Biochar-Mediated Control of Metabolites and Other Physiological Responses in Water-Stressed Leptocohloa fusca

We investigated biochar-induced drought tolerance in Leptocohloa fusca (Kallar grass) by exploring the plant defense system at physiological level. L. fusca plants were exposed to drought stress (100%, 70%, and 30% field capacity), and biochar (BC), as an organic soil amendment was applied in two concentrations (15 and 30 mg kg−1 soil) to induce drought tolerance. Our results demonstrated that drought restricted the growth of L. fusca by inhibiting shoot and root (fresh and dry) weight, total chlorophyll content and photosynthetic rate. Under drought stress, the uptake of essential nutrients was also limited due to lower water supply, which ultimately affected metabolites including amino and organic acids, and soluble sugars. In addition, drought stress induced oxidative stress, which is evidenced by the higher production of reactive oxygen species (ROS) including hydrogen peroxide (H2O2), superoxide ion (O2−), hydroxyl ion (OH−), and malondialdehyde (MDA). The current study revealed that stress-induced oxidative injury is not a linear path, since the excessive production of lipid peroxidation led to the accumulation of methylglyoxal (MG), a member of reactive carbonyl species (RCS), which ultimately caused cell injury. As a consequence of oxidative-stress induction, the ascorbate–glutathione (AsA–GSH) pathway, followed by a series of reactions, was activated by the plants to reduce ROS-induced oxidative damage. Furthermore, biochar considerably improved plant growth and development by mediating metabolites and soil physio-chemical status.

of ROS-MG-induced oxidative damage in L. fusca and its mitigation by BC-amended soil.
The objectives of the current study are: to investigate the physiological, biochemical, and metabolic responses of L. fusca under drought stress; to examine the role of bioactive metabolites in alleviating drought stress in L. fusca; to explore the ameliorative effects of BC under ROS-MG-induced oxidative damage.

Experimental Setup
A 4-month-long pot experiment was conducted at a private agriculture farm near Lodhran-Khanewal Expressway, Punjab, Pakistan (29.79 N, and 71.74 E) using garden soil, and L. fusca (Kallar grass) was used as plant material. Organic amendment (biochar) was used as soil amendment. Before the start of the experiment (1st week of July 2021), two levels of biochar (BC-1 = 15 mg/kg soil and BC-2 = 30 mg/kg soil) were taken and thoroughly mixed with the garden soil. The mixture of BC + garden soil (6 kg of soil/pot) was filled in plastic bins. The pots were kept in normal semi-field condition under direct sunlight for 4-5 weeks. Soil physio-chemical properties are mentioned in Table 1. During the 6th week (2nd week of August 2021), L. fusca seeds were sown in plastic pots and properly irrigated until they reached the stage of 2-3 true leaves. During the 8th week of the experiment, plants were thinned to 3 plants per pot and drought stress (100%, 70%, and 30% FC) was applied to the plants. Pots containing growing medium and plants were inspected and irrigated daily in measured quantity of water to maintain respective FC throughout the experiment. The following were the treatments of the drought stress: FC-1 = 100% (control), FC-2 = 70%, FC-3 = 30%. A total of 9 treatments with different combinations were applied as follows: T0 = 100% (FC-1), T1 = 70% (FC-2), T2 = 30% (FC-3), T3 = FC-1 + BC-1, T4 = FC-1 + BC-2, T5 = FC-2 + BC-1, T6 = FC-2 + BC-2, T7 = FC-3 + BC-1, T8 = FC-3 + BC-2. During the 16th week, plants were harvested, and data was collected as per the protocols described below. The physiological and biochemical analyses were performed at the agriculture extension and research office (29.81 N, and 71.73 E), Pakistan.

Soil Preparation and Analysis
Soil samples were air-dried for 2 days, and impurities were removed by sieving the samples to ensure a constant particle size of 2 mm. Soil physico-chemical properties including pH, electrical conductivity (EC), total soluble salts (TSS), and soil salinity level were measured by using a pH meter (Kent Eil 7015, Amesbury, MA, USA), conductivity meter (Model 4070, U.S. Salinity Lab, Riverside, CA, USA) by using the digital Jenway method, and salinity sensor (for NaCl content) (Soil Moisture Equipment Corporation, Goleta, CA, USA), respectively (Table 1). Total nitrogen (TN) and soil total carbon (TC) were determined through the Elemental analyzer (Thermo Scientific™, Waltham, MA, USA).

