1 Introduction

The accretion of heavy metals (HMs) in the soil and atmosphere is primarily because of manmade actions such as mining, irrigation, sludge, fertilizer application, and business waste. These activities have led to augmented levels of HMs in the soil, which can cause toxicity in the soil and negatively affect the physiological and biochemical activities of plants [17, 19]. Among HMs, cadmium (Cd) is most dangerous for plants as it is highly mobile in soil [43]. Cd can easily be transferred from one plant part to another, resulting in impaired nutrient acceptance and disrupted ion homeostasis in plants, thus restraining the plant enlargement [74]. In plants, Cd stress include chlorosis, necrosis, wilting, leaf rolling, and root browning, as well as a decrease in photosynthesis, respiration, nitrogen metabolism, and ATPase activity [71]. The quantities of hydrogen peroxide (H2O2) and reactive oxygen species (ROS), and malondialdehyde (MDA) are all elevated in cadmium toxicity, thereby disrupts chloroplast ultrastructure and stomatal morphology, resulting in reduced transpiration, and gaseous exchange [71] and consequently restraint plants yield [28, 74, 86]. Therefore, Cd toxicity not only causes damage to agricultural land but also undermines sustainable agriculture. It is crucial to implement measures to restrict accretion of toxic metal in soil and negative effects on plants to ensure sustainable agriculture and environmental conservation.

Different methods are used to mitigate the harmful effects of Cd and exogenous supplementation of plant growth regulators (PGRs) is one such technique [65]. SA has been shown to enhance germination, photosynthesis, and the production of proline, glycine betaine, and total sugars, as well as glutathione reductase (GR), superoxide dismutase (SOD), peroxidase (POX), ascorbate peroxidase (APX), and catalase (CAT). These antioxidant molecules help to maintain the osmotic balance of the cell and scavenge ROS, which can mitigate environmental stressors such as Cd exposure [4]. Brassinosteroids (BRs) also mitigate the hazardous effect of Cd and improves plant development. BRs enhance chlorophyll content, photosynthesis and relative water content; it modulates other metabolic activity and promotes production of antioxidants during different abiotic stress [76]. Additionally, BRs application modulates ion influx in soil, enhances membrane stability, reduces ROS and diminishes accumulation of HMs. Thus, BRs application enhances stress tolerance and adaptation in plant [80]. Nitric oxide (NO) is a vital player in improving the morpho-physiological and biochemical characteristics of plants under abiotic stress. It is engaged in a number of signal transduction pathways in plants. Specifically, it promotes the antioxidant defense system by stimulating the activities of enzymes such as CAT, POX, SOD and proline. To achieve this effect, exogenous NO is often administered to plants as a donor of sodium nitroprusside (SNP). This approach helps to keep the appropriate germination, plant growth under adverse circumstances [60].

SA provides tolerance against Cd stress when supplied through various methods, such as pre-soaking, hydroponic exposure, or spraying [24]. It mitigates Cd-induced toxic responses by sustaining plant growth, Cd absorption and translocation in plants, cellular redox status, membrane stability and good performance of photosynthesis [55]. Similarly, EBL alleviate the damaging effect of Cd in plants by regulating growth, nutrient status photosynthetic pigment and antioxidative defense system [39, 69]. NO increases the activity of antioxidant enzymes, which confers resistance against cadmium exposure. It also improves seed yield and reduces Cd accumulation, and thus protects plants from Cd stress [53]. SA-mediated alleviation of Cd stress not only depends on antioxidants but by also affects other machineries of Cd reclamation [57]. SA decreases Cd assimilation and root-to-shoot translocation [24]. For instance, application of SA restricts Cd absorption and mitigated Cd-triggered growth inhibition in radish [64]. The activation of element translocators mediated by SA may be the cause of the restricted absorption and translocation of Cd into vacuoles [73] such as members of ABC transporter family. SA modifies the transcription of ABC transporters in Arabidopsis (Bovet and Martinoia). SA induces these transporters to enhance vacuolar sequestration of Cd in plants. EBL alters nutrients uptake to reduce Cd accumulation in plant tissues [39]. Similarly, EBL also limits Cd accretion by increasing absorption of calcium as well as regulating ionic homeostasis [13, 81]. Moreover, EBL promotes the uptake of Mg2+, Ca2+and K+ in roots which gets transported to leaves to minimize Cd translocation. Furthermore, EBL promotes ion homeostasis, diminishes the absorption of harmful ions, and heightens the interest in inorganic ions [79]. However, reason seems to be different in case of stress mitigation by NO. It pays to Cd2+ toxicity by augmenting Cd2+ against Ca2+ uptake [8] rather relieves Cd toxicity via alternative pathway (AP) which is not concerned in reducing the uptake of Cd [34].Therefore, plant hormones like SA, BRs and NO had the capabilities to ameliorate the environmental stress and provide plant sustenance.

