Mitigating cobalt toxicity in linseed (Linum usitatissimum L.): the role of ascorbic acid in enhancing pigments formation, antioxidant defense and gene expression

Abstract

Background

Elevated cobalt (Co) levels induce cellular toxicity and reduce overall plant growth and development. Ascorbic acid is known to enhance heavy metal tolerance in plants; however, its potential to reduce Co stress in linseed remains poorly understood. In this study, we investigated the role of ascorbic acid in mitigating Co toxicity in linseed, focusing on its influence on morpho-physiological and biochemical attributes, along with regulation of stress-responsive genes.

Results

The results revealed that ascorbic acid (0.2 mM) decreased plant Co concentration by 9.36% in Roshni and 7.03% in Chandni under Co stress compared to plants under Co stress without ascorbic acid. Plant dry weight and number of grains per plant increased by 29.11% and 34.61% while chlorophyll content improved by 37.50% and 15.78% in both cultivars. Under Co stress, AsA also improved plant calcium contents by 23.28% and 15.50%, potassium contents by 18.02% and 20.02%, amino acids by 27.57% and 16.95% and total soluble protein by 29.74% and 36.15% in Roshni and Chandi over non-AsA Co stress. In addition, AsA alleviated Co toxicity by reducing oxidative stress markers including malondialdehyde by 35.56% and 36.84%, superoxide radical by 26.18% and 51.30%, and hydrogen peroxide by 26.53% and 29.72% and increased antioxidant enzymes, including superoxide dismutase: 36.95% and 29.64%; catalase: 34.05% and 31.23%; and peroxidase: 35.29% and 24.01% and osmolytes accumulation (proline: 18.18% and 45.09%) in Roshni and Chandni. Interestingly, qRT-PCR confirmed its role in enhancing the gene expression of antioxidants (CuZnSOD1, 2, CAT1, 2, POD1, 2, APX1, 2) and proline (p5CR and P5CS) in linseed.

Conclusion

Collectively, our results showed that ascorbic acid decreased cobalt-induced toxicity by modifying morpho-physiological and biochemical aspects as well as gene expression of linseed.

Graphical abstract

Supplementary Information

The online version contains supplementary material available at 10.1186/s12870-025-07661-w.

Introduction

Heavy metals are major abiotic stressors that adversely affect plants, soil fertility and crop productivity [1]. With the rapid increase in global population, industrialization and urbanization has become a primary sources of heavy metal pollution, poising serious risk to agricultural systems [2]. The response of plants to heavy metal contaminants depends largely on uptake, translocation, and accumulation of these metals within plant tissues [3]. Although the term “Heavy metals” is often used to interchangeably ‘toxic metals’ and some light metals can also be exhibit toxicity. Then main toxic metals are arsenic, lead, cadmium, mercury, chromium, and aluminum are known to significantly reduce soil productivity [4]. Soil physiochemical properties and metal ion concentrations are strongly influence the mobility and bioavailability of heavy metals, thereby affecting their uptake by plants [5]. Excessive accumulation of heavy metals can impair soil health, reduce biodiversity and pose significant threats to both fauna and flora including humans [6]. Similar to other toxic metals, elevated cobalt (Co) concentrations in the soil can impede plant growth and development by disrupting essential physiological and biochemical processes in plants [7].

Cobalt is an heavy metal in the fourth row of the periodic table, and it is crucial for humans, prokaryotes, and other species [7]. While not mandatory for all plants, it is beneficial for legume crops [8]. It is involved in N fixation processes. As a toxic and mobile heavy metal, Co is commonly found in industrial wastewater and is released through emissions from airplanes and automobiles, leading to soil contamination because of to its nonessential role in plants [9, 10]. Cobalt-containing sludge is commonly detected in urban areas, where soil pH is reduced by higher levels of manganese and ferrous oxides promote its availability and uptake by plants [11]. At elevated concentrations, Co causes severe plant damage, including root browning, leaf necrosis, and inhibition of photosynthesis [12, 13]. The degree of cobalt sensitivity varies considerably among higher plant species and genotypes [14]. Excess Co exposure also impairs carbon uptake, transpiration, and photosynthetic efficiency, resulting in reduced CO₂ assimilation in plants [10]. However, relatively few studies have explored both the beneficial and harmful effects of Co on plants [15]. According to Ali et al. [16], Co-induced toxicity disrupts the antioxidant defense system, thereby reducing plant growth, biomass, and yield. The toxicity of Co primarily arises from excessive generation of reactive oxidative species (ROS), which triggers oxidative stress [10, 17]. Cobalt toxicity in plant cells contributes in a Fenton reaction, which enhances the production of reactive oxidative species, leading to the breakdown of biomolecules via lipid peroxidation, enzyme deactivation, or degradation of DNA [10, 18]. According to [17], antioxidant enzyme-based defense systems are activated against ROS in plants against Co stress; however, the effect on linseed is still limited.

