Ascorbic acid as a mitigator of salt stress effects on radish¹

Ácido ascórbico como mitigador dos efeitos do estresse salino em rabanete

ABSTRACT

Using water with high salt concentrations in irrigated agriculture can affect sensitive plants’ metabolic and biochemical functions. Therefore, strategies are needed to minimize the adverse impacts of salt stress. This study aimed to evaluate radish plant growth, water relations, and biochemical and nutritional responses as a function of irrigation water salinity and exogenous ascorbic acid (AsA) application. The experiment was conducted in a controlled greenhouse under a completely randomized design in a 3 × 2 factorial scheme with four replicates, using one plant per experimental unit. Treatments consisted of three levels of irrigation water salinity (0.5, 3.0, and 5.0 dS m^-1) and ascorbic acid application [without (0 mM) and with (1 mM)]. Increased salinity (3.0 and 5.0 dS m^-1) affected growth, water status, biochemical traits, and nutrient accumulation in radish plants. Stem diameter (55.0%), shoot mass ratio (14.5%), relative growth rate (9.0%), and soluble sugars in shoot (29.8%) and root (47.6%) increased with 1 mM AsA application, especially at 5.0 dS m^-1. Applying 1 mM AsA reduced Na⁺ and Cl⁻ cont 0ents in both shoot and root at all salinity levels and increased the Mg²⁺ content in shoot and root and Ca²⁺ content at 3.0 and 5.0 dS m^-1.

Key words:
Raphanus sativus L.; abiotic stresses; salt stress; mitigating

HIGHLIGHTS:

The attenuating effect of ascorbic acid is dose-dependent on the concentration of salts in the irrigation water.

Ascorbic acid with a concentration of 1 mM improved growth and sugars under a 5.0 dS m^-1 salinity.

Ascorbic acid reduced Na⁺ and Cl^- and increased Mg²⁺ and Ca²⁺ in roots and shoots under salinity.

RESUMO

O uso de água com altas concentrações de sais no manejo da agricultura irrigada pode afetar funções metabólicas e bioquímicas de plantas sensíveis. Diante disso, torna-se necessário adotar estratégias que minimizem os impactos adversos do estresse salino. Objetivou-se, com este estudo, avaliar o crescimento, as relações hídricas, bioquímicas e nutricionais de plantas de rabanete em função da salinidade da água de irrigação e da aplicação exógena de ácido ascórbico (AsA). O experimento foi conduzido em casa de vegetação sob delineamento inteiramente casualizado, em esquema fatorial 3 × 2, com quatro repetições e uma planta por parcela experimental. Os tratamentos consistiram em três níveis de salinidade da água de irrigação (0,5; 3,0 e 5,0 dS m^-1) e aplicação de AsA [sem (0 mM) e com (1 mM)]. O aumento da salinidade (3,0 e 5,0 dS m^-1) afetou variáveis de crescimento, relações hídricas, bioquímicas e nutricionais do rabanete. A aplicação de 1 mM de AsA promoveu incrementos no diâmetro do caule (55,0%), razão da massa da parte aérea (14,5%), taxa de crescimento relativo (9,0%) e teores de açúcares solúveis na parte aérea (29,8%) e na raiz (47,6%), especialmente em 5,0 dS m^-1. Além disso, o AsA mitigou os teores de Na⁺ e Cl⁻ na parte aérea e raiz em todos os níveis salinos, e aumentou os teores de Mg²⁺ na parte aérea e raiz e de Ca²⁺ na parte aérea nas salinidades de 3,0 e 5,0 dS m^-1.

Palavras-chave:
Raphanus sativus L.; estresses abióticos; estresse salino; atenuante

Introduction

Radish (Raphanus sativus L.) is a valuable root vegetable due to its medicinal properties, high nutritional value, and antioxidant action (Amin, 2023). Cultivated mainly in small areas, it relies on frequent irrigation, often with low-quality water (Putti et al., 2022). Its rusticity, short cycle, and early harvest make it popular among small and medium-sized farmers, ensuring quick financial returns (Dias et al., 2022).

Although rustic, radish is moderately sensitive to soil salinity (Amin, 2023). High salt concentrations in irrigation water disrupt metabolic functions, inhibiting germination, reducing root and shoot growth, and decreasing yield (Liang et al., 2018).

Ascorbic acid (AsA) application is a promising strategy to mitigate salinity effects (Mishra et al., 2024). In plants, AsA participates in non-enzymatic antioxidant metabolism, and its application reduces oxidative stress, limits lipid peroxidation, and enhances nutrient uptake, benefiting radish growth under water deficit (Henschel et al., 2023). Additionally, AsA regulates photosynthetic enzymes and boosts antioxidant biosynthesis, neutralizing reactive oxygen species (ROS) and improving plant tolerance to salt stress (El-Beltagi et al., 2022).

