Exploring Salt Stress Tolerance in Horsegram (Macrotyloma uniflorum): Insights from Growth, Physiology and Biochemical Approaches

Exploring Salt Stress Tolerance in Horsegram (Macrotyloma uniflorum): Insights from Growth, Physiology and Biochemical Approaches | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Exploring Salt Stress Tolerance in Horsegram (Macrotyloma uniflorum): Insights from Growth, Physiology and Biochemical Approaches Narayan Singh, Vasudha Maurya, Ashutosh Sharma, Rahul Kumar This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5114057/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Soil salinity is one of the significant challenges affecting seed germination, growth, physiology, and crop productivity worldwide. Horsegram is known for its higher tolerance to stress conditions than other legumes and therefore is a potential pulse and fodder crop in salt-affected areas. During this study, 25 horsegram genotypes were screened under a controlled environment to identify salt-tolerant and sensitive genotypes based on the salt tolerance index and membership function value of 10-day-old seedlings. DH-22 and DH-29 were identified as salt-tolerant, while DH-11 and DH-12 were identified as salt-sensitive genotypes. These genotypes were further analyzed under 0, 50, 100, and 150 mM NaCl treatments to examine various growth, physiological, and biochemical parameters. The analysis revealed that the tolerant genotypes exhibited higher root and shoot length, dry and fresh weight, relative water content, chlorophyll and carotenoids content, free proline and phenolic content, and enhanced activity of antioxidant enzymes such as catalase, ascorbate peroxidase, glutathione reductase, superoxide dismutase, and guaiacol peroxidase. Further, lipid peroxidation, hydrogen peroxide content, and percent ion leakage decreased in the tolerant genotypes than in the sensitive genotypes. Additionally, the tolerant genotypes displayed less cell death and lower accumulation of H 2 O 2 and superoxide ions in histochemical staining, which may play a vital role in cellular protection during salt stress tolerance. antioxidant enzymes proline lipid peroxidation histochemical staining NaCl stress Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Introduction Salt stress is among the top abiotic stresses, posing a serious limitation to global crop productivity (Singh et al. 2024 ). As per recent estimates, the global salt-affected area has grown to up to 1,128 million hectares (Mandal et al. 2018 ). Salt stress leads to osmotic stress due to ionic imbalances, thereby leading to nutritional imbalances and oxidative bursts (Deinlein et al. 2014 ). The osmotic stress is the major constraint imposed by salt stress, which hampers the process of water uptake (Flowers et al. 2015 ). Ion toxicity and nutritional imbalances in plants follow the osmotic imbalance in plants (Munns and Tester 2008 ). Plants tolerate salt stress through mechanisms such as limiting the uptake of harmful ions like Na + and Cl-, sequestering them in vacuoles, excluding ions, translocating toxic ions from shoots to roots, scavenging reactive oxygen species (ROS), maintaining adequate K + ion levels, and enhancing their antioxidant defense system (Liang et al. 2018 ). However, the degree of tolerance varies among the genotypes (Munns and Tester 2008 ). Legumes, in particular, demonstrate a higher tolerance to abiotic stress and are often cultivated by marginal farmers in low-soil fertility or rainfed conditions, contributing significantly to global food security due to their adaptability (Zhu et al. 2005 ; Bohra et al. 2015 ). The high protein and essential mineral content in legumes makes them vital for managing protein-energy metabolism (PEM) issues. (Foyer et al. 2016 ; Considine et al. 2017 ), and are considered important in achieving global food and nutrition security. Legumes improve soil health by fixing atmospheric nitrogen, thereby contributing to the sustainability of cropping systems centered around them. (Foyer et al. 2016 ). Orphan legumes, being better adapted to extreme soil and climatic conditions than major legume crops, offer a valuable source of tolerance-related genes for combating various abiotic stresses (Cullis and Kunert, 2016 ). Horsegram [ Macrotyloma uniflorum (Lam.) Verdc.], an underutilized legume belonging to the family Fabaceae, is native to South Asia, Australia, the West Indies, and Africa (Chahota et al. 2013 ). Its significant contribution to the diet of the marginal Indian population has earned it the alternative name, ‘poor man's food’ (Kadam and Salunkhe 1985 ). Horsegram is rich in antioxidants and has high calcium, iron, phosphorus, and vitamin content. It is an important medicinal legume used to treat a wide range of human diseases, including common colds and fevers, renal calculi, leucorrhoea, artery disease, constipation, and many other nutraceutical values (Kaundal et al. 2019 ; Kaundal and Kumar 2020 ). Besides its use as a pulse, it is also grown entirely as a forage crop in several Indian states. Horsegram germplasm shows considerable variability in its morpho-agronomical characteristics in addition to the biotic and abiotic stress tolerance (Neelam et al. 2014 ; Sudhagar et al. 2022 ). Past studies have explored its genetic characterization through the use of molecular markers (Chahota et al. 2017 ; Kaldate et al. 2017 ; Kumar et al. 2020 ). Mitigating the harmful effects of salinity on crops requires identifying salt-tolerant genotypes in the germplasm and incorporating them into crop improvement strategies. Conventional selection and breeding techniques have recently been applied to study salinity tolerance in legume crops (Atieno et al. 2021 ; Kumar et al. 2021 ). However, the limited understanding of salinity tolerance mechanisms and the lack of effective screening methods hinder the progress in breeding for salinity tolerance (Wu et al. 2019 ). Moreover, selections based on physiological characteristics can enhance salt tolerance in crops more effectively than selections solely based on agronomic traits (Yousef et al. 2020 ). Since the biochemical characterization of salinity stress tolerance was lacking in horsegram, it was necessary to evaluate these parameters in this crop, and hence, the current study aimed to assess various horsegram genotypes for their salt tolerance in order to identify both salt-stress-resistant and susceptible genotypes. Keeping this in mind, our investigation examined the morphological, physiological, and biochemical alterations in salt stress-tolerant and susceptible horsegram genotypes. After the identification of two salt stress-tolerant and two susceptible lines, they were subjected to salt stress of varying concentrations (0, 50, 100, and 150 mM), and a comparison was made at the morphological, physiological, and biochemical levels among these genotypes. This is the first study involving a direct comparison of salt stress-tolerant and susceptible genotypes in terms of growth, physiological, and biochemical