Flax seeds, also known as linseed, belong to the genus Linum and the family Linaceae. They are widely utilized and classified into two forms: linseed and the flax fibers [19]. Linseed has diverse industrial applications, including in the chemical, food, animal feed, pharmaceuticals, and oilseed sectors, as well as in the production of paints, varnishes, and waterproof products. In recent years, various plant species, including linseed, have been employed for the phytoextraction of several heavy metals, such as Zn, Cu, Hg, and As [20]. Although, numerous research projects have highlighted the potential of linseed for the phytoremediation of toxic heavy metals; however, research specifically addressing cobalt toxicity in linseed remains limited [21].

Foliar application technology minimizes chemical fertilizer loss and enhances soil micronutrient efficiency, enabling faster and more direct nutrient delivery compared to conventional soil applications [22]. Ascorbic acid reacts non-enzymatic with various free radicals, including hydrogen peroxide, oxygen, and superoxide, thereby protecting plants by reducing oxidative damage and acting as an antioxidant [23]. However, low levels of ascorbate in some plants may negatively impact their growth and development, hindering their ability to tolerate stress [24]. Ascorbate plays various roles in plant physiology, including cell division, cell wall expansion, as well as other developmental processes [25]. Moreover, ascorbic acid plays crucial role in mitigating heavy metal (HM) stress [26]. This study hypothesized that the application of ascorbic acid via foliar methods could effectively alleviate the adverse effects of cobalt chloride stress on flax production. The main objectives of this study were (i) to determine the cobalt uptake and translocation in linseed and their detrimental effects and (ii) to evaluate the potential of exogenously applied ascorbic acid in reducing cobalt uptake and translocation through modulating physiological, biochemical aspects and gene expression in linseed.

Materials and methods

This experiment was conducted at the Old Botanical Garden, University of Agriculture, Faisalabad, Pakistan during the 2022–2023 growing season. Seeds of two linseed varieties (Roshni and Chandni) were obtained from the Oilseeds Section of Ayub Agricultural Research Institute (AARI), Faisalabad, Pakistan. These varieties were selected due to widespread cultivation in Pakistan. No permission was required for allocation of seeds from institute. These varieties have previously been used as test varieties in experiments investigating various abiotic stresses [19, 27, 28]. The seeds were surface sterilized in 0.1% sodium hypochlorite solution and rinsed three times with deionized water. Pots measuring 22 cm × 20 cm were filled with 5 kg clayey loamy soil. Pots were randomly arranged and re-randomized periodically to minimize positional effects. Ten seeds were placed in each pot and subsequently covered with a small amount of sand. The initial nutrients (N: 19 mg kg⁻¹ soil; P: 19 mg kg⁻¹ soil and K: 12 mg kg⁻¹ soil) were applied at the time of sowing. The initial soil physico-chemical properties were presented in table S1. Germination commenced one week after planting, and thinning were applied 15 days after planting to keep five seedlings per pot. Plants were irrigated with tap water on alternate days as per water requirement of plants. The 300 µM cobalt solution was prepared by dissolving 0.9 g cobalt chloride in 1 L of distilled water. The cobalt dose at 300 µM was selected based on preliminary studies and carefully evaluating previous studies [12, 29–31]. After 3 weeks of germination, cobalt chloride (Co) was applied to the linseed roots. The 2nd and 3rd Co treatments were applied at 15 days intervals following the initial application. Ascorbic acid (AsA) solution at 0.2 mM was prepared by dissolving 0.035 g of AsA in 1 L of distilled water. One day after the 3rd stress application, AsA was foliar applied to the leaves of all pots for both varieties. The AsA concentration was evaluated after preliminary experimentation as presented in table S1. In addition, various previous work has been evaluated before final dose selection [32–35]. This experiment was arranged in completely randomized design (CRD) with a factorial arrangement including three biological replications. Factor 1 was cobalt stress and factor 2 was ascorbic acid treatment. All pots were randomly positioned regularly to minimize experimental error.

Morphological parameters

The plant height (shoot and root) was measured after carefully uprooting from soil using a meter rod. Fresh weight of whole plant, including roots and shoots, was recorded using an electrical weight balance immediately after uprooting from pots. The dry weight was obtained by placing the plants in an oven at 70℃ for a week, and then dried plants were weighed using an electrical balance. The number of grains per plant was also counted manually.

Photosynthetic parameters

The levels of chlorophyll a, b, carotenoids, and total chlorophyll were measured using the method given by Arnon et al. [36]. Fresh leaves were chopped and ground with mortar and pestle, then the pigments were extracted using 80% acetone. Then, absorbance of the extract was read with spectrophotometer at 645 nm and 663 nm for chlorophyll a and b and at 480 nm for carotenoids.

Amino acids, protein and proline

Amino acids were analyzed using a ninhydrin colorimetric assay. The ninhydrin solution was prepared by dissolving 2 g of ninhydrin in 100mL of distilled water, while the pyridine solution was prepared by mixing 10mL of pyridine and 90mL of distilled water. To measure amino acids, 500 µL of sample was mixed in 500 µL of pyridine and ninhydrin in test tubes. The tubes were then placed in a water bath for incubation at 95℃ for 30 min. After that, 10mL distilled water was added and readings were taken using phosphate buffer as a blank at 570 nm [37]. Total soluble protein content was estimated by using the Bradford method. Fresh leaf samples were homogenized and centrifuged to obtain a clear extract. The protein content was determined by adding Bradford reagent to the extract, followed by incubation at room temperature for 10 min. Absorbance was then measured at 595 nm, and concentration was calculated using a standard curve prepared with bovine serum albumin [38]. Proline concentration was determined by homogenizing fresh leaf sample in 3% sulfosalicylic acid followed by centrifugation at 12000 rpm. The supernatant was mixed in acidic ninhydrin and glacial acetic acid. The mixture was boiled in water bath for 60 min and kept for cooling. Then toluene was added and mixture was vortexed. The upper layer was moved to a cuvette, and spectrophotometer readings were taken at 520 nm [39].

