Zinc application mitigates the adverse effects of NaCl stress on mustard [Brassica juncea (L.) Czern & Coss] through modulating compatible organic solutes, antioxidant enzymes, and flavonoid content

ABSTRACT This study examined the protective effect of Zn on salt-stressed Brassica juncea plants using some key morphological and biochemical attributes at different developmental stages (30, 60, and 90 days after treatment [DAT]). Salt stress (200 mM) caused suppression in plant height, root length, and dry weight by 58.35%, 41.15%, and 53.33%, respectively, at 90 DAT, but Zn application improved these variables by 15.52%, 16.59%, and 11.45%, respectively. Furthermore, 200 mM NaCl decreased total chlorophyll by 45.32% and relative water content by 27.62% at 90 DAT, whereas Zn application compensated the decrease in the levels of both variables. NaCl (200 mM) increased H2O2, malondialdehyde, and electrolyte leakage by 70.48%, 35.25%, and 68.39%, respectively, at 90 DAT, but Zn supplementation appreciably reduced these variables. Except for catalase, enzymatic antioxidant activity increased under NaCl stress. Zn application with salt further increased the activities of superoxide dismutase, catalase, ascorbate peroxidase, glutathione reductase, and glutathione-S-transferase by 33.51%, 9.21%, 10.98%, 17.46%, and 12.87%, respectively, at 90 DAT. At 90 DAT, salt stress increased flavonoids by 24.88%, and Zn supply by a further 7.68%. Overall, Zn mitigated the adverse effects of salt stress through osmotic adjustment, as well as by modulating the oxidative defense system and flavonoid contents.


Introduction
Salinity is a major environmental cue, which considerably suppresses crop production worldwide through its injurious effects on plant growth and development (Ashraf and McNeilly 2004;Ahanger and Agarwal 2017). Globally, high salinity affects over 20% of arable land; it exacerbates industrialization and urbanization in most parts of the world. However, poor irrigation practices are further aggravating the problem (Gupta and Huang 2014). Salinity stress hampers plant growth via diminishing cell division and expansion, reducing photosynthetic efficiency, modifying metabolic processes, as well as causing ion toxicity, osmotic stress, oxidative stress, genotoxicity, and other physiological disorders (Ibrahim et al. 2012;Wani et al. 2013;Yan et al. 2013;Ahmad et al. 2016b;Anjum et al. 2015;Hussein et al. 2017). Cumulatively, these adverse effects perturb growth and alter normal metabolism in plants (Wani et al. 2013;Rasool et al. 2013;Ahmad et al. 2014;Iqbal et al. 2015;Tang et al. 2015;Hussein et al. 2017).
Several chemical, biological, and physical strategies are being adopted to obtain a maximum economic yield from crops. For example, supplementation of mineral macroand micro-nutrients at optimal concentrations allays adverse effects of stressors, leading to growth maintenance and yield increases (Ahanger et al. 2015;Ahmad et al. 2015b;Siddiqui et al. 2015;Ahanger and Agarwal 2017). Among microelements, Zn is vital for regulating enzyme activity, the protein and membrane stabilization; it is a component of Zn finger, a major structural motif critical for DNA binding (Tavallali et al. 2009). Zinc is a cofactor of over 300 enzymes and associated proteins involved in carbohydrate metabolism, nucleic acid and protein synthesis, as well as cell division (Osredkar and Sustar 2011;Singh et al. 2015). Moreover, Zn can impart stress tolerance through the formation of Cu/Zn-SOD, a major antioxidant (Ahmad et al. 2010).
Brassica juncea (L.) Czern & Coss (family Brassicaceae) is an important oil-seed crop that often experiences salinity stress because of its vast cultivation in arid and semi-arid regions (Wani et al. 2013). Addition of Zn salts directly to soil solution has been demonstrated to improve crop growth and ameliorate stress in B. juncea (Prasad and Saradhi 1995). However, only a few reports exist in the literature on how foliar application of Zn may prevent stress-induced oxidative damage through regulating antioxidative and osmoregulatory components. Thus, in this study, we have tried to explore if an exogenous application of Zn to the foliage of salt-stressed B. juncea plants could effectively regulate the oxidative defense system and the levels of osmotically active metabolites.