Linum usitatissimum L. also called linseed oil crop or flax (common name alsi) belongs to Linaceae family. It is widely used for oil, fibber, medicine and food. Its seeds contain essential α–linolenic acid; additionally it is also a rich source of oil (41%), proteins (20%), fibber (28%), and moisture (6%), ash (4%) and trace amount of phenols and lignins. This makes it highly nutritious and helps in production of several healthy products. It also contains polysaccharides and phytochemicals which make it a valuable ingredient in health enhancing trait and nutraceutical industry [63]. Linum is also used in linen manufacturing, linoleum, cloth production and raged bags [26].

Combined application of SA, EBL, and SNP in reducing the toxicity of Cd in Linum usitatissimum has received very little attention in study so far. Determining the possible defensive interactions among these three phytohormones in the instruction of Cd lenience in Linum usitatissimum was the goal of the current study.

2 Material and methods

2.1 Plant

We got uniformly sized flaxseed or linseed (Linum usitatissimum) cv. RLC-6 seeds from IARI, New Delhi, India. Sodium hypochlorite was used for sterilization of seed.

2.2 Preparation of SA, EBL and SNP

Sigma-Aldrich Chemicals Pvt. Ltd. in India was the source of SA, EBL, and SNP. In a 100 ml volumetric flask, the appropriate amount of SA, EBL, and SNP were dissolved in 5 ml of ethanol to create the stock solution. After dilution, the stock solution was used to prepare the necessary concentrations of SNP (10–5 M), EBL (10–8 M), and SA (10–5 M).

2.3 Sources of cadmium stress

Sigma–Aldrich Chemicals Pvt. Ltd. in India provided the cadmium chloride that was employed as a Cd stress (50 mg/kg soil).

2.4 Experimental design and treatments

At the Department of Botany, Aligarh Muslim University, the experiment was carried out in November through January, during the year 2019–2020 under simple randomized complete block design. The flaxseed or linseed seeds were sown in grounded pots (diameter and length of 12 and 15 inches, respectively) containing soil and manure (3:1). Pots were setup at their natural sorroundings with 11/12 h day/night length. Overall 36 pots were used during the experiment, three pots were maintained for each treatment indicating three replicates (n = 3) for each treatment. The treatments given were (1) control (supplied with DDW only and without Cd), (2) Foliar SA treatment (10–5 M), (3) Foliar EBL treatment (10–8 M), (4) Foliar SNP treatment (10–5 M), (5) Cd treatment (50 mg/kg of soil), (6) SA + Cd, (7) EBL + Cd, (8) SNP + Cd, (9) SA + EBL + Cd, (10) SA + SNP + Cd, (11) EBL + SNP + Cd, (12) SA + EBL + SNP + Cd. As Linum is a slow growing plant and to investigate results clearly at 30 days after sowing (DAS) Cd was supplemented in soil. At 45 – 50 DAS (26th – 30th December, 2019), foliar spray of different treatments was given whereas at 60 DAS (11 January 2020), sampling was done to investigate several growth and physio–biochemical traits. Additionally, all the plants were well irrigated with tap water (300 mL) during early morning hours for healthy growth and development. Furthermore, foliar spray of different plant hormone was given in evening, one hour after sun–set with the help of the sprayer.

2.5 Growth traits

Measuring scale was used to measure SL and RL. Electronic weighing machine was used to measuring the FW. Dry weight measurement, shoot and root materials were store in oven at 80 °C for 24 h. Leaf area was assessed by drawing and evaluating the area of leaf replica on the graph paper having 1 cm X 1 cm grids.

