Menadione sodium bisulfite neutralizes chromium phytotoxic effects in okra by regulating cytosolutes, lipid peroxidation, antioxidant system and metal uptake

KEYWORDS : Trace metals; lipid peroxidation; oxidative defense system; secondary metabolites; MSB; Cr uptake

Introduction

Increasing population puts extreme pressure on agricultural production around the world. Irrigation of vegetables and crops with industrial effluents is the major cause for contam- ination of human food chain with organic and inorganic pol- lutants. Trace metals are carcinogenic non-essential entities that deteriorate the environment as a result of their extensive application in different industrial units such as painting, tex- tile, steel, tanning, catalytic and polishing units (Farid et al. 2018). Chromium is predominant among common heavy metals that deteriorate plant growth and productivity. Cr (III) and Cr (IV) are stable forms of Cr (Farid et al. 2017). Cr (VI), due to carcinogenic properties and high stability, is the cause of dermatitis, asthma, bronchitis, and necrosis in humans besides the adverse effects on agricultural production (Farid et al. 2018). Cr is recognized as the seventh abundantly found mineral on the earth’s crust (Mohammadi et al. 2018; Ahmad et al. 2020). Cr (III) bound to soil organic matter is less mobile and toxic (Barton et al. 2000). Although some plant species show growth improvement under low Cr levels, the elevated Cr concentration is damaging for majority of plants (Davies et al. 2002; Zaheer et al. 2020). Heavy metals are known to cause substantial decrease in shoot and root growth (Shanker et al. 2005; Mallhi et al. 2020). Plants grown under Cr toxicity have weak root structures that inhibit nutrient and water uptake, thereby suppressing plant growth (Mohammadi et al. 2018). Cr stress disturbs metabolism that, in turn, inhibits growth. Cr causes significant damage to important physio- biochemical processes such as mineral nutrition, metabolic pathways, water relations, hormone functions and photosyn- thesis (Shanker et al. 2005). Cr bears a structural resemblance with the key elements and thereby inhibits mineral nutrition, enzyme activities and metabolites production. Cr generates excessive reactive oxygen species (ROS) that produce oxida- tive injury. The structural as well as functional abnormalities arise because of oxidative stress produced by ROS (Shanker et al. 2005). Cr stress also interferes with the activities of nitrate reductase, cytochrome oxidase, peroxidases, catalase and disturbs ions homeostasis (Yadav 2010).

Higher ROS levels in cells due to Cr cause a noteworthy alteration in plant morphology and physiology (Farid et al. 2018). The oxidative damage in plants is counteracted by a well-defined protective antioxidant system. Antioxidant compounds and antioxidant enzymes comprise the antioxi- dant system. Ascorbic acid, carotenoids, tocopherols, flavo- noids, phenolics, and glutathione are essential antioxidant compounds, whereas peroxidase, superoxide dismutase, cata- lase, and ascorbate peroxidase are some of the critical antioxidant enzymes (Rasheed et al. 2018). Plants use the antioxidant system as an essential defense mechanism against Cr toxicity (Ehsan et al. 2014; Stambulska et al. 2018). The higher concentration of MDA and H2O2, reflect- ing the degree of oxidative damage, tends to enhance in Cr stressed plants (Jabeen et al. 2016; Zaheer et al. 2020).
Different approaches, conventional and genetic engineer- ing, are being used for plant protection from phytotoxic impacts of metals (Adrees et al. 2015; Keller et al. 2015; Rasheed et al. 2018). Such approaches help improve plant defense mechanisms, including enhanced biosynthesis of organic solute as proline. Besides shotgun approaches, the exogenous application of inorganic and organic compounds has been customized for plant protection against such stresses (Adrees et al. 2015; Rasheed et al. 2018). MSB, which is an acronym of menadione sodium bisulfite, is a derivative of vitamin K that protects plants from biotic and abiotic stresses (Jim´enez-Arias et al. 2015; Carrillo-Perdomo et al. 2016; Jim´enez-Arias et al. 2019). MSB generates small oxidative damage that develops intricate ROS signaling net- works and thereby enhanced the production of defense- related proteins (Rasheed et al. 2018; Jim´enez-Arias et al. 2019). To the best of our knowledge, there exists no infor- mation on the role of MSB in okra under Cr toxicity. The major objective of the present investigation is to ascertain the impact of MSB application in okra exposed to Cr stress and evaluate important components that mediate plant responses to Cr stress. The study was planned to investigate how far MSB application improved plant growth through alterations in secondary metabolites accumulation, oxidative defense system and degree of oxidative damage. The find- ings of the present investigation would highlight the poten- tial of MSB as plant growth regulator to improve Cr tolerance in okra and other important horticultural crops.

