Introduction

The estimated increase in the world population by the year 2050 is 9.3 billion (Ishikawa and Shabala 2018). There is dire need to increase agricultural production by 50% to fulfill the food requirements of the increasing human population (Rengasmay 2006). Plant growth and yield is significantly impeded in saline soils. Therefore, mitigation of salinity is central to increase agricultural productivity in sustainable way (Heuer 2003). Yield reductions are significant in crop plants when soil EC reaches 4 dS m−1. The agriculture sector faces annual losses worth more than US$ 27 billion due to soil salinization (Qadir et al. 2014). According to FAO, more than 800 million ha land is saline with 32 million ha of dryland agriculture. Soil salinization occurs due to anthropogenic and natural factors such as poor agricultural practices, salt fluctuations due to seasonal variability and soil processes in soils with deep water tables (Greene et al. 2016).

Crop plants are usually glycophytes with limited potential to tolerate salinity compared with halophytes. Osmotic damage and specific ion toxicity, being the major salinity components, impedes yield production in glycophytes (Munns and Tester 2008). Plant growth rate, cell expansion, and water relation are impaired due to the initial exposure of salinity (Ferchichi et al. 2018). Plants under long-term salinity face ionic stress that leads to oxidative injury and nutritional imbalance (Munns and Tester 2008). Osmotic factor of salinity inhibits photosynthesis, enzyme activities, and protein synthesis that lead to chlorosis, necrosis, and premature senescence of old leaves (Ferchichi et al. 2018). Glycophytes accumulate substantial amounts of organic osmolytes. Compatible solutes are low molecular weight nitrogen-containing compounds such as betaines, amines, amino acids, and sugars (Zhu et al. 2015; Gharbi et al. 2017; Nguyen et al. 2017). These compatible compounds also mitigate oxidative damage in addition to their function as osmotic adjustment in cytosol (Annunziata et al. 2017). Salinity causes oxidative stress, nutrient imbalance, osmotic damage, as well as specific ion injury (Ashraf and Ashraf 2012; Ashraf et al. 2015). Oxidative damage is induced by enhanced production of ROS (Ashraf and Ashraf 2016; Ashraf et al. 2018). Plants possess antioxidant defense system which includes compounds with antioxidant potential (anthocyanins, ascorbate, tocopherols, flavonoids, glutathione, and phenolics) and antioxidant enzymes [APX (ascorbate peroxidase), GR (glutathione reductase), POD (peroxidase), CAT (catalase), and SOD (superoxide dismutase)] (Ahmad et al. 2011; Akram et al. 2018; Ashraf et al. 2018). This antioxidant system is upregulated under salinity enabling plants to scavenge ROS more efficiently. Enhanced detoxification of ROS reduces oxidative damage which can be measured in terms of H2O2 and MDA levels (Akram et al. 2018; Kaya et al. 2018a, b).

Menadione sodium bisulfite (MSB) being derived from vitamin K displays significant solubility in the aqueous medium. MSB is used to study oxidative stress in plants due to its redox-active nature (Borges et al. 2014). The redox properties of vitamin K enable this compound to perform physiological functions. Naphthoquinones (menadione), quinones, and benzoquinones are able to either donate or attract electrons and these properties render them active in biological systems (Borges et al. 2014). However, these properties depend upon the concentration of vitamin K (Castro et al. 2007). Vitamin K induces the production of ROS that in turn result in enhanced accumulation of ROS-scavenging proteins (Borges et al. 2014). Vitamin K is an important element of photosystem I and functions as an essential electron carrier. Vitamin K protects plants from environmental constraints through its functions as mobile electron carrier and maintenance of redox state of membrane proteins (Lüthje et al. 2013). Phylloquinone (vitamin K1) is also an important metabolite produced during the shikimate pathway. Vitamin K can be oxidized and reduced in cycles through enzyme pools and several substances (Lüthje et al. 1998). Vitamin K derivative increases the activity of H+-ATPase and thereby maintains acid pH and negative membrane potential. This electrochemical gradient across plasma membrane mediates transport of amino acids and sucrose through symporters as well as regulates cell turgor and stomatal closure (Elmore and Coaker 2011). Exogenous MSB increases growth, chlorophyll, tissue K+ and Ca2+ contents and decrease tissue Na+ contents in Arabidopsis challenged with salinity (Jiménez-Arias et al. 2015).