2.6 SPAD chlorophyll

A non-invasive approach was used to estimate the amount of chlorophyll. The SPAD chlorophyll meter (SPAD–502; Konica, Minolta sensing, Inc., Japan) was used to measure the total chlorophyll in the leaf.

2.7 Photosynthetic attributes

Photosynthesis and related attributes viz; net photosynthetic rate (PN), internal carbon dioxide concentration (Ci), transpiration rate (E), and stomatal conductance (gs) of undamaged fresh leaves were measured using an infrared gas analyzer (IRGA) (LI–COR 6400, LICOR, and Lincoln, Nebraska, USA).

2.8 CA activity

We calculated the CA activity in leaves by applying the method described by Dwivedi and Randhawa [14].

2.9 NR activity

The method used to test nitrate reductase activity was Jaworski's [41].

2.10 Proline content estimation

Bates et al. [6] method was applied to evaluate the proline content.

2.11 Antioxidant enzyme activity

The Aebi [1] approach was used to measure the CAT activity. With minor adjustments, the Chance & Maehly [9] process was used to quantity the POX activity. However, the Kono [52] approach was used to measure SOD activity.

2.12 Reactive oxygen species estimation

2.12.1 Superoxide anion content

The superoxide anion content was determined using the Wu et al. [82] technique.

2.12.2 Superoxide anions localization

The location of the superoxide anions in the leaves was investigated using the Kaur et al. [44] approach.

2.12.3 Estimation of Hydrogen peroxide

The amount of hydrogen peroxide (H2O2) in the leaves was measured using Patterson et al.'s [61] method.

2.12.4 Hydrogen peroxide localization

The Kaur et al. [44] approach was utilized to study the localization of hydrogen peroxide in the leaves.

2.13 Lipid peroxidation

Using the approach of Heath and Packer [35], lipid peroxidation was related to malondialdehyde (MDA) level.

2.14 Histochemical detection of lipid peroxidation

After being submerged in Schiff's reagen for 60 min, the samples were rinsed with sulphite water (10% K2S2O3 + 1N HCl and DDW). A stero microscope was used to take the images [62].

2.15 Statistical analysis

SPSS 17.0 for Windows (SPSS, Chicago, IL, USA) was used to analyze the current data. Using standard error computations and analysis of variance (ANOVA) at a significance level of p < 0.05, the least significant difference (LSD) between treatments was determined.

3 Results

3.1 Growth characteristics

Cadmium stress in soil statistically hampered Linum growth parameters in respect of height, fresh and dry mass and leaf area. The values decreased for length of shoot by 26.95%, root by 35.89%, shoot fresh mass by 38.53%, root fresh mass by 47.46%, shoot dry mass by 29.61%, root dry mass by 44.06% and leaf area by 38.91% over their respective control (Fig. 1, D–G).However, the data depicted in the figures also shows that exogenous application of phytohormones like SA, EBL and SNP improved the above growth parameters in presence/absence of stress and EBL was superior over other two hormones in both the conditions. Furthermore, it was also noted that the stress generated by Cd is fully neutralized with combined treatment of phytohormones (Fig. 1). Amongst them SA + EBL + SNP is showed to be good and boost the length of shoot by 44.27%, root by 77.98%, fresh mass of shoot by 60.18%, root by 82.5% and leaf area by 33.33% as compared to control and even better than the Cd stressed plant.

Fig. 1
figure 1

Effect of plant hormone individually and in combination under cadmium stress and impact of cadmium on L. usitatissimum cv. RLC-6 (flax) Growth attributes that is A Shoot length, B Root length, C Leaf area, D Shoot fresh weight, E Root fresh weight, F Shoot dry weight, G Root dry weight at 60 days after sowing (DAS)

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3.2 Photosynthetic performance

All the photosynthetic traits like PN, gs, Ci, E and SPAD level (chlorophyll content) were increased by the foliar application of SA, EBL, and SNP alone and jointly in non-stressful situations and Cd stress condition and triple combination (SA + EBL + SNP) was most active. However, the application of Cd through soil decreased the above characteristics. The respective decrease in PN value by 15.71%, gs by 50.01%, Ci by 39.44%, E by 36.53% and SPAD value by 18.95% was observed in Cd stressed plant relative to control. Furthermore application of SA + EBL + SNP improved the performance of above parameters by 62.7% (PN), 83.58% (gs), 33.33% (Ci), 7.14% (E) and 11.84 (SPAD value) respectively under Cd stress (Fig. 2 A–E).

