Citric acid–modified coconut shell biochar mitigates saline–alkaline stress in Solanum lycopersicum L. by modulating enzyme activity in the plant and soil

2.1 Functionalization of BioC

Coconut shell BioC (CSB; obtained by slow pyrolysis at a maximum temperature of 500°C with a slow heating rate and over a prolonged period until a high degree of carbonization was achieved, pH 10.3 and EC of 4.3 dS m⁻¹. According to the manufacturer, the maximum moisture content of the biomass was 18%, and the particle size corresponded to <30 US Std. mesh (≈0.6 mm), bulk density 0.55–0.70 g cm⁻³, maximum ash content 3.96%, water soluble 2–3%, surface area 80 m² g⁻¹, average pore radius 0.78 nm, iodine number 70 mg g⁻¹, and BET surface area 70 m² g⁻¹; proximate and elemental analysis: fixed carbon, 70%; volatile matter, 16.5%; organic carbon, 55.7%; C/N ratio, 502; organic matter, 96%; total nitrogen, 0.11% [Dumas]; P, 0.06%; K, 0.74%; Ca, 0.14%; Mg, 0.04%; Na, 0.11%; S, 0.03%; Fe, 657 ppm; Cu, 51.73 ppm; Mn, 36.07 ppm; Zn, 24.05 ppm; and B, 14.9 ppm (Carbotecnia S.A de C.V., Jalisco Mexico) was functionalized following the incipient moisture impregnation method [17]. Briefly, CSB was placed in a solution of citric acid (CA, 10 mM) or ascorbic acid (AA, 10 mM) in a 1:20 (m/v) ratio for 24 h with sporadic agitation. This concentration of organic aids was selected based on previous studies [20], because this dose, in addition to being less expensive, is effective in modifying the functional groups and pH of BioC.

Finally, the solid was recovered by decantation and dried to constant weight at room temperature. During this step, approximately 200 g of material was lost, corresponding to the removal of excess solution and non-adsorbed fractions. The functionalized CSB was characterized using scanning electron microscopy (Philips, XL30 ESEM model equipped with an EDAX Genesis EDS system) and Fourier transform infrared spectroscopy (FTIR; Frontier FT-IR/NIR 110711, PIKE Technologies In.).

2.2 Treatments

To verify the response of the functionalized CSB to the mitigation of salt–alkali stress, low doses were studied. The treatments tested consisted of 1.25 g CSB functionalized with AA kg⁻¹ soil (BAA1), 2.50 g CSB functionalized with AA kg⁻¹ soil (BAA2), 5.00 g CSB functionalized with AA kg⁻¹ soil (BAA3), 10.00 g CSB functionalized with AA kg⁻¹ soil (BAA4), 1.25 g CSB functionalized with CA kg⁻¹ soil (BCA1), 2.50 g CSB functionalized with CA kg⁻¹ soil (BCA2), 5.00 g BioC functionalized with CA kg⁻¹ soil (BCA3) and 10.00 g CSB functionalized with CA kg⁻¹ soil (BCA4). In addition, there was an unstressed treatment without CSB (CK) and a saline–alkaline stressed treatment without CSB (SAS).

Saline–alkaline stress consisted of a solution of 50 mM NaCl and 10 mM NaHCO₃, which was prepared in a nutrient solution container and applied via drench starting at stage BBCH-60 (second flower cluster; Biologische Bundesanstalt, Bundessortenamt, and Chemical Industry) and until the end of the experiment. This stress level was selected to simulate moderate to severe saline–alkaline conditions commonly reported in calcareous soils of arid and semi-arid regions [21,22]. This allows the protective effects of citric acid-functionalized BioC to be assessed under conditions that significantly hinder plant growth, while remaining within physiologically relevant ranges. This allows future studies to explore milder or gradient stress levels to evaluate the efficacy of BioC under more variable field conditions.

2.3 Plant material and management

‘Adrianna’ F1 saladette tomato seedlings were transplanted 26 days after germination into black polyethylene containers with 3 kg of calcareous soil (loam texture, 7.34% total carbonates, 0.99% organic matter, pH 8.03, EC 1.13 dS m⁻¹). Before transplantation, the functionalized CSB was homogeneously mixed with soil. For crop nutrition, Steiner solution [23] at pH 6.35 and EC 2.45 dS m⁻¹ was used, which was applied through a directed irrigation system according to crop water demand. After preparing the saline–alkaline stress, the nutrient solution presented a pH of 7.52 and an EC of 10.63.

2.4 Petiole cell extract (PCE)

When SAS treatment plants showed visual damage due to salt–alkali stress (chlorosis, necrosis, stiffer, brittle and smaller shoots and leaves, leaf curl and general reduction of vigor) (corresponding to the BBCH-72 stage; presence of recently set young fruit) petiole samples were collected and macerated to extract the juice where total soluble solids (TSS) (with Hanna Instruments HI96801 digital refractometer), total salinity (with LAQUAtwin-Salt-11 S071, HORIBA Scientific), as well as K⁺, Na⁺, NO₃ ⁻ and Ca²⁺ concentration were measured with a nutrient monitoring kit (LAQUAtwin-K-11 S030, LAQUAtwin-Na-11 S022, LAQUAtwin-NO3-11 S040 and LAQUAtwin-Ca-11 S050 of HORIBA Scientific, respectively).