Plant materials and growth conditions
B. juncea (L.) Czern. & Coss. seeds of varuna cultivar were sown in Petri dishes and placed in a growth chamber. Five germinated seedlings were shifted to each pot (5 kg capacity with 23-cm diameter) containing peat, perlite, and sand (1:1:1, v/v/v) and the pots were supplied with Hoagland nutrient solution (200 mL per pot) for 2 weeks under average day/ night temperatures of 26°C/16°C. After this thinning was done manually and 3 seedlings were allowed to grow in each pot. After 18 days, varying NaCl concentrations (0, 100, and 200 mM) dissolved in Hoagland nutrient solution were applied on alternate days to all pots except the control till 90 days after treatment (DAT  (Hoagland and Arnon 1940). The pH of nutrient solution was adjusted to 6.5 with 0.1 mM KOH.
Zinc (ZnSO 4 ·7H 2 O, 1 mM) dissolved in distilled water was sprayed on plant foliage after every 2 days with a manual sprayer (10 mL/plant), from day 7 (25-day-old plant) to day 90 (115-day-old plant) of NaCl treatment. The experimental design was completely randomized with five replicates. Leaf samples (secondary leaves) were collected for analysis after salt treatment for 30 (55-day-old plant), 60 (85-day-old plant), and 90 (115-day-old plant) days.

Growth and biomass
The length of shoot and root was measured manually with a scale. The samples were dried at 65°C for 72 h for the assessment of dry biomass expressed in grams per plant.

Estimation of chlorophyll content
Chlorophyll pigments were estimated following Hiscox and Israelstam (1979). The tissue samples were extracted from fresh leaves in dimethyl sulfoxide (DMSO); the absorbance of the resultant supernatant was measured spectrophotometrically at 645 and 663 nm (Beckman 640 D, USA), using DMSO as a blank.

Estimation of leaf relative water content
Leaf discs were punched from the samples of each treatment and their fresh weight (FW) determined. The discs were floated on water for up to 4 h to record turgid weight. Subsequently, the discs were oven-dried at 85°C to estimate dry weight (DW) (Smart and Bingham 1974). Leaf relative water content (LRWC) was calculated as follows:

Determination of electrolyte leakage
Electrolyte leakage in leaf tissues was determined following Dionisio-Sese and Tobita (1998). The leaf discs were kept in 10 mL deionized water and electrical conductivity observed at time point 0 (EC 0 ). The leaf discs were heated in a water bath at 60°C for 25 min and then measured EC 1 . Thereafter, the samples were boiled at 100°C for 10 min for EC 2 measurement. The formula used to calculate the electrolyte leakage was: Electrolyte leakage(%) = (EC 1 − EC 0 )/(EC 2 − EC 0 ) × 100.

Determination of leaf free proline content
Proline content was determined following Bates et al. (1973).
The absorbance of the sample mixture treated appropriately with specific reagents was recorded spectrophotometrically at 520 nm (Beckman 640 D, USA), using toluene as a blank.

Estimation of H 2 O 2 and malondialdehyde
Hydrogen peroxide content was determined following Velikova et al. (2000). Fresh leaves (500 mg) were homogenized in 5 mL of 0.1% (w/v) trichloroacetic acid (TCA). The homogenate was centrifuged at 12,000g for 15 min; thereafter, 0.5 mL of the supernatant was mixed with 0.5 mL of 10 mM potassium phosphate buffer (pH 7.0) and 1.0 mL of 1.0 M potassium iodide solution. The supernatant absorbance was read at 390 nm.
To determine lipid peroxidation (a measure of malondialdehyde [MDA] formation), fresh leaf tissue was first macerated in 1% TCA and then centrifuged at 10,000g for 5 min. The supernatant (1.0 mL) was reacted with 4.0 mL of 0.5% thiobarbituric acid for 30 min in a water bath at 95°C, cooled in an ice-bath, and centrifuged again at 10,000 rpm for 5 min. The optical density of the samples was recorded at A 532 . Unspecific turbidity was corrected via subtracting the optical density at 600 nm from the A 532 reading. An extinction coefficient of 155 mM −1 cm −1 (Heath and Packer 1968) was used to calculate MDA concentration.

