Soil Sulfur Sources Differentially Enhance Cadmium Tolerance in Indian Mustard ( Brassica juncea L.)

: The effect of four soil-applied sulfur (100 mg S kg − 1 soil (100S) and 200 mg S kg − 1 soil (200S)) in different sources (elemental S, ammonium sulfate, gypsum or magnesium sulfate) in protecting mustard ( Brassica juncea L. (Czern & Coss.)) from cadmium effects was studied. Based on the observed reduction in growth and photosynthesis in plants subjected to 100 and 200 mg Cd kg − 1 soil, B. juncea cv. Giriraj was selected as the most Cd-tolerant among ﬁve cultivars (namely, Giriraj, RH-0749, Pusa Agrani, RH-406, and Pusa Tarak). Sulfur applied to soil mitigated the negative impact of Cd on sulfur assimilation, cell viability, and photosynthetic functions, with a lower lipid peroxidation, electrolyte leakage, and contents of reactive oxygen species (ROS: hydrogen peroxide, H 2 O 2 , and superoxide anion, O 2 •− ). Generally, added S caused higher activity of antioxidant enzymes (ascorbate peroxidase, catalase and superoxide dismutase), contents of ascorbate (AsA) and reduced glutathione (GSH); increases in the activities of their regenerating enzymes (dehydroascorbate reductase and glutathione reductase); as well as rises in S assimilation, biosynthesis of non-protein thiols (NPTs), and phytochelatins (PCs). Compared to the other S-sources tested, elemental S more prominently protected B. juncea cv. Giriraj against Cd-impacts by minimizing Cd-accumulation and its root-to-shoot translocation; decreasing cellular ROS and membrane damage, and improving Cd-chelation (NPTs and PCs), so strengthening the defense machinery against Cd. The results suggest the use of elemental S for favoring the growth and development of cultivated plants also in Cd-contaminated agricultural soils.


Introduction
The issues concerning the continuous accumulation of metals in agricultural soils because of the anthropogenic activities have been widely reviewed and discussed over the past 30 years [1][2][3]. Particularly, cadmium (Cd), a hazardous heavy metal pollutant, has wide distribution, short half-life, and higher solubility in water, without a known biological function in plants. Cadmium enters the human food chain by getting accumulated in agricultural crops, thus causing severe threats to human and animal health [4].
In nature, Cd is accumulated in soil primarily through phosphatic fertilizers, irrigation with polluted water, and weathering of parent material, and also through volcanic eruptions [5]. The presence of Cd potentially stimulates the major plant enzymes, like NADPH oxidases, whose activity produce reactive oxygen species (ROS), such as singlet oxygen ( 1 O 2 ), hydroxyl radicles (•OH), superoxide anion (O 2 •− ), and hydrogen peroxide sodium hypochlorite (NaClO) and then rinsed repetitively with double distilled water. Surface-sterilized seeds were sown in 23-cm diameter pots filled with 5 kg of reconstituted soil with peat:compost:sand (4:1:1, w/w). The pots used in the experiments had holes, however, the plants were watered by calculating field capacity, so that there was minimum leaching. Before sowing, soil samples were collected randomly from different pots for selected soil-characteristics analysis. Soil texture; pH in water; electrical conductivity; and S, N, P, and K contents were measured by soil standard methods, according to Pansu and Gautheyrou [22]; soil Cd content was determined by atomic absorption spectrophotometry (GBC, 932 Plus; GBC Scientific instruments, Braeside, Australia) ( Table 1). The pots were kept in the greenhouse with day/night temperatures of 23/16 ± 2 • C, a 16/8 h light/dark period, and relative humidity of 60 ± 4%. In the first experiment, five Brassica cultivars were screened for their tolerance to different Cd levels (0, 100 and 200 mg Cd kg −1 of soil), based on growth and photosynthetic attributes. The source of Cd was cadmium chloride (CdCl 2 ), and it was added to pots at the time of sowing.
In the third experiment, plants were grown either with 200 mg Cd kg −1 soil, S 0 , [(NH 4 ) 2 SO 4 ], CaSO 4 ·2H 2 O or MgSO 4 , or in combined treatment of Cd + S 0 , Cd + MgSO 4 , Cd + CaSO 4 ·2H 2 O, and Cd + MgSO 4 . A control group of plants without sulfur was maintained. Elemental S was given 15 days before sowing, while all the other sources were supplied at the time of sowing.
Treatments in all the experiments were arranged in a factorial randomized block design, and the number of replicates for each treatment was four (n = 4). At 30 days after sowing (DAS), analyses were made to study gas exchange, photosynthetic efficiency, growth characteristics, oxidative stress, antioxidant levels ad activity, S and N metabolism, and antioxidant system.

Growth Parameters
Growth attributes were measured at 30 DAS for experiments 1, 2, and 3. Plants were uprooted carefully from the pots and washed to remove the soil and dust from roots. Then, plants were weighed to determine the fresh biomass and were later kept for drying in an oven at 80 • C to record dry biomass. Leaf area was measured using leaf area meter (LA211, Systronics, New Delhi, India).

Estimation of Cadmium and Sulfur Content
The concentration of Cd was analyzed using atomic absorption spectrophotometer. Root and leaf samples were dried in an oven for 2 days at 80 • C. The dried tissue was weighed, ground to powder, and the resulting powder was digested with concentrated HNO 3 :HClO 4 (3:1; v/v). Cd content was determined by atomic absorption spectrophotometry (GBC, 932 Plus; GBC Scientific Instruments, Braeside, Australia). Cd translocation factor (TF) was calculated as shoot to root ratio of Cd concentration [4]. Tolerance index (TI) was calculated as the ratio of dry weights of plants exposed to Cd to that under control conditions [20].
Sulfur content for experiment 3 was determined in oven-dried leaves (0.1 g) and roots (0.5 g) digested in a mixture of concentrated HNO 3 and 60% strength HClO 4 (85: 15; v/v), using the turbidimetric method of Chesnin and Yien [23] with some modifications. A 5 mL reaction mixture was made up by adding 1.0 g BaCl 2 and 2.5 mL gum acacia, and the final volume was made up to 25 mL by adding distilled water and contents were thoroughly shaken to dissolve completely BaCl 2 . The values were recorded spectrophotometrically at 415 nm from the time of turbidity development, and a blank was run after determining each set simultaneously.