Fig. 2
figure 2

Effect of plant hormone (individually and combination) and cadmium on A Photosynthetic rate, B Internal CO2 concentration, C Stomatal concentration, D Transpiration rate, E SPAD value (total chlorophyll) in L. usitatissimum cv. RLC-6 (flax) at 60 days after sowing (DAS)

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3.3 Biochemical attributes

3.3.1 CA and NR

Plants emerged in Cd treated soil possess a significant influence and lead to reduce the content of CA (17.64%) and NR (18.42%) in respect to control. Additionally, plant applied with phytohormones SA, EBL and SNP only or in combined manner improved the activity of CA and NR. The extreme CA and NR activity was detected in combined application of SA + EBL + SNP which was about 81.23% (CA) and 22.36% (NR) more under stress. Furthermore, the adverse impact generated by Cd is mitigated by the adding of EBL, SA and SNP alone as well as in combination (Fig. 3, A–B).

Fig. 3
figure 3

Effect of plant hormone (individually and in combination) and cadmium on biochemical traits and antioxidant enzymatic machinery that is A Carbonic anhydrase activity, B Nitrate reductase activity, C Proline content, D Catalase activity, E Peroxidase activity, F Superoxide dismutase in L. usitatissimum cv. RLC-6 (flax) at 60 days after sowing (DAS)

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3.3.2 Proline content

Proline is an osmoprotectant and its content was increased by 6.52% in response to Cd stress than control. However, maximum rise in proline amount during stress was observed in the presence of SA + EBL + SNP (54.34%) followed by EBL + SNP (45.65%) in comparison to control. It is observed that EBL alone gave the best results than other phytohormone (Fig. 3, C).

3.3.3 Activities of antioxidant enzyme

In contrast to the control, the data shown in figures (Fig. 3, D–E) clearly demonstrated that Cd treatment increased the CAT, POX, and SOD activity by 4.65%, 9.12%, and 6.25%, in that order. Additionally, the leaves treated with SA + EBL + SNP and receiving Cd through soil showed the greatest increases in CAT, POX, and SOD activity. The respective surge in CAT was 29.56%, POX 61.25% and SOD 36.6% over control.

3.4 Reactive oxygen species

3.4.1 Superoxide anion content and H2O2 content

The Cd stress lead to substantial increase in superoxide anion (O2·–) and accretion of H2O2 in leaves which was about 27.77% and 47.94% more, in comparison to control. After submission of SA, EBL and SNP alone or in combination production of reactive oxygen species was abridged. However, EBL was extra efficient in this regard (Fig. 4, F and F’).

Fig. 4
figure 4

Effect of plant hormone (individually and in combination) and cadmium on (F) superoxide anion content and A–E Superoxide anion localization on leaves of L. usitatissimum cv. RLC-6 (flax) images, F’ hydrogen peroxide content and A’–E’ Hydrogen peroxide localization on leaves of L. usitatissimum cv. RLC-6 (flax) images

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3.4.2 Localization of superoxide anion and H2O2

Superoxide anion (O2·–) level was appeared by blue staining on leaves (Fig. 4, A–E) and H2O2 was exhibited by brownish spots on leaves (Fig. 4, A’–E’). In Cd treated plant, spots on leaves were more prominent than control. Moreover, accretion of O2·– and H2O2 in leaves was reduced after the application of phytohormone.

3.5 Lipid peroxidation (MDA content)

The application of Cd through soil caused substantial increase in MDA content (47.22%) in contrast to control. However, application of phytohormone like SA, EBL and SNP reduced the MDA amount. The foliar spray of the combination of SA + EBL + SNP was most beneficial (Fig. 5, F).

Fig. 5
figure 5

Effect of plant hormone (individually and in combination) and cadmium on A–E MDA content on roots of L. usitatissimum cv. RLC-6 (flax) images, A’–E’ MDA content on roots of L. usitatissimum cv. RLC-6 (flax) images, F MDA content in leaves of L. usitatissimum cv. RLC-6 (flax) images

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3.6 Identification of lipid peroxidation by histochemical means

MDA level in roots and leaves were appeared by pink and pinkish brown stains. Higher level of Cd stress exhibited prominent dark pink colour. However, loss of lipid peroxidation in roots and leaves was detected in the plants received phytohormone as foliar spray and the combination of SA + EBL + SNP was best (Fig. 5, A–E and A’–E’).