Okra (Abelmoschus esculentus Moench) is an important nutritious vegetable crop extensively grown around the globe (Abid et al. 2002). It is rich in nutrients including unsaturated fatty acids (oleic and linolenic acids), carbohydrates, calcium, potassium, and vitamins considered essential for human health (Asare et al. 2016). It is ranked as salt-sensitive vegetable crop by Qureshi and Barret-Lennard (1998). However, Esan et al. (2017) reported that response of okra could be variable from very salt sensitive to moderately salt sensitive. In addition, Jeyapraba et al. (2016) reported that okra is particularly sensitive to salinity at the initial developmental stages. Okra suffers significant yield losses due to salinity (Tanji and Neeltje 2002). For example, Minhas and Gupta (1993) reported 90%, 75%, and 50% fruit reduction in okra subjected to 6.7, 3.9, and 2.1 dS m−1, respectively. Keeping in view the adverse effects of salinity in vegetables, this study was undertaken to alleviate salinity effects in okra through foliar-applied MSB. Our hypothesis is that exogenous application of MSB may enhance plant salt tolerance by improving oxidative defense system and ion homeostasis. We also assessed the role of exogenous MSB in regulating key metabolic processes in okra subjected to salinity. This study was executed to appraise the influence of exogenous MSB on okra growth, oxidative defense system, and specific ion toxicity under salinity.

Materials and methods

A pot experiment was performed under natural environmental conditions to appraise the role of MSB in mitigating salinity effects in okra. The climatic conditions during the entire course of experiment were: average photosynthetically active radiation (PAR) 1110–1395 µmol m−2 s−1, 34% relative humidity, 33 °C temperature, and photoperiod of 12 h. Two okra cultivars (Arka Anamika and Shabnam-786), provided by AARI (Ayub Agricultural Research Institute, Faisalabad) Pakistan, were disinfected with 10% sodium hypochlorite for 5 min followed by thorough washing with distilled water. Seeds were sown in plastic pots (27 cm in diameter and 30 cm in height) filled with 8 kg of washed river sand. Thirteen seeds were sown in each pot and thinned to three plants per pot after 7 days of germination. Hoagland’s nutrient solution (full strength) was used to supplement nutrition in the sand medium. Salinity stress treatment (100 mM of NaCl) was applied 23 days after seed germination. Two liters of the nutrient solution with 100 mM NaCl was applied for the salt-treated plants every 5 days, while control plants were treated with the same nutrient solution without NaCl. Salinity treatments were applied using NaCl (analytical grade, Merck, Germany). Menadione sodium bisulfite (MSB) (analytical grade, Sigma-Aldrich) was applied as foliar spray. Different doses of MSB [CK (control), WS (water spray), 50, 100, 150 and 200 µM MSB] were prepared in distilled water with 0.1% Tween-20 (v/v) for foliar application. Ten days after the imposition of salt stress, MSB doses were applied as foliar application until drenching of leaves. After 15 days of treatment, data for morphological and biochemical attributes were recorded.

Growth attributes

Plants were carefully uprooted, washed with distilled water, and blotted dry. Shoot and root fresh weights and lengths were measured. Then plants were placed in an oven at 70 °C for 74 h to measure shoot and root dry weights. Leaf area was calculated with the help of the following formula: maximum leaf length × maximum leaf width × 0.68, where 0.68 is a correction factor (Hussain et al. 2017).

Chlorophyll

Fresh leaf tissue (0.1 g) was homogenized with aqueous acetone (80%) to measure chlorophyll (a and b) and carotenoids. The absorbance of the extract was read at 480, 645, and 663 nm with the help of a spectrophotometer (U-4000, Hitachi, Japan) (Arnon 1949).

Hydrogen peroxide (H2O2)

The method of Velikova et al. (2000) was followed to measure H2O2. 5 mL of 0.1% TCA (trichloroacetic acid) was used to grind fresh leaf (0.5 g). The resultant ground material was centrifuged at 10,000×g for 10 min to collect the supernatant. 1 mL of potassium iodide and 0.5 mL of 100 mM potassium phosphate buffer (pH 7.5) were added in 0.5 mL of the supernatant. The mixture was allowed to remain at 25 °C for 20 min before reading the absorbance at 390 nm with a spectrophotometer.