2.5 Soil solution, soil interstitial water (SS)

The SS samples were collected at the BBCH-80 stage with suction lysimeters (Irrometer Company, Inc. Riverside, CA, USA) placed at a depth of 15 cm at a distance of 5 cm from the stem at an angle of ≈ 60° and with a suction of –50 kPa. After 24 h, the samples were collected, and the electrical conductivity (EC), hydrogen potential (pH) (HI 98130-Hanna Instruments), redox potential (ORP, HM Digital ORP-200), total salinity (LAQUAtwin-Salt-11 S071), and concentrations of K⁺, Na⁺, NO₃ ⁻, and Ca²⁺ were measured in situ using a LAQUAtwin HORIBA nutrient monitoring kit. In addition, the total carbonate (carbonates and bicarbonates) content was quantified by colorimetry [24].

2.6 Agronomic parameters

Crop growth and development were evaluated at the end of the experiment by measuring the height with a flexometer, stem diameter with a digital caliper, number of leaves, number of clusters, yield per plant, and fresh and dry biomass of stems, leaves, and roots (Adventurer Pro digital scale, OHAUS).

2.7 Biochemical parameters in leaf tissue

During the BBCH-72 stage, leaf tissue samples were collected (between 10:00 and 12:00 h), placed on ice, frozen, and freeze-dried (model ECO-FD10PT, Biobase Meihua Trading Co., Ltd. Shandong, China) for 36 h. Finally, the samples were ground using a mortar and pestle and stored until further analysis.

Photosynthetic pigments (chlorophyll a, chlorophyll b, and total chlorophyll) [25], carotenoids [26], vitamin C content [27], total phenols [28], and total flavonoids [29] were determined in leaf tissue. Similarly, reduced glutathione, GSH [30], catalase, CAT [31], ascorbate peroxidase APX [32], glutathione peroxidase (GSH-Px [33] adapted from Ref. [30]), phenylalanine ammonium lyase (PAL) [34], Ribulose-1,5-bisphosphate carboxylase/oxygenase, RuBisCO [35,36], phosphoenolpyruvate carboxylase, PEPC [37] with modifications according to [38], and β-carbonic anhydrase, β-CA [39]. In the same tissue, some stress indicators, such as proline content [40], MDA [41], and H₂O₂, [41] were quantified. Except for β-carbonic anhydrase, readings were performed at the corresponding absorbances using a UV-Vis spectrophotometer (ME-UV1800; MesuLab Instruments Co., Ltd., Guangzhou City, China).

2.8 Soil enzyme activity

Soil sampling was carried out during the BBCH-72 stage, during which approximately 150 g of soil was collected at a depth of 10 cm and stored at 4°C. Alkaline phosphatase activity, ALP [42]), β-glucosidase, β-GLU [43], β-N-acetylglucosaminidase, NAG [44], urease [45], and fluorescein diacetate (FDA) hydrolysis [46] were determined on these samples using UV–Vis spectrophotometer. The data are expressed as the dry weight of the soil.

2.9 Experimental design and statistical analysis

The experiment was organized according to a randomized complete block design with ten treatments and four replicates per treatment (two plants per replicate). The blocking criterion was the location within the greenhouse to minimize potential effects of light or temperature gradients. However, a preliminary analysis indicated that the block effect was not significant; therefore, the data were subsequently analyzed according to a completely randomized design. Data was subjected to normality and homogeneity of variance tests, followed by one-way analysis of variance and a mean test Fisher’s least significant difference (p < 0.05) using the statistical program InfoStat 2020.

3.1 SEM and FTIR analysis of the CSB and functionalized CSB

SEM analysis revealed significant modifications in the surface morphology of the CSB after functionalization (Figure 1). The image captured at a scale of 50 µm shows that the CSB has a relatively compact surface with irregular morphology and the presence of some low-definition pores, lamellar structures, and fractures, indicating the lignocellulosic nature of the original biomass. In addition, areas with higher brightness intensities were observed, suggesting the possible presence of mineral residues such as Ca²⁺, K⁺, and Si (Figure 1a). The micrograph of CSB functionalized with ascorbic acid (BAA) (Figure 1b) shows a more fragmented and porous structure, with greater surface roughness and areas with greater structural openness. Finally, the micrograph of the CSB functionalized with citric acid (BCA) showed a more disuniform surface with a greater formation of aggregates with possible modifications in its structure (Figure 1c).

Figure 1

SEM Imagenes of (a) Coconut shell BioC (CSB) taken at 500×-50 µm, (b) CSB functionalized with ascorbic acid taken at 400×-50 µm, and (c) CSB functionalized with citric acid taken at 400×-50 µm. EDX (Energy-dispersive X-ray spectroscopy coupled to SEM) elemental distribution maps of (d) CSB, (e) CSB functionalized with ascorbic acid (BAA), and (f) CSB functionalized with citric acid (BCA). Source: Authors’ original work.