Cobalt estimation

Cobalt concentration in plant (shoot, root and leaves) was determined following an acid digestion procedure. Dried plant samples were finely ground and digested using a mixture of concentrated nitric acid and hydrogen peroxide to complete mineralization. After digestion, the solution was diluted with distilled water and filtered. The cobalt content in the digested extract was measured using atomic absorption spectrophotometry [40].

Mineral nutrients

The study measured mineral nutrients from roots and shoots using a digestion process. The dried plant material was ground and transferred to a digestion flasks, followed by addition of H₂SO₄ and 35% H₂O₂. The flask was first heated at 150℃, then further heated to 250℃ until fumes were appeared. The process was repeated until the material was colorless. The digested material volume was maintained at 50 mL. The extract was filtered and used to determine cations (K⁺, and Ca²⁺) in shoots, leaves, and roots using a flame photometer [40, 41].

Relative water content

Relative water content (RWC) was determined by measuring the fresh weight, turgid weight (after rehydration in distilled water for 4 h) and dry weight (after oven-drying at 70 °C). RWC was calculated by the formula given by Weatherley et al. [42].

Oxidative stress markers

Membrane stability was evaluated by measuring electrolyte leakage from leaf samples immersed in distilled water. Initial and final electrical conductivity readings were taken before and after heat treatment, respectively and membrane stability was calculated from these conductivity values [43]. The damage to membranes caused by cobalt-induced oxidative stress by measuring the malondialdehyde (MDA) content [44]. Leaf samples were emulsified in 0.1% TCA (trichloroacetic acid), 0.5% thiobarbituric acid. The absorbance is determined at 532 nm and 600 nm, and nonspecific absorbance corrected at 600 nm. The MDA level was calculated using the absorption coefficient of 156 mmol⁻¹ cm⁻¹. The method [45] was used to determine hydrogen peroxidase in plant material. The mixture was ground, trichloroacetic acid added, and centrifuged. The enzyme extract was separated, potassium iodide and potassium phosphate buffer added, and absorbance was measured at 390 nm to estimate hydrogen peroxide content.

Antioxidants

For antioxidant measurement, 0.5 g fresh leaves were homogenized in 50 mM phosphate buffer containing 1 mM EDTA and 1% PVPP. Superoxide dismutase (SOD) was assessed using photo-reduction method, including L-methionine, Triton X, riboflavin, and nitroblue tetrazolium [46]. A 2 mL plastic cuvette was filled with 400 µL of water, 250 µL of buffer solution with a pH of 7.0, 100 µL of methionine, triton, nitroblue tetrazolium NBT, enzyme extraction, and 50 µL of riboflavin. The cuvette was placed under a lamp for 15 min, and absorbance was measured at 560 nm using a spectrophotometer based on inhibition of NBT photoreduction. Catalase (CAT) was determined by preparing reaction mixture containing of 1.9 mL buffer, 1 mL hydrogen peroxide, and 100 µL sample with enzyme extracts [47]. The catalase reaction involved a mixture of cooled potassium phosphate buffer, enzyme extract, and 1.9 mM H₂O₂. The enzyme extraction was added to the mixture for the reaction. Absorbance was read at 240 nm where decrease absorbance corresponds to CAT activity. The Chance et al. [47] provided a method for peroxidase (POD) analysis included 750 µL of buffer with pH of 7.0, 100 µL of guaiacol, 50 µL of enzyme extract, and 100 µL of hydrogen peroxide. Guaiacol was dissolved in 15 mL of potassium phosphate buffer and hydrogen peroxide was dissolved in 20 mL of the buffer solution before combination in the reaction. Absorbance was recorded at 470 nm using a spectrophotometer. Ascorbate peroxidase (APX) was assayed following the method of [48]. The reaction mixture contained potassium phosphate buffer, hydrogen peroxide, ascorbic acid, and enzyme extract. The decrease in absorbance due to ascorbate oxidation was recorded at 290 nm using spectrophotometry.