Studies have shown that AsA enhances growth, yield, and abiotic stress tolerance in crops such as bell pepper, okra seedlings, and tomatoes (Wang et al., 2019; Alayafi, 2020; El-Beltagi et al., 2022). Despite this, the specific effects of AsA application on the growth, metabolism, and production of radish crops irrigated with saline water have been little explored.

In view of the above, it is hypothesized that ascorbic acid concentrations improve the growth, physiological, and nutritional aspects of radish plants under saline conditions. Thus, the aim of this research was to evaluate the growth, water, biochemical, and nutritional relationships of radish plants as a function of water salinity and under exogenous application of ascorbic acid.

Material and Methods

The experiment was carried out between October and November 2023 in a greenhouse belonging to the Department of Agrarian and Forestry Sciences of the Federal Rural University of the Semi-Arid (UFERSA), Mossoró city, Rio Grande do Norte state, Brazil (5° 12’ S, 37° 19’ W and altitude of 20 m). The climate of the region is characterized as BSh, hot and dry (Alvares et al., 2013). Mean air temperature and relative air humidity data were collected using a digital thermo-hygrometer (Figure 1).

Figure 1
Climate data during the experimental period from October to November 2023

The experiment followed a completely randomized design in a 3 × 2 factorial scheme with four replicates. The first factor was the salinity levels of the irrigation water (0.5, 3.0, and 5.0 dS m^-1), while the second factor was the application of ascorbic acid [without (0.0 mM) and with (1.0 mM)]. Each experimental unit consisted of one plant per pot, totaling 24 experimental units. The irrigation water electrical conductivity levels of 0.5, 3.0 and 5.0 dS m^-1 were considered low, moderate and high levels, respectively, according to Ribeiro et al. (2024). The AsA concentrations (0 and 1.0 mM) were based on research by Basílio et al. (2018).

Radish plant variety Crimson Gigante was sown in 2.6 dm³ pots containing a mixture of sieved soil and organic compost in a 3:1 (v/v) ratio, respectively (Table 1). At 7 days after sowing (DAS), thinning was performed, leaving one plant per pot. At 13 DAS, with the appearance of the first pair of leaves, the application of the treatments began. The saline solutions were prepared for a volume of 60 dm³ by adding sodium chloride (NaCl) to water, except for the level of 0.5 dS m^-1, prepared from the local-supply water. The electrical conductivity of the irrigation water was monitored every 5 days to check each salt concentration, using a portable conductivity meter (model CD-880, Instrutherm).

Table 1
Physicochemical attributes of the substrate used in the experiment

Physical	Value	Fertility	Value
Coarse sand (kg kg-1)	0.53	pH in water (1: 2.5)	7.15
Fine sand (kg kg-1)	0.29	ECse (dS m-1)	0.07
Silt (kg kg-1)	0.13	P (mg dm-3)	112.90
Clay (kg kg-1)	0.05	K+ (mg dm-3)	1132.30
Textural class	Loamy Sand	Na+ (mg dm-3)	214.60
		Ca2+ (cmolc dm-3)	4.10
		Mg2+ (cmolc dm-3)	2.30
		Al3+ (cmolc dm-3)	0.00
		H + Al (cmolc dm-3)	0.00
		SB (cmolc dm-3)	10.23
		t (cmolc dm-3)	10.23
		CEC (cmolc dm-3)	10.23
		V (%)	100
		m (%)	0
		PES (%)	1

• ECse - Electrical conductivity of saturated paste extracts; P, K⁺ and Na⁺: Extracted with Mehlich 1; Ca ²⁺ and Mg ²⁺: extracted with 1 M KCl at pH 7.0; H⁺ + Al ³⁺: extracted with 0.5 M calcium acetate at pH 7.0; SB - Sum of bases (K⁺ + Ca²⁺ + Mg²⁺ + Na⁺); t - Effective CEC - Cation exchange capacity; V - Base saturation; m - Aluminum saturation; PES - Percentage of exchangeable sodium

The AsA solution was prepared at the time of each application. The AsA concentrations were prepared by dissolution in distilled water, and spraying was carried out on the shoots of the plants with a portable manual sprayer. During the application, the plants were isolated to prevent contamination. In the AsA solutions, Tween-80 (0.05% v/v), a solubilizing surfactant, was added to increase the substance’s adhesion to the leaf. An average of 20 mL of solution was applied per plant. At 13 DAS, ascorbic acid applications were initiated, with weekly applications totaling three (13, 20, and 27 DAS).