Gas Exchange and Photosynthetic Parameters
Net photosynthesis (P N ), stomatal conductance (g s ), and intercellular CO 2 concentration (C i ) for all the three experiments were measured in fully expanded and intact topmost leaves in various treatments and replicates using an infrared gas analyser (CID-340, Photosynthesis system, Bio-Science, Washington, DC, USA). The measurements were made on a sunny day between 10:00 and 11:30 a.m. at photosynthetic active radiation (PAR) of 720 µmol m −2 s −1 and atmospheric CO 2 concentration of 415 µL L −1 . Chlorophyll content (SPAD values) was measured in fully expanded uppermost leaves with a SPAD chlorophyll meter (502 DL PLUS, Spectrum Technologies, Aurora, IL, USA).
Maximal PSII photochemical efficiency (F v /F m ) of intact leaf from the top for experiments 2 and 3 was measured using a chlorophyll fluorometer (Junior Pam, Heinz Walz, Germany). Leaf samples were kept in the dark for 30 min to measure maximum fluorescence (F m ) and minimum fluorescence (F o ). The value of F o was determined from a weak pulse (0.1 µmol m −2 s −1 ), while Fm was obtained from saturating pulse (>6000 µmol m −2 s −1 ). Variable fluorescence (F v ) was calculated by subtracting F o from F m , and F v /F m ratio was calculated, which is a measure of maximum quantum yield efficiency of PSII. The activity of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) was ascertained adopting the method by Usuda [24] by monitoring the oxidation of NADH at 30 • C at 340 nm. The activity was measured after the addition of enzyme extract followed by 0.2 mM ribulose-1,5-bisphosphate (RuBP). The detailed procedure is reported in Rather et al. [2].

Assessment of Oxidative Damage
The content of O 2 •− and H 2 O 2 was determined spectrophotometrically by adopting the method of Wu et al. [25] and Okuda et al. [26], respectively. The degree of production of O 2 •− and H 2 O 2 in vivo was validated via the histochemical method of Kumar et al. [27]. The electrolyte leakage (EC) and lipid peroxidation by thiobarbituric acid reactive substances (TBARS) were measured spectrophometrically according to Sullivan and Ross [28] and Dhindsa et al. [29], respectively.

Antioxidant Enzymes and Non-Enzymatic Antioxidants
Fresh leaf tissues (0.5 g) were homogenized in chilled mortar and pestle with an extraction buffer containing 0.05% (v/v) Triton X 100 and 1% (w/v) polyvinylpyrrolidone in 100 mM potassium phosphate buffer (pH 7.0). The homogenate was centrifuged at 15,000× g for 20 min at 4 • C. The activity of SOD (EC 1.15.1.1) was accessed according to the method of Giannopolitis and Ries [30] by observing the inhibition of photochemical reduction of nitro blue tetrazolium (NBT). One unit of SOD activity was the amount of enzyme that inhibits the NBT reduction by 50% followed at 560 nm. CAT (1.11.1.6) activity was determined by the method provided by Aebi [31] by visualizing the disappearance of H 2 O 2 at 240 nm. One unit of CAT activity was the amount that decomposes 1 µmol of H 2 O 2 min −1 at 25 • C. The activity of APX (EC 1.11.1.11) was measured according to the method of Nakano and Asada [32] by observing a decrease in absorbance of ascorbate at 290 nm. One unit of AP activity was defined as the amount required to decompose 1 µmol of substrate min −1 at 25 • C. GR (EC 1.6.4.2) activity was determined following the protocol of Foyer and Halliwell [33] with some modifications. Reaction mixture consisted of 0.5 mM oxidized GSH, 0.2 mM NADPH, phosphate buffer (25 mM, pH 7.8). One unit of GR activity was the amount necessary to decompose 1.0 µmol of NADPH min −1 at 25 • C. Dehydroascorbate reductase (DHAR, EC 1.8.5.1) activity was assayed following the method of Pinto et al. [34]. A 0.01 increase in absorbance at 265 nm was defined as one unit of DHAR activity.

Non-Protein Thiols and Total Phytochelatins Content
The content of non-protein thiols (NPTs) and total phytochelatins (PCs) was measured following the procedure of Lou et al. [36] with some modifications. NPT was extracted by homogenizing 0.2 g of leaf samples in 2 mL of 5% sulfosalicylic acid and then centrifuged at 10,000× g for 15 min at 4 • C. For NPT determination, the reaction mixture contained 0.2 mL of the supernatant, 0.15 mL of 10 mM 5,5 dithiobis [2-nitrobenzoic acid] (DTNB), and 0.2 M Tris-HCl (pH 8.2). The reaction mixture was incubated for 20 min, and absorbance was measured spectrophotometrically at 412 nm. PCs content was calculated by subtracting total GSH content from the total amount of NPTs.

S-Assimilating Enzymes and S-Containing Amino Acids
The activity of ATP-S (EC 2.7.7.4) for experiments 2 and 3 was determined according to Lappartient and Touraine [37] by following spectrophotometrically at 340 nm the molybdate-dependent formation of pyrophosphate. The activity of OASTL (EC 4.2.99.8) was determined spectrophotometrically with the method of Riemenschneider et al. [38].
Leaf cysteine (Cys) content for experiments 2 and 3 was determined spectrophotometrically at 580 nm using the method of Gaitonde [39], with some modifications. In brief, 500 mg fresh leaves were homogenised in ice-cold perchloric acid (5%; w/v). The obtained suspension was centrifuged at 2800× g for 1 h at 5 • C. Supernatant was filtered through Whatman No.1 filter paper. The content of methionine (Met) was determined according to Khan et al. [11].

Confocal Laser Microscopy to Study Root Cell Viability
Clean and thin sections of roots were dipped in 25 µM propidium iodide (PI) solution to visualize cell viability. After washing properly, root samples were analysed with a confocal microscope. The stained samples were visualized under a confocal microscope (Olympus Fluoview TM-FV1000, Olympus Life Sciences, Tokyo, Japan) with a 63× oil immersion objective, at excitation of 400-490 nm, emission ≥ 520 nm. Fluoview FV10 software, ver 1.7 (Olympus Life Sciences, Tokyo, Japan) was used to analyze and process the images. Analyses were performed on 30-day old roots.