Materials and methods

Seeds of okra cultivars (Akra Anamika and Shabnam-786) were attained from Ayub Agricultural Research Institute, Faisalabad, Pakistan. Okra seeds were surface sterilized by using NaOCl for 5 minutes and then seeds were washed thoroughly with distilled H2O. Five seeds were sown in thoroughly washed river sand contained in plastic pots and maintained three plants per pot by thinning. Hoagland’s nutrient solution containing potassium dichromate (K2Cr2O7) as a source of Cr (200 mM) was administered to pots two weeks after germination. Menadione sodium bisul- fite (MSB) solution (50, 100, 150, and 200 mM) along with Tween 20 (0.1%) was applied by foliar application one week after initiation of Cr stress. The whole experiment was car- ried out in a completely randomized design with four repli- cations of each treatment under natural field conditions, where photosynthetically active radiation (PAR) was 1119–1395 lmol m—2 s—1, average day and night tempera- tures 30.0 and 20.2 ◦C, respectively, and average relative humidity was 63%. Least significant difference (LSD) at less than 5% probability level (p 0.05) was used to compare different means. Data was measured two weeks after foliar
application of MSB for growth and physico-biochemical characteristics. Four plants were harvested, having uniform size from each treatment, followed by thorough washing with distilled water. Plants roots and shoots were separately cut for the measurement of fresh and dry weights. The remaining plants’ leaves were freeze-dried in liquid nitrogen and immediately stored at 80 ◦C to measure the following physiochemical attributes.

Relative water content (RWC)

Jones and Turner (1978) method was followed to measure RWC. Fresh juvenile leaf samples from each treatment were immersed in the distilled water for 3 h at 25 ◦C. Thereafter, turgid weight of immersed leaf was recorded. Then leaf was oven dried for the measurement of dry weight.

Total soluble proteins and amino acids

Total soluble proteins and free amino acids were measured from 0.5 g of leaf sample. Leaf samples was extracted in potassium phosphate buffer (pH 7.5) following Bradford (1976) and Hamilton and Van Slyke (1943), respectively.

Non-enzymatic antioxidants

Total phenolics

For determination of total phenolics, leaf sample (0.25 g) was extracted in 5 mL of 80% acetone. An aliquot of the fil- trate (100 mL) was mixed in folin-ciocalteu phenol reagent and distilled H2O. Thereafter, 5 mL of 20% Na2CO3 was mixed with in the mixture and made final volume up to 10 mL with distilled H2O. Optical density of the mixture was recorded at 750 nm (Julkunen-Tiitto 1985).

Flavonoids

Half gram leaf sample was extracted in 80% ethanol. One mL extract was reacted with NaNO2 (300 mL) and mixture was kept for 5 minutes at room temperature. Then 300 mL of AlCl3 was added and mixture was let to stay for 5 min. 1 M of sodium hydroxide (2 mL) was mixed and kept at room temperature for 10 minutes. After that, made final volume up to 10 mL by distilled H2O. After the incubation of 10 minutes at 25 ◦C, the absorbance was measured at 510 nm (Marinova et al. 2005).