At 34 DAS, the following growth variables were measured: plant height, measured with the aid of a ruler graduated in centimeters (cm); stem diameter, measured with a digital caliper in millimeters (mm); and the number of leaves, counted manually. Then, the plants were collected and taken to the Laboratory of Reception and Preparation of Samples, belonging to the Center for Plant Research of the Semi-Arid Region, for further analysis.

After that, the plants were separated into shoots and roots and then weighed on an analytical scale (0.001 g) to obtain the fresh weight of the shoots (SFW), root (RFW) and total (TFW). With the fresh mass values, the shoot mass ratio (SMR), root mass ratio (RMR), shoot-root ratio, and relative growth rate (RGR) were also obtained. Root length (RL) and root diameter (RD) were also evaluated with the aid of a digital caliper (mm).

The relative water content (RWC) was determined according to Irigoyen et al. (1992) with adaptations. Leaf discs (6 mm²) were collected from the central vein of the leaves to measure fresh mass (FMD), then immersed in distilled water for 4 hours. After gently drying with paper towels, the turgid mass (TMD) was recorded. The discs were then oven-dried at 80 °C for 24 hours to obtain the dry mass (DMD). RWC was calculated using Eq. 1.

$$RWC\left(\%\right)=\left(\frac{FMD-DMD}{TMD-DMD}\right)\times 100$$ (1)

where:

FMD - fresh mass of discs (g),

DMD - dry mass of discs (g); and,

TMD - turgid mass of discs (g).

With adaptations, electrolyte leakage (EL) was determined following Lutts et al. (1996). After three washes with distilled water (3 min each), ten leaf discs were immersed in 25 mL of deionized water in Falcon tubes, and the initial electrical conductivity (EC) was measured after 90 min (EC1). The tubes were then heated at 85 °C for 1 hour, cooled to 25 °C, and the final EC was recorded (EC2). EL was calculated using Eq. 2.

$$EL\left(\%\right)=\left(\frac{EC1}{EC2}\right)\times 100$$ (2)

where:

EC1 - initial EC reading; and,

EC2 - final EC reading.

Subsequently, the plant material was taken to a forced circulation oven at 65 ºC and kept until it reached constant weight. With the dried materials, the dry weights of the shoot (SDW), root (RDW) and total (TDW) were obtained on an analytical scale (0.001 g). Then, the samples were crushed in a portable mill to perform the biochemical and nutritional analyses of the shoots and roots.

Biochemical analyses of total soluble sugars (TSS) and reducing sugars (RS) were performed using the phenol-sulfuric acid method (Dubois et al., 1956) and the dinitrosalicylic acid reagent (Miller, 1959). For TSS, 50 mg of lyophilized tissue was extracted with 5 mL of distilled water, incubated at 100 °C for 30 min, and then centrifuged at 1000 rpm. The supernatant (100 µL) was mixed with 400 µL of water, 0.5 mL of 5% phenol, and 2.5 mL of H₂SO₄. After vortexing, the absorbance was measured at 490 nm after 20 min. The concentration of soluble proteins was determined using the Bradford reagent (Bradford et al., 1976), and proline was determined by the acid ninhydrin method (Bates et al., 1973).

The plant material used in the nutritional analyses was collected at the end of the experiment. It consisted of the entire vegetative biomass rather than only diagnostic leaves. This material was thoroughly dried and finely ground using a knife mill before analysis. The nutritional contents of K⁺, Na⁺, Ca²⁺, Mg²⁺, and Cl⁻ were determined by digestion in nitric acid. K⁺ and Na⁺ were determined by flame spectrometry, while Ca²⁺ and Mg²⁺ were determined by atomic absorption spectrophotometry (Chapman & Pratt, 1962); Cl⁻ was determined by the Mohr method, extracted by calcium nitrate solution (Ca(NO₃)₂·4H₂O), in the form of chloride ion, titrated with a standardized silver nitrate solution (AgNO₃), in the presence of potassium dichromate (K₂CrO₄) as an indicator.

All data were analyzed for normality using the Shapiro-Wilk test and homoscedasticity using the Levene test. The data were subjected to analysis of variance (F test), and in cases of significance, Tukey’s test (p ≤ 0.05) was used to compare the means. The analyses were conducted using the SISVAR software (Ferreira, 2019). Pearson’s correlation analysis and principal component analysis (PCA) were performed for joint interpretation between treatments and variables using the R software (R Core Team, 2023).