Morphological Characteristics
All the morphological characteristics including shoot length (SL), root length (RL), shoot fresh weight (SFW), and root fresh weight (RFW) were measured by using a standard measuring scale and digital weighing balance. After recording the fresh weights, plants samples were placed in a dry oven at 70 • C to obtain a constant root dry weight (RDW) and shoot dry weight (SDW).

Physiological Parameters
Total chlorophyll content (TC), stomatal conductance (gs), and photosynthetic rate (Pn) were analyzed by following standard procedures. The gas exchange parameters were recorded using a LiCor portable photosynthesis system (model LI-6200) calibrated at 800 mmol photons m −2 s −1 irradiance and 330-370 mmol CO 2 mol −1 . Stomatal conductance was measured on the adaxial leaf surface using a LiCor LI-1600 steady-state porometer [40]. Total chlorophyll (TC) was calculated using the previously developed equation [41].

ROS and MG Content
Hydrogen peroxide (H 2 O 2 ) content was determined by a peroxidase dependent assay adopting the earlier method [42]. The presence of hydroxyl radicals (OH − ) in the root and shoot samples was determined following the published procedure [43]. Plant samples were homogenized in 1.2 mL of 50 mM sodium phosphate buffer solution at pH 7.0, and centrifuged at 12,000 rpm for 10 min at 4 • C. Afterwards, 0.5 mL supernatant was mixed in 1 mL of 25 mM sodium phosphate buffer solution containing 2.5 mM 2-deoxyribose solution and incubated at 35 • C in the dark for 1 h. Then, the mixture was mixed with 1 mL glacial acetic acid and 1 mL of 1% thiobarbituric acid (TBA; Sigma, USA) and boiled for 10 min before immediately cooling in an ice bath. The absorbance was then measured at 532 nm.
Superoxide radicals (O 2 − ) were detected by transferring the plant samples into 0.2% nitro blue tetrazolium chloride (NBT) dissolved in 50 mM sodium phosphate buffer at pH 7.5. An insoluble formazan compound of dark-blue color appeared when NBT reacted with O 2 − . The samples were then moved to a bleaching solution to remove the chlorophyll. Afterwards, samples were ground in 0.1% acetic acid solution and centrifuged at 10,000 rpm for 10 min, with the absorbance noted at 560 nm [44].
For Methylglyoxal (MG) content, fresh samples (100 mg) were ground in distilled water and centrifuged at 11,000 rpm for 10 min. After that, 100 µL of 5 M perchloric acid solution and 250 µL of 7.2 mM and 1,2-diaminobenzene solution were added to 650 µL of supernatant. The absorbance at 336 nm was recorded with a spectrophotometer (JASCO-V 530) [45].
For the determination of the lipid peroxidation, the amount of MDA formed by the TBA reaction was measured [46]. Ground samples of shoots and roots were centrifuged at 10,000 rpm for 5 min. To 1.0 mL of supernatant, in a separate test tube, 4.0 mL of 0.5% TBA was added. The mixture was heated at 95 • C for 30 min, then cooled in ice-cold water and later centrifuged at 5000 rpm for 5 min. Absorbance was measured at 532 nm and corrected for unspecific turbidity by subtracting the value at 600 nm.