Physiological Measurements of the Guard Cells
Fresh leaves from 30-day-old plants were plugged off from each branch of various treatments and were fixed by 2.5% glutaraldehyde, and stomatal images were captured by means of a Carl Zeiss EVO 40 scanning electron microscope (Zeiss, Aalen, Germany) at extra high tension and high voltage at 20 kV. Leaf samples were first fixed with 2.5% glutaraldehyde plus 2% paraformaldehyde (v/v) in 0.1 M phosphate buffer (pH 7.0) in equal quantity for 45 h, and then washed three times with phosphate buffer for 15 min at each step. The samples were then post fixed with 1% osmium oxide in phosphate buffer (pH 7.0) for 1 h and washed three times with the same phosphate buffer for 15 min.
Then, they were dehydrated by a graded series of ethanol (50, 70, 80, 90, 95, and 100%) for about 15-20 min at each step, and transferred to the mixture of alcohol and isoamyl acetate (1:1; v/v) for about 30 min. Finally, the samples were transferred to pure isoamyl acetate for 1 h and dehydrated with liquid CO 2 . The dehydrated specimen was coated with gold-palladium and observed under the microscope. Analyses were performed on 30-day old leaves.

Statistical Analysis
Data were analysed statistically by analysis of variance (ANOVA) using SPSS v17.0 for Windows (IBM Corporation, New York, NY, USA). Least significant differences (LSDs) were calculated among treatments at p < 0.05 levels. More details are given in table and figure captions.

Screening of Cultivars for Cd Tolerance
The effects of Cd on growth parameters (fresh plant biomass, dry plant biomass, leaf area) and photosynthetic attributes (chlorophyll content measured by SPAD, net photosynthesis, stomatal conductance) in the five cultivars of B. juncea were summarized in Table 2. Irrespective of the cultivars, both 100 Cd and 200 Cd levels impaired the tested photosynthetic and plant growth traits, compared to the control. However, the maximum adverse effect of Cd on plant photosynthetic and growth attributes was observed with 200 Cd, compared to 100 Cd. The highest Cd accumulation was recorded in roots and leaves of Pusa Tarak, and the lowest in Giriraj cultivar. Tolerance index (TI) of all the cultivars, calculated using data of plant dry mass treated with Cd 200, confirmed that Giriraj had the highest tolerance index (0.789) among all the tested cultivars, whereas Pusa Tarak was the most sensitive ( Table 2). The cultivars showed tolerance to Cd in the following order: Giriraj > RH-0749 > Pusa Agrani > RH-406 > Pusa Tarak.

Response of Plants to Different S Sources and S Levels
To assess the S-requirement of the crop, we evaluated the effects of two S levels (100S and 200S) from four S sources (i.e., elemental S, S 0 ; ammonium sulfate, AS; gypsum, Gyp; and magnesium sulfate, MS) on growth and photosynthetic parameters, and S-assimilation (Table 3 and Figure 1). Plants showed differential responses to S sources, and the response was dose-dependent.  Photosynthetic and growth parameters were positively influenced by both S levels (100S and 200S). In contrast to 100S, the effects of 200S on growth and photosynthetic attributes were more conspicuous for all S sources. Among the four sources of S used, S 0 showed a more protruding effect than any other S source, both at 100 and 200S levels, and generally increased the values of growth and photosynthetic parameters. The entity of the   Photosynthetic and growth parameters were positively influenced by both S levels (100S and 200S). In contrast to 100S, the effects of 200S on growth and photosynthetic attributes were more conspicuous for all S sources. Among the four sources of S used, S 0 showed a more protruding effect than any other S source, both at 100 and 200S levels, and generally increased the values of growth and photosynthetic parameters. The entity of the effect of S 0 was followed by those of AS, Gyp, and MS. S 0 -treated plants had increased chlorophyll content, net photosynthesis, intercellular CO 2 concentration, stomatal conductance and maximal PSII efficiency, plant fresh, and dry biomass, particularly at 200S. The increases in these characteristics were by 48.9, 37.7, 75.2, 70.9, 35.2, 40.1, and 86.3%, respectively, compared to the control (Table 3).
Of the two S levels, 200S was more efficacious in increasing the activity of ATP-S and OASTL by 93.3 and 87.8% (S 0 ), 86.7 and 73.0% (AS), 67.9 and 58.1% (Gyp), and 61.9 and 47.9% (MS), respectively, over the control ( Figure 1A,B). A similar trend was observed for Cys and Met content, where 200S caused increased Cys content by two-times and Met content by 79.1%, in respect to the control ( Figure 1C,D). The higher effectiveness of 200S improved plant growth more than 100S. For this reason, 200S treatment was considered for further study.

Effects of Different S Sources on Cd and S Accumulation
The accumulation of Cd in Cd-exposed plants was significantly higher in roots than leaves (Table 4). While all S sources reduced the accumulation of Cd in roots and leaves, S 0 was the most effective. S 0 -treated plants recorded a reduction by 78.5% of Cd content in roots and by 84.3% in leaves, followed by AS, Gyp, and MS (Table 4). Moreover, plants treated with S 0 had the lowest value of translocation factor (TF = 0.590), when compared with the other S sources. The content of S was markedly reduced by 22.6% in roots and by 23.3% in leaves in Cd-stressed plants, compared to the control ( Table 4). The accumulation of S in roots and leaves increased after the application of each S form, particularly for S 0 ( Table 4).

Effects of Different S Sources on Plant Growth under Cd Stress
Plants subjected to 200Cd showed a decline in fresh biomass by 17.9%, dry biomass by 26.6%, and leaf area by 8.4%, compared to the control ( Figure 2). Plants receiving S in different forms showed an increase in all the afore-said growth parameters under no Cd stress, and elemental S showed a maximum increase of 53.6% in fresh biomass, 71.2% in dry biomass, and 56.4% in leaf area, compared to the control. Furthermore, a significant amelioration of Cd toxicity was seen in plants receiving different S sources. In Cd-exposed plants, the application of elemental S maximally enhanced fresh biomass by 20.6%, dry biomass by 28.7%, and leaf area by 24.5% compared to the control, and so conspicuously restored the damage caused by Cd ( Figure 2).

