Antioxidant enzyme responses and metabolite functioning of Pisum sativum L. to sewage sludge in arid and semi-arid environments

The productivity of plants is a direct variant of the countless biotic and abiotic stresses to which a plant is exposed in an environment. This study aimed to investigate the capabilities of leguminous plant garden pea (Pisum sativum L.) to resist water deficit conditions in arid and semi-arid areas when applied with varied doses of sludge for growth response. The effect of sludge doses was evaluated on crop yield, antioxidant enzymes, viz., ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR), superoxide dismutase (SOD), and glutathione reductase (GR), and metabolites (ascorbic acid, glutathione, and total protein content). The effective sludge concentrations with respect to seed weight and crop yield were found to be in the following trend: D2 (6.25%)>D3 (12.5%)>D1 (2.5%)>D0 (control) under organic amendment (OA). Conversely, a high dose of the sludge reduced the seed weight and total crop yield. The sludge doses D2 under arid and semi-arid conditions along with organic amendments (OA) significantly enhance the antioxidant enzyme activity, whereas sludge dose D3 with OA ominously regulates the activity of these enzymes. Besides, seeds depicted a considerable increase in ascorbic acid, glutathione, and total protein content in arid and semi-arid conditions upon the application of sludge with OA. Sewage sludge as a source of nutrients indirectly enhances crop yield, antioxidant enzymes, and antioxidant metabolites. Thus, it improves the defense mechanism, reduces abnormal protein glycation, and depletes the susceptibility of protein to proteolysis.


Introduction
Most of the plants are prone to adapt to biotic and abiotic stresses, causing prominent variation in their productivity. These plants modify their internal and external factors to cope up with environmental stresses (Rouached et al. 2015). Their adaptive strategies determine their stress tolerance capability (Costa et al. 2011), and consequently survival in these conditions (Kramer and Boyer 1995). Plants growing in stressful environments have the potential to adapt by way of changing Responsible Editor: Gangrong Shi * Khalid Rehman Hakeem kur.hakeem@gmail.com; khakim@kau.edu.sa 1 their morphological, molecular, and biochemical characteristics (Jeuffroy et al. 2012;Khaleghi et al. 2019). The antioxidative protection components in plants destruct reactive oxygen species (Pau and Lawson 2002). The stability stuck between manufacture and reduction of reactive oxygen species could be troubled by numerous factors which rapidly enhance the production of reactive oxygen species within the cells (Pau and Lawson 2002;Delfini et al. 2010) and may be responsible for the destruction of metabolites. Plants enhance the production of antioxidants to neutralize the oxidative stress effects within the cells (Lawson and Smith 2002;Meyer and Hell 2005;Colville and Kranner 2010;Delfini et al. 2010). Among the crop plants, leguminous plants are very sensitive to environmental stresses (Voisin and Gastal 2015), prominently water shortages. The alteration in molecular and in biochemical characteristics in legumes could be vital for growing these kinds of plants in stressful environments as they activate the mechanism of stress tolerance by way of enhancing the production of antioxidants (Guilioni et al. 2003;Lejeune-Hénaut et al. 2008). Therefore, the changes both intrinsic and extrinsic adapted by these plants under stressful environments are pivotal for their survival and productivity.
Considering the problems associated with a shortage of water in many regions of the world, the present research proposal shall be based on the assumption that leguminous plants having a high potential to resist water deficit environments can be grown in arid and semi-arid conditions with varied doses of sludge for growth response. It is also assumed that at different stress doses, plants shall respond efficiently at a certain concentration with respect to the physiological characteristics, antioxidant molecules, and metabolites.
Antioxidants significantly decrease the concentration of reactive oxygen species in plants and protect them from the damages caused due to oxidative stress (Matamoros et al. 2010). Among the crop plants, leguminous plants are a good source of proteins and other important metabolites and have been placed on top of the economical scale (Graham and Vance 2003). Numerous research studies confirmed that the antioxidants play a crucial role to protect the internal tissues of crop plants and have enough capabilities to diminish the likely impacts of reactive oxygen species, thereby take part intolerance from any kind of environmental stresses (Mittler et al. 2004;Van Breusegem et al. 2008). Due to oxidative stress, fruit-bearing plants reduce the crop yield due to low shelf life which is associated with low production of antioxidants (Davey and Keulemans 2004;Malacrida et al. 2006;Halliwell and Gutteridge 2007;Stevens et al. 2008). Antioxidants and antioxidant enzymes particularly (superoxide dismutases, catalases, peroxiredoxins, and glutathione peroxidases) amend the concentrations of reactive oxygen species and neutralize their toxic effects in plant cells (Dietz 2003;Matamoros et al. 2003;Mittler et al. 2004;Navrot et al. 2006). The water-soluble antioxidants especially glutathione and ascorbate create redox buffers in cells of plants and provide stress responses (Bouvier et al. 1998;Arrigoni and De Tullio 2002;Noctor et al. 2002) and growth (Matamoros et al. 2003;Palma et al. 2006;Hicks et al. 2007). Various research studies have been done globally on different crop plants, viz., Pyrus communis, Lycopersicon esculentum, and Amelanchier alnifolia, and reported that the antioxidant and antioxidant enzymes are very crucial for fruit development, maturation, and ripening under any stress (Jiménez et al. 2002a(Jiménez et al. , 2002bReddy et al. 2004;Gill and Tuteja 2010). Furthermore, fruit tissues are protected by antioxidants from reactive oxygen species and thereby resist any kind of environmental disturbance (Mittler et al. 2004;Van Breusegem et al. 2008;Davey and Keulemans 2004;Malacrida et al. 2006;Stevens et al. 2008). The use of seed inoculation and RDF with plant growth-promoting rhizobacteria (PGPR) increases the yield of leguminous crops (Mishra et al. 2010).
Water accessibility is the most vital factor for plant growth and crop productivity as well as an important factor for determining the species distribution in varied climatic zones around the world. Considering the problems associated with a shortage of water in many regions of the world, the present research proposal shall be based on the assumption that leguminous plants having a high potential to resist water deficit environments can be grown in arid and semi-arid conditions with varied doses of sludge for growth response. It is also assumed that at different stress doses, plants shall respond efficiently at certain concentrations with respect to the physiological characteristics, antioxidant molecules, and metabolites.