Gene expression analysis

Total RNA was isolated from linseed leaves using the TRIzol reagent (Invitrogen, USA) following the manufacturer’s protocol. RNA integrity and purity were assessed by agarose gel electrophoresis and Nano Drop 2000 spectrophotometer. Approximately 1 µg of RNA was tested with RNase-free DNase I (Thermo Scientific, USA) to remove genomic DNA contamination and first strand cDNA was synthesized using the revert-Aid First strand CDNA synthesis kit with oligo primers. Primers were designed based on conserved regions of L. usitatissimum sequences deposited in NCBI, or from the homologous sequences of closely related species where linseed specific sequences are unavailable. The ACTIN gene was used as an internal control for normalization of expression level. Qrt-PCR was performed using SYBR Green Master Mix (Applied biosystems) on a Real-Time PCR system. Each reaction contained 10 µL green mix, 0.5 µL forward primer, 0.5 µL reverse primer, 2 µL cDNA template and nuclease-free water up to 20 µL. Cycling conditions including initial denaturation at 95 °C fir 2 min, followed by 40 cycles of 95 °C for 15 s, annealing at 58–60 °C (primer specific) for 30 s, and 72 °C for 30 s. Melting curve analysis were performed to confirm amplification. Relative expression was calculated using the 2- ΔΔ CT method.

Statistical analysis

The collected data was analyzed by using the statistical software SPSS, version 24 [49]. The 2 way factorial ANOVA was applied to check the main effects and their interaction. The tukey HSD-test was also applied to check the mean among treatments. The R programming software was used for correlation analysis and to visualize data. The overall variety mean was also presented in the graphs.

Results

Growth and yield aspects

Cobalt (Co) stress significantly decreased key growth and yield parameters of linseed, including shoot length (SL), root length (RL), shoot fresh weight (SFW), shoot dry weight (SDW), root fresh weight (RFW), and root dry weight (RDW), and the number of grains (NOG) (p < 0.05; Table 1). The Co at 300 µM significantly decreased SL by 39.27% and 35.90%, RL by 39.08% and 31.92%, SFW by 64.80% and 65.22%, SDW by 39.95% and 43.35%, RFW by 35.90% and 32.27%, and RDW by 33.33% and 37.11% in Roshni and Chandni compared to the control. The NOG was also negatively affected by 30.04% and 32.07% under Co300 (i.e. 300 µM Co) vs. the control. Ascorbic acid (AsA) showed the most significant improvement in growth and yield parameters under Co stress (p < 0.05). The AsA at 0.2 mM improved SL by 29.39% and 22.60%, RL by 23.16% and 16.96%, SFW by 125.40% and 40%, SDW by 34.62% and 34.62%, RFW by 24% and 17.36%, and RDW by 21.43% and 28.71%, NOG by 22.09% and 28.70% under Co300 stress over non-AsA Co300 stress conditions in both varieties.

Table 1.

Foliar application of ascorbic acid on growth and yield aspects of linseed varieties (Roshni and Chandni) under Cobalt stress

Treatments	Shoot length (cm)		Root length (cm)		Shoot fresh weight plant − 1 (g)		Shoot Dry weight Plant − 1 (g)
	Roshni	Chandni	Roshni	Chandni	Roshni	Chandni	Roshni	Chandni
Cobalt (Co)
Co0	38.46 ± 3.64a	36.28 ± 3.43a	9.81 ± 0.49a	9.25 ± 0.47a	2.29 ± 0.15a	2.07 ± 0.12a	0.50 ± 0.01a	0.46 ± 0.01a
Co300	21.68 ± 0.92b	20.25 ± 1.02b	6.5 ± 0.25b	5.50 ± 0.30b	0.82 ± 0.06b	1.01 ± 0.16b	0.32 ± 0.02b	0.30 ± 0.02b
P value	< 0.001	< 0.001	< 0.001	< 0.001	< 0.001	< 0.001	< 0.001	< 0.001
LSD value	1.84	1.94	0.36	0.24	0.10	0.08	0.02	0.01
Ascorbic acid (AsA)
AsA0	25.09 ± 2.45b	23.46 ± 2.43b	7.35 ± 0.62b	6.53 ± 0.76b	1.33 ± 0.29b	1.23 ± 0.26b	0.37 ± 0.05b	0.36 ± 0.04b
AsA0.2	35.05 ± 5.13a	33.06 ± 4.84a	8.95 ± 0.88a	8.22 ± 0.92a	1.79 ± 0.37a	1.86 ± 0.22a	0.44 ± 0.04a	0.41 ± 0.03a
P value	< 0.001	< 0.001	< 0.001	< 0.001	< 0.001	< 0.001	< 0.001	< 0.01
LSD value	1.36	1.94	0.36	0.24	0.20	0.08	0.02	0.01
Co × AsA
Co0 + AsA0	30.45 ± 1.01b	28.73 ± 0.95b	8.72 ± 0.12b	8.22 ± 0.12b	1.97 ± 0.05b	1.81 ± 0.05b	0.48 ± 0.01a	0.45 ± 0.01a
Co0 + AsA0.2	46.46 ± 1.04a	43.83 ± 0.98a	10.89 ± 0.15a	10.28 ± 0.15a	2.62 ± 0.07a	2.34 ± 0.02a	0.52 ± 0.01a	0.48 ± 0.00a
Co300 + AsA0	19.73 ± 0.52d	18.2 ± 0.9d	5.99 ± 0.16d	4.83 ± 0.06d	0.68 ± 0.02d	0.65 ± 0.03d	0.27 ± 0.01c	0.26 ± 0.00b
Co300 + AsA0.2	23.63 ± 0.44c	22.3 ± 0.41c	7.01 ± 0.18c	6.17 ± 0.09c	0.96 ± 0.03c	1.37 ± 0.03c	0.36 ± 0.01b	0.34 ± 0.01c
P value	< 0.01	< 0.01	< 0.05	< 0.05	< 0.05	< 0.05	< 0.01	< 0.05
LSD value	3.61	3.81	0.70	0.48	0.20	0.15	0.04	0.03
Treatments	Root fresh weight plant − 1 (g)		Root Dry weight Plant − 1 (g)		No of Grains plant − 1
	Roshni	Chandni	Roshni	Chandni	Roshni	Chandni
Cobalt (Co)
Co0	0.52 ± 0.04a	0.49 ± 0.04a	0.28 ± 0.02a	0.26 ± 0.02a	51.71 ± 0.71a	48.78 ± 0.67a
Co300	0.32 ± 0.01b	0.29 ± 0.02b	0.17 ± 0.01b	0.16 ± 0.01b	39.78 ± 2.33b	37.35 ± 1.72b
P value	< 0.001	< 0.001	< 0.001	< 0.001	< 0.001	< 0.001
LSD value	0.02	0.02	0.01	0.01	2.36	1.93
Ascorbic acid (AsA)					.
AsA0	0.36 ± 0.03b	0.33 ± 0.03b	0.19 ± 0.02b	0.18 ± 0.02b	42.89 ± 3.67b	40.87 ± 3.27b
AsA0.2	0.48 ± 0.06a	0.45 ± 0.06a	0.25 ± 0.03a	0.24 ± 0.03a	48.6 ± 1.85a	45.27 ± 1.96a
P value	< 0.001	< 0.001	< 0.001	< 0.001	< 0.01	< 0.01
LSD value	0.02	0.02	0.01	0.01	2.36	1.93
Co × AsA
Co0 + AsA0	0.43 ± 0.01b	0.40 ± 0.01b	0.23 ± 0.00b	0.22 ± 0.00b	50.99 ± 0.98a	48.1 ± 0.93a
Co0 + AsA0.2	0.61 ± 0.01a	0.58 ± 0.01a	0.32 ± 0.01a	0.30 ± 0.01a	52.43 ± 1.01a	49.47 ± 0.96a
Co300 + AsA0	0.29 ± 0.01d	0.26 ± 0.01d	0.14 ± 0.00d	0.14 ± 0.00d	34.79 ± 0.92c	33.63 ± 0.64c
Co300 + AsA0.2	0.34 ± 0.01c	0.32 ± 0.01c	0.19 ± 0.00c	0.18 ± 0.00c	44.77 ± 1.18b	41.07 ± 0.78b
P value	< 0.01	< 0.01	< 0.01	< 0.01	< 0.05	< 0.05
LSD value	0.04	0.03	0.02	0.02	4.65	3.78

