1 Introduction
Sulphur (S) is an essential macronutrient for plant growth and development and plants generally take it up in the form of sulfate through dedicated sulfate transporters (Takahashi et al., 2011; Talukdar and Talukdar, 2014a). Sulfate is first activated by ATP sulfurylase (ATPS) to adenosine 5´-phosphosulfate (APS) which is the branching point in sulfate assimilation (Takahashi et al., 2011). APS is either reduced to sulfite by APS reductase (APR) or is phosphorylated to 3´-phosphoadenosine 5´-phosphosulfate (PAPS) by APS kinase. In the reductive assimilation pathway, sulfite is then reduced to sulfide by sulfite reductase, followed by incorporation into the amino acid skeleton of O-acetylserine (OAS) to make cysteine (Cys), which is the donor of reduced sulfur for all further metabolites like glutathione (Martin et al., 2005). PAPS is involved in the production of Met-derived (aliphatic) or tryptophan-derived (indolic) secondary metabolites such as glucosinolates involved in plants’ tolerance to biotic stresses (Takahashi et al., 2011). Therefore, the fates of APS are increasingly becoming an intense focus of current research regarding crop genomics and applied breeding. 

Generation  of reactive oxygen species (ROS) is a usual process in aerobic respiration (Noctor et al., 2011; Talukdar, 2014a,b) and one of the earliest cellular defense responses following successful pathogen recognition (Talukdar, 2013c). ROS-homeostasis i.e., balances between its generation and scavenging, may be unbalanced in favor of oxidative state by biotic and abiotic factors (Noctor et al., 2011; Talukdar, 2013a,b 2015a,b). Ascorbate (AsA)-glutathione (GSH) cycle plays key roles  in cellular detoxification of ROS, in which ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR) and glutathione reductase (GR) enzymes scavenge hydrogen peroxide (H₂O₂), and regenerate AsA and GSH, respectively (Noctor et al., 2011, 2012). Both AsA and GSH are non-enzymatic antioxidant defense components in plants and interact with diverse types of cellular and metabolic processes (Noctor et al., 2011). Besides, AsA-GSH cycle components, phenylalanine ammonia-lyase (PAL) catalyzes the first step in the phenylpropanoid pathway, by deamination of L-phenylalanine to trans-cinnamate and ammonia (Dixon et al., 2002; Kim and Hwang, 2014). In addition to its important roles in plant development, PAL is also a key enzyme in plant stress responses. Its expression and activity is stimulated by diverse types of biotic and abiotic stress factors such as pathogen attack, tissue wounding, extreme temperatures, UV irradiation, nutrient deficiency, plant signaling molecules, including jasmonic acid, salicylic acid and abscisic acid, salinity and heavy metals (De Gara et al., 2003; Li et al., 2011; Talukdar, 2013c).

 
Induced mutation techniques provide a powerful tool to study the genomic and biochemical mechanisms of plant metabolism including antioxidant defense. This technique has been previously used in lentil and other related grain legumes to develop cytogenetic and breeding tools (Talukdar, 2013d). The importance of being a stable mutant line is enhanced manifold when it can be utilized to dissect intrinsic physiological and biochemical events related to crop productivity (Tsyganov et al., 2007; Talukdar, 2012a; 2012b, 2014c). Lentil is a cool-season grain legume grown widely in the Indian subcontinent, West Asia, North Africa and parts of Europe, Oceania and North America (Erskine et al., 2011) and has tremendous health benefits with a rich source of dietary and antioxidant compounds (Erskine et al., 2011; Talukdar, 2012b). However, the crop in South and South-East Asia contributing nearly 45% of total world lentil production is increasingly under threat from drought, soil salinity, arsenic, and heavy metal toxicity along with different biotic stresses (Erskine et al., 2011; Talukdar and Talukdar, 2014a). S metabolism is the key to agricultural productivity and anti stress factors of grain legumes (Deshbhratar et al., 2010; Tabe et al., 2010; Arya and Roy, 2011; Khan and Mazid, 2011; Liao et al., 2012; Perveen et al., 2015) but its assimilation process in up-stream thiol metabolisms and subsequent incorporation into downstream antioxidant defense metabolites like glutathione, phytochelatins (PCs) and other enzymatic defense components are only partially known (Talukdar, 2014c; Talukdar and Talukdar, 2013a,b, 2014a). Considering the importance of APS in the branching point of S assimilation and subsequent response to growth conditions and stress tolerance, mutation was induced in lentil genotypes and subsequently, two novel mutant types deficient in either APR or APK activity were isolated in M₂ progeny. The aim of the present study was set to 1) assess the foliar APR and APK activity, 2) characterize the morho-agronomic traits, and 3) dissect the antioxidant defense activity in the background of deficient APR or APK activity.