Malondialdehyde (MDA)

Heath and Packer (1968) method was used to determine the MDA contents. Fresh leaf material (0.5 g) was ground in 5 mL TCA (6%). The supernatant was collected after centrifugation at 10,000×g for 10 min. Then, 0.5 mL supernatant was mixed with 2 mL TBA (thiobarbituric acid) and the resulting reaction was incubated at 95 °C for 45 min. When the reaction solution reached 25 °C, its absorbance was read at 532 and 600 nm with a spectrophotometer.

Phenolics

Julkunen-Tiitto (1985) procedure was followed to measure total phenolics from fresh leaf. 5 mL aqueous acetone (80%) was used to homogenize 0.5 g fresh leaf. The homogenized material was centrifuged (10,000×g for 10 min) to get the supernatant. The Folin–Ciocalteu phenol reagent (1 mL) and distilled water (2 mL) were mixed with the supernatant (0.1 mL). The volume of the mixture was raised to 10 mL with distilled water after the addition of 5 mL Na2CO3 (20%). The reaction solution was thoroughly shaken before getting its absorbance at 720 nm with a spectrophotometer.

Anthocyanin contents

10 mL of 100 mM potassium phosphate buffer with 7.5 pH was used to grind 0.5 g fresh leaf. The ground material was centrifuged (10,000×g for 10 min) to take out the supernatant whose absorbance was directly read at 600 nm with a spectrophotometer (Kubo et al. 1999).

Flavonoids

The method of Marinova et al. (2005) was used to measure flavonoids. One mL of the supernatant (the one used for phenolics determination) was mixed with AlCl3 and NaNO2 (300 uL of each). The reaction solution was incubated at 25 °C for 5 min and 1 M NaOH (2 mL) was added. The mixture was used to get absorbance at 510 nm after incubation at 25 °C for 10 min. A spectrophotometer (the model already mentioned) was used to measure flavonoids.

Ascorbic acid

Mukherjee and Choudhuri’s (1983) method was followed for the measurement of ascorbic acid from fresh leaf material (0.5 g) extracted in 10 mL of 6% TCA. The homogenate was filtered and 1 mL of 2% dinitrophenylhydrazine was added to 2 mL filtrate. Then one drop of 10% thiourea was added to the reaction mixture. The reaction solution was incubated at 45 °C for 20 min. Then mixture was allowed to reach room temperature. Afterward, 2.5 mL of 80% H2SO4 was added to the reaction mixture. The absorbance of the mixture was read at 530 nm.

Total soluble proteins and antioxidant enzyme assay

Total soluble proteins were extracted from 0.5 g fresh leaf in 10 mL chilled potassium phosphate buffer (100 mM) with 7.5 pH. The supernatant was collected after centrifugation of homogenate at 10,000×g for 15 min. The centrifugation of the homogenate was done at 4 °C. Bradford (1976) method was followed to determine total soluble proteins from the supernatant. This supernatant was also used for measuring the antioxidant enzyme activities.

Catalase (CAT) and peroxidase (POD) activities were measured with the help of the method by Chance and Maehly (1955). The reaction mixture containing 100 mM potassium phosphate buffer (pH 7.5), 5.9 mM H2O2, and enzyme extract (0.1 mL) was used for measuring CAT activity. Change in absorbance was read every 20 s at 240 nm. The reaction mixture for POD consisted of 100 mM potassium phosphate buffer (pH 7.5), H2O2 (40 mM), and guaiacol (20 mM). Change in absorbance was read every 20 s at 470 nm. SOD activity was measured following the method of Giannopolitis and Ries (1977). The reaction mixture contained 100 mM potassium phosphate buffer (pH 7.5), riboflavin (1.3 µM), methionine (13 mM), NBT (50 µM), and enzyme extract (0.1 mL). The activities of the antioxidant enzymes were expressed in U mg−1protein.