changes, including the activities of antioxidant enzymes, in salt stress-tolerant and susceptible horsegram genotypes. Results from this research may be instrumental in developing biotechnological approaches to boost salt tolerance in horsegram and related legumes in an environmentally sustainable way. Material and methods Collection and screening of horsegram genotypes for salt stress tolerance Horsegram genotypes were collected from various regions of India, including Himachal Pradesh, Punjab, and Jammu and Kashmir (Fig. 1). The seeds were first surface-sterilized with a 1% (v/v) sodium hypochlorite (NaOCl) solution and then thoroughly rinsed with sterile dH 2 O. Following this, the seeds were soaked in dH 2 O water for 3–4 hours and placed on Petri dishes lined with Whatman No. 1 filter paper, which had been saturated with 100 mM NaCl. To prevent the evaporation of the salt solution or dH 2 O, the Petri dishes were sealed with parafilm. These dishes were then incubated in a seed germinator under controlled conditions (temperature of 25 ± 2°C, a 16-hour photoperiod, and 80–90% relative humidity). After 10 days, seedling growth was investigated by measuring the root length (RL) and shoot length (SL). Each trial was repeated three times to ensure the accuracy and reliability of the findings. Measurement of morpho-physiological traits All collected genotypes were evaluated for their tolerance to salt stress by exposing them to 100 mM NaCl and assessing growth parameters such as root length, shoot length, and the number of germinated seeds at the seedling stage (10 days old). The seedlings were then harvested, rinsed with dH 2 O, and gently blotted on tissue paper to eliminate excess moisture prior to measurement. RL and SL were recorded using a thread for precise measurement. Germination index The sprouting seeds were counted for ten days. Using the collected data, the germination index (GI) was determined using the formula: Germination index = ∑G t /T Where G t represents the total number of seeds that have germinated by the T th day, T is the number of days since sowing (Li et al. 2020 ). Salt tolerance index To evaluate the differences in tolerance levels among the horsegram genotypes, a tolerance index was estimated for the traits, i.e ., RL, SL, and GI, studied in the current investigation using the formula given below: $$\:Salt\:tolerance\:index=\frac{{V}_{in}}{{V}_{ic}}$$ Here, STIi represents the tolerance index (TI) of trait i, while V in and V ic correspond to the values of trait i in the stressed treatment and control, respectively. The STI is specific to each trait of each genotype (Li et al. 2020 ). Membership function value (MFV) The tolerance index was evaluated using the Membership Function Value (MFV). In fuzzy set theory, the membership function extends the concept of the indicator function from classical sets, representing the degree of truth (Zadeh, 1965 ). $$\:{U}_{ij}=\frac{({U}_{i}-{U}_{jmin})}{{(U}_{jmax}-{U}_{jmin})}$$ Here, Uij is the membership function value of trait i for genotype j in terms of stress tolerance. U jmax and U jmin represent the highest and lowest STI values of trait i observed across all genotypes, respectively. The average MFV of all traits (U i ) for each genotype in terms of stress tolerance was calculated. Subsequently, the average MFV for all genotypes was estimated separately. Based on a previously described method, the stress tolerance levels of genotypes were categorised into five groups (Chen et al. 2012 ), therefore the mean value (Ū) and the standard deviation (SD) of MFV as follows: (1) Ui ≥ Ū + 1.64SD, highly stress-tolerant (HST); (2) Ū + 1SD ≤ Ui ≤ Ū + 1.64SD, stress-tolerant (ST); (3) Ū − 1SD ≤ Ui < Ū + 1SD, moderately stress-tolerant (MST); (4) Ū − 1.64SD ≤ Ui < Ū − 1SD, stress-sensitive (SS); (5) Ui < Ū − 1.64SD, highly stress-sensitive (HSS). Selection of contrasting genotypes Considering the salt tolerance index and MFV analysis, two salt-tolerant and two salt-sensitive genotypes were identified and used for subsequent experimentation at 0, 50, 100 and 150 mM NaCl stresses. Measurement of root, shoot length, dry and fresh weight To analyse the root and shoot length, fresh weight (FW) of two salt-tolerant and sensitive genotypes, they were measured at 0, 50, 100, and 150 mM NaCl salt concentrations after harvesting 10-day-old seedlings. The samples, once fresh, were placed in an oven at 80°C for 72 hours, and their dry weight (DW) was measured. Relative water content To evaluate the relative water content (RWC), the fresh weight (FW) of the seedlings was measured immediately after harvesting. The harvested seedlings were then submerged in dH 2 O in the closed petri plates. After 24 hours, the turgid weight (TW) of the seedlings was measured. Subsequently, the seedlings were placed in a hot air oven set at 80°C for 72 hours to ensure thorough drying. After drying, the dry weight (DW) of the seedlings was measured using a precise weighing machine. Finally, RWC of the 10-day-old seedlings was calculated using the method described by Barrs and Weatherley ( 1962 ). $$\:\text{R}\text{e}\text{l}\text{a}\text{t}\text{i}\text{v}\text{e}\:\text{w}\text{a}\text{t}\text{e}\text{r}\:\text{c}\text{o}\text{n}\text{t}\text{e}\text{n}\text{t}=\frac{\text{F}\text{W}-\text{D}\text{W}}{\text{T}\text{W}-\text{F}\text{W}}\times\:100$$ Total chlorophyll, carotenoid and chl a/b ratio To quantify chlorophyll and carotenoids content, the Lichtenthaler and Wellburm method was used (Lichtenthaler and Wellburm 1983). The 100 mg of freshly harvested leaves of horsegram seedlings were homogenized in liquid nitrogen, followed by the addition of 1.5 ml of 80% acetone (v/v), and centrifuged at 12,000 rpm for 10 minutes. The supernatant was collected, and the absorbance was taken at 645, 662, and 670 nm, and the 80% acetone was used as a blank. The total chlorophyll, chlorophyll a + b (Chl a + b), chlorophyll a and b ratio (Chl a/b), and carotenoids content were determined by the following formulas: Chl a (µg/gFW) = [11.75 (A 662 ) – 2.35 (A 645 )] ×20 Chl b (µg/gFW) = [18.61 (A 645 ) – 3.96 (A 662 )] ×20 Carotenoids (µg/gFW) = [(1000 × A 470 − 2.27 × chl a − 81.4 × chl b/227] ×20 Chl a + b (µg/gFW) = Chl a + Chl b Chl a/b = Chl a / Chl b Biochemical estimation Free proline content Proline content in horsegram seedlings was measured following the Bates et al. ( 1973 ) method. A 1-gram sample of 10-day-old seedlings was homogenized in 3% (w/v) sulfosalicylic acid (3.6 ml) and centrifuged at 10,000 rpm for 15 minutes. To 1.6 ml of the supernatant, 4 ml of acid ninhydrin (prepared by dissolving 1.25 g of acid ninhydrin in 30 ml of glacial acetic acid and 20 ml of 6M phosphoric acid) and 4 ml of glacial acetic acid were added and the mixture was heated in a water bath at 100°C for 1 hour. The reaction was then stopped on ice and the red color was extracted with 8 ml of toluene. The absorbance of the toluene layer was measured at 520 nm, and proline concentration was assessed using a calibration curve. Lipid peroxidation