h of equilibrium (Hailegnaw et al., 2019). The concentration of Na⁺ in all soil samples was measured by following the method of Xiao et al. (2020). Soil samples (10 g) were mixed by adding DTPA (Diethylene Triamine Penta Acetic Acid) solution (20 mL) and the extract was finally analyzed on a multi-sequential atomic absorption spectrophotometer (Thermo Scientific ICE-3000 series). Cl⁻ in soil was determined by using 5 mL soil extract obtained from the saturated paste of soil. In soil extract, 4 drops of 5% potassium chromate were added as an indicator and titrated with 0.01 N silver nitrate till the end point, i.e., the reddish-brown color appeared (Ryan et al., 2001). The volume of silver nitrate used for titration was noted and the concentration of Cl⁻ was calculated using the following formula (Ryan et al., 2001).

V = Volume used of titrant, i.e., Silver Nitrate.

N = Normality of Silver Nitrate.

V1 = Volume of Soil extract.

Percent variance analysis for incubated soil at 24^th DAI was calculated to observe the effectiveness of applied treatments to combat salinity stress in soil. It was done by proceeding with the following equation (Takarina and Pin, 2017):

The pot experiment was conducted at a greenhouse (Botanical Garden, Government College University, Lahore) located at 31°33′20.54″ N and 74°19′40.56″ E near Mall Road Lahore, Pakistan. The temperature of the greenhouse ranged from 14°C to 20°C, relative humidity was around 40 ± 12% and ambient CO₂ concentration varied from 0.028–0.067 vol%. The purpose of this experiment was to confirm the best rate of selected BCs from study 1 on wheat (Triticum aestivum L.) crops under NaCl stress. There were 20 treatments having Control, SBc, ZnBc, MnBc, and FeBc (each BC at the rate of 0.1% of soil w/w) at four levels of brackish water (BW) (0, 5, 10, and 15 dSm⁻¹) applied to the pots (7.5 × 10 cm size) having soil (3 Kg) with four replicates (n = 4). As wheat is a glycophytic crop and could not tolerate the higher concentration of salt stress that’s why we have chosen the 10dSm⁻¹ as a standard rate from previous experiment and took a higher (15dSm⁻¹) and lower rate (5dSm⁻¹). Ten seeds of wheat (cultivar name ~ Faisalabad 2008) were sown with four replicates. After 8 days of germination, four plants per pot were maintained. Then, NaCl stress was applied in the form of BW to the plants through irrigation in 12 equal intervals till harvesting. After 60 days of germination, plants were carefully harvested; then, fresh weight and root and shoot length were measured.

All parameters were statistically analyzed through Microsoft Excel 2013® (Microsoft Cooperation, Redmond, WA, USA) and Statistix@8.1® software package (Copyright 2005, Analytical Software, USA) was used to design a two-way analysis of variance (Khan et al., 2018). Least significant difference test (LSD) was performed to compare the means of different treatments at p ≤ 0.05 by using Statistix@8.1® (Steele and Grabau, 1997). Principal component analysis (PCA) was performed for incubation experiment to evaluate best-applied treatments through XLSTAT version 2021 (Rabiya et al., 2022) and a graphical representation of datasets was supported by using Sigma plot 14.0 (Ishfaq et al., 2021). All the results were the means of four replicates (n = 4) with standard error (SE).

In SBc, there was not any proper band formation occurred in the range of 4,000–2,500 cm⁻¹ (Figure 1). While, a very sharp peak (3,652.80 to 2,713.05 cm⁻¹) was observed in Fe-enriched biochar demonstrating the formation of C=O and O-H acidic functional groups (Figure 1). Similarly, slightly sharp peaks (3,662.42 to 2,812.75 cm⁻¹; 3,593.47 to 2,960.02 cm⁻¹) were also examined in the Zn and Mn enriched BCs respectively, due to the formation of functional groups (C=O and O-H) (Figure 1).