Leaf free proline

10 mL sulfosalicylic acid (3%) was used to grind 0.5 g fresh leaf. The extract was filtered and glacial acetic acid and ninhydrin (2 mL each) were added to 2 mL filtrate. The mixture was heated for an hour at 100 °C. The mixture was allowed to get to room temperature and then toluene (4 mL) was added to the mixture. The absorbance was read at 520 nm with a spectrophotometer (Bates et al. 1973).

Total free amino acids

Potassium phosphate buffer extract of 0.5 g fresh leaf was used to measure this parameter. The supernatant (1 mL) was mixed with acid ninhydrin and 10% pyridine (1 mL each). The mixture was incubated at 95 °C for 30 min. Distilled water was added to make the volume to 7.5 mL and the absorbance of the mixture was obtained at 570 nm with a spectrophotometer (Hamilton and Van Slyke 1943).

Determination of Na+, K+, and Ca2+

The oven-dried leaves, stem, and roots (0.1 g) were acid digested (Wolf 1982). Concentrated H2SO4 (2 mL) was added to the digestion flask containing dry plant material and incubated for 24 h. After incubation, 0.5 mL of H2O2 (35%) was added to the digestion mixture and then heated at 250 °C. The flasks were then removed from heating and cooled down at room temperature and put on heat after adding 0.5 mL H2O2. The procedure was repeated until a clear transparent solution was obtained. Sodium, calcium, and potassium in the digested solutions were measured with flame photometer (PFP-7, Jenway Ltd. Felsted, UK).

Statistical analysis

The experiment was laid out in a completely randomized design with factorial arrangement. There were four replicates of each treatment. Analysis of variance (ANOVA) of data was computed with the help of statistical software Costat, version 6.303 (Table 1S). The difference between means was compared with the help of least significant difference (LSD) at P ≤ 0.05.

Results

Plant growth

Growth characteristics (shoot and root lengths and fresh and dry masses) considerably (P  ≤ 0.001) diminished in two cultivars subjected to salinity stress. There was also a noticeable lessening (P  ≤ 0.001) in leaf area in okra plants challenged with salinity. However, exogenous MSB significantly increased various growth attributes in okra cultivars under salinity. Of different doses of MSB, it was seen that a lower dose of MSB (50 µM) showed maximum improvement in growth attributes under salinity, while higher MSB dose (200 µM) had negative effect on plant growth under saline conditions. Cultivar Shabnam-786 manifested higher biomass accumulation compared with cv. Arka Anamika when 50 µM MSB was applied as foliar spray under salinity (Figs. 1 and 1S; Table 1S).

Fig. 1
figure 1

Effect of menadione sodium bisulfite on growth characteristics and photosynthetic pigments of salinity-stressed okra (Abelmoschus esculentus Moench) plants (n = 4; mean ± S.E.). Different lowercase letters in each attribute are significantly different at P ≤ 0.05. CK control, WS water stress, different concentrations of MSB (50, 100, 150, and 200 µM)

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Photosynthetic pigments

Decrease in growth attributes was associated with decrease in photosynthetic pigments. Salinity caused marked (P ≤0.001) decline in photosynthetic pigments (a and b and total chlorophyll contents) in two cultivars. However, changes in carotenoids were not significant under salinity. Plants with MSB treatment displayed higher photosynthetic pigments (carotenoid content, chlorophyll a and b, and total chlorophyll). Plants fed 50 µM MSB as foliar application had maximal values of these pigments. Higher dose of MSB (200 µM) had non-significant effect on these variables under salinity. The differences between cultivars were not significant except for chlorophyll b where cv. Shabnam-786 exhibited more chlorophyll b over Arka Anamika under salinity (Fig. 1; Table 1S).

H2O2 contents

As a result of salinity exposure, endogenous levels of H2O2 markedly (P ≤0.001) increased in the two cultivars. However, exogenous MSB noticeably decreased (P ≤0.001) H2O2 contents. The cultivars did not display noticeable difference with respect to H2O2 contents and MSB doses. Plants with 50 µM MSB treatment manifested a noticeable reduction in H2O2 contents over other MSB concentrations in two cultivars under salinity. The endogenous H2O2 contents in cv. Shabnam-786 treated with 50 µM MSB was slightly lower compared with cv. Arka Anamika. The higher concentration of MSB resulted in further increase in H2O2 contents in the two cultivars under salinity (Fig. 2; Table 1S).