Results and Discussion

The summary of the analysis of variance is presented in Table 2. A significant interaction between salinity and ascorbic acid was observed for the growth variables SMR, and RMR. Salinity independently influenced PH, SD, NL, RL, and RD, while AsA affected RD and RGR. The S × AsA interaction was significant for the biochemical variables TSS and TSR. The S factor influenced reducing sugars in the shoot (RSS), reducing sugars in the root (RSR), protein content in the root (PR), proline content in the shoot (PLS), proline content in the root (PLR), RWC, and EL, while AsA affected RSS and RSR. Regarding nutritional variables, the S × AsA interaction had effects on NaS, KS, CaS, MgS, ClS, NaR, KR, MgR, and ClR, with an individual effect of S on CaR.

Table 2
Summary of analysis of variance of the variables analyzed in radish under levels of electrical conductivity of irrigation water and ascorbic acid application

SV	DF	Mean squares
		PH	SD	NL	RL	RD
Salinity (S)	2	23.56**	36.85**	21.12**	527.88**	601.26**
AsA	1	3.15ns	10.53ns	0.37ns	1.26ns	66.66*
S × AsA	2	6.00ns	2.23ns	0.37ns	37.11ns	24.52ns
Residual	18	3.68	2.96	1.40	15.67	16.21
CV (%)		15.11	23.03	20.16	11.86	14.53
		SFW	RFW	SDW	RDW	TDW
Salinity (S)	2	110.63**	912.99**	0.20ns	1.46**	2.73**
AsA	1	21.45ns	36.35ns	0.30ns	0.07ns	0.66ns
S × AsA	2	2.65ns	10.20ns	0.01ns	0.10ns	0.05ns
Residual	18	8.40	14.14	0.09	0.17	0.22
CV (%)		29.69	25.12	27.49	37.40	21.46
		SMR	RMR	STR	RGR	TSS
Salinity (S)	2	0.02**	0.02**	0.80**	1203.70**	663.32**
AsA	1	6.66ns	0.00ns	0.004ns	416.32*	0.17ns
S × AsA	2	0.01*	0.01**	0.20ns	33.79ns	1204.59**
Residual	18	0.00	0.00	0.06	69.45	78.25
CV (%)		10.04	10.42	23.07	5.67	9.16
		TSR	RSS	RSR	PS	PR
Salinity (S)	2	89123.05**	0.001**	0.03**	0.06ns	0.93**
AsA	1	23834.72*	0.001**	0.01**	0.03ns	0.23ns
S × AsA	2	35816.64**	0.0003ns	0.00009ns	0.14ns	0.02ns
Residual	18	3323.31	0.0001	0.002	0.07	0.11
CV (%)		12.04	3.93	10.67	12.34	15.94
		PLS	PLR	RWC	EL	NaS
Salinity (S)	2	2820.20**	5078.34**	1775.07**	9175.13**	8.30**
AsA	1	119.14ns	143.87ns	119.17ns	61.88ns	9.52**
S × AsA	2	46.79ns	470.44ns	34.80ns	32.75ns	1.97**
Residual	18	258.45	149.27	162.87	57.98	0.10
CV (%)		50.30	29.57	16.61	12.81	6.26
		KS	CaS	MgS	ClS	NaR
Salinity (S)	2	1.24**	4.66**	3.05**	308.82**	28.21**
AsA	1	5.22**	8.72**	18.02**	10.54**	8.03**
S × AsA	2	0.74**	2.20**	0.38**	3.02**	3.38**
Residual	18	0.009	0.01	0.01	0.04	0.01
CV (%)		4.13	1.14	3.34	1.68	2.68
		KR	CaR	MgR	ClR
Salinity (S)	2	6.95**	1.05**	0.08**	99.94**
AsA	1	1.95**	0.03ns	0.54**	27.75**
S × AsA	2	0.87**	0.13ns	0.13**	10.35**
Residual	18	0.18	0.05	0.00	0.19
CV (%)		16.83	8.82	0.43	5.30