The EDX-SEM elemental distribution maps (Figure 1d–f) showed that CSB presented scattered signals of C and O and minor traces of other elements such as Ca²⁺, K⁺, and Si, predominantly carbonaceous surface, and low heterogeneity (Figure 1d), while BAA showed increased presence and distribution of O, Cl^–, K⁺, and Na⁺ (Figure 1e) and BCA greater uniformity in the distribution of O and other functional elements (Figure 1f).

Infrared spectroscopy by Fourier transform allowed the identification of peaks of functional groups present on the surface of the materials in the range of 4,000–500 cm⁻¹ (Figure 2). In the spectrum corresponding to CSB, a broad band was observed at approximately 3,400 cm⁻¹, corresponding to O–H stretching vibrations of hydroxyl groups, and a weak band was identified near 2,920 cm⁻¹, associated with C–H stretching of aliphatic groups. In the region from 1,700 to 1,600 cm⁻¹, a more pronounced band was evident for BAA and BCA than for CSB, attributed to C═O stretching of carboxyl groups (1,700 cm⁻¹) and C═C stretching of aromatic structures (1,600 cm⁻¹). In addition, a band around 1,400–1,380 cm⁻¹, corresponding to C–H bending vibrations of aliphatic groups and/or COO⁻ symmetric stretching, which is more intense for BAA and BCA, also stands out. Finally, in the region near 1,100 cm⁻¹, signals attributable to C–O alcohols, ethers, or acids were observed.

Figure 2

FTIR Spectra of coconut shell BioC (CSB), ascorbic acid functionalized CSB (BAA), and citric acid functionalized CSB (BCA). Source: Authors’ original work.

3.2 PCE parameters of tomato plants under saline–alkaline stress grown in calcareous soil with the addition of functionalized biochar

Total salinity showed a consistent increase of up to 125% in BCA4 with respect to CK, with the BCA3 treatment promoting a reduction of 48.79% with respect to the SAS treatment. Likewise, the concentration of Na⁺ showed a notable increase in PCE under saline–alkaline stress (up to 2,000%) compared to non-stressed plants (CK). In particular, this ion showed a 23.4 and 23.1% reduction in PCE in plants treated with BAA3 and BCA1, respectively.

The NO₃ ^–, K⁺, and Ca²⁺ contents showed an increase consistent with the application of the functionalized CSB; in the case of nitrate, the highest content was documented under the BCA2 (Δ80%) and BAA4 (Δ72%) treatments compared to the SAS treatment. For K⁺, the BCA4 and BCA2 treatments showed increases of 79 and 61%, respectively. Finally, the Ca²⁺ concentration increased in all treatments with respect to the CK control, highlighting treatments BCA1, BCA3, and BAA2 with 175.08, 104.44, and 100.36%, respectively. In the case of TSS, increases of 31.85, 30.44, and 10.77% were found in the PCE of plants developed under BCA2, BCA4, and BAA4, respectively, compared to the CK control (Table 1). The CSB treatments functionalized with CA showed better responses than those functionalized with AA.

3.3 SS parameters of tomato plants under saline–alkaline stress grown in calcareous soil with the addition of functionalized biochar

The pH increased by 6.02% in BCA2 with respect to SAS; however, there was no significant difference between the treatments. The ttreatments BCA3 and BCA4 showed a slight reduction with respect to SAS of 1.35 and 0.12%, respectively. The total salinity showed an increase of 297.53% with BCA1 compared to CK; however, treatments BCA3, BAA2, and BCA2 achieved reductions of up to 29.57, 25.71, and 23.87% with respect to SAS (Table 2). The ORP increased by 14.11% with respect to CK, whereas BCA4, BAA4, and BCA2 managed to reduce it by 20.32 and 0.82%, respectively. BCA4 reduced EC by 55.66% compared to SAS. The carbonate and bicarbonate contents were significantly reduced in BAA4 (84.11 and 70.27%, respectively). TSS dissolved in the SS presented an increase of up to 138.33% with the BAA3 and BCA1 treatments compared to CK, and at the same time, 7.51% higher than that in SAS (Table 2).

Na⁺ concentration revealed a remarkable increase in SS under saline–alkaline stress (up to 1004.66%) compared to that in non-stressed plants (CK). In particular, this ion showed 34.09, 32.53, and 30.12% reductions in SS in plants treated with BCA3, BAA2, and BCA2, respectively, compared to those treated with SAS. Similarly, K⁺ and NO₃ ⁻ presented the same trend, showing a considerable increase in functionalized CSB applications with respect to the control (CK). For K⁺, treatments BAA3, BAA1, and BCA1 stood out with 60.46, 52.32, and 41.85% increases, respectively, compared to CK. BCA1, BAA3, and BAA1 showed the highest increases in NO₃ ⁻ (49.23, 38.97, and 27.18%, respectively). Finally, Ca²⁺ concentration decreased in all treatments with respect to CK by up to 31.63% (Table 2).