Effect of Different S Sources in Preventing Adverse Effects of Cd on Photosynthesis
The plants grown in the presence of Cd 200 exhibited reduced net photosynthesis by 22.2%, stomatal conductance by 19.8%, intercellular CO2 concentration by 30.6%, chlorophyll content by 21.2%, maximal PSII efficiency by 18.3%, and Rubisco activity by 27.5%, compared to the control (Figure 3). Although under unstressed conditions, photosynthetic attributes were enhanced by all the four S sources, and elemental S improved theses parameters more prominently than the other S forms. Elemental sulfur supplemental in plants grown with Cd also resulted in maximal alleviation of Cd-induced photosynthetic inhibition and promoted net photosynthesis, stomatal conductance, intercellular CO2 concentration, and chlorophyll content by 28.9, 64.2, 32.2, and 34.2%, respectively, compared

Effect of Different S Sources in Preventing Adverse Effects of Cd on Photosynthesis
The plants grown in the presence of Cd 200 exhibited reduced net photosynthesis by 22.2%, stomatal conductance by 19.8%, intercellular CO 2 concentration by 30.6%, chlorophyll content by 21.2%, maximal PSII efficiency by 18.3%, and Rubisco activity by 27.5%, compared to the control (Figure 3). Although under unstressed conditions, photosynthetic attributes were enhanced by all the four S sources, and elemental S improved theses parameters more prominently than the other S forms. Elemental sulfur supplemental in plants grown with Cd also resulted in maximal alleviation of Cd-induced photosynthetic inhibition and promoted net photosynthesis, stomatal conductance, intercellular CO 2 concentration, and chlorophyll content by 28.9, 64.2, 32.2, and 34.2%, respectively, compared to the control ( Figure 3A-D). Similarly, S 0 treatment displayed an utmost increase in maximal PSII efficiency and Rubisco activity by 11.1 and 60.5%, respectively, whereas in MS-treated plants, maximal PSII efficiency was reduced by 5.30% and Rubisco activity increased by 20.1%, compared to the control ( Figure 3E,F) to the control ( Figure 3A-D). Similarly, S 0 treatment displayed an utmost increase in maximal PSII efficiency and Rubisco activity by 11.1 and 60.5%, respectively, whereas in MStreated plants, maximal PSII efficiency was reduced by 5.30% and Rubisco activity increased by 20.1%, compared to the control ( Figure 3E,F).

Effects of Different S Sources on Oxidative Stress and Antioxidants
Cd presence led to a significant increase in H2O2 and O2 •− content by 3.5 and 2.8 times, compared to the control (Figure 4). The application of different S sources moderated the production of oxidative markers produced because of Cd toxicity and recovered the oxidative damage, with S 0 -treated plants showing the most prominent reduction. The accumulation of O2 •− was evidenced by the formation of scattered dark blue formazan in the leaves ( Figure 4A-D) and that of H2O2 by brownish colored formazan in leaves ( Figure  4E-H). Fresh leaves from Cd-stressed plants exhibited more pronounced spots when compared to control plants. Furthermore, leaves of plants treated with elemental S without Cd or Cd observed fewer spots as compared to the Cd-stressed plants.

Effects of Different S Sources on Oxidative Stress and Antioxidants
Cd presence led to a significant increase in H 2 O 2 and O 2 •− content by 3.5 and 2.8 times, compared to the control (Figure 4). The application of different S sources moderated the production of oxidative markers produced because of Cd toxicity and recovered the oxidative damage, with S 0 -treated plants showing the most prominent reduction. The accumulation of O 2 •− was evidenced by the formation of scattered dark blue formazan in the leaves ( Figure 4A-D) and that of H 2 O 2 by brownish colored formazan in leaves ( Figure 4E-H). Fresh leaves from Cd-stressed plants exhibited more pronounced spots when compared to control plants. Furthermore, leaves of plants treated with elemental S without Cd or Cd observed fewer spots as compared to the Cd-stressed plants. Cd stress significantly raised the electrolyte leakage and content of thiobarbituric acid reactive substances (TBARS) by 2.2 and 2.6 times, compared to control plants ( Figure  5A,B). Nevertheless, distinct S sources supplementation proved efficacious in curbing Cdinduced increase in electrolyte leakage and lipid peroxidation and toned down both stress biomarkers efficiently. Moreover, Cd-exposed plants with elemental S exhibited a decrease by 19.5% in electrolyte leakage and by 21.7% in TBARS content, with respect to control plants. The reduction in electrolyte leakage and TBARS content was lower in MStreated plants, which displayed a considerable increase among all the S sources under Cd stress, compared to the control ( Figure 5A,B).
The oxidative stress test results suggested that the exogenous treatment with S sources helped plants to tolerate Cd stress by deescalating Cd-induced oxidative injury caused by ROS accumulation (Figure 5C-E). Indeed, plants exposed to Cd showed an increase in the activities of CAT, SOD, and APX enzymes by 91.5, 39.1, and 52.8%, compared to the control, and this increase was significantly higher than in unstressed plants supplied with different S sources, showing the response of plants' inherent defense capability to counter oxidative stress ( Figure 5C-E). Plants treated with various S forms exhibited an increase in activity of all the three antioxidant enzymes under Cd presence. The activity of CAT, SOD, and APX was highest with S 0 (3.5, 3.8 and 6.3 times, respectively), with respect to the control ( Figure 5C-E). Cd stress significantly raised the electrolyte leakage and content of thiobarbituric acid reactive substances (TBARS) by 2.2 and 2.6 times, compared to control plants ( Figure 5A,B). Nevertheless, distinct S sources supplementation proved efficacious in curbing Cd-induced increase in electrolyte leakage and lipid peroxidation and toned down both stress biomarkers efficiently. Moreover, Cd-exposed plants with elemental S exhibited a decrease by 19.5% in electrolyte leakage and by 21.7% in TBARS content, with respect to control plants. The reduction in electrolyte leakage and TBARS content was lower in MS-treated plants, which displayed a considerable increase among all the S sources under Cd stress, compared to the control ( Figure 5A,B).
The oxidative stress test results suggested that the exogenous treatment with S sources helped plants to tolerate Cd stress by deescalating Cd-induced oxidative injury caused by ROS accumulation (Figure 5C-E). Indeed, plants exposed to Cd showed an increase in the activities of CAT, SOD, and APX enzymes by 91.5, 39.1, and 52.8%, compared to the control, and this increase was significantly higher than in unstressed plants supplied with different S sources, showing the response of plants' inherent defense capability to counter oxidative stress ( Figure 5C-E). Plants treated with various S forms exhibited an increase in activity of all the three antioxidant enzymes under Cd presence. The activity of CAT, SOD, and APX was highest with S 0 (3.5, 3.8 and 6.3 times, respectively), with respect to the control ( Figure 5C-E). In order to assess the role of the AsA-GSH cycle in Cd stress tolerance, assessment of activity of GR and DHAR and content of AsA and GSH were evaluated. The Cd-treated plants exhibited an increase in GR activity by 39.3%, while GSH content was reduced by 16.8%, compared to the control ( Figure 5F,G). Plants grown with different sources of S increased activity of GR and GSH content in normal as well as in stressed conditions compared to the control. Plants receiving S 0 with Cd maximally increased the activity of GR and content of GSH by 2.1 and 1.6 times, respectively, compared to control plants. Similarly, the activity of DHAR and AsA contents under the influence of distinct S sources increased, and the maximal increase was recorded in S 0 -treated plants, in both stressed as well as unstressed conditions ( Figure 5H,I).