• SV - Source of variation; DF - Degrees of freedom; AsA - Ascorbic acid; PH - Plant height; SD - Stem diameter; NL - Number of leaves; RL - Root length; RD - Root diameter; SFW - Shoot fresh weight; RFW - Root fresh weight; SDW - Shoot dry weight; RDW - Root dry weight; TDW - Total dry weight; SMR - Shoot mass ratio; RMR - Root mass ratio; STR - Shoot-tuber ratio; RGR - Relative growth rate; TSS - Total sugars in the shoot; TSR - Total sugars in the root; RSS - Reducing sugars in the shoot; RSR - Reducing sugars in the root; PS - Protein content in the shoot; PR - Protein content in the root; PLS - Proline content in the shoot; PLR - Proline content in the root; RWC - Relative water content; EL - Electrolyte leakage; NaS - Sodium content in the shoot; KS - Potassium content in the shoot; CaS - Calcium content in the shoot; MgS - Magnesium content in the shoot; ClS - Chloride content in the shoot; NaR - Sodium content in the root; KR - Potassium content in the root; CaR - Calcium content in the root; MgR - Magnesium content in the root; ClR - Chloride content in the root; CV - Coefficient of variation; ^ns, *, ** - Not significant, significant at p ≤ 0.05 and p ≤ 0.01 by the F test, respectively.

The overall effects of reduced growth on radish plants were observed with increasing salinity levels in irrigation water (Figures 2A-E). It was observed that the height of the plants was reduced with increasing doses of salt; however, with the application of AsA, no significant differences were observed in the salinity treatment (Figure 2A). Stem diameter (SD) was also affected by the increase in salinity, with significant reductions being observed (Figure 2B). The number of leaves was reduced by the increase in salinity (Figure 2C), regardless of the application of AsA; as the salinity increased from 3.0 to 5.0 dS m^-1, the number of leaves was reduced between 17.24 and 44.82%, respectively, compared to the control (0.5 dS m^-1). For root length (Figure 2D) and diameter (Figure 2E), the same behavior was observed for all salinity levels, where the increase in salinity led to reductions of 45.74 and 45.97%, respectively, in root length and diameter, at the level of 5.0 dS m^-1, when compared to the salinity of 0.5 dS m^-1.

Figure 2
Plant height (A), stem diameter (B), number of leaves (C), root length (D) and root diameter (E) of radish plants subjected to different levels of electrical conductivity in irrigation water

The lack of significant effect of exogenous application of AsA on plant height, stem diameter, number of leaves, and root length can be attributed to the intensity of salt stress at the levels tested, which may have exceeded the ability of AsA to mitigate damage. Under high salinity conditions, excessive accumulation of toxic ions, such as Na⁺ and Cl⁻, compromises essential physiological processes, such as water and nutrient absorption, directly affecting plant growth and development (Akram et al., 2017). In addition, AsA may have been translocated to other metabolic processes, putting it at the expense of plant tolerance to salinity (Hasanuzzaman et al., 2020). It is worth mentioning that, under extreme salinity conditions, although important, antioxidant mechanisms are insufficient to compensate for damage to essential physiological processes, such as water and nutrient absorption, and root growth can be heavily impaired (Shabala & Munns, 2017). On the other hand, the diameters of the stem and roots were positively influenced at salinity levels 5.0 and 0.5 dS m^-1, respectively, since AsA plays an important role in reducing oxidative damage caused by salt stress, helping to neutralize free radicals and promoting the recovery of plant cells (Hasanuzzaman et al., 2020).

In the case of fresh matter, the shoot was influenced only by salinity (Figure 3A). As salinity levels increased, the fresh mass of radish plants was reduced regardless of the application of AsA (Figure 3A).

Figure 3
Shoot fresh weight - SFW (A), root fresh weight - RFW (B), shoot dry weight - SDW (C), root dry weight - RDW (D) and total dry weight - TDW (E) of radish plants subjected to different levels of electrical conductivity in irrigation water

However, a gain of 63% in fresh matter was observed in the treatment that did not receive the application of AsA for the salinity level of 0.5, compared to the 5.0 dS m^-1 level. In the root, the fresh weight was gradually reduced with the increase in salinity, not influenced by the application of AsA (Figure 3B). However, about the root dry matter, the salinity level of 5.0 dS m^-1 drastically reduced the dry weight, leading to a 65% reduction without AsA and when compared to the salinity level of 0.5 dS m^-1 (Figure 3C). Regarding total dry matter, plants under salinity of 5.0 dS m^-1 showed a significant reduction in total weight, equal to 50.94% in plants without AsA and compared to the control (0.5 dS m^-1) (Figure 3D). Shoot dry matter was not influenced by the application of saline water and AsA.

These results demonstrate that the accumulation of salts in the soil and root zone interferes with the ion balance and increases the production of reactive oxygen species, which oxidize molecules and cause damage to plant tissues. This imbalance also affects the translocation of nutrients and the metabolism of carbohydrates, resulting in lower growth and yield due to decreases in biomass (Hernández-Herrera et al., 2022; Balasubramaniam et al., 2023).