 
2 Results
2.1 Plant growth and grain yield of mutants
In comparison to control genotype L 414, shoot length, root length, and dry weight of both organs increased significantly (p<0.05) in apkLc1 but decreased substantially in the mutant line aprLc1 (Table 1). While shoot biomass accumulation was 2.5-fold higher in apkLc1, root dry mass increased over that of control genotype by about 3-fold in the mutant. Per plant seed yield was increased by about 2-fold in apkLc1 but was reduced by about 3-fold in aprLc1 mutant (Table 1).

 
2.2 APK and APR activity in the mutants
Foliar and root APK activity in apkLc1 mutant line were only 5% and 6%, respectively, of those measured in control genotype L 414 (Figure 1). APR activity in the mutant increased significantly in both organs over that of L 414; while there was 3-fold increase in leaf APR level, root activity was 2-fold higher than control genotype (Figure 1). In aprLc1 mutant, APR activity was only 1% in leaves and 5% in roots compared to control (Figure 1). The APK activity in the mutant, however, exhibited significant increase over control genotype; it was 2-fold higher in leaves and registered 1.8-fold enhancement in roots (Figure 1).

 
2.3 Antioxidant defense response of the mutants
Total and reduced GSH content increased over L 414 by about 2.5-fold in leaves and 2-fold in roots of apkLc1 mutant. Total and reduced AsA as well as GSSG content, however, did not change significantly in the mutant (Table 1). Both AsA and GSH level in leaves and roots of aprLc1 mutant decreased significantly by about 3-fold in both organs. The level of dehydroascorbate (DHA) and GSSG, however, increased by about 1.8-2.5-fold in the mutant compared to L 414 (Table 1). Measurable Cys content was increased nearly 2.0-fold in leaves and 1.5-fold in roots of apkLc1 mutant but decreased by nearly similar magnitude in leaves and roots of the aprLc1 mutant (Table 1).

Among enzymatic antioxidant defense components, SOD activity was lower in both organs of the apkLc1 but markedly higher in the aprLc1 mutant in relation to L 414 (Figure 2). APX, DHAR and GR activities exhibited significant hike in apkLc1 mutant but decreased markedly in aprLc1 mutant. APX activity increased by about 2.7-fold in leaves and 3-fold in roots whereas DHAR level was 2-fold higher than L 414 in both organs of apkLc1 mutant. GR activity in leaves increased by about 1.9-fold in apkLc1 but was comparable to control in the roots of the mutant (Figure 2). Enzyme activity was declined by about 2-5-fold in aprLc1 mutant, showing highest reduction in the leaves of the mutant (Figure 2).

 
2.4 PAL activity in the mutant
PAL activity was substantially low in apkLc1 mutant but nearly 2.6-3-fold higher than L 414 in the aprLc1 mutant (Figure 2). PAL activity increased by about 2.6-fold in roots and 3-fold in leaves of aprLc1 mutant (Figure 2).

2.5 H₂O₂ content, lipid peroxidation and electrolyte leakage (EL) %
Foliar and root H₂O₂ content, lipid peroxidation and EL% were significantly higher in aprLc1 mutant (Table 1). The H₂O₂ and malondealdehyde (MDA) level increased by about 2-fold in leaves and by about 1.8-fold in roots of aprLc1 mutant. Similar trend was noticed for EL% in the mutant organs. In apkLc1, foliar and root H₂O₂ level did not change significantly but MDA and EL% decreased significantly in both organs of the mutant compared to L 414 (Table 1).

3 Discussion
Two unique lentil mutants impaired in two important branching points of S assimilation were isolated and biochemically characterized. While apkLc1 exhibited impaired activity of APK, aprLc1 showed depleted level of APR. Both the enzymes significantly use APS as their substrates. However, they sharply differed in their characteristics. The better growth rate in apkLc1 mutant was manifested by higher shoot and root length, and their dry weights while completely opposite scenario was encountered for aprLc1 mutant. Obviously, increase in seed yield in apkLc1 mutant but poor yield in aprLc1 confirmed the above said differences in growth performance of both the mutants.