Lipid peroxidation was assessed using the thiobarbituric acid (TBA) reaction with malondialdehyde (MDA), a polyunsaturated fatty acid end product (Hodges et al. 1999 ). One gram of 10-day-old horsegram seedlings was homogenized in 80% acetone (v/v), followed by centrifugation at 10,000 rpm for 10 minutes. The 1 ml supernatant was collected and divided into two test tubes: One with 1 ml of 20% (w/v) TCA (trichloroacetic acid) and the other with 20% (w/v) TCA in 0.65% (w/v) TBA. After heating both tubes in boiling water for one hour and cooling them rapidly, they were centrifuged at 10,000 rpm for 10 minutes to collect the supernatant. Absorbance readings were taken at 600, 532 and 440 nm. The MDA content was evaluated using the following formulae: [(Abs 532 (+TBA) ) - (Abs 600 (+TBA) ) - (Abs 532 (-TBA) -Abs 600 (-TBA) )] = A (Abs 440 (+TBA) - Abs 600 (+TBA) ) 0.0571 = B MDA equivalents (nmol/ml) = (A-B/157000)10 6 Hydrogen peroxide content The H₂O₂ content was measured using the procedure described by Velikova et al. ( 2000 ). Freshly collected 10-day-old seedlings were homogenized in 2 ml of 0.1% (w/v) TCA and centrifuged at 10,000 rpm for 10 minutes. A 0.4 ml sample of the supernatant was mixed with 0.4 ml of 10 mM phosphate buffer (pH 7.0), and 0.8 ml of potassium iodide was added. The absorbance of the mixture was recorded at 390 nm, and the concentration of H₂O₂ was determined from a calibration curve. Percent ion leakage To evaluate the percent (%) ion leakage (IL), freshly harvested 10-days-old seedlings were placed in test tubes where ddH 2 O (15 ml) was filled, followed by shaking for 4 hours in a shaking incubator at 30°C. The electrical conductivity of the original medium was measured initially (EC1) as described by Ahmad et al. ( 2016 ). After that, all the test tubes were autoclaved at 121°C for 20 minutes and then cooled. The electrical conductivity of every test tube was assessed again (EC2), and the % IL was calculated by following formula: Total phenolic content To evaluate total phenol content, plant extract of 10-days-old seedlings was treated with 0.5 mL of Folin and Ciocalteu reagent and 2 ml of 20% (w/v) sodium carbonate as described by Singleton's (Singleton et al. 1999 ). After 1 hour of incubation in the dark, absorbance readings at 650 nm were taken. The total phenol content was calculated using a calibration curve of gallic acid. Protein extraction and quantification Protein extraction from 10-day-old horsegram seedlings was carried out following Vyas et al. ( 2007 ). Fresh seedlings (1 gram) were homogenized in a pre-chilled mortar with 50 mM extraction buffer containing 1 mM phenylmethylsulphonyl fluoride, 1 mM dithiothreitol, 2 mM EDTA, 10% (w/v) polyvinyl polypyrrolidone, and 0.5% (v/v) Triton X-100. The homogenate was centrifuged at 10,000 rpm for 10 minutes, and the supernatant was used for assessing protein content and antioxidant enzyme activity. Total protein was quantified using the Bradford method (1976) with BSA as the reference standard. Antioxidant enzymes activity assay To evaluate the superoxide dismutase (SOD) activity, a 1.0 ml reaction mixture was prepared by adding 50 mM phosphate buffer (pH 7.8), 0.1 mM EDTA, 13 mM NBT (nitro blue tetrazolium chloride), 2.0 mM riboflavin, and 16 µl enzyme extract following the Vyas et al. ( 2007 ) method. Later, riboflavin was added, and the tubes were exposed to white fluorescent light with an intensity of about 400 µM/m 2 /min. After 15 minutes, the lights were turned off and the tubes were covered with a thick black cloth. The absorbance was subsequently measured at 560 nm. The SOD activity was expressed as the amount of enzyme needed to inhibit NBT photoreduction by 50%. To assess ascorbate peroxidase (APX) enzyme activity, a reaction mixture of 1 ml was prepared, containing 50 mM sodium phosphate buffer (pH 7.0), 0.1 mM EDTA, 0.5 mM ascorbic acid, 1 mM H₂O₂, and 10 µl of enzyme extract according to the method by Nakano and Asada ( 1981 ). The decrease in absorbance at 290 nm was monitored every 10 seconds for 1 minute, and the oxidized ascorbate was quantified using an extinction coefficient of 2.6 mM/cm. To assess guaiacol peroxidase (GPX) activity, a 1.0 ml reaction mixture was prepared with 50 mM potassium phosphate buffer (pH 7.0), 10 mM H₂O₂, 9.0 mM guaiacol, and 33 µl of enzyme extract according to Fernandez-Gracia et al. (2004). The absorbance change at 436 nm was recorded every 10 seconds over a 1-minute period at 25°C, and GPX activity was determined using an extinction coefficient of 26.6 mM/cm. One unit of GPX activity was defined as the enzyme's ability to oxidize 1.0 µM of guaiacol per minute per gram of fresh tissue. To measure catalase (CAT) activity, a 1.0 ml reaction mixture was set up with 50 mM potassium phosphate buffer (pH 7.0), 10 mM H₂O₂, and 20 µl of enzyme extract, according to the method described by Aebi ( 1984 ). The reaction, conducted at 25°C, was monitored by measuring the decrease in H₂O₂ absorbance at 240 nm every 6 seconds for 1 minute. One unit of catalase activity was defined as the enzyme's capacity to degrade 1.0 micromole of H₂O₂ per minute per gram of fresh weight. To analyze the glutathione reductase (GR) activity, a 1 ml reaction mixture was prepared by adding 50 mM potassium phosphate buffer (pH 7.8), 1 mM GSSH, 2 mM EDTA, 0.1 mM NADPH, and 30 µl of enzyme extract following the Jahnke et al. ( 1991 ) method. The oxidation of NADPH was initiated at 25°C, followed by measuring the decrease in absorbance at 340 nm for 1 minute at 6-second intervals. At pH 7.8, the one unit of GR activity was defined as the enzyme catalyzing the reduction of one micromole of GSSG per minute per gram of fresh weight. The activities of various antioxidant enzymes were expressed in terms of specific activity, calculated using the following formula: $$\:\text{S}\text{p}\text{e}\text{c}\text{i}\text{f}\text{i}\text{c}\:\text{a}\text{c}\text{t}\text{i}\text{v}\text{i}\text{t}\text{y}\:=\:\frac{\text{u}\text{n}\text{i}\text{t}\:\text{a}\text{c}\text{t}\text{i}\text{v}\text{i}\text{t}\text{y}}{\text{t}\text{o}\text{t}\text{a}\text{l}\:\text{p}\text{r}\text{o}\text{t}\text{e}\text{i}\text{n}\:\text{c}\text{o}\text{n}\text{t}\text{e}\text{n}\text{t}}$$ Histochemical detection of superoxide anion, hydrogen peroxide, cell viability Leaves from 10-day-old seedlings of both salt-sensitive and tolerant horsegram genotypes were utilized for histochemical assays to detect superoxide anion, hydrogen peroxide, and cell viability. The detection of superoxide anion (O₂⋅⁻) and hydrogen peroxide was carried out using methods described by Thordal et al. ( 1997 ) and Frahry and Schopfer ( 2001 ), respectively, while cell viability was assessed using trypan blue as per Díaz-Tielas et al. ( 2012 ). The leaves were stained overnight at room temperature in solutions of NBT (nitro blue tetrazolium chloride), DAB (3,3'-diaminobenzidine), and trypan blue. Following staining, the leaves were transferred to ethanol and boiled to eliminate chlorophyll, enabling clearer