Fig. 2
figure 2

Effect of menadione sodium bisulfite on the biochemical attributes of salinity-stressed okra (Abelmoschus esculentus Moench) plants. (n = 4; mean ± S.E.). Different lowercase letters in each attribute are significantly different at P ≤ 0.05. CK control, TSP total soluble proteins, AsA ascorbic acid, TFAA total free amino acids, WS water stress, different concentrations of MSB (50, 100, 150, and 200 µM)

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MDA contents

Imposing salinity stress in the plant-growing media led to a remarkable (P ≤ 0.001) rise in MDA content which is taken as an indirect measure of lipid peroxidation. The MDA contents were greater (P ≤ 0.05) in cv. Arka Anamika compared with cv. Shabnam-786. Foliar-applied MSB significantly altered endogenous MDA contents. For instance, MSB applied at 50 µM concentration caused significant reduction in this attribute compared with other MSB treatments [CK (control), WS (water spray), 100, 150, and 200 µM MSB] in both cultivars under salinity stress. The maximal values for MDA contents were recorded in plants with 200 µM MSB treatment under salinity (Fig. 2; Table 1S).

Non-enzymatic antioxidants

Ascorbic acid

There was a prominent increasing effect of salinity (P ≤ 0.001) on ascorbic acid contents in okra plants. Both okra cultivars significantly differed (P ≤ 0.001) with respect to this variable. In this context, a more considerable increase in ascorbic acid was recorded in cv. Shabnam-786 over cv. Arka Anamika under salinity. Exogenous supplementation of varying MSB doses (0, 50, 100, 150 and 200 µM) exhibited significant effect (P ≤ 0.001) on this variable. For instance, exogenous MSB (50 µM) showed maximal values of ascorbic acid contents over higher MSB dose (200 µM) which had negative impact on this attribute under salinity stress (Fig. 2; Table 1S).

Phenolics

Phenolics accumulation in okra plants enhanced considerably (P ≤ 0.001) with the imposition of salinity. There existed marked differences (P ≤ 0.01) between the two cultivars for phenolics accumulation. Higher phenolics contents were present in cv. Shabnam-786 over Arka Anamika. The exogenous MSB 50 µM resulted in a more considerable (P ≤ 0.001) increase in phenolics over other MSB doses under salinity. The elevated dose of MSB (200 µM) resulted in minimal values of phenolics under salinity (Fig. 2; Table 1S).

Flavonoids

A considerable rise in flavonoids accumulation (P ≤ 0.001) was observed in okra plants under salinity. Cultivar Shabnam-786 accumulated more flavonoid contents (P ≤ 0.01) than cv. Arka Anamika. Plants displayed variable (P ≤ 0.001) flavonoid accumulation in response to foliar application of MSB. In this context, maximal flavonoids were present in plants sprayed with 50 µM MSB, whereas plants treated with 200 µM MSB showed minimal flavonoid accumulation under salinity (Fig. 2; Table 1S).

Anthocyanins

In the present study, there existed significant variation (P ≤ 0.001) in anthocyanin contents in okra plants. Both cultivars displayed different anthocyanin contents (P ≤ 0.01) in response to salinity and foliar MSB applications. Cultivar Arka Anamika had more anthocyanins compared with cv. Shabnam-786 under salinity. Plants accumulated significantly higher anthocyanins when MSB was applied at 50 µM. The elevated level of exogenous MSB (200 µM) showed decreasing effect on this variable under salinity (Fig. 1; Table 1S).

Total soluble protein, leaf free proline and total free amino acids

Total soluble protein

Total soluble proteins increased remarkably (P ≤ 0.001) in plants exposed to salinity in the growth medium. Both cultivars had slightly different (P ≤ 0.05) total soluble protein contents. For example, foliar MSB (50 µM) resulted in more endogenous levels for this variable in cv. Shabnam-786 than that of cv. Arka Anamika. The exogenous MSB considerably affected (P ≤ 0.001) this variable in both cultivars under salinity. MSB-induced changes in total soluble protein were conspicuous in plants sprayed with 50 µM of plant growth regulator under salinity (Fig. 2; Table 1S).