The physicochemical properties of soil were significantly affected by the application of BW (Table 2). Brackish water with excessive Na⁺ and Cl⁻ is a major environmental stress that not only increased the availability of Na⁺ and Cl⁻ in soil but also in the cytosol of plants at a toxic level (Tanveer and Yousaf, 2020). Furthermore, Zhang et al. (2019) reported that the soil EC was significantly enhanced with the increase in the level of BW. Due to the unique properties of FeBc, it acts as an incredible soil conditioner; hence it significantly influenced the pH, EC, and Na⁺ of soil (Table 5). Similarly, the application of SBc and remaining MECBs also improved the physicochemical properties of soil by mitigating the adverse impacts of saline stress as shown in the PCA biplot (Figure 2; Supplementary Table S1). A similar study reported that biochar is a powerful adsorbent of Na⁺ and Cl⁻ ions mitigating salinity stress in soil (Patel et al., 2017) as it provides electrostatic interactions by forming OH⁻ groups (Saffari et al., 2016). Thus, the retention of Na⁺ and Cl⁻ ions is significantly attributed by van der walls forces (physical adsorption) under the addition of minerals-enriched biochar (Parkash and Singh, 2020). Moreover, MECBs may provide additional benefits to soil health and plant growth by providing essential nutrients such as Fe, Zn, and Mn (Cao et al., 2016). However, the specific effects of MECBs on bioavailable Na⁺ in soil may depend on various factors such as soil type, Bc composition, and management practices. Some studies have investigated the effects of Bc on soil Na⁺ levels. For example, Zhang et al. (2016) found that adding Bc to saline-alkali soil reduced soil Na⁺ levels and increased soil organic matter content. Similarly, Zhao et al. (2018) reported that applying biochar reduced soil Na⁺ levels in sodic soil. Overall, while there is limited research on the specific effects of MECBs on bioavailable Na⁺ in soil, Bc has been shown to improve soil properties and may have potential to reduce soil Na⁺ levels in certain contexts. Similarly, it was clearly shown in the PCA biplot, the variation in values of pH and EC were non-significant among the different time intervals (8^th, 16^th, and 24^th day of incubation) (Figure 2; Table 2). The overall scenario of EC revealed that the movement of ions (Na⁺ and Cl⁻) efficiently increased with time which might be due to the increased availability of essential nutrients in soil treated by SBc and MECBs under salinity stress (Table 2). Likewise, a similar study reported that the salinity and biochar application increased the pH of the soil, but this change in pH remained non-significant (Zhang et al., 2019).

Moreover, results revealed that K⁺/Na⁺ homeostasis in wheat roots were significantly (p ≤ 0.05) influenced by the addition of MECBs especially FeBc (Table 3). Although, the presence of optimized biochar improved soil properties; so, more uptake of ions was examined by the roots of T. aestivum L.; however, there was non-significant change in shoot K⁺ concentration reported with applid treatments (Figure 3; Table 3). Different studies suggested that Na⁺ accumulation is a common response of plants to salt stress, while K⁺ concentration remains relatively constant in plant’s shoot (Flowers and Colmer, 2008; Wang et al., 2012). The exact mechanisms underlying this response are still under investigation, but it is thought to involve selective ion transporters and channels in the plasma membrane of root and shoot cells. Zhang et al. (2018) examined that K⁺ ions are essential for plants and oxidative stress is efficiently decreased by the enhanced K⁺ content in plant roots. Generally, Na⁺ and K⁺ balance effectively regulates the salt stress which might be due to the presence of high concentrations of Fe, Zn, and Mn in soil and T. aestivum L. (Figure 3; Supplementary Table S1); thus, endorsing ion balance (Zhang et al., 2018). As, MECBs are acidic in nature; so, its application significantly (p ≤ 0.05) influenced the pH (acidic) of soil which played a vital role in activating the effect of biochar in NaCl sorption (Supplementary Table S1). Although, EC is decreased in soil with biochar addition which might cause a decrease in soil evaporation through high water holding capacity but increased salt leaching, soil porosity, and hydraulic conductivity (Dahlawi et al., 2018). Interestingly, the same trend was investigated in this proposed study under salinity stress (Supplementary Table S1).