visualization of blue or dark brown staining. The stained leaves were then placed on a glass plate and photographed with a white background. Statistical analysis All experiments were conducted in triplicate and results were presented as means ± SE (standard error). Statistical analysis was performed using two-way ANOVA in SPSS statistics 22.0 software for Windows. Tukey's HSD test was used for pairwise comparisons between treatments and between genotypes at 5% level of significance (p ≤ 0.05). Results Assessment of morpho-physiological traits The collected 25 genotypes of horsegram germplasm showed variation in the morphology of seeds ( i.e. , shape, size and colour), as presented in Fig. 1. The seeds of these horsegram genotypes exhibited colour like dark-brown, peruvian-brown or orange-brown and reddish-brown. Assessment of stress tolerance index of horsegram genotypes at 100 mM NaCl salt stress Results showed that GI of genotypes DH-22, DH-3, DH-35, DH-29, DH-9 and DH-33 exhibited slightly higher value of STI as compared to other genotypes when subjected to 100 mM NaCl stress, i.e. , 1, 0.95, 0.95, 0.94, 0.93, 0.90 respectively while DH-11, DH-12, and DH-30 exhibited lowest STI i.e. , 0.47, 0.48, 0.50 respectively (Fig. 2a), amongst all genotypes. This suggests that the GI of DH-22, DH-3, DH-35, DH-29, DH-9, and DH-33 was more resistant to salt stress, while DH-11, DH-12, and DH-30 were more sensitive. It was found that genotypes DH-10, DH-29, DH-22, DH-18 exhibited the highest STI value of root length as compared to other genotypes at 100 mM NaCl salt stress (1.00, 0.93, 0.92, 0.91, respectively), while DH-11, DH-12, DH-35, DH-4 exhibited the least STI (0.32, 0.35, 0.37, 0.39) among all genotypes under consideration (Fig. 2b), suggesting that root length of DH-10, DH-29, DH-22, DH-18 was tolerant and DH-11, DH-12, DH-35, DH-4 was sensitive towards salt stress. It was observed that genotypes DH-29, DH-7, DH-22, DH-38 exhibited the highest STI value of shoot length as compared to other genotypes at 100 mM NaCl salt stress (0.86, 0.82, 0.79, 0.71, respectively), while DH-4, DH-12, DH-11 exhibited the least STI (0.12, 0.14, 0.19) among all genotypes under consideration (Fig. 2c), suggesting that shoot length of DH-29, DH-7, DH-22, DH-38 was tolerant and DH-4, DH-12, DH-11 was sensitive towards salt stress. Assessment of MFV and categorisation of genotypes Assessment of stress tolerance was done on the basis of MFV. Based on the tolerance index, MFV for each trait of each genotype and mean MFV were calculated separately for salt stress. Mean MFV reflects the tolerance level. The higher the mean MFV, the greater will be the tolerance level of the genotype. Results of table 1 indicated that the highest mean MFV under salt-stressed environments was found in DH-29, DH-22, DH-9, and DH-10 (0.928, 0.854, 0.811, and 0.802, respectively), whereas the lowest mean MFV was found in DH-12, DH-11, DH-4, and DH-2 (0.224, 0.255, 0.287, and 0.345, respectively). Five categories of horsegram genotypes were identified based on their ability to tolerate 100 mM NaCl stress i.e. , (1) U i ≥ Ū + 1.64SD, highly stress-tolerant (HST); (2) Ū + 1SD ≤ U i ≤ Ū + 1.64SD, stress-tolerant (ST); (3) Ū − 1SD ≤ U i < Ū + 1SD, moderately stress-tolerant (MST); (4) Ū − 1.64SD ≤ U i < Ū − 1SD, stress-sensitive (SS); (5) U i < Ū − 1.64SD, highly stress-sensitive (HSS). As depicted in table 1, under 100 mM NaCl stress, 2 horsegram genotypes i.e. , DH-22, DH-29, were HSS, 3 genotypes i.e. , DH-7, DH-9, DH-10 were ST, 15 genotypes i.e. , DH-3, DH-13, DH-15, DH-16, DH-17, DH-18, DH-19, DH-21, DH-23, DH-24, DH-30, DH-33, DH-35, DH-37, DH-38 were MST, 3 genotypes i.e. , DH-2, DH-4, DH-32 were SS, and 2 genotypes i.e. DH-11 and DH-12 were HSS. Selection of contrasting genotypes Based on the salt tolerance index and MFV analysis, two highly salt stress-tolerant genotypes (DH-22, DH-29), and two highly sensitive genotypes (DH-11, DH-12), were selected for the evaluation of morpho-physiological and biochemical traits. Growth characteristics Root shoot length Increasing concentrations of salt led to a higher reduction in plant growth (in terms of root/shoot length) in the sensitive genotypes (DH-11 and DH-12) than the tolerant genotypes (DH-22 and DH-29) in 10-day-old seedlings (Fig. 3). The shoot length of tolerant genotypes DH-22 and DH-29 decreased by 16% and 13%, respectively, under the 150 mM NaCl salt treatment. In contrast, the shoot length of sensitive genotypes DH-11 and DH-12 showed reductions of 35% and 23%, respectively, at the same salt concentration. Under 50 mM NaCl salt treatment, the tolerant genotypes DH-22 and DH-29 showed reductions in shoot length of 6% and 2.5%, respectively. Meanwhile, the sensitive genotypes DH-11 and DH-12 exhibited reductions of 11% and 14%, respectively (Fig. 4a). At 150 mM NaCl salt treatment, the root length of tolerant genotypes DH-22 and DH-29 decreased by 31% and 29%, respectively. In contrast, sensitive genotypes DH-11 and DH-12 experienced more severe reductions, with decreases of 53% and 57%, respectively. Additionally, at 50 mM NaCl salt treatment, root length significantly decreased by 36% and 37% in the sensitive genotypes (DH-11 and DH-12), Therefore, in the tolerant genotypes, root length remained relatively less unaffected, as shown in Fig. 4b. Effect of salt stress on fresh weight (FW), dry weight (DW) (gm) It was observed that in all genotypes, both FW and DW consistently decreased as salt concentrations increased. Under normal conditions, tolerant genotypes exhibited higher FW and DW compared to sensitive genotypes. Specifically, when exposed to 150 mM NaCl salt stress, the FW of tolerant genotypes decreased by 13% (DH-22) and 15% (DH-29), while the FW of sensitive genotypes decreased by a larger margin, 30% (DH-11) and 40% (DH-12) (Fig. 4c). However, under low salt treatment (50 mM NaCl), the FW of tolerant genotypes decreased by 2% (DH-22) and 3% (DH-29), while the FW of sensitive genotypes decreased by 13% (DH-11) and 22% (DH-12). When subjected to high salt treatment (150 mM NaCl), the DW decreased by 15% and 14% in tolerant genotypes DH-22 and DH-29, respectively; a significantly higher decline of 44% and 37% was observed in sensitive genotypes DH-11 and DH-12, respectively (Fig. 4d). Physiological traits Relative water content In this investigation, it was observed that a decrease in relative water content (RWC) in both genotypes with increased salt concentration. The tolerant genotypes maintained a higher RWC compared to the sensitive genotypes. Specifically, in the salt-sensitive genotypes DH-11 and DH-12, the RWC decreased by 3% and 4%, respectively, when treated with 50 mM NaCl. However, in the tolerant genotypes DH-22 and DH-29, the RWC only decreased by 1.6% and 1%, respectively, under the same treatment conditions. When exposed to 150 mM NaCl salt treatment, RWC decreased by 9% and 11% in sensitive genotypes DH-11 and DH-12, respectively, therefore, a lower decrease of 6% and 2% was observed in tolerant genotypes DH-22 and