Regarding the proportions of shoot and root dry weight ratio (Figures 4A and B), it was observed that, under the salinity of 5.0 dS m^-1, the application of ascorbic acid increased the proportion of shoot in the whole plant, compared to the other treatments with salinity (Figure 4A).

Figure 4
Shoot mass ratio - SMS (A), root mass ratio - RMR (B), shoot/tuber ratio - STR (C) and relative growth rate - RGR (D) of radish plants subjected to different levels of electrical conductivity in irrigation water and under exogenous application of ascorbic acid

Furthermore, in proportional terms, the application of AsA led to a lower root fraction in plants under higher salinity (Figure 4B). These values show that the increase in salinity compromised root growth more severely than shoot growth, verified by higher values of shoot-root ratio (Figure 4C). The relative growth rate (RGR) was reduced more strongly in plants under high salinity (5.0 dS m^-1), and the application of AsA increased the growth rate by 9.06% in the treatment without AsA (Figure 4D).

Salinity impairs all essential processes, such as photosynthesis, protein synthesis, growth, and biomass production (Adhikari et al., 2020). However, the exogenous application of AsA favored an increase in growth and biomass, given that its application increases cell division and enlargement (Hassan et al., 2021), which affects the morphological variables SMR, RMR, and RGR.

There was significant interaction for total sugars (TS), while reducing sugars (RS) showed only an individual effect of the factors in radish subjected to salinity and application of ascorbic acid (Figure 5). The TS content was affected by the salinity in the shoot and roots; however, at the salinity level of 5.0 dS m^-1, the application of AsA promoted increments of 29.83 and 47.60%, respectively, in shoots and roots, compared to the treatment that did not receive the application of AsA (Figure 5A). For the RS content, the increase in salinity caused reductions in its content of 12.5 and 27.7%, respectively, in the shoots and roots, at the salinity level of 0.5 dS m^-1 and when compared to the salinity level of 0.5 dS m^-1 (Figure 5B). Furthermore, the exogenous application of AsA (1 mM) promoted an increase of 11.76% in the RS content compared to the concentration of 0 mM (Figure 5B).

Figure 5
Total sugars - TS (A) and reducing sugars - RS (B) of the shoot and root parts of radish plants subjected to different levels of electrical conductivity in irrigation water and under exogenous application of ascorbic acid

The observed increases in sugars may be due to the activation of carbohydrate-synthesizing enzymes caused by the application of AsA and salicylic acid, which increase the accumulation of these macromolecules, and also by the hydrolysis of polysaccharides (Dawood et al., 2022). In addition, soluble sugars are important in signaling nutrients, osmoprotectants, and metabolites involved in diverse responses to abiotic stresses (Afzal et al., 2021).

Different effects were observed on proteins and proline levels in shoots and roots (Figure 6). In the shoot, salinity and AsA application had no effect on total protein content. On the other hand, there was a significant reduction in proteins in the roots, especially under the salinity level of 5.0 dS m^-1, showing a reduction of 36.02% compared to the salinity level of 0.5 dS m^-1.

Figure 6
Proteins (A) and proline (B) of the shoot and root parts of radish plants subjected to different levels of electrical conductivity in irrigation water

This reduction is due to the proteolysis of this macromolecule, changes in the protein structure, and disintegration of peptide chains (Perveen & Hussain, 2021). Salinity also induces the accumulation of reactive oxygen species (ROS), causing lipid peroxidation, protein oxidation, enzyme inactivation, DNA changes, and interaction with essential constituents of plant cells (Shafiq et al., 2020).

It is notorious that the increase in salinity (3.0 to 5.0 dS m^-1) increased the accumulation of proline in both the shoot and the roots (Figure 6B). No effect of AsA application was observed for the proline content in both plant parts. The increase in this stress-related amino acid is because proline is a compatible solute that helps in osmotic balance, making plants withstand adverse conditions; in addition, proline mediates stress signaling, eliminating ROS and maintaining the osmolarity of cells under stressful conditions (Elkelish et al., 2020).

The relative water content (RWC) and electrolyte leakage were influenced only by salinity (Figures 7A and B). At the 5.0 dS m^-1 level, the RWC showed a reduction of 28.80% compared to the level of 0.5 dS m^-1 (Figure 7A). In contrast, the electrolyte leakage showed membrane damage of 70.49 and 74.22%, respectively, at salinity levels of 3.0 and 5.0 dS m^-1 compared to the level of 0.5 dS m^-1 (Figure 7B).