Ascorbic acid

0.25 g of fresh leaf sample was ground in trichloroacetic acid (6%) and the mixture filtered. Filtrate (2 mL) was reacted with 1 mL of 2% 2, 4 – DNPH (2, 4 dinitrophenylhydrazine) alongside 10% of thiourea (one drop). The reaction mixture was heated for 20 minutes at 98 ◦C. Thereafter, mixture was cooled down and 80% of H2SO4 (2.5 mL) was mixed on ice. The absorbance was measured at 530 nm (Mukherjee and Choudhuri 1983).

Antioxidant enzyme activities

About 0.5 g of leaf sample was ground in 10 mL of potas- sium phosphate buffer (pH 7.5) on ice, and centrifuged at 4 ◦C for 20 minutes at 12,000 g. After centrifugation, supernatant was taken and stored at 80 ◦C for measurement of antioxidant enzymes activities.

Superoxide dismustase (SOD)

Giannopolitis and Ries (1977) method was followed for the determination of SOD activity. 3 mL of reaction mixture consisted of 13 mM methionine, 1.3 mM riboflavin, 50 mM nitro-blue tetrazolium, 75 mM EDTA (ethylene diamine tet- raacetic acid) and 50 mL enzyme extract. The reaction mix- ture was then irradiated for 15 minutes under fluorescent lamps alongside the control without enzyme extract. The absorbance of the mixture was recorded at 560 nm. Activity of SOD was expressed as Units mg—1 protein.

Peroxidase (POD)

The measurement of POD activity was executed following Chance and Maehly (1955). Three mL mixture consisted of 1.8 mL potassium phosphate buffer (50 mM; pH 7.5), 20 mM guaiacol (100 mL), 1 mL 40 mM H2O2 and enzyme extract (100 mL). The variation in optical density was noted at 470 nm for 3 minutes, with every 20 s interval.

Catalase (CAT)

The activity for CAT was measured by following the method of Chance and Maehly (1955). Three mL reaction mixture consisting of 1.9 mL of 50 mM potassium phosphate buffer (pH 7.5), 1 mL of 5.9 mM H2O2 and 100 mL of enzyme extract. The alteration in optical density was noted for 3 minutes, with every 20 s interval.

Ascorbate peroxidase (APX)

The measurement of APX activity was performed with the help of Nakano and Asada (1981). Three mL of reaction mixture contained 0.5 mM ascorbic acid, potassium phos- phate buffer (pH 7.5), H2O2 (1 mM) and enzyme extract (200 mL). The absorbance of the reaction was measured at 290 nm for 2 minute, with every 20 s interval.

Oxidative stress markers

Hydrogen peroxide (H2O2) About 0.25 g leaf sample was extracted with ice cold TCA (5%) and centrifuged for 5 minutes at 10,000 × g. 500 mL of supernatant was mixed to 500 mL of 50 mM potassium phos- phate buffer (pH 7.5) and potassium iodide (1 mL). The reac- tion mixture was incubated for 20 minutes room temperature. The absorbance was noted at 390 nm (Velikova et al. 2000).

Malondialdehyde (MDA)

Leaf (0.25 g) sample was homogenized in 5% TCA (5 mL) and homogenate centrifuged for 5 minutes at 10,000 g. After centrifugation, supernatant was mixed to thiobarbitu- ric acid (2 mL) and reaction mixture was heated for 30 minutes at 95 ◦C. After incubation, the mixture was
cooled down at room temperature. The absorbance was measured at 532 and 600 nm (Heath and Packer 1968).

Total soluble sugars (TSS)

An aliquot (100 mL) from 80% ethanol extract of leaf sample (0.25 g) was mixed with anthrone reagent. The mixture was heated at 95 ◦C for 15 minutes. Thereafter, reaction mixture was cooled down at room temperature and absorbance read at 625 nm (Yemm and Willis 1954).

Reducing sugars (RS)

One mL of the ethanol extract (used for TSS) was added in 6% of O-toluidine (5 mL) and the mixture was heated for 20 minutes at 95 ◦C. The mixture was kept at room tempera- ture and absorbance read at 630 nm (Nelson 1944).