DH-29, respectively (Fig. 5a). Total Chlorophyll (a + b) content, ratio of a/b and carotenoid content The results indicated a consistent decreasing pattern in chlorophyll a + b and carotenoid content across different genotypes and salt stress treatments. Salt-tolerant genotypes DH-22 and DH-29 maintained higher chlorophyll a + b and carotenoid content as compared to salt-sensitive genotypes DH-11 and DH-12 under all salt stress concentrations (Fig. 5b, d). As the NaCl concentration increased from 50 mM to 150 mM, there was a noticeable reduction in chl a + b for all genotypes, indicating salt-induced chlorophyll degradation. In sensitive genotypes, DH-11 and DH-12, total chlorophyll content decreased by 74% and 73%, respectively, while in tolerant genotypes DH-22 and DH-29, it decreased by 62% and 59%, respectively, at the 150 mM NaCl salt treatment. Initially, under control conditions, the ratio of chl a/b showed no significantly variation between the genotypes. However, as salt concentration increased, the ratio of chl a/b decreased in tolerant genotypes and increased in sensitive genotypes (Fig. 5c). The ratio of chl a/b increased by 18% and 15% in sensitive genotypes DH-11 and DH-12, respectively, while in tolerant genotypes DH-22 and DH-29, it decreased by 18% and 9%, respectively, at the 150 mM NaCl salt treatment. Tolerant genotypes DH-22 and DH-29 exhibited a decrease in carotenoid content of 26% and 17% at 50 mM NaCl, 39% and 42% at 100 mM NaCl and 60% and 54% at 150 mM NaCl salt stress, respectively, therefore, sensitive genotypes DH-11 and DH-12 experienced reductions of 36% and 27% at 50 mM NaCl, 61% and 56% at 100 mM NaCl and 73% and 70% at 150 mM NaCl salt stress, respectively (Fig. 5d). Biochemical traits Free proline content It was observed that the salt-tolerant genotypes DH-22 and DH-29 consistently exhibited higher levels of free proline accumulation compared to the salt-sensitive genotypes DH-11 and DH-12 across all salt stress concentrations. As the concentration of NaCl increased from 50 mM to 150 mM, there was a clear trend of increasing proline accumulation in salt-tolerant genotypes, followed by sensitive genotypes. Under 100 mM NaCl salt stress, free proline accumulation increased by 40% in the tolerant genotypes DH-22 and DH-29, and by 40% and 37% in the salt-sensitive genotypes DH-11 and DH-12, respectively. In the sensitive genotypes DH-11 and DH-12, proline accumulation increased by 45% and 49%, respectively, under 150 mM NaCl salt treatment. However, in tolerant genotypes DH-22 and DH-29, it increased by 53% and 53%, respectively, under 150 mM NaCl salt stress (Fig. 6a). Lipid peroxidation (MDA Content) and cell viability Genotypes DH-22 and DH-29, which are salt-tolerant, showed reduced MDA content relative to the salt-sensitive DH-11 and DH-12. This suggests a more effective antioxidant defence system or lower susceptibility to oxidative damage. The MDA content showed a significant increasing trend with higher salt concentrations, indicating a dose-dependent response to salt stress. With rising salt concentrations, salt-sensitive genotypes (DH-11 and DH-12) was experienced more significant oxidative stress than the tolerant genotypes. Specifically, in the sensitive genotypes DH-11 and DH-12, MDA content increased by 29% and 22% at 50 mM NaCl, 65% and 86% under 100 mM NaCl and 136% and 128% at 150 mM NaCl salt stress, respectively. Conversely, it was found out that the tolerant genotypes DH-22 and DH-29 showed increases of 15% and 16% at 50 mM NaCl, 66% and 54% at 100 mM NaCl and 91% and 81% at 150 mM NaCl salt stress, respectively (Fig. 6b). Furthermore, Cell viability in horsegram leaves, visualized with trypan blue staining, was depicted in Fig. 10. The salt-tolerant and sensitive seedlings grown in salt-stress conditions showed different staining patterns with trypan blue, reflecting varying levels of cell viability. Tolerant genotypes DH-22 and DH-29 displayed a light staining pattern with increased salt concentration, while sensitive genotypes DH-11 and DH-12 exhibited a dark staining pattern. The findings revealed that sensitive genotypes were more prone to oxidative stress due to salt stress compared to the tolerant genotypes. Hydrogen peroxide content In the present investigation, Salt-tolerant genotypes (DH-22 and DH-29) showed significantly lower hydrogen peroxide content as compared to salt-sensitive genotypes (DH-11 and DH-12) under both normal and salt stress conditions (Fig. 6c). In the salt-tolerant genotypes, H 2 O 2 content increased by 0.12% (DH-22) and 0.46% (DH-29). In contrast, in the sensitive genotypes, it increased by 0.62% (DH-11) and 0.07% (DH-12) when subjected to 50 mM NaCl salt stress. However, at 150 mM NaCl salt stress, the increase was 0.84% (DH-22) and 0.09% (DH-29) for the salt-tolerant genotypes, while in the sensitive genotypes, it increased by 0.66% (DH-11) and 0.78% (DH-12). At salt stress of 150 mM NaCl, the increase in DH-22 was 0.84%, in DH-29 was 0.09%, in DH-11 was 0.66% and in DH-12 was 0.78%. The NBT and DAB staining indicated a lower accumulation of O2 − and H 2 O 2 in tolerant genotypes as compared to the sensitive genotypes (Fig. 11, 12). This suggests that the tolerant genotypes had lower accumulation of ROS and H 2 O 2 as compared to the sensitive genotypes. Percent ion leakage Our investigation showed that the percent (%) ion leakage increased significantly with rising salt concentrations in both tolerant and sensitive genotypes. Tolerant genotypes DH-22 and DH-29 generally demonstrate lower % ion leakage as compared to sensitive genotypes DH-11 and DH-12 (Fig. 6d). The lowest % ion leakage was observed at 50 mM NaCl salt stress in salt-tolerant genotypes DH-22 (28%) and DH-29 (32%) as opposed to sensitive genotypes DH-11 (47%) and DH-12 (43%). The highest % ion leakage occurred at 150 mM NaCl salt treatment in sensitive genotypes DH-11 (81%) and DH-12 (78%) as compared to tolerant genotypes DH-22 (65%) and DH-29 (69%). Additionally, It was observed through trypan blue staining that the tolerant genotypes exhibited reduced cell death relative to the sensitive genotypes (Fig. 10). Total phenolic content The increase in salt concentrations led to a significant rise in Total Phenolic Content (TPC) in both sensitive and tolerant genotypes. In tolerant genotypes DH-22 and DH-29, the TPC levels increased by 27% and 38% respectively, while in sensitive genotypes DH-11 and DH-12, it was increased by 19% and 16% respectively at 100 mM NaCl. This suggested that the TPC content in tolerant genotypes has increased significantly within the treatment as well as between the genotypes. At 150 mM NaCl salt stress, TPC content increased by 34% (DH-22) and 36% (DH-29) as compared to 31% (DH-11) and 29% (DH-12) in sensitive genotypes (Fig. 7a). The findings showed that tolerant genotypes had higher TPC levels than sensitive genotypes, implying that they might have boosted their antioxidant defense mechanisms in response to salt stress by elevating TPC. Total protein content The total