Effect of Different Sources of S on Variations in S Assimilation under Cd Stress
Plants grown with Cd exhibited increases in the activity of ATP-S and OASTL by 29.4 and 37.5%, respectively, compared to the control ( Figure 6A,B). The activity of both the Sassimilation enzymes was significantly elevated by all the S sources, in unstressed as well as in stressed plants. Furthermore, plants treated with Cd plus distinct S forms showed a significant and marked increase in the activities of ATP-S and OASTL by 3.2 and 3.8 times using S 0 , 2.8 and 3.4 times with AS, 2.5 and 3.2 times with Gyp, and 2.3 and 2.7 times by supplying MS, respectively, compared to the control ( Figure 6A,B). The content of Cys and Met increased with all the S sources, with or without Cd ( Figure 6C,D). Plants grown under Cd showed increased Cys content by 53.2%, whereas Met content was reduced by 24.9%, compared to control plants ( Figure 6C,D). Elemental S maximally improved the content of Cys and Met by 2.8 times in unstress plants, with respect to the control. Further, In order to assess the role of the AsA-GSH cycle in Cd stress tolerance, assessment of activity of GR and DHAR and content of AsA and GSH were evaluated. The Cd-treated plants exhibited an increase in GR activity by 39.3%, while GSH content was reduced by 16.8%, compared to the control ( Figure 5F,G). Plants grown with different sources of S increased activity of GR and GSH content in normal as well as in stressed conditions compared to the control. Plants receiving S 0 with Cd maximally increased the activity of GR and content of GSH by 2.1 and 1.6 times, respectively, compared to control plants. Similarly, the activity of DHAR and AsA contents under the influence of distinct S sources increased, and the maximal increase was recorded in S 0 -treated plants, in both stressed as well as unstressed conditions ( Figure 5H,I).

Effect of Different Sources of S on Variations in S Assimilation under Cd Stress
Plants grown with Cd exhibited increases in the activity of ATP-S and OASTL by 29.4 and 37.5%, respectively, compared to the control ( Figure 6A,B). The activity of both the S-assimilation enzymes was significantly elevated by all the S sources, in unstressed as well as in stressed plants. Furthermore, plants treated with Cd plus distinct S forms showed a significant and marked increase in the activities of ATP-S and OASTL by 3.2 and 3.8 times using S 0 , 2.8 and 3.4 times with AS, 2.5 and 3.2 times with Gyp, and 2.3 and 2.7 times by supplying MS, respectively, compared to the control ( Figure 6A,B). The content of Cys and Met increased with all the S sources, with or without Cd ( Figure 6C,D). Plants grown under Cd showed increased Cys content by 53.2%, whereas Met content was reduced by 24.9%, compared to control plants ( Figure 6C,D). Elemental S maximally improved the content of Cys and Met by 2.8 times in unstress plants, with respect to the control. Further, S sources reversed the Cd-induced reduction in Cys and Met content and improved it maximally by 4.3 and 1.9 times, respectively, using S 0 .
S sources reversed the Cd-induced reduction in Cys and Met content and improved it maximally by 4.3 and 1.9 times, respectively, using S 0 . NPT and PCs content was analyzed in leaves of both stressed and unstressed plants. Both NPT and PCs content in Cd-stressed plants was significantly higher than in control plants (9.2 and 83.4%, respectively) ( Figure 6EF). Plants treated with different forms of S under Cd stress showed a significant and conspicuous rise in the content of both NPT and PCs. NPT content, which was maximal in the plants treated with S 0 + Cd (1.7 times), was followed by AS-treated plants (1.5 times), whereas the plants treated with Gyp/MS + Cd displayed no significant difference over control plants ( Figure 6E). Similarly, PC content also showed a significant and remarkable increase by 1.8 times following S 0 + Cd treatment, while Gyp + Cd ranked lowest (1.5 times) in increasing PC content among the four S sources used, compared to control plants ( Figure 6F).