4 Discussion

A wide range of anthropogenic activities consequences in the accretion of HMs such as Cd in the soil. Cd is highly toxic that severely reduced the physiological and morphological attributes of the plant [16, 20, 37]. It hinders the intake of nutrients, the photosynthetic apparatus, the antioxidant apparatus, and plant growth [75]. In the current research, exposure of Linum to Cd resulted in stunted growth and reduced plant biomass along with leaf area (Fig. 1). Decrease in plant growth and biomass is one of the most common symptoms of Cd toxicity [29, 31, 33, 42]. The reduction of growth and biomass could be an after effect of decline in chlorophyll and photosynthetic values (Fig. 1A–G). Chlorosis is regarded as the main symptom which appears due to Cd toxicity [36] via exchange of Cd with Fe or Mg and affects the stability as well as biosynthesis of chlorophylls. Chlorophyll estimation is one of the important indicators of photosynthesis [11]. In the present outcome, the amendment of soil with Cd resulted in decrease of chlorophyll (Fig. 2E) which is in corroboration with the observation of Zargar et al. [88] who found a drop in SPAD values in Cd treated plants.

Plant growth and biomass directly depends on the photosynthates synthesized via photosynthesis [18]. A steep decline in gaseous exchange parameters was noted in plants raised in Cd amended soil (Fig. 2) which might be an outcome of Cd– induced stomatal deformity such as closure of stomata and reduction in stomatal density [84]. Comparable response was described by Zargar et al. [88] in mustard plants growing under Cd stress. Carbon dioxide binds with rubisco (major enzyme of Calvin cycle) in the attendance of CA to mark the commencement of C3 cycle [5]. Since, a decrease in Ci along with CA action was observed in Cd treated plants (Figs. 2B and 3A), this resulted in inducing a brake on efficient functioning of Calvin cycle, thereby reducing the photosynthate production [75]. NR is a vital enzyme linking the carbon and nitrogen assimilation in plants [58]. NR activity is closely linked to the concentration of CO2 in the environment, which is influenced by the availability of carbohydrates. Conversely, when carbon storage decreases, the uptake of nitrate decreases as well [66]. Supplementing the soil with Cd results in the inhabitation of NR activity (Fig. 3B) which suggests the reduction in protein synthesis [7] and ultimately, overall growth of the plant (Fig. 1A–G). The outcomes are lined with the study of Hayat et al. [30] where Cd mediated decrease in NR activity and growth was observed in tomato plants.

Phytohormones are important regulating molecules and hampered the development of the plants [21, 78]. In this work, the application of phytohormones (SA, SNP and/or EBL) to Linum usitatissimum resulted in enhancement of growth, SPAD value and photosynthetic attributes (Figs. 1, 2, 3) under presence/absence of Cd. The results align with previous research published by Shi et al. [73], Yuanjie et al. [85], Lu et al. [56] and Sami et al. [68]. Furthermore, the treatment of all three hormones together demonstrated the greatest efficacy in reducing the growth and photosynthetic properties' toxicity symptoms caused by Cd. (Figs. 1, 2, 3). SA + SNP, SA + EBL, SNP + EBL or SA + SNP + EBL minimized Cd–induced reduction of photosynthetic pigments (Fig. 2E), suggesting that SA– and/or NO– induced growth improvement partially relied on to their protective responses on photosynthetic pigments under Cd stress. The findings are in coherence with previous studies where combined application of SA, NO or BR enhanced resistance against abiotic stress [40, 72]). Combined and individual application of BR, NO or SA on rice under stress boosted the morphological parameters and further improved stress tolerance [22]. The stress alleviation by the application of SA and SNP might depend on their potential to control Cd–homeostasis by restraining Cd accumulation in maize and wheat plants [23, 46]. Hayat et al. [32] proposed that SA and/or BRs treatment on Brassica juncea improved CO2 assimilation inside the plant cells by enhancing CA activity. NO also participate in enhancing CA activity and mitigating abiotic stress [67]. BRs enhanced the activity of CA in several crops under stressed and non–stressed environment [3]. Our investigations are lined with the results of several researchers where treatment of SA, NO or BRs in combination or alone under stressed or non–stressed condition enhanced NR activity in several crops like mustard, tomato, mung, this further increased nutrient assimilation and membrane integrity [15, 88].