protein content in salt-tolerant genotypes DH-22 and DH-29 was significantly higher than in salt-sensitive genotypes DH-11 and DH-12 under control conditions, as shown in Fig. 7b. As salt stress increased, the protein content decreased in both the salt-tolerant and sensitive genotypes. In the tolerant genotypes, the total protein content decreased by 11% in DH-22 and 7% in DH-29, while in the sensitive genotypes, the greater reduction was observed, with reductions of 21% in DH-11 and 12% in DH-12 at the 100 mM NaCl salt treatment. Furthermore, at the 150 mM NaCl salt treatment, the total protein content decreased by 13% in DH-22 and 20% in DH-29 among the tolerant genotypes, while it decreased by 28% in DH-11 and 14% in DH-12 among the sensitive genotypes. Tolerant genotypes maintain higher protein content as compared to sensitive genotypes as salt concentrations increase. Activity of antioxidant enzymes Superoxide dismutase Superoxide dismutase (SOD) is a crucial antioxidant enzyme involved in scavenging superoxide radicals, offering insights into the plant's response to oxidative stress. It was observed that the specific activity of SOD was significantly higher in tolerant genotypes DH-22 and DH-29 under control conditions and increased significantly with rising salt concentrations in both tolerant and sensitive genotypes (Fig. 8a). In tolerant genotypes, the specific activity of SOD increased by 60% (DH-22) and 84% (DH-29), while in sensitive genotypes, it increased by 116% (DH-11) and 105% (DH-12) at 150 mM NaCl salt treatment. At 50 mM NaCl salt treatment, the specific activity of SOD increased by 15% and 19% in tolerant genotypes DH-22 and DH-29, respectively, but in sensitive genotypes DH-11 and DH-12, it increased by 34% and 18%, respectively. The higher SOD activity in tolerant genotypes indicates a more efficient ROS scavenging system, contributing to their ability to survive oxidative stress in salt stress conditions. Ascorbate peroxidase The specific activity of APX did not increase significantly at 50 mM NaCl. However, at 100 mM NaCl, the specific activity of APX increased significantly in both salt-sensitive genotypes (DH-11, DH-12) and salt-tolerant genotypes (DH-22, DH-29. In the tolerant genotypes, the specific activity of APX increased by 317% (DH-22), and 322% (DH-29) and in sensitive genotypes, it increased by 281% (DH-11), 231% (DH-12) under 100 mM NaCl salt treatment. Furthermore, under 150 mM NaCl salt treatment, specific activity increased by 587% (DH-22), 622% (DH-29) in tolerant genotypes, and 427% (DH-11), 370% (DH-12) in sensitive genotypes. In particular, under conditions of elevated salt stress, genotypes that were tolerant showed higher APX activity in comparison to sensitive genotypes (Fig. 8b). This shows that to efficiently scavenge hydrogen peroxide and lessen salt-induced oxidative stress, the tolerant genotypes may strengthen their antioxidant defense mechanisms by upregulating APX activity. Guaiacol peroxidase The specific activity of GPX was significantly higher in salt-tolerant genotypes DH-22 and DH-29 compared to salt-sensitive genotypes DH-11 and DH-12. This elevated activity was noted as salt concentrations increased. At 150 mM NaCl salt treatment, the activity of GPX increased by 145% (DH-11), 187% (DH-12) in salt-sensitive genotypes and 98% (DH-22), 120% (DH-29) in salt-tolerant genotypes. Additionally, at 50 mM NaCl salt treatment, GPX activity increased by 20% (DH-22), 23% (DH-29) in tolerant genotypes and 53% (DH-11), 72% (DH-12) in sensitive genotypes. The analysis shows that the maximum GPX activity was increased in the sensitive genotypes compared to the tolerant genotypes, the tolerant genotypes still maintained higher GPX activity overall (Fig. 8c). Catalase Higher CAT specific activity was detected in the salt-tolerant genotypes DH-22 and DH-29 compared to the salt-sensitive genotypes DH-11 and DH-12. It was observed that CAT activity was increased significantly with higher salt concentrations in both salt-tolerant and salt-sensitive genotypes. In salt-tolerant genotypes, CAT activity increased by 388% (DH-22) and 395% (DH-29) at 150 mM salt treatment and also in salt-sensitive genotypes, it increased by 444% (DH-11) and 482% (DH-12). The 50 mM NaCl treatment produced the smallest boost in CAT activity, with an increase of 77% (DH-22), 72% (DH-29) in salt-tolerant genotypes, and a 70% (DH-11), 72% (DH-12) increment in salt-sensitive genotypes. These results indicate that there is no significant change in both categories of genotypes, but salt-tolerant genotypes exhibit higher CAT activity as compared to salt-sensitive genotypes at all salt concentrations, as illustrated in Fig. 8d. Glutathione reductase In plants, Glutathione reductase (GR) is an important enzyme involved in replenishing reduced glutathione, which helps in the antioxidant protective mechanism when plants are under stress from their environment. In our results, the specific activity of GR was significantly increased as salt concentrations rose in both salt-tolerant genotypes (DH-22, DH-29) and sensitive genotypes (DH-11, DH-12) (Fig. 8e). However, the activity of GR was higher in the tolerant genotypes compared to the sensitive genotypes in all treatments. For instance, at 50 mM NaCl salt treatment, GR activity was increased by 34% (DH-22), 37% (DH-29) in tolerant genotypes 26% (DH-11) and 30% (DH-12) in sensitive genotypes. At 150 mM NaCl salt treatment, GR activity increased by 89% (DH-22), 108% (DH-29) in tolerant genotypes and 75% (DH-11), 73% (DH-12) in sensitive genotypes. This analysis revealed that tolerant genotypes may elevate their antioxidant protective mechanism by increasing glutathione reductase activity to maintain the reduced state of glutathione and counteract salt-induced oxidative stress. Correlation analysis The correlation plot in Fig. 9 shows the results of Pearson's correlation analysis of oxidative stress markers and oxidative defence molecules. It includes various factors such as growth-related parameters and enzymatic and non-enzymatic components. Root length (RL), shoot length (SL), dry weight (DW), relative water content (RWC), chlorophyll a + b, carotenoid and protein levels were all strongly positively correlated. On the other hand, hydrogen peroxide (H 2 O 2 ), total phenolic content (TPC) and guaiacol peroxidase (GPX) show weak positive correlations. Moreover, the chlorophyll a/b ratio, percentage of ion leakage, and malondialdehyde (MDA) levels exhibited clear negative correlations. It was further observed that proline content and all antioxidant enzymes (SOD, CAT, APX, and GR) showed weak negative correlations, excluding GPX. Histochemical visualization of superoxide anion, H 2 O 2 accumulation and cell viability To visualize cell death in in-situ , we used the trypan blue staining method. We observed the highest level of blue color in trypan blue staining (Fig. 10) in the salt-sensitive genotypes (DH-11, DH-12) as compared to the salt-tolerant genotypes (DH-22, DH-29) when treated with 150 