The presented data on bars are mean of three biological replications and the error bar on each bar are standard error of three replications. The alphabet lettering on error bar showed significant or non-significant difference among means (Tukey HSD). ASA: ascorbic acid; Co: cobalt

Total chlorophyll and carotenoids

The effect of cobalt (Co) stress on the total chlorophyll contents (Chl) and carotenoids (Caro) of linseed was statistically significant (p < 0.05). A significant negative effect was observed under 300 µM Co, reducing Chl by 57.32% and 42.42% and Caro by 41.67% and 34.80% in Roshni and Chandni as compared to the control (0 µM Co) in Roshni and Candni (Fig. 1). Ascorbic acid (AsA) showed significant improvement on these parameters of linseed under Co stress (p < 0.05). Interestingly, AsA at 0.2 mM caused a significant improvement in Chl by 37.50% and 15.78% and Caro by 28.57% and 25% under Co300 stress over non-AsA (0 mM) stressful conditions (Fig. 2).

Fig. 1.

Effect of foliar applied ascorbic acid on a total chlorophyll contents and b carotenoid contents of two linseed varieties (Roshni and Chandni) under cobalt stress. The presented data on bars are mean of three biological replications and the error bar on each bar are standard error of three replications. The alphabet lettering on error bar showed significant or non-significant difference among means (Tukey HSD). Chl: chlorophyll: Caro: carotenoids; AsA0.2: ascorbic acid at 0.2 mM; Co: cobalt at 300 µM. Co0 and AsA0 are considered as control (no application)

Fig. 2.

Effect of foliar applied ascorbic acid on a Amino acids and b Total soluble protein of two linseed varieties (Roshni and Chandni) under cobalt stress. The presented data on bars are mean of three biological replications and the error bar on each bar are standard error of three replications. The alphabet lettering on error bar showed significant or non-significant difference among means (Tukey HSD). A.A: amino acids; TSP: total soluble protein; AsA0.2: ascorbic acid at 0.2 mM; Co: cobalt at 300 µM. Co0 and AsA0 are considered as control (no application)

Cobalt contents

Under the influence of cobalt (Co) stress, there was a significant enhancement in the shoot Co, leaves Co, and root Co in both linseed varieties, with statistical confirmation (p < 0.05). Under 300 µM Co, there was significant rise in shoot Co by 33.02% and 28.49%, leaves Co by 32.67% and 30.92%, and root Co by 31.80% and 28.09% in Roshni and Candni, as compared to the control (Co at 0 µM) as shown in Fig. 3. Moreover, the ascorbic acid (AsA) under Co stress had a significant negative effect (p < 0.05). Intriguingly under 300 µM Co, when treated with 0.2 mM AsA, there was a substantial decrease in shoot Co by 17.24% and 20.83%, leaves Co by 17.07% and 11.43%, and root Co by 49.02% and 47.43% over non-AsA Co stress at 300 µM in both varieties.