Metabolite Extraction
All extraction of metabolites such as amino acids, organic acids, and sugars in both roots and shoots of L. fusca grass were carried out by the earlier documented procedure [56]. Sample volumes of 1 µL were analyzed with a Trace GC gas chromatograph coupled to a PolarisQ ion trap mass spectrometer equipped with an AS2000 auto sampler (Thermo Electron, Dreieich, Germany). Derivatized metabolites were evaporated at 250 • C in splitless mode and separated on a 30 m × 0.25 mm RTX-5MS capillary column with a 0.25 mm coating equipped with an integrated 10 m guard column (Restak, Bad Homburg, Germany). A helium carrier gas flow was adjusted to 1 mL/m. The interface temperature was set to 250 • C and the ion source temperature to 220 • C. The oven temperature was kept constant for 3 min at 80 • C after each analysis. Mass spectra were recorded at 1 scan/s with a scanning range of 50 to 750 m/z. Metabolites were identified by comparison with pure standard (Sigma-Aldrich, Saint Louis, MO, USA). In addition, the freely available Golm Metabolome Database [57] was of particular help in identifying several metabolites. All identified compounds matched the references by mass spectral data and chromatographic retention time. Relative levels of selected metabolites were determined automatically by integrating the peak areas of selective ions [58] with the processing setup implemented in Xcalibur 1.4 software (Thermo Electron, Dreieich, Germany). Relative response ratio was calculated by normalizing the respective peak areas to the peak area of the internal standard ribitol and dividing the value by the dry weight of the sample. Measurements were performed in technical duplicates for each of the three replicates of control and the BC-amended and non-amended plants.

Statistical Analysis
A pot experiment was conducted with 9 treatments in total (3 replications each). All the data were analyzed statistically and significant differences were calculated by the oneway analysis of variance technique (ANOVA) under complete randomized design (CRD) using the SPSS software version 16.0 (Chicago, IL, United States). The least significance difference (LSD) test was applied to compare means at a 5% probability level. Duncan's Multiple Range Test (DMRT) was used as a post hoc mean-separation test (p < 0.05) using SPSS statistics (16.0) software.

Biochar-Dependent Changes in Antioxidant Activities under Drought Stress
According to hierarchical clustering, the control plants made separate group than the treated plants, which showed the obvious effect of treatments in both roots and shoots of L. fusca plants (Figure 4). The studied antioxidants showed different trends since Gly I, Gly II, SOD, CAT, POD, GPX, GSH, AsA, GR, DHAR, and DHA reduced in roots by 44,16,24,5,39,14,38,25,46,17, and 35% following 70% FC, and 88,73,58,25,66,24,60,45,69,30, and 63% after 30% FC, respectively. However, GST and MDHAR showed a slight increment of 2-and 1.3-fold at 30% FC and 3.1-and 1.6-fold at 30% FC, respectively. Contrary to others, APX and GSSG in roots showed a different pattern of growth under both stress conditions. APX and GSSG increased under 70% FC, whereas both of them were down-regulated at 30% FC. However, BC amendment enhanced the antioxidant activity in roots with few exceptions (Gly II under 70% FC and GST under 30% FC in the case of BC-1). The plants treated with BC-1 presented 1.