mM NaCl salt. It was observed that, as the salt treatment decreased, the blue color gradually faded, reaching its maximum in the salt-tolerant genotypes compared to the salt-sensitive genotypes. As a result, the least amount of blue color was reported in the control groups of both sensitive and tolerant genotypes. Thus, it suggests the lack of cell death in these groups. To detect superoxide ions and H 2 O 2 accumulation in both sensitive and tolerant genotypes, we used NBT (Nitro-blue Tetrazolium chloride) and DAB (Diaminobenzidine) staining. When exposed to 150 mM NaCl salt treatment, maximum blue spots were observed in NBT staining and a dark brown color in the salt-sensitive genotypes (DH-11, DH-12) compared to the salt-tolerant genotypes (DH-22, DH-29) (Fig. 11). In the control group and both sensitive and tolerant genotypes, minimal blue spots and dark brown colour were observed. However, as salt stress increased, there was a gradual rise in blue color spots and dark brown color, indicating an accumulation of superoxide ions and H 2 O 2 , with the maximum accumulation found in the salt-sensitive genotypes, in contrast to the salt-tolerant genotypes. Discussion Addressing salt stress is crucial for overcoming one of the main challenges to crop productivity and global food security. The pulses like horsegram (poor man’s pulse) contribute greatly to ensure protein rich food, specifically for the unprivileged human population. Moreover, there is considerable variability in horsegram germplasm for salt stress tolerance, which can be utilized for crop improvement. Hence, identifying the biochemical changes related to salt stress tolerance and susceptibility is crucial. The current investigation was carried out to identify salt-stress tolerant and susceptible genotypes in horsegram and unravel the biochemical mechanisms underlying tolerance and susceptibility. During long-term evolution, plants have designed sophisticated responses to various environmental stresses. Within the same species, different germplasms exhibit varying responses to identical stresses following their growth under diverse environmental forms or breeding for specific purposes (Li et al. 2020 ). Identifying highly salt-tolerant and sensitive genotypes requires the screening of multiple genotypes. Salinity tolerance, a complex, polygenic, and quantitative trait, is significantly influenced by environmental conditions, making genotype screening for salt tolerance challenging (Atieno et al. 2021 ). The 10-days old seedling screening provides preliminary insights into the variation in salt tolerance among different genotypes (Sikder et al. 2020 ). Previous studies by Ramadan et al. ( 2023 ), Kaur et al. ( 2023b ), Kaur et al. ( 2022b ) and Shaheenuzzamn, ( 2015 ), identified root length (RL) and shoot length (SL) as critical traits under salinity stress, suggesting their use in genotype screening for salt tolerance. Horsegram, considered moderately salt-tolerant, exhibits variability in response to saline environments across different genotypes (Pantola et al. 2020). Salt stress negatively impacts the normal growth and development of chickpeas, affecting traits like plant length, fresh and dry weight, germination index, and leaf area (Alom et al. 2016 ). To evaluate the effects of salt stress on different horsegram genotypes, RL, SL, and GI were employed in the current study. As a result, through the analysis of phenotypic and physiological traits, a reliable and efficient method was used to identify horsegram genotypes tolerant to salt stress. The initial step of this analysis was to assess the STI values for RL, SL, and GI. STI is a prevalent tool for selecting superior genotypes adapted to stress (Jha et al. 2022 ). The higher the value of STI at the seedling stage of plants, the greater the plants' tolerance to salt stress, which can be attributed to greater accumulation of compatible solutes such as proline, an elevation in the K+, Ca2+, and Mg2+, as well as greater retention of K + in photosynthetic tissues, thereby reducing sodium ion uptake and efficient Na + sequestration in vacuoles (Boussora et al. 2024 ; Singh et al. 2024 ; Rahman et al. 2017 ). Further, the MFV was calculated for all the genotypes for all traits for further validation of results obtained from STI. The MFV value acts as an indicator of plant stress tolerance (Gholizadeh et al. 2022 ). Greater the MFV, higher will be the tolerance, and vice-versa. Results show that among 25 genotypes under study, DH-22 and DH-29 displayed the highest mean MFV, and DH-11, DH-12 displayed the least MFV value under salt stress. Thus, in the present study involving 25 genotypes, two genotypes, viz ., DH-22 and DH-29, were identified as salt stress-tolerant, while the other two genotypes, viz ., DH-11 and DH-12, were recognized as salt stress-susceptible. Effect of salt on growth parameters in tolerant vis-à-vis sensitive genotypes The salt sensitive genotypes exhibited higher reduction in shoot and root length, in contrast to the tolerant genotypes at seedling stage under 100 mM NaCl salt stress conditions. (Fig. 4a, b). Assessing growth-related parameters is a key method for evaluating salt tolerance in various plant genotypes, at seedling stage of 10 days. Other studies by researchers were also used 10-days seedling for screening of tolerant and sensitive genotypes of different crops (Mi et al. 2020 ; Alom et al. 2016 ; Khatun et al. 2013 ; Mano et al. 1996 ). Generally, the salt stress affected seedlings exhibit slow growth or reduced mobilization of food, thereby delaying the cell division and damaging the growing hypocotyls (Müller et al. 2010 ). This leads to a metabolic reprogramming so that most of the metabolites are utilized for protection against salt-induced damage rather than promoting growth (Negrão et al. 2017 ). In some similar analysis involving pulses, it was analysed that the salt tolerant genotypes of Pisum sativum and Cajanus cajan revealed a lesser decrease in root-shoot length, fresh weight, and dry weight when compared with the salt sensitive ones. (Khan et al. 2022 ; Joshi et al. 2022 ). In the present study also, the tolerant genotypes displayed a lesser reduction in dry and fresh weight, when compared to sensitive genotypes (Fig. 4c, d). Comparative study on physiological traits RWC is an important physiological trait which can be employed as an important indicator to discriminate the salt tolerance ability among genotypes (Sánchez-Rodríguez et al. 2010 ). The present analysis revealed that RWC declined in both genotypes, yet the tolerant genotypes preserved higher RWC levels compared to the sensitive ones with increasing salt concentrations. Likewise, in pea and chickpea, sensitive genotypes showed a greater decrease in RWC than tolerant genotypes (Khan et al. 2022 ; Kaur et al. 2014 ). Chlorophyll, a pervasive natural green pigment, is found in the leaves and other parts of plants and is necessary for photosynthesis. (Humphrey 2004 ; Zahra et al. 2022 ). When chlorophyll absorbs a photon of