Influence of Different S Sources on Cell Viability and Stomatal Studies under Cd Stress
Propidium iodide (PI) is a staining dye that penetrates damaged cell membranes and stains nucleic acids, which become visible inside the dead cell as red fluorescent spots. Root cells of Cd-stressed plants were less viable and showed more stain. However, Cdinduced cell death was reversed by S application, and S 0 -treated plants exhibited a similar response to control plants ( Figure 7A-D). NPT and PCs content was analyzed in leaves of both stressed and unstressed plants. Both NPT and PCs content in Cd-stressed plants was significantly higher than in control plants (9.2 and 83.4%, respectively) ( Figure 6EF). Plants treated with different forms of S under Cd stress showed a significant and conspicuous rise in the content of both NPT and PCs. NPT content, which was maximal in the plants treated with S 0 + Cd (1.7 times), was followed by AS-treated plants (1.5 times), whereas the plants treated with Gyp/MS + Cd displayed no significant difference over control plants ( Figure 6E). Similarly, PC content also showed a significant and remarkable increase by 1.8 times following S 0 + Cd treatment, while Gyp + Cd ranked lowest (1.5 times) in increasing PC content among the four S sources used, compared to control plants ( Figure 6F).

Influence of Different S Sources on Cell Viability and Stomatal Studies under Cd Stress
Propidium iodide (PI) is a staining dye that penetrates damaged cell membranes and stains nucleic acids, which become visible inside the dead cell as red fluorescent spots. Root cells of Cd-stressed plants were less viable and showed more stain. However, Cd-induced cell death was reversed by S application, and S 0 -treated plants exhibited a similar response to control plants ( Figure 7A-D). Electron microscopy was used to examine the stomatal behavior in response to Cd stress and S supply. Stomatal analysis depicted a noteworthy change in stomatal aperture in Cd-stressed plants. Stomata were normal in the leaf samples of control, S 0 and Cd + S 0treated plants. However, Cd-treated plants showed semi-closed stomata with distorted guard cells ( Figure 7E-H).

Discussion
The present study aimed to scrutinize the efficacy of different sources and levels of S in influencing growth and photosynthesis and antioxidant metabolism, and to investigate the potential of the S sources in the alleviation of Cd-induced stress in B. juncea. Dosedependent S requirement revealed that 200 mg kg −1 S (200S) was more effective for achieving maximum growth and photosynthesis than 100S. The S sources used showed a differential effect in mitigating Cd-induced toxicity in plants. In general, S 0 was the most effective among all the four S sources used, followed by AS, then Gyp and lastly MS.

S-Assimilation Plays a Central Role in Enhancing Defense against Cd Stress
Sulfate uptake followed by S-assimilation is presumed to be a critical determinant for plant's survival to a wide range of environmental cues since S-containing compounds confer protection to both biotic and abiotic stresses [9][10][12][13][14]. In the present study, the use of all four different S sources increased S-assimilation enzymes, ATP-S and OASTL, and S-containing amino acid content (Cys and Met), which were able to detoxify Cd (Table  4 and Figure 6). Our results agree with the studies of Liang et al. [15] and Lou et al. [36] in Brassica chinensis, Khan et al. [6] in wheat, Per et al. [40,41], Asgher et al. [42], Masood et al. [21] in mustard, and Flávio et al. [43] in maize, where S assimilation was significantly involved in lowering Cd-induced toxicity by increasing contents of enzymatic and nonenzymatic antioxidants and S-containing metabolites. It is also known that S-assimilation is upregulated in response to various biotic and abiotic factors [44]. Elemental S was also observed to be effective in enhancing the S-assimilation in some previous studies focused on salt-stressed mustard [45,46] and mung bean [47]. Elemental sulfur is a slow S releasing fertilizer with high S content and, when compared with other S forms, it increases the Electron microscopy was used to examine the stomatal behavior in response to Cd stress and S supply. Stomatal analysis depicted a noteworthy change in stomatal aperture in Cd-stressed plants. Stomata were normal in the leaf samples of control, S 0 and Cd + S 0 -treated plants. However, Cd-treated plants showed semi-closed stomata with distorted guard cells ( Figure 7E-H).

Discussion
The present study aimed to scrutinize the efficacy of different sources and levels of S in influencing growth and photosynthesis and antioxidant metabolism, and to investigate the potential of the S sources in the alleviation of Cd-induced stress in B. juncea. Dosedependent S requirement revealed that 200 mg kg −1 S (200S) was more effective for achieving maximum growth and photosynthesis than 100S. The S sources used showed a differential effect in mitigating Cd-induced toxicity in plants. In general, S 0 was the most effective among all the four S sources used, followed by AS, then Gyp and lastly MS.

S-Assimilation Plays a Central Role in Enhancing Defense against Cd Stress
Sulfate uptake followed by S-assimilation is presumed to be a critical determinant for plant's survival to a wide range of environmental cues since S-containing compounds confer protection to both biotic and abiotic stresses [9,10,[12][13][14]. In the present study, the use of all four different S sources increased S-assimilation enzymes, ATP-S and OASTL, and Scontaining amino acid content (Cys and Met), which were able to detoxify Cd (Table 4 and Figure 6). Our results agree with the studies of Liang et al. [15] and Lou et al. [36] in Brassica chinensis, Khan et al. [6] in wheat, Per et al. [40,41], Asgher et al. [42], Masood et al. [21] in mustard, and Flávio et al. [43] in maize, where S assimilation was significantly involved in lowering Cd-induced toxicity by increasing contents of enzymatic and non-enzymatic antioxidants and S-containing metabolites. It is also known that S-assimilation is upregulated in response to various biotic and abiotic factors [44]. Elemental S was also observed to be effective in enhancing the S-assimilation in some previous studies focused on salt-stressed mustard [45,46] and mung bean [47]. Elemental sulfur is a slow S releasing fertilizer with high S content and, when compared with other S forms, it increases the soluble SO 4 2− content in soil due to microbial oxidation [48]. Among essential elements, mobility of S is intermediate, as it is less mobile compared to N, P, and K, but mobile compared to Ca and most micronutrients. So, a continuous supply of S is required for emergence to crop maturity. Moreover, S 0 has a long time of residence in soil and can supply till later stages of growth, while other forms of S release SO 4 2− , which is mostly available at the earlier stages of growth and gets easily depleted [48]. S assimilation in plants is the principal biosynthetic pathway leading to amino acids, which are required for the synthesis of various metabolites, such as GSH, NPT, and PCs, which contribute in mechanisms of biochemical adaptation to metal stress [3]. These S-containing amino-acid pool also participate in enhancing the biosynthesis of antioxidants [17,18]. Thus, S 0 -assisted rises in antioxidants, along with S-metabolites, are the key players in conferring Cd tolerance to B. juncea plants. The enhanced S assimilation could be the reason for mitigating Cd toxicity, as it contributed in the production of GSH ( Figure 5G), Cys, and Met ( Figure 6C,D), and also increased the activity of ATP-S and OASTL ( Figure 6A,B). Moreover, ATP-S overexpression was also reported to improve the phytoextraction capability of B. juncea plants for Cd [49]. A study by Xiang and Oliver [50] in Arabidopsis showed that, in response to Cd stress, plants responded by overexpressing genes involved in S-assimilation and in the synthesis of GSH and PCs. Hence, our results showed that supplemental S nutrition upregulated S-assimilatory pathway and increased the status of antioxidant metabolites, such as GSH ( Figure 5G) and of PCs ( Figure 6F), which are oligomers of GSH and act as heavy metal chelators and/or detoxifiers, and play a prominent role in the mitigation of Cd-induced stress.