Fig. 3.

Effect of foliar applied ascorbic acid on a Shoot cobalt, b Root cobalt and c Leaves cobalt of two linseed varieties (Roshni and Chandni) under cobalt stress. The presented data on bars are mean of three biological replications and the error bar on each bar are standard error of three replications. The alphabet lettering on error bar showed significant or non-significant difference among means (Tukey HSD). AsA0.2: ascorbic acid at 0.2 mM; Co: cobalt at 300 µM. Co0 and AsA0 are considered as control (no application)

Nutrient contents

The results revealed that cobalt (Co) stress significantly affected the plant calcium (Ca) and potassium (K) of both linseed varieties (p < 0.05). Under Co stress at 300 µM, shoot Ca was reduced by 35.56% and 37.11%, root Ca by 12.50% and 49.80%, leaves Ca by 37.15% and 32.88%, shoot K by 50% and 48.54%, root K by 40% and 28.71% and leaves K by 34.15% and 35.57% in Roshni and Candni rather than the control (Co0) as shown in Fig. 4. Ascorbic acid (AsA) had a significant impact on these parameters of linseed under Co stress (p < 0.05). Under Co300, 0.2 mM AsA caused the most significant increase in shoot Ca by 13.79% and 16.60%, root Ca by 22.58% and 63.90%, leaves Ca by 25.16% and 17.19%, shoot K by 31.82% and 37.50%, root K by 48.28% and 37.11% and leaves K by 18.52% and 21.14% without AsA application in Roshni and Candni, respectively.

Fig. 4.

Effect of foliar applied ascorbic acid on a Shoot calcium, b Root calcium, c Shoot potassium, d Root potassium, e Leaves calcium and f leaves potassium of two linseed varieties (Roshni and Chandni) under cobalt stress. The presented data on bars are mean of three biological replications and the error bar on each bar are standard error of three replications. The alphabet lettering on error bar showed significant or non-significant difference among means (Tukey HSD). Ca: calcium; K: potassium; AsA0.2: ascorbic acid at 0.2 mM; Co: cobalt at 300 µM. Co0 and AsA0 are considered as control (no application)

Water status, membrane stability and osmolytes

Cobalt (Co) stress significantly affected the relative water content (RWC), membrane stability (MS), and proline content of both linseed varieties (p < 0.05). Under Co stress (300 µM Co), the most significant negative effects were in RWC, with reductions of 32.99% and 30.05%, and MS with decreases of 42.71% and 40.11%, while proline significantly in-creased by 36.09% and 34.13% in Roshni and Candni as compared to the control where no Co applied (Fig. 5). Ascorbic acid (AsA) had a significant effect on these parameters under Co stress (p < 0.05). Notably under Co300, AsA at 0.2 mM caused the most significant increase in 27.57% and 16.95% for RWC, 42.84% and 39.40% for MS and 18.18% and 45.09% for proline over non-AsA Co300 in both varieties.

Fig. 5.

Effect of foliar applied ascorbic acid on a relative water content, b membrane stability, and c Proline of two linseed varieties (Roshni and Chandni) under cobalt stress. The presented data on bars are mean of three biological replications and the error bar on each bar are standard error of three replications. The alphabet lettering on error bar showed significant or non-significant difference among means (Tukey HSD). RWC: relative water content; MS: membrane stability; AsA0.2: ascorbic acid at 0.2 mM; Co: cobalt at 300 µM. Co0 and AsA0 are considered as control (no application)

Stress markers

Cobalt stress (Co) significantly increased malondialdehyde (MDA), superoxide radical (O₂⁻), and hydrogen peroxide (H₂O₂) in linseed (p < 0.05). It was noticed that MDA was significantly increased by 61.81% and 27.87%, O₂⁻ by 41.68% and 28.85% and H₂O₂ by 32.67% and 117.97% in both varieties under Co stress at 300 µM as compared to the control (without Co application) as displayed in Fig. 6. The effect of Ascorbic acid (AsA) on MDA, O₂⁻ and H2O2 under Co stress was statistically significant (p < 0.05). Interestingly, AsA at 0.2 mM showed a significant decrease in MDA by 35.56% and 36.84%, O₂⁻ by 26.18% and 51.30% and H2O2 by 26.53% and 29.72% as compared to non-treated Co stress conditions.

Fig. 6.