The growth and physiology of T. aestivum L. were significantly (p ≤ 0.05) affected under salinity stress (Table 4). It might be due to the more uptake of Na⁺ and Cl⁻ ions from root to shoot which inhibit cell division because excess of Na⁺ and Cl⁻ ions have the ability to attach with the plant cell wall ultimately reducing its overall growth (Muro-González et al., 2020). Furthermore, it leads to the inhibition of K⁺ influx which adversely affects the photosynthetic machinery of the plant thus causing stomatal closure, damaged thylakoid membranes, disturbed ionic and osmotic homeostasis, oxidative damage, and disruption in the synthesis of secondary metabolites; a similar trend for photosynthetic rate was investigated in this research study (Table 4; Khoshbakht et al., 2018; Muro-González et al., 2020; Zhao et al., 2020). However, the transpiration rate also significantly (p ≤ 0.05) decreased in T. aestivum L. through the addition of MEBs; thus, growth and physiology were improved efficiently by increasing the soil’s water holding capacity under salinity stress (Table 4). Thus, salt stress is positively regulated by the activation of Medicago sativa GSK-₃ kinase (present in plastid) through moderating sugar metabolism (Zhao et al., 2020). Salt stress is also efficiently tolerated through two major enzymes (Fad6 and GPAT) which localized in the plastid to balance the fluidity of thylakoid membrane thus moderating fatty acid metabolism (Suo et al., 2017). Recovery of PSII is also associated with some genes such as RUB and RCI present in chloroplasts through more K⁺ influx under high salinity; however, the production of ROS mainly in the apoplast, chloroplast, mitochondria, and peroxisomes also significantly scavenged due to normalization of photosynthetic machinery (Calderon et al., 2013). Similarly, the results of photosynthetic activities were highly consistent with this mode of action (Table 4). However, Farooq et al. (2020) has been reported salinity tolerance (30%) in Vigna unguiculata L. (Walp. Plant) with application of simple organic-originated biochar when compared to control. Moreover, Taqdees et al. (2022) also reported increased chl. a and b (70% and 76%) through the application of Zn and Si amended biochar, respectively under salinity stress.

Salinity stress significantly (p ≤ 0.05) caused oxidative damage in the wheat plant through uncontrolled production of reactive oxygen compounds (Figure 4). Salinity stress causes stomatal closure in the shoot of plants which leads to burst production of ROS under high Na⁺ concentration (Rejeb et al., 2015). Thus, oxidative damage induced in plants by the Mehler reaction (PSI), production of singlet oxygen (PSII) in the thylakoid membrane, and H₂O₂ production due to the inhibition of water splitting manganese-complex which allow more electrons to leak from PSI-II resulting high rates of CMP (Lee et al., 2013). Similarly, this trend of electrolyte leakage has also been reported in this proposed study (Figure 4) and also previous studies under salinity stress (Dong et al., 2017; Kotagiri and Kolluru, 2017; Hniličková et al., 2019).

The application of MECBs significantly (p ≤ 0.05) triggered the activities of antioxidant enzymes which efficiently scavenged ROS production under salinity stress (Figure 5). The presence of minerals (Zn, Fe, Mn) act as structural components of APX and CAT which prevent the plant from oxidative damage; moreover, Zn and Fe as a cofactor also trigger the activities of antioxidants to scavenge the free radicles which ultimately protects the proteins and lipids from being further damaged (Marreiro et al., 2017). Thus, reduced Na⁺ and increased K⁺ uptake efficiently stimulates the ascorbate mutant (vtc2-I) which converts H₂O₂ into an H₂O molecule; which further triggers the activities of APX and CAT through efficiently scavenging production of ROS in plants (Haruta et al., 2014) and the findings of this proposed study were highly consistent to this mechanism (Figure 5). Interestingly, Kul et al. (2021) reported that oxidative damage in tomato plants was efficiently recovered by the application of rice husk biochar (5%) by efficiently influencing SOD (6.1%) and CAT (6.9%) under NaCl stress.

As to cope the oxidative damage; non-enzymatic mechanisms were also triggered inside the plant body which further lowered the effects of ROS by producing a greater number of hydroxyl (OH⁻) groups under salinity stress (Feng et al., 2018). The application of MECBs significantly increased the content of proteins and total phenolic; while the results were well pronounced in T. aestivum L. with the application of FeBc under different rates of BW (Table 4; Figure 6). Thus, more salt stress is tolerated by the activation of mitochondrial genes (AOX_I and Mn-SOD) causing improvement in metabolic activities and flow of water (reduced osmotic stress) in plants, which efficiently improved the overall growth and physiology of plants (Zhao et al., 2020). A similar study examined the increase in contents of total phenolics (1.7-folds) in summer savory plants treated with organic material pyrolyzed biochar (2%) under NaCl stress (Mehdizadeh et al., 2019a,b). Moreover, similar results with the application of ZnO nanoparticles have also been reported by Noohpisheh et al. (2021) in cultivars of Trigonella foenum-graecum under salinity stress.