Significant improvement in growth performances in apkLc1 in comparison to L 414 genotype might be related to enhanced level of Cys, reduced GSH and comparable AsA level in both organs of apkLc1 mutant. Remarkably enough, elevated APR activity in apkLc1 mutant might be orchestrated through metabolic diversion of sulfate pool to be utilized by the enzyme because that pool presumably is minimal used by APK. Stimulated APR level plays significant role in sulphate assimilation towards Cys which is the building block of GSH (Arya et al., 2011; Noctor et al., 2012). Enhanced Cys generation in apkLc1 mutant might have ensured its steady supply to generate enough GSH pool in both organs of the mutant. Reduced APK activity in apk1 apk2 mutants reportedly resulted in an increased flux/assimilation primarily through Cys biosynthesis pathway and then to accumulation of other reduced S compounds, particularly GSH (Mugford et al., 2009, 2011). In additional consequences, the apk1 apk2 mutants possess low levels of sulfated secondary compounds glucosinolates and are also affected in growth (Mugford et al., 2011). The present apkLc1 mutant in contrast exhibited better growth but surprisingly showed low PAL activity which is linked with phenylpropanoid pathway and often related to many secondary metabolites utilized by plants against pathogenic invasions, insecticides and other biotic stress factors (Dixon et al., 2002). Preliminary results pointed out sensitivity of apkLc1 mutant to Fusarium wilt infection (Talukdar, unpublished). Contrastingly, the aprLc1 mutant showing high PAL activity in both organs exhibited poor growth performances but showed tolerance to wilting (Talukdar, unpublished). Obviously, primary reduced S compounds are more important for plant growth and seed yield rather than secondary metabolites and APR rather than APK in the branch point of S assimilation pathway plays pivotal roles in the process. However, it is noteworthy that the present mutants have not been challenged by any external stress factors, and the duets of APR and APK in the S assimilation branch point would then be more obvious in lentils. In fact, attenuated or modulated expressions of different APR isoforms under varying degrees of S availability and mutants revealed stiff competition between APR and APK at the branch point for utilization of sulfate and its subsequent down-stream utilization into different primary or secondary metabolites (Grant et al., 2011). Transcriptional up-regulation of APR in myb51 mutant corresponded with increased enzyme activity and accumulation of GSH (Yatusevich et al., 2010). MYB is an important transcriptional regulator of APR activity (Sonderby et al., 2007; Gigolashvili et al., 2008; Malitsky et al., 2008). Presumably, increased APR activity in the present apkLc1 mutant might be an adaptation to low accumulation of secondary compounds in the mutant through increase GSH content as an alternative defense compound. Conversely, high APK activity in the aprLc1 mutant might have complemented loss of primary S compounds to some extent through production of enough pool of secondary metabolites. Interestingly, both APR and APK is under cellular redox regulation but they sharply differed in regulation; while APR is feedback inhibited by Cys and GSH (relieved in the presence of BSO, a specific inhibitor of GSH biosynthesis), APK substrate inhibition is alleviated by presence of reductants (Ravilious et al., 2012). As both the enzymes uses APS as their common substrates at the branch point of S assimilation, their completely opposite manifestations in two present lentil mutants confirmed partitioning of S between primary and secondary metabolisms of plants, and the switch towards a particular pathway is clearly evidenced when the other circuit is crippled. Further study, however, is needed to dissect APR/APK roles in legumes crops like lentil exposed to external stress conditions.

Coupled with the increase in non-enzymatic antioxidant components, enzymatic constituents in AsA-GSH cycle also showed enhanced level in the mutant over those in L 414. Higher APX activity suggested elevated levels of ROS-scavenging capacity of the mutant which is fuelled by increased activity of DHAR and concomitant production of AsA, the exclusive co-factor of APX activity. Furthermore, enhanced foliar GR level and comparable activity in roots readily recycled GSSG to GSH in both organs of the mutant. Interestingly, reduced SOD activity in both leaves and roots of apkLc1 suggested low superoxide radicals generation. SOD is an important generator of H₂O₂ during dismutation of superoxide radicals. H₂O₂ is a diffusible ROS within cell and attacks enzymes and other proteins rich in sulphydryl groups (Quan et al., 2008; Upadhyay, 2014). Low SOD level definitely might be responsible for reduced H₂O₂ level in both organs of apkLc1 mutant. H₂O₂ level is often related to membrane lipid peroxidation and oxidative damage (Talukdar, 2013a,b). Significant decrease in MDA, the lipid peroxidation product, and low electrolyte leakage, signifying reduced oxidative damage of membrane might be attributed to low H₂O₂ accumulation in both organs of apkLc1 mutant. 
 