Cd Accumulation, Translocation, and Role of Sulfur
Cadmium concentration was highest in roots and lower in leaves of B. juncea, as roots are the prime organ of Cd accumulation (Table 3). This is in accordance with previous findings by Zhang et al. [51], Per et al. [41] and Yamaguchi et al. [52]. Among the four S sources used, S 0 caused the lowest Cd levels in roots and leaves, as confirmed by the low values of translocation factor (TF) ( Table 4). This decrease in Cd accumulation after the application of S 0 could be due to immobilization of Cd in soil, which reduces its availability to mustard plants [17]. The addition of S 0 to alkaline soils, compared to other S sources, reduces soil pH through microbial oxidation [53,54]. This increases the solubility of micronutrients, which becomes readily available to plants. On the other side, ammonium sulfate, gypsum, and magnesium sulfate have a little or no considerable effects on soil pH. Moreover, in alkaline soils, free SO 4 2− of ammonium sulfate, gypsum, and magnesium sulfate can form complexes with Cd, forming SO 4 2− -Cd complexes in Cd-polluted soils [55]. Therefore, SO 4 2− in the form of ammonium sulfate, gypsum, and magnesium sulfate become less available for plant uptake. Moreover, S 0 ensures enough availability of SO 4 2− by microbial action to be used by plants for longer times. On the contrary, Zhang et al. [19] found that gypsum was more effective in reducing Cd accumulation than S 0 in rice plants. However, there are many mechanisms by which a plant minimizes Cd toxicity, including immobilization (binding of Cd to cell wall), compartmentalization (vacuolar sequestration), prevention (decreasing Cd entry into roots), and chelation (Cd-PCs mediated) [12]. In the present study, S supply enhanced tolerance to B. juncea plants against Cd by accumulating more Cd in roots than shoots (Table 4), reducing Cd translocation by the involvement of heavy metal chelators, like NPTs and PCs ( Figure 7E,F) ( Table 3). These observations coincide with the works of Yamaguchi et al. [52,56] and Liang et al. [15], where the authors reported the S-mediated induction of limited translocation Cd in the tested plants. Besides, there are also several reports that have shown that S supply induces sulfate uptake transporters and the level of their transcripts, which resulted in increased S content in Cd-exposed plants [52,57,58]. Besides, it has been reported that S 0 -assisted microbial oxidation increases the availability of other minerals, such as Fe, N, Cu, Ca, and Mn in soil [59]. Therefore, S 0 supplement prevents the deficiency of mineral elements, particularly in Cd-stressed conditions. S mediated uptake of sulfate along with other nutrients could also be another major strategy for reducing Cd uptake from soil and lower Cd toxicity.

Sulfur Increases Plant Growth by Mitigating Cd-Induced Toxicity
In the present study, Cd-induced stress resulted in the reduction of the major growth parameters like plant fresh biomass, plant dry biomass, and leaf area (Figure 2). S supplementation modulated Cd-induced toxicity in mustard and improved growth biomarkers, with S 0 being the most effective among the four S sources used (Figure 2). The increase in plant biomass specifically with S 0 could be attributed to the reduction in Cd-induced phytotoxicity [60]. Notably, soil pH is the most important parameter controlling mobility of cationic heavy metals in soils [61]. Lowering the soil pH increases the solubilization of Cd into the soil. Microbial oxidation of S 0 introduced into the soil leads to the production of H 2 SO 4 . This process precipitates divalent cationic metals such as Cd [62,63]. This could also be one of the reasons for higher plant growth with S 0 supplementation. In contrast, Singh et al. [64] reported that rice plants supplied with bentonite S exhibited greater biomass and outperformed other S sources, namely S 0 , ordinary super phosphate and gypsum. However, in our study, the enhancement in growth with S under Cd stress is parallel to previous studies reported for B. campestris [17], Oryza sativa [6], Fagopyrum tararicum [12], and B. juncea [41]. Sulfur can amend the growth through inhibiting oxidative stress and restraining Cd transport from root to shoot [16]. Besides this, Asgher et al. [42] showed that plants exposed to Cd and exogenous S possessed more plant dry biomass, compared to Cd that only stressed plants. Treatment with ammonium phosphate and S fertilizers were used for reduction in Cd translocation, which advanced shoot growth in rice plants [9]. Moreover, S treatment enhanced total plant fresh weight, plant height, and root length in B. chinensis, thereby decreasing the inhibitory effects of Cd toxicity on plant growth [36]. These findings suggest that diminished plant growth is an indicator and a direct measure of Cd toxicity and that S has a positive role in influencing plant growth under Cd stress.