Antioxidant enzyme assay
Fresh leaves (10 g each) were homogenized in a buffer of 100 mM Tris-HCl (pH 7.5) containing 5.0 mM DTT, 10 mM MgCl 2 , 1.0 mM EDTA, 5.0 mM magnesium acetate, 1.5% PVP, 1.0 mM PMSF, and 1 μg/mL aproptinin. The extract was centrifuged for 15 min at 10,000 rpm to obtain the supernatant for enzyme assays. Two millimolar ascorbate was added to the buffer for extracting APX. Soluble protein content was determined following Bradford (1976), using bovine serum albumin as the standard.
SOD (EC 1.15.1.1) activity was determined following van Rossum et al. (1997), after an initial photoreduction of nitroblue tetrazolium (NBT) at 560 nm. One SOD unit was defined as the amount of protein that caused a 50% decrease in SOD-inhibitable NBT reduction and was expressed as Unit mg −1 protein.
Catalase (EC 1.11.1.6) activity was assayed by monitoring the decrease in absorbance at 240 nm for 3 min (Luck 1971) and was expressed as EU mg −1 protein.
Fresh leaves (0.5 g each) were homogenized in 3.0 mL meta-phosphoric acid (5%) containing 1.0 mM EDTA; the homogenate was centrifuged at 11,500g and 4°C for 15 min. The resultant supernatant was used for ASA and GSH determination, following Huang et al. (2005) and Anderson (1985), respectively. Calculations were performed with ASA and GSH standards.
GST (EC 2.5.1.18) activity was estimated by the method of Hasanuzzaman and Fujita (2013). The absorbance was read at 340 nm for 1 min using a spectrophotometer (Beckman 640 D, USA) and was expressed as EU mg −1 protein.

Estimation of total flavonoid content
Flavonoid content was determined using the colorimetric method (Zhishen et al. 1999). Absorbance was read at 510 nm and flavonoid content expressed as mg catechin equivalents g −1 of extract (mg g −1 ).

Statistical analysis
Data were presented as mean ± SE of five replicates and subjected to one-way analysis of variance (ANOVA), followed by Duncan's multiple range test for examining the differences among the values. Significance was set at P ≤ 0.05.  Under 100 mM NaCl, proline content increased over the control by 41.06%, 50.43%, and 57.82% at 30, 60, and 90 DAT, respectively (Table 3). Under 200 mM NaCl, the increase went up to 52.08%, 59.61%, and 65.42% at 30, 60, and 90 DAT, respectively. A further increase of 7.75%, 16.14%, and again 16.14% was recorded at 30, 60, and 90 DAT, respectively, due to Zn supplementation (Table 3).
MDA content reflects the degree of lipid peroxidation and is widely accepted as an indicator of oxidative damage. Leaf MDA was higher in salt-stressed seedlings than in the control plants (Figure 1(B)). Under 100 mM NaCl, MDA content increased by 16.38% (30 DAT), 16.77% (60 DAT), and 22.42% (90 DAT), whereas under 200 mM NaCl, the increase was 34.93% (30 DAT), 33.89% (60 DAT), and 35.25% (90 DAT). In general, Zn application decreased MDA content in salt-treated plants compared with that in salt-treated plants (Figure 1(B)). Figure 2 presents enzymatic antioxidant activity under NaCl and Zn treatments. Both NaCl treatments increased SOD activity over the control with a maximal increase of 22.53%, 23.52%, and 28.97% at 30, 60, and 90 DAT, respectively, under 200 mM NaCl (Figure 2(A)). Zinc application further increased SOD activity in salt-stressed plants by 19.87% (100 mM NaCl + Zn) and 33.51% (200 mM NaCl + Zn). Note: Data presented are the means ± SE (n = 5). Different letters next to the number indicate significant difference (P < .05) among the treatments within a developmental stage. Symbols $, £, and ¥ denote significant changes among the different developmental stages in the same treatment for the given parameter. DAT, days after treatment. Note: Data presented are the means ± SE (n = 5). Different letters next to the number indicate significant difference (P < .05) among the treatments within a developmental stage. Symbols $, £ and ¥ denote significant changes among the different developmental stages in the same treatment for the given parameter. DAT, days after treatment.

Antioxidant in NaCl-treated plants
Increasing NaCl concentrations consistently decreased CAT activity. A 20% decrease with reference to the control was observed in all the three samples (Figure 2(B)). However, Zn supplementation enhanced the CAT activity over that in salt-stressed plants, by 4% at 30 DAT, 9.48% at 60 DAT, and 9.21% at 90 DAT.
At 90 DAT, the GR activity under 100 and 200 mM NaCl increased significantly over the control by plants maximally by 19.71% and 25.50%, respectively (Figure 2(D)). Application of Zn further increased the GR activity by 15.31% (30 DAT), 11.02% (60 DAT), and 10.29% (90 DAT) over that in the 200 mM NaCl-treated plants.
Zinc application increased the ASA content by 13.79% and end with relative to the salt-stressed plants. Figure