Figure 7
Relative water content - RWC (A) and electrolyte leakage - EL (B) of radish plants subjected to different levels of electrical conductivity in irrigation water

It is known that salinity drastically affects plant-water relationships, as there is a reduction in soil water availability, impairing the overall water content in the plant (Singh et al., 2022). Singh et al. (2022) also mention that electrolyte leakage is induced by stress, causing increased lipid peroxidation, plasma membrane rupture, and the release of essential ions and compatible solutes.

The nutritional composition of the shoots and roots of radish plants cultivated under different salinity levels and AsA application showed a significant interaction between the factors salinity and AsA concentrations for all elements analyzed (Na⁺, K⁺, Ca²⁺, Mg²⁺, Cl^-) (Table 3).

Table 3
Nutritional composition of the shoot and root of radish plants grown under different salinity levels and exogenous application of ascorbic acid

Treatments	0.5 dS m-1	0.5 dS m-1 + AsA	3.0 dS m-1	3.0 dS m-1 + AsA	5.0 dS m-1	5.0 dS m-1 + AsA
	Shoot
Na+ (g kg-1)	4.09 aC	3.96 aB	6.33 aB	4.18 bB	7.42 aA	5.33 bA
K+ (g kg-1)	3.80 aA	1.93 bA	2.61 aB	1.79 bA	2.26 aC	1.73 bA
Ca2+ (mg L-1)	8.80 bC	9.59 aC	10.49 bA	11.09 aB	9.26 bB	12.05 aA
Mg2+ (mg L-1)	2.60 bB	4.42 aC	2.67 bB	5.24 aB	4.10 bA	5.71 aA
Cl- (%)	6.20 aC	5.25 bC	14.75 aB	11.60 bB	20.31 aA	19.82 bA
Root
Na+ (g kg-1)	2.75 aC	2.08 bC	3.60 aB	3.32 bB	8.11 aA	5.05 bA
K+ (g kg-1)	4.55 aA	3.02 bA	1.93 aB	1.82 aB	2.13 aB	1.80 aB
Ca2+ (mg L-1)	3.08 aA	2.86 aA	2.09 aB	2.20 aB	2.51 aB	2.89 aA
Mg2+ (mg L-1)	1.50 bA	1.59 aB	1.31 bC	1.59 aB	1.35 bB	2.02 aA
Cl- (%)	4.10 aC	3.80 aC	9.75 aB	8.00 bB	14.75 aA	9.35 bA

• Means followed by the same capital letters in the row do not differ statistically for salt stress and means followed by the same lowercase letters in the row do not differ statistically for ascorbic acid concentrations by Tukey’s test (p ≤ 0.05)

In the shoot, Na⁺ and Cl^- contents were increased with salinity severity, showing increases of 35.38 and 44.88% (Na⁺) and 57.96 and 69.47% (Cl^-), respectively, for the salinity levels 3.0 and 5.0 dS m^-1, compared to the salinity level of 0.5 dS m^-1. However, the application of AsA led to significant reductions to the ions mentioned, with decreases of 33.96 and 28.17% (Na⁺), respectively, at salinity levels of 3.0 and 5.0 dS m^-1 and 15.32, 21.35 and 2.41% (Cl^-), respectively, at salinity levels of 0.5, 3.0 and 5.0 dS m^-1, and both in comparison to the treatment that did not receive the application of AsA (Table 3).

K⁺ content in the shoot was reduced with the increase in salinity; the application of AsA did not influence this nutrient. On the other hand, Ca²⁺ and Mg²⁺ contents increased in the shoot as the salinity levels intensified, and there were increases in their contents of 8.23, 5.41 and 23.15% (Ca²⁺) and 41.17, 49.04 and 28.20% (Mg²⁺) with the exogenous application of AsA, all for the salinity levels 0.5, 3.0 and 5.0 dS m^-1, respectively (Table 3).

In the roots, the contents of Na⁺ and Cl⁻ increased with the intensification of salinity, showing increases of 42.22 and 74.35% (Na⁺) and 57.94 and 72.20% (Cl⁻), respectively, for the salinity levels of 3.0 and 5.0 dS m^-1, compared to the level of 0.5 dS m^-1 (Table 3). On the other hand, the increase in salinity levels reduced the K⁺ and Ca²⁺ contents up to the level of 3.0 dS m^-1, with decreases of 57.58 and 32.14%, respectively, when comparing the levels of 0.5 and 3.0 dS m^-1 (Table 3). The exogenous application of AsA did not influence the root contents of Na⁺, Cl⁻, K⁺ and Ca². However, the application of AsA promoted significant increases in the content of Mg²⁺ as the salinity levels increased, with increases of 6.13, 17.82 and 33.16%, respectively, for the levels of 0.5, 3.0 and 5.0 dS m^-1.