3.4 Agronomic parameters of tomato plants under saline–alkaline stress grown in calcareous soil with the addition of functionalized biochar

Agronomic parameters showed an increase in tomato plants under saline–alkaline stress treated with functionalized CSB compared to those in SAS. The number of bunches increased by 9.52 and 7.23% in the BCA1, BCA2, and BAA2 treatment groups, respectively. Fresh weight was 29.41, 22.58, and 18.80% higher in BCA2, BCA4, and BCA1, respectively. The dry weight increased by 48.3, 41.5, and 38.6% with BCA2, BCA3, and BAA3, respectively. Relative root length showed a slight increase in BAA3, BCA3, and BCA1 treatments by 14.44, 10.79, and 10.64%, respectively, compared to SAS. Yield, fresh weight, and root dry weight were reduced with respect to CK and increased with respect to SAS by up to 43.03% with BCA1, 13.14% with BCA1, and 13.75% with BCA1 (Table 3).

3.5 Biochemical parameters of tomato plants under saline–alkaline stress grown in calcareous soil with the addition of functionalized biochar

The use of CSB functionalized with citric acid at a dose of 10 g kg⁻¹ of soil (BCA4) promoted higher contents of chlorophyll a, chlorophyll b, and total chlorophyll (23.96, 29.02, and 27.20%, respectively). The chlorophyll a/b ratio was lower in all plants treated with functionalized CSB than in the absolute control and SAS, with BCA1 being 12.72% lower than that of CK and 15.78% lower than that of SAS. The presence of red carotenoids did not show significant differences; however, BCA1 was 20.65% lower than SAS, BCA4 was 8.95% higher than SAS, and 33.52% higher than CK (Table 4).

Vitamin C concentration showed no significant difference, but treatments BAA1, BAA4, and BAA2 showed reductions of 8.73, 7.66, and 7.18%, respectively, compared to SAS. The use of CSB induced a higher production of total phenols with treatments BAA2, BAA4 and BAA1, by 7.42, 5.14 and 3.42% respectively, compared to SAS. In contrast, total flavonoids did not present a significant difference; BCA2 presented a greater reduction of this compound with respect to SAS by 59.65 and 68.95% compared to CK. BCA4 showed a different result, being 72.91% higher than SAS and 33.09% higher than CK. In the same sense, GSH concentration did not show significant differences; however, it showed that higher doses (BCA4) apparently promoted a higher concentration since it presented an increase of 152.42% with respect to SAS and 177.78% to CK (Table 5).

The BAA3, BCA2, and BCA3 treatments increased GSH-Px activity by 774.58, 687.29, and 687.29%, respectively, with respect to SAS. Catalase activity was increased by 75.00, 62.12, and 51.51% with BCA2, BCA4, and BCA3 treatments, respectively, compared with SAS. In contrast, the use of CSB functionalized with citric acid at a dose of 1.25 g kg⁻¹ reduced APX activity by 75.81% compared to SAS and 76.65% compared to CK. In contrast, PAL activity of BCA4 was 94.54% higher than that of SAS (Table 6).

Regarding stress indicators, H₂O₂ showed a reduction among treatments (p > 0.05), but not with respect to SAS, with BAA2 being the treatment with the lowest concentration among CSB-treated plants (27.27% lower than CK and equal to SAS) and BCA2 being the treatment with the highest concentration (218.78 and 84.82% higher than CK and SAS, respectively) (Figure 3a). For MDA content, BCA2 treatment showed the lowest accumulation, with a content 34.31% lower than SAS, while BCA4 presented the highest accumulation (55.88% higher than SAS), but the difference was not significant (p = 0.3148) (Figure 3b). Finally, proline results indicated that BAA4 reduced 61.38% as a function of SAS, whereas BCA4 promoted higher production by 19.01% compared to CK (Figure 3c).

Figure 3

Stress indicators quantified in the leaf tissue of tomato plants under saline–alkaline stress grown in calcareous soil with the addition of functionalized biochar. (a) Malondialdehyde (MDA), (b) hydrogen peroxide (H₂O₂), (c) proline. Mean values ± standard error (n = 3) within columns followed by the same letter are not significantly different (p > 0.05), T = treatment, CK = unstressed treatment, SAS = saline–alkaline stress without CSB, BAA1 = 1.25 g CSB functionalized with ascorbic acid kg⁻¹ soil, BAA2 = 2.50 g CSB functionalized with ascorbic acid kg⁻¹ soil, BAA3 = 5.00 g CSB functionalized with ascorbic acid kg⁻¹ soil, BAA4 = 10.00 g CSB functionalized with ascorbic acid kg⁻¹ soil, BCA¹ = 1.25 g CSB functionalized with citric acid kg⁻¹ soil, BCA2 = 2.50 g CSB functionalized with citric acid kg⁻¹ soil, BCA3 = 5.00 g CSB functionalized with citric acid kg⁻¹ soil, BCA4 = 10.00 g CSB functionalized with citric acid kg⁻¹ soil.

The activity of β-CA increased (p > 0.05) in BCA3, BAA4, and BAA3 by up to 47.65, 16.83, and 16.51%, respectively. RuBisCO activity was not significantly different (p = 0.1893); however, under BCA4 treatment, an increase of 22.85% with respect to SAS and 163.26% with respect to CK was observed. PEP carboxylase activity did not show significant differences (p = 0.5922); however, it was observed that lower doses of BAA (BAA1) resulted in a greater increase of 40.65% with respect to SAS, and with increasing doses (BAA3) resulted in a greater reduction of 64.83% with respect to SAS (Figure 4).