Upon encountering stressful conditions, plants tend to generate excessive levels of ROS that oxidize nucleic acids, protein and lipids, causing cellular disruptions [10, 12, 87]. Current article, outburst of O2·– and H2O2 level was triggered in plants under Cd stress which eventually resulted in higher MDA content (Figs. 4, 5) suggesting occurrence of oxidative damage. The outcomes are consistent with the earlier experiment conducted by Yan et al. [83] on Brassica napus, showing that Cd stress triggers ROS and MDA deposition in root and leaves. Against the ROS production, plants activate their defence mechanisms, including the generation of CAT, POX and SOD, and proline [45]. The documented rise in antioxidant enzymes (as shown in Fig. 3D–F) and proline levels (as illustrated in Fig. 3C) is anticipated to be a direct result of the ROS-induced response. Irfan et al. [38] reported similar response in mustard crop upon encounter with Cd stress. Current findings presented that follow–up treatment of phytohormones such as SA, NO or BRs alone or in combination to Cd–stressed reduced the ROS production and MDA accumulation (Figs. 4 and 5). SA, BRs or NO augmented antioxidant system which reduced O2·– and H2O2 content which in turn controlled the MDA level [25, 47, 54, 59, 70]. Agami [2] reported that SA and/or EBL supplementation mitigated stress by improving antioxidant machinery in maize. Similarly, application of SA and/NO on wheat unregulated the activity of CAT, POX and SOD and decreases ROS production to reduce stress [77]. Additionally, in tomato it was observed that EBL and/ or SNP treatment enhanced antioxidant enzymes. However, EBL showed best results in enhancing antioxidant enzymatic machinery [29, 33]. The osmotic adjustments done by proline help in maintaining plant turgor, membrane stability and antioxidant machinery [32]. Spray of plant hormone separately or in combination promoted proline production and its accumulation in Cd treated plants. Furthermore, combination of three plant hormones (SA + SNP + EBL) boosted up proline content followed by combination of two plant hormones. Results were supported by the investigations of Kohli et al. [50], where SA and/ or EBL application enhanced osmolyte content in mustard and reduced toxic effect of stress. Combined and individual application of SA and/or NO maintained cell turgor and osmotic balance in wheat during stress by increasing the proline content [77]. Considering the physiological aspects it could be inferred that all the three hormones altered the parameters in a similar pattern however, the difference lied amongst their efficiency in producing the response (Figs. 1, 2, 3, 4, 5).

Significant reduction of Cd toxicity in the flax plant was observed in the plants received SA, SNP, and EBL together. Kohli et al. [51] clarified that SA and EBL act combinedly to enhance crop resistance against abiotic stress moreover, BAK1 which is regarded as the receptor of BR signaling, its overexpression results in higher SA production [48]. Similarly, NO and SA collaborate to improve the accumulation of SA and jointly transmit the resistance signal by influencing the identical effectors proteins or their respective genes [49]. According to a recent finding [27], NR–reliant NO synthesis participates in the EBL–regulated mitigation of abiotic stress. Hence, simultaneous application of the three selected hormones creates a loop of signals that triggers the pronounced resistance in Linum usitatissimum against Cd stress (Fig. 6).

Fig. 6
figure 6

Schematic representation showing the effect of SA, EBL, and SNP under cadmium stress on plant

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5 Conclusions

The present study's findings unequivocally showed that the damaging effects of Cd stress inhibited photosynthesis and the yield of Linum plant. However, by boost-up of antioxidant enzymes and detoxifying MDA, H2O2, and ROS in stressed plants, foliar administration of SA, SNP, or EBL, either alone or in grouping, to the Cd-treated Linum plants has reduced the toxicity. When applied in combination, SA, SNP, or EBL had a greater impact on reducing Cd stress in Linum plants than when applied separately. Furthermore, comprehensive research concentrating on the cellular level must be clarified in later investigations.