Leaf free proline

There existed noticeable variation (P ≤ 0.05) in both okra cultivars for accumulation of leaf free proline under salinity stress and varying doses of foliar-applied MSB (0, 50, 100, 150 and 200 µM). Higher proline contents were observed in cv. Shabnam-786 over Arka Anamika under salinity. The exogenous MSB markedly affected (P ≤ 0.001) proline levels in plants under salinity. In this regard, more proline accumulation was found in plants when 50 µM MSB was applied as foliar spray under salinity. By contrast, exogenous application of higher concentration of MSB (200 µM) led to minimal values of proline under salinity stress (Fig. 2; Table 1S).

Total free amino acids

Exposure of okra plants to salinity resulted in a more considerable improvement (P ≤ 0.001) in total free amino acids. Cultivar Shabnam-786 accumulated more total free amino acids than cv. Arka Anamika under salinity. However, maximal increase in this variable was seen only in those plants sprayed with 50 µM MSB, while other concentrations of MSB did not influence the endogenous levels of this variable (Fig. 2; Table 1S).

Antioxidant enzyme activities

SOD activity

Exogenous supplementation of MSB significantly affected (P ≤ 0.001) SOD activity in two cultivars under salinity. Cultivar Shabnam-786 had markedly higher SOD activity over Arka Anamika when MSB was applied at 50 µM under salinity. Foliar application of lower MSB dose (50 µM) had marked increasing effect on this variable compared with those of other MSB doses under salinity stress (Fig. 2; Table 1S).

POD activity

When two okra cultivars were subjected to salinity stress, a noticeable enhancement (P ≤ 0.001) in POD activity was recorded. Foliar-applied MSB-induced noticeable (P ≤ 0.001) changes in this variable under salinity. In this context, plants with MSB treatment (50 µM) showed maximal POD activity in cv. Shabnam-786 compared to Arka Anamika under salinity. Higher MSB doses (100, 150 and 200 µM) did not affect POD activity under stress conditions (Fig. 2; Table 1S).

CAT activity

Salinity considerably enhanced (P ≤ 0.001) CAT activity in okra plants. There existed marked differences (P ≤ 0.001) between two cultivars with reference to this variable. Cultivar Shabnam-786 displayed maximal CAT activity over cv. Arka Anamika under salinity. Foliar-applied varying doses of MSB (0, 50, 100, 150 and 200 µM) markedly (P ≤ 0.001) influenced CAT activity under salinity. Plants administered MSB (50 µM) exhibited more CAT activity than those of other MSB doses under salinity (Fig. 2; Table 1S).

Uptake of Na+, K+ and Ca2+

To examine the interactive effect of MSB and salinity on nutrient uptake, we measured Na+, Ca2+ and K+ in leaf, stem and root tissues. Figure 3 shows that salinity caused a noticeable accretion (P ≤ 0.001) in the accumulation of toxic Na+ in the root, stem and leaf tissues between the cultivars. However, cv. Shabnam-786 manifested lower Na+ content in different plant tissues compared to cv. Arka Anamika. Plants fed with 50 µM MSB showed remarkable (P ≤ 0.001) abatement in tissue Na+ content of roots, stem, and leaf under saline condition (Fig. 3; Table 1S).

Fig. 3
figure 3

Effect of menadione sodium bisulfite on nutrient contents of salinity-stressed okra (Abelmoschus esculentus Moench) plants (n = 4; mean ± S.E.). Different lowercase letters in each attribute are significantly different at P ≤ 0.05. CK control, WS water stress; different concentrations of MSB (50, 100, 150, and 200 µM)

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Salinity caused distinguishable (P ≤ 0.001) enhancement in the accumulation of Ca2+ in root, stem, and leaf of both okra cultivars. The accumulation of Ca2+ was more in cv. Shabnam-786 compared to Arka Anamika under salinity. We further recorded the maximal values of tissue Ca2+ in plants treated with 50 µM MSB and this effect was more pronounced (P ≤ 0.001) in cv. Shabnam-786 (Fig. 3; Table 1S).

Tissue K+ content in root, stem, and leaf diminished considerably (P ≤ 0.001) due to salinity stress. Decrease in tissue K+ was many folds greater in cv. Arka Anamika over Shabnam-786 under salinity. Plants administered with MSB (50 µM) manifested increase (P ≤ 0.001) in root, stem and leaf K+ content under salinity. Foliar application of 200 µM MSB had negative effect on tissue K+ content under salinity stress (Fig. 3; Table 1S).