Figure 4

Enzymes associated with photosynthesis were quantified in the leaf tissue of tomato plants under saline–alkaline stress grown in calcareous soil with the addition of functionalized biochar. (a) Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), (b) β-carbonic anhydrase (βCA), (c) Phosphoenolpyruvate carboxylase (PEPC). Mean values ± standard error (n = 3) within columns followed by the same letter are not significantly different (p > 0.05), T = treatment, CK = unstressed treatment, SAS = saline–alkaline stress without CSB, BAA1 = 1.25 g CSB functionalized with ascorbic acid kg⁻¹ soil, BAA2 = 2.50 g CSB functionalized with ascorbic acid kg⁻¹ soil, BAA3 = 5.00 g CSB functionalized with ascorbic acid kg⁻¹ soil, BAA4 = 10.00 g CSB functionalized with ascorbic acid kg⁻¹ soil, BCA¹ = 1. 25 g CSB functionalized with citric acid kg⁻¹ soil, BCA2 = 2.50 g CSB functionalized with citric acid kg⁻¹ soil, BCA3 = 5.00 g CSB functionalized with citric acid kg⁻¹ soil, and BCA4 = 10.00 g CSB functionalized with citric acid kg⁻¹ soil.

3.6 Enzymatic parameters of calcareous soil grown with tomato plants under saline–alkaline stress with the addition of functionalized biochar

Soil enzyme activity had a positive impact under CSB treatments. The ALP activity was significantly higher in the BAA3, BAA1, and BAA2 treatments, with increases of 108.31, 87.09, and 73.71%, respectively, compared to SAS (p = 0.0209) (Table 7). The activity of β-glucosidase (β-GLU) did not show significant differences between treatments (p = 0.1093); however, the activity of BAA2 was 40.21% lower than that of SAS. For β-N-acetylglucosaminidase (NAG), BAA1, BAA3, and BAA2 treatments increased by 20.01, 13.37, and 2.27%, respectively, although there were no statistically significant differences with respect to SAS (p > 0.05) (Table 7). The BAA1, BAA2, and BCA4 treatments increased FDA activity by 155.72, 64.92, and 23.67%, respectively, with respect to SAS; however, no significant differences were observed (p = 0.3749). Finally, the BCA3, BCA2, BAA2, and BCA4 treatments increased urease activity by 35.89, 17.94, and 15.38%, respectively, with respect to SAS, but the differences were not significant (p = 0.2116) (Table 7).

4.1 SEM and FTIR analysis of CSB and functionalized CSB

The type of soil in which plants grow is a determining factor in their physiological responses to salt–alkali stress. In this study, the calcareous soil used seems to have increased the negative effects of saline–alkaline stress in plants without functionalized CSB by showing lower accumulation of antioxidant compounds and lower transient nutrient content in the CSB. In this context, the application of functionalized CSB at low doses may be a strategy to mitigate the damage caused by this type of stress [47]. This response may be associated with the fact that functionalization with CA and AA modified the biochar surface, promoting the addition of oxygenated functional groups, such as hydroxyl (−OH) and carbonyl (C═O), (C−O), carboxyl anion (COO−), and an alkyl (CH₃) functional group on the CSB matrix (Figure 2), which amplified the nutrient retention capacity, cation exchange, and selective adsorption of Na⁺ [48]. Moreover, the functionalization of CSB with CA reduced the pH from 10.30 to 7.90 and with AA to 7.17, which favored nutrient bioavailability and decreased soil alkalinity, promoting better responses in tomato plants.

However, although SEM and FTIR characterization provided useful information on surface morphology and functional groups, the BET surface area, pore volume, and elemental composition of the functionalized BioC were not measured. According to the manufacturer, unmodified BSC had an iodine value of 70 mg g⁻¹, a BET surface area of 70 m² g⁻¹, and an average pore radius of 0.78 nm. Therefore, the lack of evaluation of these parameters after functionalization represents a limitation of the present study, as changes in surface area, porosity, or C:N ratio could affect nutrient retention, adsorption capacity, and overall plant response under saline–alkali stress. Therefore, future studies should include these analyses to better understand the mechanisms underlying BioC performance.

4.2 PCE parameters of tomato plants under saline–alkaline stress grown in calcareous soil with the addition of functionalized biochar

High concentrations of soluble and alkaline salts, such as NaCl, NaHCO₃, and Na₂CO₃, increase the Na⁺ content and decrease the uptake of K⁺, Ca²⁺, Mg²⁺, NO₃ ⁻, P, Zn, Fe, Mn, Cu, and B, causing osmotic, ionic, and oxidative stress [49]. In response to this environment, plants promote cytosolic Ca²⁺ signatures to activate protein kinases that modulate osmotic stress signaling [4]. In this study, an increase in Ca²⁺ was observed in all treatments compared with unstressed plants, highlighting BCA2 (175.08%), BCA3 (104.44%), and BAA2 (100.36%), which may indicate that these treatments promoted greater Ca²⁺ accumulation in the PCE, possibly modulating osmotic stress signaling.