In a sharply contrasting scenario, reduced growth performance and low seed yield in the aprLc1 mutant was accompanied by declined level of GSH and AsA level and concomitant increase in their oxidized states. Coupled with this, decreasing levels of APX, DHAR and GR activities resulted in severe impediment in ROS-scavenging capacity, and low regeneration of AsA and GSH from their oxidized states. Along with this, higher SOD activity in both organs in the mutant generated huge H₂O₂ which over-accumulated, and might be responsible for elevated level of oxidative damage in the form of membrane lipid peroxidation and electrolyte leakage of the leaves and roots of the mutant.

Present result clearly pointed out differential behavior of two lentil mutants and effects of mutations in vital branching point of plant S assimilation pathway. The results indicated partitioning of S between APR and APK which has cascading effects on down-stream primary and secondary metabolic pathways of lentil mutants. Mutations in APR crippled growth but conferred high PAL activity whereas mutations in APK improved growth performances through partitioning of higher S and concomitant reduction to Cys and GSH. Further study of APR/APK duet in S assimilation pathway would be carried out once their double mutants are available. 

4 Materials and Methods
4.1 Induction and identification of mutants
Fresh seeds of lentil (Lens culinaris Medik. cv. L 414) were presoaked in water for five hours and treated with freshly prepared 0.10%, 0.15% and 0.5% aqueous solution of ethyl methane sulfonate (EMS) for six hours with intermittent shaking at 25 ± 2°C keeping a control (distilled water), as described earlier (Talukdar, 2014). After thorough washing with running tap water, seeds were sown in the field to raise M₁ progeny and self-pollinated in next season to grow M₂ progeny following earlier protocol (Talukdar, 2014; Taukdar and Talukdar, 2013). Out of about 1000 M₂ individuals screened during winter of 2013 and 2014, four variant plants were identified and isolated in 0.15% EMS-treated M₂ progeny. Among the four plants, three plants exhibited vigorous growth with high yield but low Fusarium wilt tolerance while the rest one showed poor growth and low yield with high wilt tolerance (Table 1). The four plants were self-pollinated, and advanced to M₃ generation to confirm the trait inheritance. Progeny plants were harvested, and morpho-agronomic traits were recorded. Preliminary biochemical estimation suggested that the three plants with high growth potential were deficient in APK activity while the rest one with weak growth showed deficient APR activity. Based on the obtained result at M₃, the former one was designated as apkLc1 while the later one was designated as aprLc1. Plant parts were oven-dried at 60°C for two days to measure dry weight. Further biochemical analysis was done in leaves. Variety L 414 was used as control throughout the experiment.

4.2 Assay of APR and APK enzymes
Enzymatic activity of APR was measured using a radiometric assay that monitors formation of acid volatile [35S]-sulfite (Berendt et al., 1995). Tissue extracts were prepared in 100 mM Tris (pH 8.5), 2 mM DTT, and 1 mM EDTA. Homogenates were centrifuged at 4°C and the supernatant collected. Assay was performed following Phartiyal et al., (2008) at 30°C and was initiated by adding 0.5 µg of protein. Reactions were quenched after 20 mins with the addition of 5 µL 0.84 M Na₂SO₃, followed by the addition of 45 µL of H₂SO₄. Reaction rates were linear from 5 to 30 mins. Uncapped reaction tubes were placed into 6 mL scintillation vials capped and incubated overnight containing 1 mL trioctylamine. Reaction tubes were removed from the vials, and 3 mL Ecolume scintillation fluid was added, and the radioactivity was counted by scintillation counting. All reaction rates were corrected for nonenzymatic rates (Phartiyal et al., 2008). APK assay was carried out following Burnell and Whatley (1975) spectrophotometrically. Briefly, the reaction mixture contained 10 µmol of MgCl₂, 0.4 µmol of PEP, 2.5 µmol of ATP, 10 µmol of NAF, 200 µmol of Tris-HCl (pH 8.0), 5 µmol of KCN, 10 U lactate dehydrogenase, 10 U pyruvae kinase, and 0.1 mL of crude extract in a total reaction mixture of 0.95 mL. Both endogenous rates of NADH oxidation and in the presence of APS were monitored for 1 to 2 mins. The rate of endogenous NADH oxidation was subtracted from that observed in presence of APS to get the APS-dependent rate of NADH oxidation.