Discussion

Vitamins are important due to their functions as enzyme cofactors and as regulators of metabolism in plants. Vitamins, both lipid soluble (A, E and K) and water soluble (B and C), act as antioxidants (Asensi-Fabado and Munné-Bosch 2010). In our study, we evaluated the functions of water-soluble derivative compound of vitamin K, menadione sodium bisulfite (MSB), in mediating important metabolic reactions in okra plants under salinity. MSB was first reported as important regulator of plant growth in 1985 (Rama Rao et al. 1985). However, until recently, very few reports are present in the literature showing MSB-mediated improvement in plant defense responses under salinity (Borges et al. 2014; Jiménez-Arias et al. 2015). In the present study, salinity led to a noticeable decrease in growth of okra plants. Salt-induced growth suppression was related to a decrease in chlorophyll and increase in MDA and H2O2 levels along with enhanced tissue Na+ and limited tissue K+ concentration. Foliar MSB (50 µM) application defended plants from salinity-induced oxidative injury by increasing the concentrations of antioxidant compounds (ascorbic acid, phenolics, flavonoids, and anthocyanins) and antioxidant enzyme activities (CAT, POD, and SOD). Better antioxidant system readily scavenged ROS (H2O2) and protected membranes, which is evident in terms of minimal MDA accumulation. Consequently, plants with minimal oxidative damage, lower Na+ content, and higher chlorophyll had better growth under saline condition. Our results manifested minimal tissue Na+ level in plants treated with MSB (50 µM) under salinity. Likewise, MSB-treated plants had more K+ and Ca2+ content that could have contributed to better plant growth under salinity.

Salinity reduces plant growth in several other plants such as wheat (Siddiqui et al. 2017; Wang et al. 2018), honeysuckle (Cheng et al. 2018), Acacia gerrardii Benth (Abd-Allah et al. 2018), maize and rice (Ashraf et al. 2018; Yasmeen and Siddiqui 2017), tomato (Siddiqui et al. 2017), and okra (Habib et al. 2012). Root growth is inhibited first due to the ionic and osmotic effects of salinity. Consequently, water and mineral absorption, distribution, and accumulation are retarded (Cheng et al. 2018). Plants challenged with salinity showed growth suppression due to buildup of higher Na+ and lower K+ contents which inhibit important metabolic reactions (Ashraf et al. 2018). Plants require optimum chlorophyll for maintaining normal photosynthesis. However, salinity considerably diminished chlorophyll contents (Kaya et al. 2018a) as evident in the present study. Salt-induced decrease in chlorophyll could have been due to altered chlorophyll biosynthesis, enhanced degradation of chlorophyll by chlorophyllase enzyme, and changes in chloroplast membranes and chlorophyll protein complex (Ashraf and Harris 2013; Kaya et al. 2018a). Decline in chlorophyll content is positively associated with suppression in plant growth as found in maize (Ashraf et al. 2018), wheat (Ashraf and Ashraf 2012), sunflower (Santos 2004), bean (Taïbi et al. 2016), and tomato (Li et al. 2015). In our study, MSB-treated plants had greater chlorophyll contents due to lower oxidative damage. It is reported that chlorophyll content usually enhanced in plants with minimum oxidative damage (Taïbi et al. 2016). Similar to our results, exogenous application of MSB was reported to improve growth by increasing chlorophyll and proline concentration under salinity (Jiménez-Arias et al. 2015). Our results manifested greater proline in plants administered exogenously applied MSB. Moreover, enhanced proline levels regulate the activity of chlorophyllase enzyme and thereby more chlorophyll concentration is found in plants with greater endogenous levels of proline (Elsheery and Cao 2008). In our study, more chlorophyll contents due to exogenous application of MSB could be attributed to MSB-induced increased proline accumulation.