In addition, exposure of plants to salinity and alkalinity conditions reduces NO 3 − uptake to promote cellular ionic balance between Cl⁻, HCO₃⁻, CO₃ ²⁻, K⁺, Ca²⁺, and Mg²⁺ and reduce oxidation mechanisms [50]. However, reports mention that BioC has a high cation exchange capacity (CEC) that allows it to adsorb and retain K⁺, Ca²⁺, Mg²⁺, and NH₄⁺ on its porous and negatively charged surface, in addition to regulating soil EC, avoiding “salinity peaks” that can generate osmotic or ionic toxicity, which allows greater ease of water and nutrient absorption by root hairs, which promotes chlorophyll content [51].

Furthermore, Barrow and Hartemink [52] emphasized that the effect of pH on nutrient availability depends not only on their chemical behavior in the soil, but also on the plant’s ability to absorb them, and that some nutrients, such as NO₃ ⁻ and PO₄ ³⁻, may increase their concentration in the SS with increasing pH, but their uptake by the plant may decrease due to changes in transport mechanisms or ionic competition. Thus, the pH of functionalized CSB is considerably lower than that of unfunctionalized CSB, which may have favored the bioavailability and uptake of nutrients in the soil by reducing the pH of the rhizosphere medium to a level more suitable for nutrient solubility.

In addition to the effects of BioC on the soil matrix, CA itself might have contributed to stress mitigation since, as a mobile organic acid, it can chelate cations such as Ca²⁺, Mg²⁺, and Fe³⁺, improving their availability to plants under saline–alkaline conditions [20]. Furthermore, CA can transiently reduce soil pH and improve CEC, promoting nutrient uptake and alleviating ionic and osmotic stress [53].

Furthermore, our results suggest a synergistic effect between biochar and CA: while BioC alone improved soil structure and cation retention, the addition of CA enhanced nutrient availability and uptake, exceeding the levels observed with BioC alone. This synergy likely contributed to the increased NO₃ ⁻, K⁺, and Ca²⁺ contents observed in PCE, improving stress signaling, chlorophyll synthesis, and overall physiological performance of tomato plants under saline–alkaline stress.

These effects were observed in the results of this study by increasing the NO₃ ⁻, K⁺, and Ca²⁺ content in the PCE by up to 80, 79 and 175.08%, under the BCA2, BCA4 and BCA2 treatments, respectively, indicating a higher efficiency in the uptake of essential nutrients even under adverse conditions. This improvement may be due to the functional groups added through BCA functionalization, which also provided a greater number of cation exchange sites, pore spaces, and specific surface area and generated a lower pH (7.90) than the unfunctionalized CSB, which together facilitated the retention of toxic cations such as Na⁺ and controlled nutrient release and improved root structure and microbial activity. As observed by the FDA, treatments with CSB functionalized with AC and AA, except for BCA3, BAA4, and BAA3, were superior to SAS. These characteristics together improve the rhizosphere environment thereby favoring K⁺, Ca²⁺ and NO 3 − uptake by the plant even under adverse conditions. In addition, enhanced Ca²⁺ uptake may be due to its involvement in stress signaling while NO 3 − promotes the synthesis of nitrogenous compounds, both of which are associated with increased chlorophyll production, since NO₃ ^– is a fundamental component of the chlorophyll tetrapyrrole ring, and Ca²⁺ facilitates chloroplast integrity and photosynthesis, in addition CSB promotes a more stable rhizospheric ionic environment allowing better water and nutrient uptake.

The increase in NO₃ ^– in the PCE of CSB-treated plants agrees with the findings of Tang et al. [54], who reported an increase in N uptake in plants grown in CSB-amended soils under saline conditions. Similarly, K⁺ concentrations increase due to plant efforts to regulate osmosis, membrane potential, and enzyme activity [55]. An increase in TSS concentration was also observed in treatments BCA2, BCA4, and BAA4, which may indirectly reflect the capacity for active osmotic regulation as part of the plant’s acclimatization mechanism to salt–alkaline stress [56].

4.3 Agronomic parameters of tomato plants under saline–alkaline stress grown in calcareous soil with the addition of functionalized biochar

The agronomic parameters of tomato plants (number of clusters, fresh and dry weight of vegetative part, length, fresh and dry weight of root) were improved by the addition of CSB compared to plants treated with SAS; these results are consistent with those reported by Soothar et al. [51], who added 30 and 45 g kg⁻¹ of BioC in soil with 1–5% salts, and were able to improve plant height, leaf number, leaf area, shoot and root fresh and dry weight compared to the control in maize plants under salinity stress conditions. In this study, these improvements resulted in a performance increase of up to 43%, which interestingly corresponds to the lowest dose BCA1 treatment (1.25 g kg⁻¹), while BCA4 treatment (10 g kg⁻¹) mainly improved physiological characteristics such as chlorophyll content, antioxidant capacity, and stress response. This contrasting response could be related to differences in the modulation of the rhizosphere environment: low doses could provide sufficient functional groups and surface activity to improve nutrient availability and ionic homeostasis without excessively altering soil properties, thus directly favoring reproductive development and fruit set. Conversely, higher doses of BioC could have induced greater improvements in physiological stress signaling capacity, antioxidant responses, and ionic compartmentalization, which alleviated stress symptoms at the foliar and cellular levels, but did not translate proportionally into yield increases. Similar dose-dependent effects of BioC have been reported in other crops, where moderate or low applications maximize productivity, while higher applications are more effective in improving physiological stability than yield [57]. These improvements reflect an indirect effect of the enhanced characteristics of CSB after functionalization, which allows it to locally modulate the rhizosphere environment, even in soils with an alkaline reaction pH, probably by increasing the CEC and uptake of nutrients such as NO₃ ^–, K⁺, and Ca²⁺ [58] as found in the CEP.