In general, high concentrations of Na⁺ and Cl^- in the soil reduce the activities of nutrient ions; with this, there is an increase in salinity, causing osmotic stress in cultivated plants, reduction in water availability, stress and changes in cellular ionic balance, as well as deficiency and toxicity of some nutrients, but in addition, the increase in salinity causes a reduction in the absorption of N , P, K⁺, Ca²⁺, and Mg²⁺ (Ehtaiwesh et al., 2022). Hassan et al. (2021) mention that applying AsA reduces the Na⁺ content and increases K⁺ and Ca⁺ contents in plants in soils subjected to salinity. In addition, the plant’s water absorption is limited when there is an increase in ions, putting nutrient absorption at the expense (Caetano et al., 2024); in addition, the accumulation of Na⁺ impairs the absorption of K⁺ around plant roots due to the similar chemical properties of these ions (Kumari et al., 2021).

The Na⁺ content showed moderate negative correlations with NL (-0.76), RL (-0.75), RD (-0.77), RFW (-0.76) and TFW (-0.79), as well as strong negative correlations with PH (-0.85), SD (-0.87), SFW (-0.85), RDW (-087) and SDW (-0.87) for the shoot of the radish (Figure 8). Regarding the root part, Na⁺ showed negative correlations from strong to very strong that ranged from -0.82 to -0.96, with RFW and RGR, respectively. On the other hand, the Cl^- content showed negative correlations from strong to very strong with the growth variables (PH, SD, NL, RL, RD, RFW, SFW, TFW, RDW, SDW, TDW and RGR), with variations from -0.80 to -0.97 for the shoot and from -0.88 to -0.97 for the root part. In addition, the growth variables mentioned showed negative correlations ranging from moderate to very strong with RWC (-0.75 to -0.95) and EL (-0.73 to -0.98).

Figure 8
Pearson’s correlation between the variables analyzed

The sum of the principal components (PC), PC1 and PC2, contributed with 73.45% of the total variability (Figure 9). PC1 resulted in 60.17% of the total variability and obtained positive correlations with Ca - shoot, Mg²⁺ (shoot and root), Cl^- (shoot and root), Na⁺ (shoot and root), EL, RWC, SDW/RDW and Proline (shoot - root), mainly for ECw 3.0 dS m^-1, ECw 5.0 dS m^-1 and ECw 5.0 dS m^-1 + 1 mM AsA. PC2 resulted in 13.28% of the total variability and obtained negative correlations with the growth variables (PH, SD, NL, RL, RD, SFW, RFW, TFW, SDW, RDW, TDW, SDW/RDW, RGR), Ca-root, K⁺ (shoot and root), Protein (shoot and root), RS (shoot and root) and TSS (shoot and root), essentially for ECw 0.5 dS m^-1 and ECw 0.5 dS m^-1 + 1 mM AsA treatments. Thus, it can be stated that the highest salinity in the irrigation water (ECw 5.0 dS m^-1) resulted in the highest concentration of salts, such as Na⁺ and Cl^-, which negatively influenced the biometric and biochemical variables related to PC2.

Figure 9
Principal component analysis (PCA) between variables and treatments used

Conclusions

1. The increase in salinity (3.0 and 5.0 dS m^-1) affected the growth variables and water, biochemical, and nutritional relationships of radish plants.

2. Plant height, stem diameter, shoot mass ratio, shoot and root ratio, relative growth rate and soluble sugars were increased with exogenous application of ascorbic acid (1 mM), mainly at the salinity level of 5.0 dS m^-1.

3. Applying 1 mM of ascorbic acid reduced Na⁺ and Cl^- contents in the shoots and roots at all salinity levels and increased the contents of Mg²⁺ (shoot and root) and Ca²⁺ (shoot) at the tested salinity levels of 3.0 and 5.0 dS m^-1.

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• ¹ Research developed at Universidade Federal Rural do Semi-Árido, Mossoró, RN, Brazil

Supplementary documents

• There are no supplementary sources.

Financing statement

• This study was funded by the Coordination for the Improvement of Higher Education Personnel - Brazil (CAPES) - Financing Code 001.

Edited by

• Editors: Geovani Soares de Lima & Carlos Alberto Vieira de Azevedo

Data availability

There are no supplementary sources.

Publication Dates

• Publication in this collection
  25 Aug 2025
• Date of issue
  Dec 2025

History

• Received
  31 Dec 2024
• Accepted
  12 May 2025
• Published
  07 July 2025