Results

Growth attributes

A significant decrease in shoot and root fresh and dry weights from untreated plants was evident in plants raised under Cr stress. However, MSB application increased growth attributes under Cr toxicity. Maximum increase in growth characters was recorded from plants treated with 50 mM MSB. Cultivar Shabnam-786 was superior to cv. Arka Anamika for growth characteristics under Cr stress (Figure 1A–D; Table 1S).

Relative water contents (RWC)

Cr stress markedly (p 0.001) decreased RWC in both okra cultivars. There was no significant variation in RWC of two cultivars under stress and non-stress conditions. However, upon MSB treatment, there was a significant (p 0.001) increase in RWC in Cr-stressed plants. Plants treated with 50 mM MSB had maximal values for RWC when subjected to Cr stress (Figure 1E; Table 1S).

Photosynthetic pigments

Contents of chlorophyll significantly (p 0.001) altered in both okra cultivars due to Cr stress. However, Cr stress did not affect anthocyanin contents and neither did MSB treat- ment have any influence on anthocyanins. Cultivar Shabnam-786 had higher anthocyanins from plants of cv. Arka Anamika. MSB application (0, 50, 100, 150 and 200 mM) significantly (p 0.001) enhanced Chl. a, b and total Chl. in Cr-stressed okra plants. Higher chlorophyll contents were measured in plants treated with 50 mM MSB under Cr toxicity (Figure 1F–J; Table 1S).

Total free amino acids, soluble proteins and soluble sugars

Cr caused a significant (p 0.001) increase in total free amino acids and soluble proteins in two okra cultivars. Higher levels of these variables were measured in cv. Shabnam-786 than those in cv. Arka Anamika. MSB treat- ment at 50 mM significantly increased total free amino acids and soluble proteins. Total soluble sugars, reducing sugars and non-reducing sugars did not contribute significantly to Cr tolerance in okra neither did MSB application had any impact on the endogenous levels of these variables (Figure 2A–C, Figure 3A–C; Table 1S).

Proline

Plants subjected to Cr stress had a significant (p 0.001) increase in proline contents in both okra cultivars. However,the levels of proline contents were more in cv. Shabnam-786 in comparison to cv. Arka Anamika. MSB application had a marked effect on proline contents of Cr-stressed plants. Maximal values for proline contents were measured from plants treated with 50 mM MSB. Higher MSB levels resulted in a consistent decline in the studied variable (Figure 2B; Table 1S).

Non-enzymatic antioxidants

Cr stress resulted in a significant rise in the levels of non- enzymatic antioxidants viz. phenolics, flavonoids and ascorbic acid in comparison to plants under non-stress con- ditions. Plants treated with different doses of MSB showed significant changes in the levels of non-enzymatic antioxi- dant compounds. MSB treatment at 50 mM produced max- imal increase in ascorbic acid, flavonoids and phenolics contents under Cr stress. Cultivar Shabnam-786 accumu- lated greater phenolics and flavonoids, while two cultivars did not differ for endogenous levels of ascorbic acid (Figure 2D–F; Table 1S).

Antioxidant enzymes activities

A significant (p 0.001) increase in the activities of antioxi- dant enzymes (APX, CAT, POD and SOD) was measured in two okra cultivars exposed to Cr toxicity. Cultivar Shabnam- 786 showed higher activities of antioxidant enzymes in com- parison to cv. Arka Anamika. Plants treated with MSB had conspicuous (p 0.001) increase in the activities of antioxi- dant enzymes under Cr stress. However, lower concentration of MSB (50 mM) was more effective in stimulating the anti- oxidant enzyme activities under Cr toxicity (Figure 2G–J; Table 1S).

Oxidative damage

MSB significantly (p 0.001) circumvented Cr-induced oxi- dative damage in both okra cultivars. The degree of oxida- tive damage measured in the form of H2O2 and MDA was more in cv. Arka Anamika in comparison to cv. Shabnam- 786. MSB treatment at 50 and 100 mM resulted in a signifi- cant (p 0.001) decrease in the accumulation of H2O2 and MDA, a measure of lipid peroxidation (Figure 3D,E; Table 1S).