Effect of foliar applied ascorbic acid on a hydrogen peroxide, b malonaldehyde, c superoxide dismutase d catalase e peroxidase and f ascorbate peroxidase of two linseed varieties (Roshni and Chandni) under cobalt stress. The presented data on bars are mean of three biological replications and the error bar on each bar are standard error of three replications. The alphabet lettering on error bar showed significant or non-significant difference among means (Tukey HSD). H₂O₂: hydrogen peroxide; MDA: malonaldehyde; O₂⁻: superoxide radical; SOD: superoxide dismutase; CAT: catalase; POD: peroxidase; APX: ascorbate peroxidase; AsA0.2: ascorbic acid at 0.2 mM; Co: cobalt at 300 µM. Co0 and AsA0 are considered as control (no application)

Enzymatic antioxidants

The activities of antioxidant enzymes including superoxide dismutase (SOD), catalase (CAT), peroxidase (POD) and ascorbate peroxidase (APX) were significantly increased under cobalt stress (300 µM Co) in both linseed varieties (p < 0.05). However, it was noticed that Co stress significantly increased SOD by 34.99% and 12.12%, CAT by 62.50% and 54.54%, POD by 41.13% and 89.12%, and APX by 21.34% and 27.12% in Roshni and Candni as compared to the control (Fig. 6). The interaction of ascorbic acid (AsA) and Co was statistically significant (p < 0.05). Interestingly, the AsA at 0.2 mM gave the most significant increase in SOD by 36.67% and 29.64%, CAT by 35.90% and 31.30% and POD by 36.36% and 25.77% under Co300 over non-AsA stressful conditions.

Gene expression

According to Fig. 7, application of AsA under Co stress significantly enhanced the expression of all tested antioxidant and stress-related genes compared to Co stress alone. Under Co stress (300 µM), the highest induction was observed in CuZnSOD2 (72.9%) followed by CAT2 (55.42%), P5CR (55.13%) and POD2 (49.88%) in response to AsA0.2 over non-AsA Co300 in linseed. Among peroxidases, POD1 and APX1have 52.13% and 44% while pro-line biosynthesis related genes (P5CS: 45.94%) and moderate increased was observed in APX2 (30.18%) in linseed in response to AsA0.2 under Co300 over non-AsA Co stress.

Fig. 7.

Effect of ascorbic acid (AsA) on gene expression level of antioxidants and proline in two linseed varieties (Roshni and Chandni) under cobalt stress (Co)

Correlation analysis

The analysis of the correlation among linseed parameters across various treatments demonstrated significant differences in the correlation strength (Fig. 8). In the control (a), robust positive correlations were noted between POD and CAT (r = 0.91), SOD and RWC (r = 0.87), as well as chlorophyll and carotenoids (r = 0.89). Conversely, proline exhibited a negative correlation with RWC (r = −0.62). In the context of ascorbic acid treatment, the correlation between POD and CAT was notably high, recorded at 0.92; additionally, the positive correlation between proline and SOD reached a value of 0.76. In the cobalt treatments, a notable negative correlation was observed between chlorophyll and proline, quantified at r=−0.79, in conjunction with RWC-oxidative stress markers, which exhibited a correlation of r=−0.74. The introduction of ascorbic acid in the context of cobalt stress re-instated beneficial correlations, such as that between superoxide dismutase (SOD) and relative water content (RWC), which enhanced to r = 0.83, as well as the relationship be-tween chlorophyll and catalase (CAT), which exhibited an improved r = 0.81 value. Con-currently, this led to a diminishment of cobalt’s effects, as evidenced by a less pronounced negative correlation between proline and chlorophyll, with a correlation coefficient of r=−0.52.

Fig. 8.

The correlation analysis of all parameters under a control b ascorbic acid c cobalt and d ascorbic acid under cobalt stress

Discussion

Excessive cobalt (Co) uptake poses significant hazard to plant growth and development and subsequently decreases agricultural productivity [50]. In this study, we hypothesized that ascorbic acid (AsA) application could decrease cobalt uptake and translocation in two different linseed varieties. We observed that cobalt stress significantly decreased linseed growth and yield aspects. Previously, Ejaz et al. [51] stated that heavy metals move upwards from the roots and are translocated to the other plant sections along the xylem stream. The excessive influx of Co stress through the roots accumulates in various plant parts, resulting in diverse symptoms that vary by species [52]. Cobalt toxicity disrupts chlorophyll biosynthesis, causing decreased chlorophyll concentrations in plant tissues, and impairs photosynthesis [7]. Our results showed that chlorophyll contents were decreased under cobalt stress, which eventually resulted in a decrease in overall photosynthesis in linseed. Previously, Wang et al. [31] stated that excessive Co disrupted chlorophyll production and protein content by interfering with stomatal guard cells, limiting CO₂ absorption for photosynthesis in plants. According to Abbass et al. [53], plant photosynthetic activity declined due to reduced chlorophyll biosynthesis, which consequently impaired plant growth and development. In the present study, the linseed growth and yield aspects were reduced under Co stress which correlated with decreased chlorophyll activities as evidenced through correlation analysis. This response is commonly associated with oxidative stress (from overproduction of ROS) induced by elevated Co concentrations [54]. The negative correlation between chlorophyll content and ROS strongly suggests that the reduction in linseed growth is a consequence of oxidative stress manifested through elevated levels of oxidative stress markers.