Experimental design
The experiment was laid out in a randomized complete block design (RCBD) with three replications for each environmental condition (arid and semi-arid) to evaluate different sewage sludge treatments. Besides, an experimental unit with respect to environmental conditions was further divided into two blocks, viz., organic amendment (OA) and without OA. Each experimental unit consisted of 9 plants, planted in three columns and two rows with a spacing of 50 cm × 25 cm (size of experimental unit 1.50 × 1.25 m 2 ). Four-week-old pea seedlings were transplanted on the fourth week of April with proper care. Generally, soils in Srinagar (J&K) belong to the Inceptisol soil category (FAO 2015).

Collection of seed samples
The seed samples were collected from randomly selected plants from each treatment of every replication for analysis.

Preparation of samples
After collecting fresh seed samples, they were washed thoroughly with tap water and then dipped in dilute HCl and further washed with single-and double-distilled water. The moisture was whipped with filter paper and muslin cloth. Treatment wise samples from each replication were then analyzed for quality parameters, viz., antioxidant enzymes, and essential metabolites.

Antioxidant enzymes
Total superoxide dismutase (SOD) concentration was analyzed on the spectrophotometric method adopted by Rubio et al. (2002). About 40 μl of enzyme extract was transferred in test tubes to which 50 mM phosphate buffer (pH 7.8), 55 μM NBT, 9.9 mM L-methionine, 2 mM EDTA, and 0.02% Triton X-100 were added. To this reaction, the mixture riboflavin was added lastly in complete dark condition. The activity of SOD depends upon its ability to decrease the photochemical reduction of nitroblue tetrazolium. SOD activity was calculated by reading the OD at 560 nm for 2 min at 25°C.

Ascorbate peroxidase (APX)
The plant sample tissue was extracted in 20 mM potassium phosphate (pH 7.4). The mixture was homogenized with Polytron, incubated on ice for 20 min, and vortexed for every 2-min interval. Next, the mixture was centrifuged at 15,000×g at 4°C for 15 min. The resultant supernatant was collected and dialyzed before the enzyme assay. The final reaction mixture consists of 1.0 ml that comprised 20 mM of potassium phosphate buffer (pH 7.0) and 2.5 mM ascorbic acid. The 10 μl of enzyme extract is added to initiate the reaction. Due to ascorbate oxidation, the decrease in absorbance is monitored for 3 min at an absorbance rate of 265 nm and calculated by using extinction coefficient, 14 mM −1 cm −1 .