4.4 Biochemical parameters of tomato plants under saline–alkali stress with the addition of functionalized biochar

To counteract the adverse effects of salt–alkaline stress, such as excessive ROS accumulation and antioxidant imbalance, plants activate enzymatic and non-enzymatic defense systems, including APX, GSH, GSH-Px, CAT, vitamin C, phenolic compounds, proline (Pro), and flavonoids, which can neutralize, eliminate, or transform ROS [8,59]. In this study, the application of functionalized CSB favored the accumulation of GSH (BCA4 = 152.42%), GSH-PX (BAA3 = 774.58%), CAT (BCA2 = 75.00%), phenols (BAA2 = 7.42%), flavonoids (BCA4 = 152.42%), and PAL (BCA4 = 94.54%), suggesting that functionalized CSB promotes the overall antioxidant capacity of plants under saline–alkaline stress conditions.

In this experiment, BCA4 was superior to the other doses of BCA and BAA in increasing Pro content in leaf tissue by 19.01% compared to CK, although this value was 19.01% lower than that observed in SAS. The accumulation of Pro suggests an activation of defense mechanisms against stress, acting as an osmoprotector; therefore, the increase observed in BCA4 could be associated with a better capacity of plant tissue to resist stress compared to the other doses of functionalized CSB, although its efficacy was surpassed by SAS. These results agree with those reported by El-Sharkawy et al. [60] who mentioned that cotton stalk BioC modified with sulfuric acid (0.1 M; 1:100, w/v) increased Pro content in maize by 53.05% and wheat with 50.17% grown in saline-sodic soils. They also reported that CAT increased by 30.16 and 7.38% in both crops under the same conditions, similar to the data obtained in this study.

Likewise, the functional groups present in BioC probably play a determining role in the behavior of Pro and flavonoids, as they may have acted as chemical signals or modulators of the cellular redox state that promoted the production of compounds such as GSH, GSH-Px, CAT, and phenols, which are related to abiotic stress tolerance through the positive regulation of secondary metabolism under stress conditions [8]. This could be attributed to an increased stability of antioxidant enzymes and the preservation of cell membrane integrity promoted by the neutralization of ROS through the ascorbate-glutathione cycle, favored by the presence of phenolic (OH, COOH) and carboxylic (C═O, OH) groups on the surface of the functionalized biochar, which can act as direct donors or redox cofactors; in addition, they can improve the bioavailability and uptake of Fe, Cu, and Zn, which are essential for the activity of enzymes such as CAT and GSH-Px [60].

Similarly, BAA4 and BCA2 treatments achieved the highest reduction in MDA (32.35 and 31.37%, respectively) as a function of SAS, which may be due to the improved characteristics of CSB after functionalization, which maintained the integrity of cell membranes by reducing oxidative damage and lipid peroxidation. These results agree with those found by Zhang et al. [9] who when applying 377.9 g of BioC-based organic fertilizer 10 kg⁻¹ of soil observed that the MDA content in the final growth stage of sugar beet under saline–alkaline stress conditions was reduced by 26% with respect to plants without BioC. Highlighting that, the treatments with better response correspond to considerably lower doses of CSB (10 g kg⁻¹ in BAA4 and 2.5 g kg⁻¹ in BCA2), which represents approximately 26.5 and 6.6% of the dose applied by the previously cited authors, respectively. These results can be attributed to the effect exerted by BioC on nutrient bioavailability, an efficient antioxidant response that protects against salt–alkaline stress [61].

H₂O₂ is one of the main indicators of oxidative stress in plants. In this experiment, it was observed that although the BAA2 treatment presented the lowest H₂O₂ accumulation, it did not have a direct correlation with the activity of APX and GSH-Px, GSH, Pro, and flavonoids, indicating that the reduction of H₂O₂ was mediated by other ROS detoxification compounds different from the ascorbate-glutathione cycle, such as phenolic compounds, since they presented a higher content in this treatment (7.42% > to SAS), because these molecules have a high capacity to donate electrons and stabilize free radicals, thus contributing to the reduction of oxidative stress without requiring direct intervention of the ascorbate-glutathione cycle. Thus, the decrease in H₂O₂ observed in BAA2 could be attributed to the protective effect of phenols, which represent an alternative defense mechanism against induced salt–alkali stress. These results agree with the findings of Hasanuzzaman et al. [8], who reported H₂O₂ reduction in jute plants under salt stress treated with 2 g of BioC + chitosan kg⁻¹ of soil.