Discussion
In the present study, salt stress caused a decline in plant growth through reducing plant height, root length, and shoot DW, which substantiates some earlier reports on different plant species (Husen et al. 2016). The negative effects of NaCl on growth and biomass yield might have been due to osmotic stress and low uptake of essential elements (Hashem et al. 2014). In the present investigation, it is amply clear that Zn foliar sprays allayed NaCl stress effects in B. juncea. Weisany et al. (2014) reported that Zn application reduces saltstress damage considerably because of positive effects on the uptake and partitioning of important mineral elements. Our study shows that foliar spray of Zn allayed NaCl-caused effects in B. juncea. Zinc application considerably reduces the salinity-caused damage because of its positive effects on the uptake and partitioning of important mineral elements (Weisany et al. 2011(Weisany et al. , 2014. It improves plant growth by increasing the natural auxin (indole acetic acid) production and, consequently, activating the cell division and enlargement (Ali and Mahmoud 2013), maintaining membrane structural integrity (Weisany et al. 2014), accumulating phospholipids (Jiang et al. 2014), improving protein synthesis (Ebrahimian and Bybordi 2011), scavenging free oxygen radicals (Jiang et al. 2014), mediating nutrient translocation from developing cells (Jiang et al. 2014), and restricting excessive Na + and Cl − uptake (Weisany et al. 2011;Siddiqui et al. 2015;Yousuf et al. 2016aYousuf et al. , 2016b. Salt stress clearly reduced total chlorophyll content of B. juncea leaves, which is in agreement with earlier findings of Weisany et al. (2011), Hayat et al. (2012, and Iqbal et al. (2015) in different plant species. Salt stress is known to hamper the synthesis of enzymes involved in the generation and protection of photosynthetic pigments (El-Tayeb 2005). Destruction of these enzymes via salt stress triggers changes in the structure and stability of the pigment-protein complex, leading to chlorophyllase up-regulation and enhanced chlorophyll degradation; this process may be the cause for reduced chlorophyll content in salt-stressed plants (Fang et al. 1998). Additionally, Zn application may increase photosynthetic pigment synthesis through direct positive effects on the uptake of magnesium, a central chlorophyll component (Weisany et al. 2011(Weisany et al. , 2014. Our data showing chlorophyll increase with Zn application are in agreement with the findings of Samreen et al. (2017) for Vigna radiata and Novo et al. (2014) for B. juncea. Zinc-induced protection of sulfhydryl group may be another reason for amelioration of the drastic chlorophyll decline by salt stress (Weisany et al. 2011).
High concentrations of toxic ions such as Na + in the soil solution cause osmotic stress, resulting in a reduced water uptake. In the salt-stressed plants, this lowered water uptake decreases the stomatal conductance (Tavallali et al. 2009). Zinc application proved beneficial in maintaining the tissue water content of B. juncea even under saline conditions. Tavallali et al. (2009) demonstrated that a reduced water potential in the salt-stressed Pistacia vera led to cellular dysfunction. Our results regarding the effect of Zn application on  RWC in salt-stressed plants find support from Weisany et al. (2011).
In our study, application of NaCl as well as Zn increased proline accumulation. Increased proline accumulation is believed to maintain tissue water potential under environmental stresses, thus offering an important tolerance strategy (Ahanger et al. 2014(Ahanger et al. , 2015Ahmad et al. 2015aAhmad et al. , 2015b. Saltstressed plants maintain higher proline content through enhancing the activity of key enzymes involved in proline synthesis . Proline thus protects enzymatic function and helps in free radical scavenging (Rajendrakumar et al. 1997). Zinc application may improve proline accumulation via regulating solute potential, and therefore water uptake from the soil.
In our study, salt stress induced H 2 O 2 over-accumulation in B. juncea plants, but Zn application inhibited its production. Under certain threshold levels, H 2 O 2 has a protective effect due to its role in stress-induced signaling processes. However, H 2 O 2 overproduction causes excessive peroxidation of membrane fatty acids, protein denaturation, and negative effects on DNA/RNA integrity (Tuteja et al. 2009;Habibi 2014). The accumulation of H 2 O 2 in salt-stressed plants may be due to reduced RWC, which limits H 2 O 2 diffusion from its generation site (Weisany et al. 2014). Stressmediated H 2 O 2 production, subsequent membrane leakage, and MDA formation in combination determine the intensity of oxidative stress (Tavallali et al. 2009;Ahmad et al. 2014Ahmad et al. , 2015a. Loss of membrane integrity from increased ROS production causes leakage of important mineral ions from cellular organelles (Tuna et al. 2007). Specifically, membrane integrity is compromised through the generation of lipid hydroperoxides via ROS-membrane interactions (Hashem et al. 2014). Zinc-treated mustard seedlings exhibited reduced MDA content and increased membrane stability, confirming the positive role of Zn in avoiding ROS-induced oxidative damage under salt stress.