Glutathione reductase
The procedure for determining the concentration of glutathione reductase in plant tissues was analyzed by the oxidation of NADH and NADPH (Schaedle and Bassham 1977). The enzyme extract was prepared before the enzyme assay; 200 mg of fresh samples was homogenized by mortar and pestle in 5 ml of 50 mM Tris-HCl buffer at pH 7.6. The resultant supernatant was collected after being centrifuged at 22,000×g for 4 min and dialyzed prior to enzyme assay.
The final reaction mixture (1 ml) was composed of 200 μl enzyme extract, 50 mM Tris-HCl buffer (pH 7.6), 1 mM glutathione disulfide (GSSG), 0.15 mM NADPH, and 3 mM MgCl 2 . A decrease in NADPH absorbance was observed at 340 nm. The specific activity of the enzyme is expressed as a unit per milligram of protein.

Antioxidant metabolites
Ascorbic acid was estimated from fresh green seeds using 2, 6-dichlorophenolindophenol dye and expressed as per the  AOAC (1995) and Nielsen (1998). Glutathione was measured as per the method (Matamoros et al. 1999) using HPLC fluorescence detection. The reduced and oxidized concentrations of glutathione were measured spectrophotometrically by utilizing enzymatic cycling (Griffith 1980). Proteins were determined by the extraction method (Loscos et al. 2008).

Statistical analysis
Data for calculating the sludge and organic amendment effects were evaluated with one-way ANOVA, followed by a significance test using Tukey's post hoc test. The level of significance was set at p ≤ 0.05 for all the evaluating plant attributes. All other tables, graphics, and calculations were created using Microsoft Excel 2010. Statistical analysis was performed using GraphPad Prism Version 8.01 (San Diego, USA).

Seed weight
The recorded observations on seed weight exhibited different values with respect to sludge concentration. Moreover, environmental conditions (arid and semi-arid) and organic amendments significantly influence the seed yield. Plants responded quite effectively and exhibited the highest seed weight (1.13 g FW) at sludge concentration D 2 (6.25%) under organic amendment (OA). However, some reverse effects were also observed at very high sludge concentration D 3 (12.5%) in both environmental conditions and depicted the values with a range of 0.70-0.90 g FW in arid conditions and 1.00-1.12 g FW in the semi-arid environment (Fig. 2). Overall, the effective sludge concentrations with respect to seed weight were found to be in the following trend: D 2 (6.25%)>D 3 (12.5%)>D 1 (2.5%)>D 0 (control).

Crop yield
The results indicated that crop yield per plant showed different values with the supplementation of different sludge doses. Moreover, the data signposted that plants amended with sludge dose of D 2 (6.25%) under organic amendment (OA) exhibited higher yield per plant in both arid and semi-arid environs (Fig. 3). Among the sludge doses, D 2 proved to be a better concentration, while D 3 significantly declines the crop yield per plant in both conditions. Organic amendments (OA) more precisely influence crop production in both arid and semi-arid conditions. However, the effect was observed dominantly in the semi-arid environment (623.20 g) at D 2 (OA). Interestingly, a decrease of crop yield was exhibited at the high dose of sludge (D 3 ), mostly in arid conditions (205 g FW) than in the semi-arid environments (299.10 g FW).

Total protein content
Total protein content (TPC) fluctuated considerably in seeds, but variability was found low at dose concentrations D 1 and D 3 with or without OA in all conditions. However, high concentrations (23 ± 2.01 and 26.5 ± 2.4) account 70-80% increase in TPC in comparison to control (Table 1). The overall trend of TPC in seeds follows the trend D 3 >D 1 >D2>D 0 . Less pronounced capriciousness in seed TPC could be due to a low supply of essential nutrients (D 1 ).

Ascorbic acid
The effect of different sludge doses on ascorbic acid content (AAC) both in arid and semi-arid environs with or without OA is depicted in Table 1. The findings revealed that sludge Fig. 2 Effects of various concentrations of sludge on the mean seed yield/plant under arid and semi-arid conditions. ns, non-significant. ***P < 0.001 significantly raises the levels of AAC in seeds up to 104.43 ± 12.23 mg/100 g for D 2 . However, the negative effect was noticed at a high concentration of sludge (D 3 ), which decreases the 30% AAC level with respect to D 2 .