Cr uptake and accumulation

Cr accumulation in roots and leaves was significant (p 0.001) in okra plants under Cr toxicity. Plants treated with different MSB doses manifested a remarkable alteration (p 0.001) in root Cr contents. For instance, 50 mM MSB induced a considerable increase in root Cr contents. However, in leaf Cr, minimal values for leaf Cr were seen in plants treated with 50 mM MSB. Likewise, minimal aerial translocation of Cr was present in plants with 50 mM MSB applications. MSB restricted Cr to roots and significantly prevented its aerial transport (Table 1S; Figure 3F–H).

Figure 1. Effect of menadione sodium bisulfite on growth, RWC (relative water content) and leaf pigments in okra subjected to chromium stress. MSB 1 ¼ 50 lM; MSB2 ¼ 100 lM; MSB3 ¼ 150 lM; MSB4 ¼ 200 lM.

Discussion

In the present study, Cr stress produced a significant decline in the growth characteristics. A number of reports are avail- able in the literature where Cr stress-induced decrease in plant growth (Adrees et al. 2015; Gill et al. 2015; Francisco et al. 2017). The decrease in plant growth in our study was attributed to Cr-induced oxidative damage. Besides, Cr con- tents in plants exhibited a significant negative correlation with plant growth (Figure 4). Similar results were also described in barley (Ali et al. 2013) and in Brassica napus (Gill et al. 2015). Foliar-applied MSB produced pronounced improvement in plant biomass under Cr toxicity. MSB increased plant growth in Arabidopsis (Jim´enez-Arias et al. 2015) and tomato (Jim´enez-Arias et al. 2019) under saline conditions. Our results displayed an increase in plant growth due to MSB-mediated strengthening of oxidative defense system under Cr stress. Decrease in oxidative damage due to MSB-stimulated antioxidant system has been recorded under Cd stress in okra plant (Rasheed et al. 2018). In our study, a significant reduction was observed in RWC in okra plant.

Figure 2. Effect of menadione sodium bisulfite on TFAA (total free amino acids), proline, TSP, total soluble proteins, and oxidative defense in okra subjected to chromium stress. MSB 1 ¼ 50 lM; MSB2 ¼ 100 lM; MSB3 ¼ 150 lM; MSB4 ¼ 200 lM.

Figure 3. Effect of menadione sodium bisulfite on total soluble sugars (TSS), reducing sugars (RS), non-reducing sugars (NRS), hydrogen peroxide (H2O2), malondial- dehyde (MDA) and chromium (Cr) uptake and accumulation in okra under Cr toxicity. MSB 1 ¼ 50 lM; MSB2 ¼ 100 lM; MSB3 ¼ 150 lM; MSB4 ¼ 200 lM.

Decrease in RWC due to Cr stress has been reported previ- ously in Brassica napus (Gill et al. 2015). Okra plants sub- jected to Cd stress also displayed a significant decrease in RWC (Rasheed et al. 2018). MSB significantly increased RWC in Cr-stressed okra plants. A proposed Cr tolerance mechanism mediated by MSB is given in Figure 5. Our find- ings are in keeping with earlier reported data in Cd-stressed okra where MSB significantly improved RWC (Rasheed et al. 2018). Similar results also reported MSB-mediated improved RWC in tomato under salinity (Jim´enez-Arias et al. 2019).

Chlorophyll contents significantly decreased in okra under Cr stress. Reduction in chlorophyll contents in Brassica napus under Cr toxicity was reported by Gill et al. (2015). Decrease in chlorophyll contents might have been due to heavy metal-induced improvement in chlorophyllase activity (Hegedu€s et al. 2001). Furthermore, deterioration in chlorophyll content was attributed to degradation of thyla- koid structures and related proteins in chloroplast (Mohanty et al. 1989). In our study, MSB increased chlorophyll con- tents in okra exposed to Cr stress. Further, we recorded a significantly negative association of chlorophyll molecules with H2O2 and MDA (Figure 4). In this regard, Ahmad et al. (2020) reported that oxidative damage due to ROS production degrades chlorophyll molecules in Brassica oleracea L. under Cr toxicity. Increases in chlorophyll con- tents due to MSB was reported in salinity stressed Arabidopsis (Jim´enez-Arias et al. 2015), tomato (Jim´enez- Arias et al. 2019) and Cd-stressed okra (Rasheed et al. 2018). In our results, MSB improved the antioxidant system to neutralize ROS and increase chlorophyll contents under Cr stress. Rasheed et al. (2018) also reported that bettered chlorophyll contents were due to MSB-strengthened antioxi- dant system in okra under Cd toxicity.