Sulfur Prevents Negative Effects of Cd on Photosynthesis
Cd may exert its inhibitory effects on photosynthesis directly by interacting with its enzymes [5], or indirectly by obstructing the synthesis of photosynthetic pigments or by causing their degradation [8]. In our study, Cd-treated plants showed a considerable reduction in chlorophyll content, leaf gas exchange parameters, maximal PSII efficiency, and Rubisco activity (Table 3 and Figure 3). The reversal of damage on photosynthetic characteristics and photo-inhibition caused by Cd was modulated by exogenous application with various S sources (Table 3 and Figure 3). The results show that all S sources increased various photosynthetic attributes, which were earlier reduced because of Cd-induced phytotoxicity. Even in this case, S 0 showed the highest beneficial effects. Elemental S has been used in many studies in improving photosynthesis under various types of abiotic stresses [19,45]. The reason for increased photosynthesis under Cd stress with elemental S involves S-mediated allocation of N to Rubisco protein and through increase in stomatal conductance [41]. Sulfur starvation aggravates chlorophyll content, maximal PSII efficiency, Rubisco activity, and causes alterations in photosynthetic electron carriers, loss of grana, while S deficiency under Cd stress resulted in severe disintegration of thylakoids, suggesting the importance of S nutrition in photosynthesis [65]. Nazar et al. [66] reviewed the involvement of excess S in improving quantum yield, leaf gas exchange parameters, chlorophyll content, and Rubisco activity due to the enhanced synthesis of GSH, which provides a shielding effect to photosynthetic apparatus against salt stress. This finding suggests that the application of S can enhance photosynthesis by reversal of damage on common attributes associated with photosynthetic efficiency. This beneficial effect of S was visible in the response of guard cells to S, which allowed stomatal opening also in the presence of Cd ( Figure 7H).

Sulfur Is Involved in the Reversal of Cd-Induced Oxidative Burst
Cd exerts its harmful effects by causing oxidative injury to plants triggered by ROS accumulation [6,41,42,67]. Our results show that Cd exposure in surplus quantity severly increased ROS formation (O 2 •− , H 2 O 2 ), electrolyte leakage ( Figure 5A) and lipid peroxidation ( Figure 5B) in leaves of B. juncea plants (Figure 4). A significant reduction in oxidative biomarkers was observed in plants treated with different S forms + Cd, suggesting that S supply protected plant cells from oxidative injury. Antioxidant enzymes are used by plants to scavenge excess ROS production under harsh environmental conditions, thus preventing cells from oxidative damage. The current study showed that Cd supply caused a small increase in antioxidant enzyme activity (CAT, SOD, APX), however, the application of S boosted this upsurge in antioxidant defense, which is particularly clear in plants treated with S 0 + Cd ( Figure 5C-E). Earlier reports have also shown that S treatment triggered the stimulation of antioxidants (SOD, CAT, APX), which played an active role in defending various environment cues like Cd toxicity [6,18,40,41], salt stress [45,46], and Cr (VI) excess [68].
The AsA-GSH cycle, also known as Foyer-Halliwell-Asada cycle, is a major pathway in maintaining a proper balance between generation and quenching of excessive ROS and regulating cellular redox status inside the cell in plants facing various environmental challenges [17,68]. In our study, GR and DHAR activities increased in response to Cd stress in plants supplied with various S sources ( Figure 5F,H), and this was consistent with the increase GSH and AsA content ( Figure 5G,I). The maximal beneficial effect to plants was obtained with the application of S 0 , where it completely outperformed the other three sources (ammonium sulfate, gypsum and magnesium sulfate), both in the presence or absence of Cd. A plethora of literature scrutinizes the efficiency of AsA-GSH cycle in alleviating Cd-induced oxidative stress in response to S supply [12,15,17]. GSH acts as the first line of defense in response to Cd-induced toxicity before the synthesis and arrival of PCs [69]. The reversal of Cd-induced oxidative burst is clear by the visibly increased cell viability test of roots cells in plants subjected to S treatment and Cd exposure, compared to the roots of only Cd-stressed plants ( Figure 7D).
Our study also revealed that Cd exposure reduced GSH levels in only Cd-treated plants, compared to control plants ( Figure 5G), and this agrees with the study of Liang et al. [15]. This depletion in the GSH pool can be correlated to demand in the synthesis of PCs, which is an integral component of NPTs and can be reversed by application with S [36], which is in harmony with our results (Figure 6E,F). Therefore, we can affirm that S application relieved Cd-induced oxidative stress by upregulating plant's antioxidant machinery, by improving the AsA-GSH pathway and by inducing the biosynthesis of heavy metal chelators, like NPT and PCs, which detoxified Cd and hence lowered oxidative stress ( Figure 8).

Conclusions
In this study, 200S treatment was more effective than 100S in counteracting Cd stress in B. juncea. The S supply reduced Cd uptake and Cd accumulation in root and leaves, with plants showing lower oxidative stress symptoms. Sulfur given to plants significantly increased enzymatic and non-enzymatic antioxidant defense, and the levels of PCs and NPTs, which are both useful for the detoxification and sequestration of Cd, respectively. The results also showed the role of the different S sources (elemental S, ammonium sulfate, gypsum, and magnesium sulfate) and their response towards Cd, with S 0 showing

Conclusions
In this study, 200S treatment was more effective than 100S in counteracting Cd stress in B. juncea. The S supply reduced Cd uptake and Cd accumulation in root and leaves, with plants showing lower oxidative stress symptoms. Sulfur given to plants significantly increased enzymatic and non-enzymatic antioxidant defense, and the levels of PCs and NPTs, which are both useful for the detoxification and sequestration of Cd, respectively. The results also showed the role of the different S sources (elemental S, ammonium sulfate, gypsum, and magnesium sulfate) and their response towards Cd, with S 0 showing the more significant effects among all S sources in detoxifying Cd-induced phytotoxicity. Elemental S as a source of S was used in many studies to scrutinize its efficiency in Cd-exposed plants [41,55,60] and it is recommended over other organic S-based fertilizers since it (i) has a longer time of residence in soil, (ii) can activate soil microbiome, (iii) can enhance the productivity and the nutritional quality of crops, and (iv) can transform into reactive sulfur species and volatile sulfur species that can, in turn, enhance plant tolerance to environmental stresses [48].
The problem of Cd contamination of agricultural soil is increasing continuously because of its addition through phosphatic fertilizers, contaminated irrigation or other sources. It is so urgent and essential to find strategies and mechanisms that can lower the effects of Cd toxicity in crops. The application of sulfur could be promoted in soils contaminated with subtoxic or toxic Cd levels to improve the growth and development of cultivated plants. The mechanisms on which this higher defense and tolerance of plants against Cd is based may be explored further through biotechnological and genetic tools.