Reduced glutathione (GSH)
Treatment of sludge was at three different doses D 1 , D 2 , and D 3 with organic amendments (OA) under arid and semi-arid conditions. These doses of sludge showed a decrease in GSH content; however, D 2 was found more significant and effective in both arid and semi-arid conditions to modulate the GSH content (27.23 ± 4.3 and 30.23 ± 3.3), which accounts 70-80% increase in GSH content in comparison to control. The overall trend of GSH follows the trend D 3 >D 1 >D2>D 0 .

Ascorbate peroxidase (APX)
The current study revealed that environmental stress affects the enzyme activity in garden pea (Pisum sativum L.). The sludge doses, viz., D 1 , D 2 , and D 3 , under arid and semi-arid conditions along with organic amendments (OA) significantly enhance the ascorbate peroxidase (APX) activity. Among the sludge concentrations, D 2 proved to be the best effective dose Fig. 3 Effects of various concentrations of sludge on the crop yield/plant under arid and semi-arid conditions. ns, non-significant. *P < 0.05, **P < 0.01, and ***P < 0.001 Data represent mean ± SD (n = 03). The alphabetical letters are significantly different within each group at P ≤ 0.05 using Tukey's post hoc test BFP, banana fruit peel to exhibit high value (36.7 ± 9.8) in semi-arid conditions with OA and lowest (19.5±2.2) in an arid environment (Table 2).

Dehydroascorbate reductase (DHAR)
Treatment of sludge with OA under arid and semi-arid conditions ominously regulates the activity of dehydroascorbate reductase (DHAR). However, D 2 showed a significantly higher enhancement of DR (1997 ± 12.7) under semi-arid conditions with OA.

Superoxide dismutase (SOD)
Pisum sativum showed a considerable increase in the activity of SOD under the joint influence of sludge and OA, more in a semi-arid environment. However, the influence of the D 2 dose (with OA) showed the highest increase SOD activity (156.81 ± 18.9) under semi-arid conditions.

Glutathione reductase (GR)
The sludge concentration exposure in Pisum sativum showed a significant increase in the activity of the GR enzyme. The D 2 dose showed significantly higher restoration under semi-arid conditions (29.87 ± 6.2).

Physiological characteristics
Nevertheless, garden pea (Pisum sativum L.) is an imperative leguminous crop and significantly enriches the soil nutrient environment by fixing atmospheric N 2 (Soumare et al. 2020). However, arid and semi-arid conditions predominantly oppose plant production due to the lack of essential nutrients.  Data represent mean ± SD. The alphabetical letters are significantly different within each group at P ≤ 0.05 using Tukey's post hoc test BFP, banana fruit peel photosynthesis, accumulation, transfer, and distribution of biomass (Zou et al. 2015;Lorenzoni et al. 2016). Pertinently, at some stage of sludge concentration along with OA, the plant responds proportionally with respect to seed weight and overall crop yield (Choi 2020). Conversely, a high dose of sludge decreases the seed weight and total crop yield. High nutrient supply supports enormous stem length and decreases the root length (Razaq et al. 2017;Balawejder et al. 2020). Besides, it decreases the pH of the soil to more acidic (Bloom et al. 2006) Antioxidant metabolites

Total protein content
Moreover, high sludge concentration (D 3 ) in all environmental conditions causes toxicity and suppresses plant growth. Nutrient supply enhances soil physical environment and pertinently enhances the plant metabolites than un-amendment ones (Dheeba et al. 2015). The reduction in the nutrient content may be due to the inhibition of enzymes involved in protein synthesis (Balashouri and Devi 1994).

Ascorbic acid
Significantly higher levels of ascorbic acid content in garden pea could be due to increased accumulation of carbohydrates and conversion of more organic acids into sugars with sludge doses. Similar findings were reported by Aminifard et al. (2012) and Dhotre et al. (2018).

Reduced glutathione (GSH)
High sludge concentrations of D 3 cause toxicity under all environmental conditions. Less prominent capriciousness in seed GSH might be due to less supplementation of important nutrients and toxicity suppression due to high the sludge concentration D 3 under all environmental conditions. Glutathione itself is a vital non-enzymatic antioxidant; it keeps other antioxidant components active and improves their activities (Hasanuzzaman et al. 2017). Nutrient doses (sludge) increased glutathione concentration in fruits (Gutiérrez-Gamboa et al. 2017).