Salt stress enhances ROS generation, thereby increasing antioxidant enzyme activity (Tavallali et al. 2009;Ahmad et al. 2016b;Weisany et al. 2014). In the present study, salt stress up-regulated the activities of SOD, APX, GR, and GST. Furthermore, zinc application caused rapid ROS elimination, thereby further enhancing antioxidant enzyme activity coupled with better plant growth. Previous research supports our findings that supplementation of optimal mineral concentrations keeps ROS at nontoxic levels (Ahanger et al. 2015;Iqbal et al. 2015). SOD acts on superoxide radicals, which are further scavenged by either CAT or APX through the ascorbate-glutathione pathway; as a result, superoxide radicals are removed and prevented from forming more toxic radicals (Ahmad et al. 2010;Bose et al. 2014). Additionally, SOD prevents metal-mediated formation of toxic OH − . In the ascorbate-glutathione pathway, APX, GR, GSH, and ASA are key members that mediate removal of H 2 O 2 through the transfer of electrons from NADPH to H 2 O 2 , with GSH and ASA acting as mobile redox buffers. Our findings suggest that Zn application elevated GSH and GR concentrations, contributing to efficient H 2 O 2 scavenging and lower lipid peroxidation. Increased APX activity can overcome salt-stress-induced decreases in CAT activity; both molecules are active H 2 O 2 scavengers (Ahmad et al. 2010;Gill and Tuteja 2010). Here, we demonstrated that CAT activity was decreased by salt stress, but increased by Zn supplementation, suggesting that Zn can optimize CAT activity to improve the processes of H 2 O 2 scavenging under stress. GST mediates detoxification via conjugating xenobiotics with the non-enzymatic antioxidant, tripeptide glutathione (Ahmad et al. 2010;Gill and Tuteja 2010). Zn-induced enhancement in the GST activity can mediate quick detoxification of radicals, in addition to its role in hormone homeostasis, vacuolar sequestration of anthocyanins, cell apoptosis, and stress responses (Dixon et al. 2010;Gill and Tuteja 2010). Our results showing increased GST activity in salt-stressed B. juncea plants concur with the findings of Gapińska et al. (2008) in Lycopersicon esculentum.
Flavonoids are polyphenols with low molecular weight and are vital to the protection of photosynthesizing cells. Plants maintaining higher flavonoid content show improved photosynthetic efficiency due to rapid, flavonoid-mediated scavenging of superoxide radicals (Majer et al. 2014). It is not clear if the Zn-induced enhancement of NaCl tolerance is related to the metal's involvement in flavonoid synthesis. However, in salt-stressed plants, flavonoids are thought to act as chelators (Winkel-Shirley 2002). Mineral supplementation has been shown to enhance polyphenol accumulation in Avena sativa, leading to growth maintenance (Ahanger et al. 2015). Besides their role as antioxidants, flavonoids function in plant metabolism and are likely involved in various signaling mechanisms, as suggested by their interaction with protein kinases involved in initiating cell growth and differentiation (e.g. mitogen-activated protein kinases) (Brunetti et al. 2013).

Conclusion
Salt (NaCl) imposed osmotic and oxidative stress, leading to decreases in chlorophyll and RWC, along with increases in H 2 O 2 production, lipid peroxidation, and electrolyte leakage. To combat salt stress, the B. juncea plants increased proline, ASA, and GSH accumulation, as well as SOD, APX, GR, and GST activities. Flavonoid content also increased with increasing NaCl concentrations and with developmental stages (30, 60, and 90 DAT). Zinc foliar application to NaCl-stressed plants decreased H 2 O 2 production, lipid peroxidation, and electrolyte leakage while maintaining chlorophyll levels and RWC. It also further enhanced proline content, activities of antioxidant enzymes, and levels of non-enzymatic antioxidants. The increase in antioxidants and flavonoid content is thought to enhance ROS scavenging efficiency, thereby improving B. juncea tolerance to salt stress.

Disclosure statement
No potential conflict of interest was reported by the authors.