Discussion
Drought stress is one of the hazardous environmental factors influencing plant growth and development by modifying physiological and biochemical systems [59,60]. However, the use of fodder or forage grasses in such neglected soils is the ideal approach to make use of barren or water-stressed soils. Leptocohloa fusca has been considered an ideal plant for saline soils but it was not previously studied under drought-stress conditions. Hence, the current research was carried out to explore the potential of L. fusca under water stress and the effects of BC amendments on morpho-physiological and biochemical characteristics.
The results of the current study revealed that, under drought stress, morpho-physiological attributes substantially reduced with a progression in drought severity. These findings were parallel with previous studies [21,[61][62][63][64][65]. In addition, organic-soil amendment (BC) significantly improved plant growth including SL, RL, RFW, SFW, RDW, and SDW. These results were found indirectly in accordance with the previous findings, wherein organic soil amendments considerably reduced the toxic effects of abiotic stress, including heavy metals, drought, and salinity in different plant species [21,32,[66][67][68][69]. All the physiological parameters such as TC, Pn, and gs reduced in the current experiment. Some previous studies were found in line with the current results [65,70,71]. BC amendment considerably reduced drought stress and improved the physiological attributes of L. fusca. Previous studies on Triticum aestivum L. [72], Beta vulgaris [35], and Zea mays [73] were in accordance with our current results.
Mineral nutrients are the building blocks of essential organic molecules, for example, amino acids, enzymes, proteins, organic acids, DNA, and RNA [2]. Water scarcity impairs the nutrient uptake in plants and hence affects many biological functions which ultimately influence plant overall growth and development [59]. Some research also showed that under extreme or persistent drought-stress conditions, nutrient supplementation could not improve the plant's nutritional state [74]. Soil fertility and plant nutrient uptake has been a topic of discussion among researchers. However, recent studies suggested that nutrient supply under drought stress does not have any significant impacts on plant nutritional status, but it mainly depends on water availability [75]. The current results showed a visible difference in nutrient uptake such as Na + , Cl − , Ca 2+ , Mg 2+ , K + , and NO 3 − in L. fusca roots and shoots following drought stress. Some previous studies were consistent with the current results [21,59,[63][64][65]71,76,77]. In contrast, BC application significantly improved Na + , Cl − , Ca 2+ , Mg 2+ , K + , and NO 3 − uptake in L. fusca'sroots and shoots. Previous studies confirmed the current findings, wherein BC helped in plant nutritional uptake by improving soil physio-chemical properties and soil water-holding capacity [33,36,78,79]. With limitations to gene involvement in response to water stress, our results suggested that the significant decline in physiological, biochemical, and metabolic response in L. fusca might be due to the decrease in plant nutrient uptake. However, stress-responsive genes and their mechanisms in plant physiological, biochemical, and metabolic response should be investigated to better understand responses of L. fusca against drought stress.
The accumulation of ROS and their damaging effects on cellular organelles are the core elements of plants exposed to abiotic stressors [80]. ROS such as MDA, H 2 O 2 , O 2 − , OH − , 1 O 2 , etc., are the primary products in plant metabolism. However, in higher amounts these reactive species cause serious damage to plant cells which ultimately leads to cell death. In the current experiment, the accumulation of ROS caused oxidative damage to L. fusca plants under water-stress conditions and showed toxicity symptoms such as leaf wilting, necrosis, and denaturation of light-dependent photosynthetic systems. A plethora of studies have confirmed our current findings where MDA, H 2 O 2 , O 2 − , and OH − increased with an increase in drought severity [7][8][9]17,19,28,70]. In contrast, BC application substantially reduced ROS-mediated stress in L. fusca, which was also confirmed by the earlier studies [15,32,35,68,[81][82][83]. As well as ROS, recent studies revealed that, as a result of excessive lipid peroxidation, some reactive carbonyl species (RCS) are also produced, which cause oxidative injury to plant cells [11,84]. Excessive MG production in roots and shoots of L. fusca confirmed that RCS was produced downstream of ROS and caused irreversible oxidative damage to plant cells in response to drought stress, and similar outcomes were found in previous experiments [11,[84][85][86]. To combat ROS-RCS-induced oxidative damage, plants are well-equipped with protective mechanisms including antioxidants. In the current study, antioxidants including SOD, CAT, POD, APX, GSH, AsA, GST, MDHAR, DHAR, DHA, GPX, Gly I, and Gly II played a crucial role in alleviating ROS-MG-induced oxidative damage in L. fusca plants. Along with other antioxidant enzymes, GSH-AsA-cycle-related enzymes ( Figure 6) substantially reduced H 2 O 2 , O 2 − , OH − , and MDA, levels. Gly I, and Gly II are two-step MG-detoxification pathways that convert MG to D-lactate using glutathione as a co-factor. Our results are broadly in line with previous findings [11,18,19,28,29,85,86], whereas, BC application considerably improved the antioxidants in L. fusca roots and shoots.
These results are indirectly in line with previous studies wherein BC application alleviated oxidative stress in Brassica chinensis, Carthamus tinctorius, Zea mays, Setaria italica, and Eisenia fetida under different abiotic-stress conditions [34,66,[87][88][89]. However, the current study provides new insights into ROS-MG-induced oxidative stress and its alleviation by BC application. However, MG-induced oxidative stress and its alleviation by organic soil amendments, especially BC application, is still at rudimentary stages and needs more scientific knowledge in future.
Another promising aspect of this research was its metabolomics profile, which revealed a drop in the majority of metabolites under water stress. Some previous studies showed that the metabolites have signaling effects toward stress tolerance in plants. However, this entirely depends on the plant species, type, and intensity of stress and soil fertility [2,22,23,84]. ROS oxidize amino acids and the alteration of this key organic component result in loss of given protein-mediated functions including metabolic, structural, transport, and regulatory activities, which ultimately leads to cell death in plants [2,90,91]. Three essential amino acids-tryptophan, tyrosine, and phenylalanine-produced by the shikimate pathway are essential for plant development, stress tolerance, and pest resistance [92]. Studies have demonstrated that, under water stress, maize and chickpea plants hyper-accumulated tryptophan, phenylalanine, proline, and tyrosine [83,93]. Contrary to these findings, the current study revealed that amino acid substantially reduced under drought stress in L. fusca plants [2,87,[94][95][96][97][98]. Sugars are the most abundant group of organic molecules in plants [2,99]. Soluble sugars are the integral part of plant cells and take part in many vital activities such as antioxidant defense systems, and mechanically shape and support plant cells [2,90]. Furthermore, sugars such as sucrose, maltose, fructose, glucose, sorbitol, and deoxyribose may act as ROS-scavenging molecules. Their ROS-scavenging order is as follows: maltose > sucrose > fructose > glucose > deoxyribose > sorbitol [100,101]. However, the direct link between oxidative stress-induced modification to sugar and plant physiology under abiotic stress is still missing. In future, sugar-stress-related genetic study is also needed to better understand the scavenging or signaling role of sugars in plants under water-stress conditions. In many plant species, organic acids are crucial for their ability to withstand drought stress [2,83]. Some earlier studies elaborated that lowered levels of organic acids result in long-term drought resistance in plants [102,103]. On the other hand, in the current experiment, a significant reduction in organic acids such as CA, FA, LA, SA, BA, MA, PA, OA, GA I, and GA II in both roots and shoots of L. fusca was noticed after drought stress [24,71,89,95,[104][105][106][107]. This might be due to the high sensitivity of L. fusca plants to water-stress conditions. However, in future, a stress-responsive genetic study associated with the exceptional behavior of L. fusca is needed to have better understanding. With limitations to the stress-responsive genetic study of L. fusca plants under drought stress, the current findings confirmed that the L. fusca plant is a drought-sensitive plant species, as evidenced by the remarkable reduction in metabolites, which were then improved by BC amendment. These findings revealed a strong correlation between BC amendment and drought-stress tolerance in plants. These results confirmed the findings of some previous studies [32,[66][67][68]88,[108][109][110].