Another plant response to stress is the synthesis and accumulation of secondary metabolites, such as phenolic compounds and flavonoids, and the activation of the enzyme PAL [62]. In this study, the accumulation of flavonoids was increased by applying BCA3 and BCA4 which could indicate that higher doses decrease oxidative stress and improve nutrition by having higher capacity to adsorb toxic salts and ROS by the modified structure of the functionalized CSB. Deng et al. [63] reported an increase in phenol and flavonoid contents (4.4 and 19.5%, respectively) when applying BioC from pruning residues pyrolyzed at 500°C in tomato plants. In the same sense, it has been reported that citric acid applied to the soil can reduce the harmful effects of abiotic stress by stimulating the activity of antioxidant enzymes such as PAL [64]. Consistently, in this study, a greater increase in PAL activity was observed in the BCA4 treatment, which was 94.54% higher than that in SAS, suggesting that the functionalization of CSB with CA could promote the activation of this enzyme as part of the defense mechanism against saline–alkaline stress.

Net photosynthesis can mitigate oxidative and osmotic stress by providing the carbon skeletons necessary for the activation of primary and secondary metabolism [65]. In this sense, the BioC application can indirectly contribute to this process by improving soil physical and chemical conditions, favoring organic carbon sequestration, and reducing ionic toxicity. Under high-salinity conditions, Na⁺ accumulation hinders cellular ionic balance and reduces RuBisCO activity [66], causing an increase in photorespiration and a reduction in the rate of carbon fixation. To minimize this event, the enzymes β-CA and PEP carboxylase participate in atmospheric carbon fixation and CO₂ channeling [67]. In this study, RuBisCO and PEP carboxylase increased with the use of CSB, suggesting a relative enhancement of the photosynthetic process under saline–alkaline stress conditions, which agrees with the increase in chlorophyll a, b, and total chlorophyll a in the BCA4 treatment (23.96, 29.02, and 27.20%, respectively). This response coincides with that reported by Zhang et al. [9], who described higher chlorophyll b concentrations and similar trends in chlorophyll a and total chlorophyll in sugar beets with BioC-based organic fertilizer applications under saline–alkaline stress, probably because BioC has the ability to enhance Mg and N uptake, which is fundamental in chlorophyll biosynthesis and the functioning of the photosynthetic system [68].

It is important to recognize that the reproducibility of the observed effects may vary depending on soil type, composition, and seasonal environmental fluctuations. Since saline–alkali stress is influenced by multiple soil and climatic factors, further validation under various conditions is required to confirm the general applicability of the results.

4.5 Enzymatic parameters of calcareous soil grown with tomato plants under saline–alkaline stress with addition of functionalized biochar

BioC contributes to the modulation of the physical, chemical, and biological properties of the soil, influencing enzymatic activity, mineral nutrition, and the microbiome [69]. Thus, BAA1, BAA2, and BCA4 treatments promoted soil enzymatic activity with respect to saline–alkaline stress conditions, suggesting that CSB could promote ALP, NAG, FDA, and urease activity, whereas β-GLU remained stable in soil under saline–alkali stress, suggesting that the treatments did not affect soil carbon transformation processes. This fact coincides with that reported by Egamberdieva et al. [70], who reported a 42% increase in FDA activity when BioC was added as an amendment.

The application of CSB functionalized with CA and AA promoted urease and ALP activity in saline–alkaline soils, as both enzymes are closely related to nutrient availability. Urease catalyzes the hydrolysis of urea to ammonium (NH₄ ⁺), which facilitates nitrogen utilization, whereas ALP hydrolyzes organic phosphorus compounds to release inorganic phosphate that will be available to the plant. These enzymes show their maximum catalytic speed in environments with slightly alkaline pH (between 7.5 and 9.0 for ALP and between 6.5 and 8.5 for urease), but their activity can be limited in conditions of high salinity or high EC. Thus, the decrease in EC, pH, and soil alkalinity observed with CSB application could have generated a favorable environment for its functioning, contributing to the improvement in the mineralization of essential nutrients [71]. The increase in ALP suggests a positive effect of the treatments on soil phosphorus mineralization. A recent study showed that BioC functionalization of bougainvillea pruning waste with CA could improve enzyme activity in heavy metal-contaminated soil by up to 145% urease activity and 59% ALP [72].

Similarly, the increase in β-N-acetylglucosaminidase activity observed in this study suggests a positive stimulation in the recycling of nitrogen compounds and in the activity related to chitin degradation. This is particularly beneficial under saline–alkaline stress conditions, where nitrogen mineralization is usually limited. The results obtained here agree with those described by Kracmarova-Farren et al. [73], who indicated that adding BioC from wood chips increased the activity of this enzyme. Similarly, BioC has the ability to protect extracellular enzymes from denaturation and degradation during prolonged exposure to saline–alkaline stress due to physical adsorption or covalent binding of enzymes to the porous surface of BioC [74], which leads to the promotion of soil health under saline–alkaline stress conditions in calcareous soils.

It is important to emphasize that this experiment covers only one single growth cycle, and long-term evaluations are needed to determine the persistence of the beneficial effects of citric acid-functionalized biochar in agricultural soils. Potential negative impacts, such as changes in microbial community structure or risks of organic acid leaching, were not assessed and should be addressed in future studies to ensure the environmental safety of large-scale applications.