4.3 Estimation of Cys, glutathione and ascorbate content
Cys content was determined following Gaitonde (1967). Reduced (GSH) and oxidized glutathione (GSSG) and ascorbate (AsA) content was measured following Griffith (1980) and Law et al. (1983), respectively.

 
4.4 Assay of antioxidant defense enzymes
Leaf tissue (250 mg) was homogenized in 1 mL of 50 mM K-phosphate buffer (pH 7.8) containing 1 mM EDTA, 1 mM DTT, and 2 % (w/v) polyvinyl pyrrolidone using a chilled mortar and pestle kept in an ice bath. The homogenate was centrifuged at 15,000 g at 4 °C for 20 mins. Clear supernatant was used for enzyme assays. For measuring APX activity, the tissue was separately ground in homogenizing medium containing 2.0 mM AsA in addition to the other ingredients. All assays were done at 25 °C. Soluble protein content was determined using BSA as a standard (Bradford, 1976). Superoxide dismutase (SOD, EC 1.15.1.1) activity was determined by nitro blue tetrazolium (NBT) photochemical assay (Beyer and Fridovich, 1987) and was expressed as unit min^-1mg^-1 protein. One unit of SOD was equal to that amount causing a 50 % decrease of SOD-inhibited NBT reduction. Ascorbate peroxidase (APX, EC 1.11.1.11) activity (nmol AsA oxidized min^-1 milligram^-1 protein) was assayed following Nakano and Asada (1981). Three milliliters of the reaction mixture contained 50 mM K-phosphate buffer (pH 7.0), 0.1 mM EDTA, 0.5 mM AsA, 0.1 mM H₂O₂ and 0.1 mL of enzyme extract. The H₂O₂-dependent oxidation of AsA was followed by a decrease in the absorbance at 290 nm (ε = 2.8 mM⁻¹ cm⁻¹). APX activity was expressed as nmol AsA oxidized min^-1 mg^-1 protein. DHAR (EC 1.8.5.1) activity was measured following Nakano and Asada (1981). The complete reaction mixture contained 50 mM potassium phosphate buffer (pH 7.0), 2.5 mM GSH, 0.2 mM DHA and 0.1 mM EDTA in a final volume of 1 mL. Reaction was started by adding suitable aliquots of enzyme extract and the increase in absorbance was recorded at 30 s intervals for 3 mins at 265 nm. Enzyme activity was expressed as nmol AsA formed min^-1 mg^-1 protein. Glutathione reductase (GR, EC 1.6.4.2) activity was determined by monitoring the glutathione dependant oxidation of NADPH, as described by Carlberg and Mannervik (1985). In a cuvette, 0.75 mL 0.2 M potassium phosphate buffer (pH 7.0) containing 2 mM EDTA, 75 μL NADPH (2 mM), and 75 μL oxidized glutathione (20 mM) were mixed. Reaction was started by adding 0.1 mL enzyme extract and the decrease in absorbance at 340 nm was monitored for 2 mins. GR specific activity was calculated as nmol NADPH oxidized min^-1 mg^-1 protein. Phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) activity was determined following the direct spectrophotometric method (Cavalcanti et al., 2007). Two hundred microlitres of the crude enzyme extract previously dialyzed overnight with 100 mM Tris-HCl buffer, pH 8.8, were mixed to a solution containing 200 μL 40 mM phenylalanine, 20 μL 50 mM β-mercaptoethanol and 480 μL100 mM Tris-HCl buffer, pH 8.8. After incubation at 30 °C for 1 h, the reaction stopped by adding 100 μL 6 N HCl. Absorbance at 290 nm was measured and the amount of trans-cinnamic acid formed was evaluated by comparison with a standard curve (0.1-2 mg trans-cinnamic acid/ml) and expressed as μg of trans-cinnamic acid h^-1 mg^-1 protein.

4.5 Estimation of H₂O₂ content, lipid peroxidation and electrolyte leakage (EL) %
The H₂O₂ content and membrane lipid peroxidation rate were determined following Wang et al. (2007) and by measuring the malondialdehyde (MDA) equivalents (Hodges et al., 1999), respectively. Electrolyte leakage (EL %) was measured according to Dionisio-Sese and Tobita (1998). 

4.6 Statistical analysis
Data are means ± standard error (SE) of at least three replicates. Multiple comparison of means were performed by variance analysis on all experimental data, and statistical significance (p<0.05) of means was determined by Duncan’s multiple range tests using SPSS software (SPS Inc., USA v. 10.0).

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