Conclusions
The findings revealed that water stress had a negative impact on the nutrient intake of L. fusca, which in turn hampered overall growth and development of L. fusca. Reduced nutrient uptake in L. fusca plants during drought stress interfered with several physiological and biochemical systems, altering overall plant growth and development. However, biochemical studies demonstrated that the AsA-GSH cycle, along with other antioxidants, had a substantial impact on physiological modifications and ROS-detoxification in L. fusca in response to drought stress (Figure 7). Another finding of this study is the presence of MG, a highly reactive dicarbonyl molecule that is formed downstream to ROS and is responsible for oxidative damage in plants under water stress. The glyoxalase system, which is involved in MG detoxification, is comprised of two enzymes, Gly I and Gly II, which completed the conversion of MG to D-Lactate/lactic acid by utilizing GSH as a co-factor ( Figure 7). Furthermore, it was found that BC application substantially reduced ROS-MG-induced oxidative damage by promoting antioxidant activity, protein, and amino and organic acids, and reducing ROS and MG content, which in turn improved morphological and physiological growth in L. fusca under drought stress. However, the concept of ROS-MG-induced oxidative stress in plant molecular studies is still at a rudimentary stage, and further studies are needed at genetic levels which will unfold the roles of proteins, antioxidants, and genes involved in MG-induced oxidative stress and its detoxification mechanism in L. fusca under drought stress.

Acknowledgments:
The authors wish to thank the laboratory staff members for their assistance during the physiological and biochemical analyses.

Conflicts of Interest:
The authors declare no conflict of interests.