We observed that Co stress reduced cell membrane stability by increasing membrane permeability, thereby inducing oxidative stress-mediated membrane damage. These results are consistent with findings of Samet et al. [50], who reported similar effects of heavy metal stress in iceberg lettuce. Likewise, Mansoor et al. [55] demonstrated that excessive Co-induced accumulation of ROS, particularly hydrogen peroxide (H₂O₂) disrupts cellular membranes and adversely affects plant physiology, metabolic processes, and nutrient uptake. In this study, elevated MDA levels under Co toxicity further confirmed oxidative damage to linseed cells. Elevated MDA levels lead to lipid peroxidation and protein denaturation under HMs stress, as documented by [56, 57]. Plants strengthen their self-defense mechanisms by mitigating ROS accumulation and activating antioxidant enzymes in response of various HMs [58]. In the present study, AsA markedly reduced oxidative injury by lowering H₂O₂ and MDA levels. Furthermore, AsA enhanced the activities of antioxidant enzymes under stressful conditions, which are essential for ROS detoxification and maintaining cellular redox homeostasis. Specifically, AsA treatment increased the activities of antioxidant enzymes (SOD, CAT, POD and APX), thereby minimizing oxidative damage through the reduction of ROS accumulation in linseed exposed to stress. According to Ali et al. [59], SOD catalyzed the conversion of oxygen species (O₂) into hydrogen peroxide (H₂O₂), while other antioxidant enzymes catalyzed the conversion of hydrogen peroxide (H₂O₂) into water molecules (H₂O) and oxygen (O₂) in oat under stressful conditions.

Cobalt stress markedly decreased relative water content (RWC), likely due to disturbances in cellular water balance that led to cellular dehydration. Similar findings were reported by Zahid et al. [18], who observed a decline in RWC in wheat under Co stress. Notably, the application of AsA effectively improved the RWC of linseed under Co stress. Wang et al. [60] further demonstrated that AsA regulates stomatal behavior, minimizing water loss while sustaining transpiration rates and reducing oxidative stress in guard cells during heavy metal exposure, thereby enhancing root water conductivity. In the present study, a direct correlation between RWC and proline was observed, suggesting that the improvement in RWC may result from osmotic adjustment that alters water potential. Ali et al. [61] also reported a positive correlation between RWC and proline (osmoregulator) indicting efficient water uptake by plants under stressful conditions. Proline, a potent osmolytes, plays a crucial role in maintaining osmotic balance within plant cells under Co stress, enabling water retention and sustaining cellular metabolism [50]. Moreover, increased proline accumulation in response to AsA under Co stress clearly reflected enhanced stress tolerance in linseed [62]. Additionally, the positive interaction of proline with antioxidants enzymes and its negative association with H₂O₂ and MDA further emphasize its the positive role in mitigating Co-induced oxidative stress [50]. A similar trend reported for Hibiscus rosa-sinensis L under HMs stress [63]. In the present study, amino acids (AA) levels increased following the application of AsA in linseed subjected to Co stress. According to Wu et al. [64], AsA plays a pivotal role in nitrogen metabolism and is directly involved in amino acids synthesis in plants. Likewise, Savarese et al. [65] highlighted that AA biosynthesis contributes to nutrient uptake, ion transportation, and protein metabolism in plants. In our study, AsA application enhanced the levels of Ca²⁺ and K⁺, which were closely associated with improved AA metabolism. Moreover, an increase in protein synthesis was observed, suggesting reduced protein oxidation in linseed under Co stress [66]. The positive correlations among chlorophyll content, growth, and yield with micronutrients, amino acids, and proteins collectively indicate that AsA effectively mitigated Co toxicity and enhanced the physiological performance of linseed.

Conclusions

This study investigated the effects of ascorbic acid on the morphophysiological, biochemical traits and gene expression of two linseed varieties, Roshni and Chandni under cobalt stress. Ascorbic acid reduced cobalt accumulation in both varieties, increased plant dry weight and grain number, and chlorophyll content. These improvements were associated with elevated levels of calcium, potassium, amino acids, and protein levels. Additionally, ascorbic acid improved cell water status and membrane stability by decreasing malondialdehyde and hydrogen peroxide accumulation. Enhanced antioxidant activities of enzymes including superoxide dismutase, catalase, and peroxidase, along with greater osmolytes accumulation, further alleviated oxidative stress. Overall, ascorbic acid effectively mitigated cobalt-induced toxicity by modulating the physiological and biochemical properties, leading to improved growth and productivity of linseed. Importantly, these findings suggest that foliar application of ascorbic acid can be considered a practical and low-cost approach to improve linseed performance under heavy metal stress. However, further validation under field conditions and across diverse agro-climatic regions is required to establish its agronomic viability and to explore its potential use in other crops facing similar stress challenges.

Authors’ contributions

Conceptualization: H.A; Methodology: H.A, and M.B.J; Formal analysis: H.A, and G.A; Investigation: H.A, and I.M; Validation: H.A; Visualization: H.A and F.T; Resources: M.B.J, S.B, A.M.A, K.S, J.B, and A.O.U; Writing original draft, H.A, H.A, M.J and I.M; Writing review & editing: G.A, S.B, F.T, G.A and M.A.S. All authors have read and agreed to the published version of the manuscript.

Funding

Not available.

Data availability

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

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.