Salinity-mediated accretion in H2O2 contents caused peroxidation of lipids measured as MDA contents, thereby disturbing membrane permeability (Ashraf et al. 2018; Mandhania et al. 2006). In our study, we recorded enhanced H2O2 levels that in turn increased MDA accumulation and thus degraded membranes. Salt-induced greater oxidative injury in the form of more H2O2 and MDA generation has been documented in various plant species such as maize (Kaya et al. 2015), cucumber (Wang et al. 2016), tomato (Siddiqui et al. 2017), sorghum (Surender Reddy et al. 2015), and rice (Habib et al. 2016). A better antioxidant system in the form of non-enzymatic and enzymatic antioxidants protects plants from oxidative damage (Abid et al. 2018; Ali et al. 2018; Pehlivan 2018). In our study, we found minimal MDA and H2O2 levels in MSB-treated plants under salinity stress. This could have been due to higher concentration of non-enzymatic compounds (ascorbic acid, phenolics, flavonoids and anthocyanins) and activities of antioxidant enzymes (SOD, POD and CAT) in plants fed with foliar MSB. Our results are corroborated with previous report in Arabidopsis where exogenous MSB enhanced the expression of ROS-scavenging genes (Jiménez-Arias et al. 2015). MSB supplementation as foliar spray or seed priming induced a slight oxidative burst in the form of ROS production which in turn triggered the accumulation of antioxidant compounds and enhanced the activities of enzymes involved in ROS detoxification, thereby conferring salinity tolerance to plants (Borges et al. 2014). In the present study, MSB application resulted in greater accumulation of ascorbic acid, phenolics, flavonoids, and anthocyanins along with enhanced activities of SOD, POD, and CAT. This change in oxidative defense system could have been attributed to a slight oxidative burst caused by MSB (Borges et al. 2014).

Plants challenged with salinity face ionic imbalance due to buildup of Na+ content which in turn interfered with the uptake and accumulation of K+ and Ca2+. In the present study, we recorded increased tissue Na+ that decreased tissue K+. However, we have noted increase in tissue Ca2+. Salt-induced increase in Na+ and interference of toxic Na+ with the accumulation of K+ and Ca2+ have been documented in wheat (Ashraf and Ashraf 2016), maize (Ashraf et al. 2018), okra (Habib et al. 2012), and Arabidopsis (Jiménez-Arias et al. 2015). In the present study, exogenous application of MSB significantly diminished Na+ with concomitant accretion in tissue K+ and Ca2+. Our results are in keeping with earlier study where MSB-treated Arabidopsis plants had shown less Na+ and more tissue K+ and Ca2+ concentration (Jiménez-Arias et al. 2015). More Na+ in growth medium actively competes with K+ uptake and resultantly causes decline in tissue K+ (Ashraf and Harris 2004; Ashraf and Ashraf 2016). In contrast, salinity stress significantly increased Ca2+ which was maximal in plants administered foliar MSB. This could have been due to the function of Ca2+ as a signaling molecule in plants under salinity stress (Kong et al. 2015; Tuteja and Mahajan 2007).

Conclusion

In the current study, salinity considerably diminished growth, chlorophyll, and tissue K+ alongside the increase in tissue Na+ and Ca2+. Reduction in growth was associated with salt-induced decline in chlorophyll and increase in tissue Na+. Moreover, H2O2 and MDA accumulation also revealed greater intensity of oxidative damage that in turn degraded chlorophyll and suppressed plant growth. However, MSB application as foliar spray defended plants from oxidative injury by arousing the antioxidant system and altering nutrient uptake. Plants treated with MSB accumulated increasing concentrations of phenolics, flavonoids, ascorbic acid, and anthocyanins under salinity. Furthermore, the activities of antioxidant enzymes (SOD, POD, CAT) substantially increased in plants sprayed with MSB. Better antioxidant system in MSB-treated plants resulted in lower oxidative damage that in turn increased chlorophyll and improved growth. We measured Na+, K+, and Ca2+ in MSB-treated plants and found more tissue K+ and Ca2+ and lower Na+ under salinity. MSB protected plants from specific ion toxicity effect of salinity. Among MSB doses (0, 50, 100, 150, and 200 µM), 50 µM MSB was found to be effective as it protected plants from oxidative damage and specific ion toxicity under salinity stress.

Author contribution statement

MAA and MI planned the experiment and prepared the manuscript. MI supervised the whole experiment; MAA and HFA did sampling and analyses. All authors read and approved the manuscript.