Antioxidant enzymes
Ascorbate peroxidase (APX) The activity of APX gets modulated by various environmental factors including OA. Sludge doses supply essential nutrients and cause an alteration in nutrient uptake. The present study coincides with earlier findings of Han et al. (2009) and Maheshwari and Dubey (2009), who reported that APX activity increases with abiotic stresses.

Dehydroascorbate reductase (DHAR)
This up-regulation of DHAR activity in Pisum sativum might be due to the suppression of the production of free radicals under the influence of sludge and OA (Fig. 4). This increase in DHAR activity in Pisum sativum might be due to the constant up-regulation of the DHAR encoding genes with sludge dose in OA, which may increase the tolerance in Pisum sativum against the environmental stress. DHAR plays an important role in suppressing the oxidant (H 2 O 2 ), and its expression is activated by several abiotic stress factors (Ali et al. 2005;Lu et al. 2008;Fan et al. 2014). Additionally, DHAR plays an important role in plant growth and development (Chen and Gallie 2006). The lack of DHAR can swiftly decrease the ascorbic acid content and lead to a slower rate of leaf expansion, consequently affecting plant growth and development (Ye et al. 2000).

Superoxide dismutase (SOD)
This increase in the SOD activity might be the nutritive potency of sludge metallic minerals and nitrogen as a source for amino acid biosynthesis and the production of proteins. Sludge treatment containing various metallic ions increased SOD activity (Devi and Prasad 2005;Yadav 2010).

Glutathione reductase (GR)
This up-regulation in the GR activity might be due to the scavenging of ROS because sludge contains various nutrients especially nitrogen components which is important for the synthesis of proteins/enzymes, whereas D 3 showed an abnormal decline of GR due to toxicity of high sludge. Modulation in the expression profile of various GR isoforms has been known to occur under various stress conditions (Yousuf et al. 2012;Gill et al. 2013).

Conclusion
Based on the findings, sludge treatment in association with organic amendments (OA) contributes to conferring resistance and adaptation of garden pea (Pisum sativum L.) to reduce water stress and has the potential role in solving the effects of environmental inflections. Moreover, sludge doses given to the garden pea act as a source of nutrients, which enhances crop yield, antioxidant enzymes (SOD, CAT, APX, and GR), and antioxidant metabolites (GSH) in arid and semi-arid conditions. Besides, it increases the reductive powers such as NADPH of plant cells to neutralize the free radicals and also suppress the ROS down-regulatory pathways. Thereby, it improves the defense mechanism, reduces abnormal protein glycation, and diminishes programmed cell death in arid and semi-arid environs. Consequently, optimum sludge treatment (6.25%) in association with OA showed a significant reduction in protein or lipid peroxidation as well as inhibits the generation of free radicals. Therefore, the primary conclusion is that the sludge treatment (6.25%) can work as an adequate source of nutrients and may support the cultivation program of this essential crop to increase productivity, especially under arid and semi-arid conditions. Fig. 4 Environmental conditions induce modulation in various physiological systems including the defense system. Oxidative stress is one of the conditions induced due to the changing environment. (a) The oxidative stress leads to the modulations in the defense systems (enzymatic as well as non-enzymatic) which decreases the efficiency of antioxidative enzymatic and non-enzymatic systems and leads to molecular, cellular, and programmed cells deaths. However, these alterations can be combated by supplying the nutrients such as sludge (nutrients rich) doses to the Pisum sativum. Sludge dose supplies nutrients to reactivate the antioxidative enzymes, metabolites, and also non-enzymatic ROS scavenging biomolecules (such as GSH). (b) Sludge dose can also increase the reductive powers such as NADPH of plant cells to neutralize the free radicals and also suppress the ROS down-regulatory pathways (Ca 2+ Calmodulin or MAPK (MPK3 or MPK6). Hence, it can protect the plant cells against oxidative stress-induced damages. (c) Superoxide dismutase (SOD) is one of the primary antioxidative enzymes, which helps in the neutralization of free radicals; however, stress conditions inhibit SOD activity. Sludge treatment as a source of nutrients can indirectly enhance the activity of SOD and can also upregulate the other reductive power-generating sources such as GSH. Thus, it improves the defense mechanism, reduces abnormal protein glycation, enhances enzymatic activation, and depletes susceptibility of protein to proteolysis