Figure 4. Relationship of root and shoot Cr and TF with ASA (ascorbic acid), MDA (malondialdehyde), H2O2 (hydrogen peroxide), CAT (catalase), APX (ascor- bate peroxidase), POD (peroxidase), SOD (superoxide dismutase), pro (proline), Anth (anthocyanins), Flav (flavonoids), Phen (phenolics), TSS (total soluble sug- ars), TChl. (total chlorophyll), RWC (relative water contents), SDW (shoot dry weight), SDW (shoot dry weight), SFW (shoot fresh weight).

ROS production increased under Cr stress that ultimately caused oxidative damage (Chatterjee et al. 2015; Gill et al. 2015). Our results displayed raised H2O2 levels and lipid peroxidation measured as MDA in okra under Cr stress. Cr contents manifested a strong positive association with lipid peroxidation and H2O2 generation (Figure 6). MSB- mediated increase in antioxidant enzymes activities (SOD, POD, CAT and APX) and levels of non-enzymatic antioxi- dant compounds (ascorbic acid, flavonoids and phenolics) decreased H2O2 and MDA contents. Protection against oxi- dative damage in plants by MSB has been reported in okra (Rasheed et al. 2018) and tomato (Jim´enez-Arias et al. 2019) under Cd and salinity, respectively. Furthermore, increase in chlorophyll contents are reflected in the form of an MSB- strengthened antioxidant defense system in the pre- sent study.
Plants accumulated osmolytes of lower molecular weight like proline that also functions as a radical scavenger (Szabados and Savoure 2010). In our study, okra plants showed a significant increase in proline accumulation under Cr stress. MSB induced further increase in proline levels of Cr-stressed plants. Similar effect of MSB was recorded in salinity stressed Arabidopsis (Jim´enez-Arias et al. 2015) and Cd-stressed okra (Rasheed et al. 2018). Besides proline, Cr stress resulted in a significant increase in total free amino acids and soluble proteins in MSB-treated okra plants.

Figure 5. Proposed mechanism for MSB-mediated improvement of Cr tolerance in okra.

Figure 6. Principal component analysis (PCA) displaying relationship of root and shoot Cr and TF (translocation factor) with oxidative stress markers (MDA and H2O2), growth attributes and antioxidant enzyme activities.

Similar effect of MSB on these variables was recorded in okra under Cd stress (Rasheed et al. 2018). Increase in sol- uble sugars and total free amino acids has been reported earlier in grapes (Zonouri et al. 2014) and in quinoa (Aziz et al. 2018) under salinity and drought stress, respectively. Enhanced accumulation of total amino acids and total sol- uble proteins provide tolerance to the plants against abiotic stresses (Aziz et al. 2018). Plants pretreated with MSB exhib- ited greater endogenous levels of soluble protein and total free amino acid under Cr stress. Similar effect of MSB has been recorded in Cd-stressed okra plants (Rasheed et al. 2018).

Conclusion

Taken together, MSB significantly mitigated Cr effects in okra reflected in the form of improved growth characteris- tics and chlorophyll contents. MSB increased oxidative defense system which provide protection to plants against Cr-induced oxidative damage measured as H2O2 and MDA levels. Proline accumulation alongside higher levels of amino acids as well as total soluble protein contents due to MSB improved growth in okra under Cr. MSB treated plants also depicted a significant increase in internal concentration of flavonoids, ascorbic acid and phenolics. Thus, it is clear from findings of the present study that cv. Shabnam 786 possessed higher Cr stress tolerance as compared to cv.