light (quantum), it rapidly migrates through the pigments to the reaction center, where photochemical conversion of light energy to chemical energy occurs (Najar et al. 2019 ). In the present investigation, the total chlorophyll (chl a + b) and carotenoid content decreased significantly with the increased salt concentration; The tolerant genotypes of horsegram, however, sustained higher chl a + b and carotenoid content than their sensitive genotypes (Fig. 5c, d). Moreover, an increase in salt concentrations led to a rise in the Chl a/b ratio in sensitive genotypes, while it dropped in tolerant genotypes. (Fig. 5c). The decrease in the chl a/b ratio could be linked to the greater extent of chl b degradation occurring in the sensitive genotypes. Further, in a study conducted on mungbean, it was observed that chl and carotenoid content decreased sharply in sensitive genotypes than the tolerant ones, under salt stress (Alharby et al. 2019 ; Sehrawat et al. 2019 ). Here, it is important to mention that the decreases in chlorophyll contents due to higher chlorophyllase activity associated with decline in plant growth under salt stress (Khan et al. 2007 ). The decrease in chlorophyll observed in this analysis was associated with reduced growth. Comparative evaluation of biochemical parameters The present revealed that salt stress led to higher levels of MDA, H 2 O 2 , and percent ion leakage, although the extent of these increases varied between genotypes. Plants often experience an oxidative burst, involving a significant increase in ROS levels under stress (Kaur et al. 2014 ). Salt stress induces the formation of ROS, including superoxide radicals (O 2 ⋅ − ) hydrogen peroxide (H 2 O 2 ), and hydroxyl radicals (OH⋅) (ElSayed et al. 2021 ). These ROS lead to increased membrane lipid peroxidation, resulting in membrane decay and therefore, an enhanced ion leakage. Moreover, Damage caused by ROS extends to important macromolecules such as lipids, nucleic acids, proteins and photosynthetic pigments (Rezayian et al. 2019 ). In the present analysis, the tolerant genotypes displayed the lesser increase in MDA content, H 2 O 2 and percent ion leakage, than the susceptible genotypes with increase salt concentration (Fig. 6b, c, d). Further, mungbean sensitive genotypes demonstrated significantly higher levels of MDA content and ROS compared to the tolerant genotypes (Rohman et al. 2019 ). In another experiment on barley and rice, it was observed that % ion leakage increased sharply in sensitive genotypes than the tolerant ones under salt stress (Mahlooji et al. 2018 ; Singh et al. 2018 ). Generally, plants accumulate phenols and osmoprotectant like proline under abiotic stress including salt stress. Free proline and phenols have multiple functions in plant which include regulation of osmotic pressure, protection of membrane integrity, stabilization of enzymes/proteins, maintenance of appropriate NADP + , NADPH ratios, scavenger of ROS, and free radicals (Mohamed and Aly 2008 ; Misra and Saxena 2009 ; Tiwari et al. 2010 ). In the present analysis the tolerant genotypes accumulated higher proline (Fig. 6a) and phenolics (Fig. 7a) in comparison to sensitive genotypes. The synthesis of phenolic compounds through the phenylpropanoid pathway is enhanced by salinity through the stimulation of the enzyme phenylalanine ammonia lyase (PAL) (Gao et al. 2008 ), and thus leads to a higher TPC (Lim et al. 2012 ; Falcinelli et al. 2017 ). Kiani et al. proposed that proline and total phenolic content accumulation helps in enhancing stress tolerance under salt stress (Kiani et al. 2021 ). It is reported that salt tolerant genotypes of chickpea and soyabean accumulated higher proline and phenolic content rather than the sensitive genotypes (Kaur et al. 2022a ; El-Esawi et al. 2018 ). As polymers of amino acids, proteins are essential for various functions in plant structure and various plant functions including plant defense (Jain et al. 2022 ). In the present investigation, the protein content decreases with increase in salt concentration in both salt-sensitive and tolerant genotypes (Fig. 7b), but in the tolerant genotype’s protein content exhibited greater stability than than sensitive ones. In the previous studies in common bean, a similar reduction in the protein content was reported under stress. However, the salt tolerant genotypes displayed a lesser reduction in protein content than the sensitive genotypes (Azimychetabi and Sabokdast 2021 ; Farhangi-Abriz and Torabian, 2017 ). Comparative analysis of antioxidant enzymes’ activities To reduce the ROS-triggered oxidative damage under various abiotic stress, plants possess a wide array of antioxidants. Besides the non-enzymatic antioxidants in the plant cells like phenolics, plant cells possess enzymatic antioxidants like SOD, APX, GR, and CAT etc (Sharma et al. 2021 ). In the present investigation, the activities of CAT, APX, GPX, GR, and SOD were analyzed under salt stress. Superoxide dismutase (SOD) is an important enzyme which catalyzes the conversion of O 2 ⋅ − into H 2 O 2 . Its activity generally increases under the salt stress conditions (Rasool et al. 2013 ; Khoshbakht et al. 2018 ). In the present investigation, the activity of SOD significantly increased under the salt stress; however, the pattern of increase differed among the salt sensitive and tolerant genotypes. In the present analysis, the tolerant genotypes showed a higher rise in SOD activity than the sensitive. Similarly, the activity of SOD in the salt-tolerant genotypes of Vigna radiata was recorded to be higher than the sensitive genotypes (Rohman et al. 2019 ). This enhanced activity of SOD under stress seems to provide tolerant genotypes with better protection against salinity including oxidative damage of cell membranes, as judged by the lower rates of lipid peroxidation (Fig. 8a). APX and GR are two key enzymes of the ascorbate-glutathione interaction pathway (Noctor and Foyer 1998 ). APX converts ascorbate into dehydroascorbate, which is employed as a crucial substrate for H 2 O 2 detoxification (Anjum et al. 2016 ; Pang et al. 2010). Both APX and GR are involved in the oxidation and reduction of ascorbate, glutathione and NADPH in the ascorbate-glutathione cycle (Kunert and Foyer, 2023 ). In the present study, The rise in APX and GR activities under salt stress was greater in the tolerant genotypes compared to the sensitive ones. A corresponding trend was observed in some other investigations on salt stress. Chakraborty et al. ( 2019 ) and Kaur et al. ( 2023a ) reported that the tolerant genotypes of peanut and chickpea had higher activity of APX and GR than the sensitive genotypes facing salt stress (Figs. 8 and 18). The higher APX and GR activity may be able to elevate NADP + concentrations to gain electrons emerging from the electron transport chain of photosynthesis, consequently reducing ROS generation (Reddy et al. 2004 ). Catalase scavenges H 2 O 2 by directly converting it to H 2 O and O 2 in the peroxisomes as well as glyoxysome (Mittler, 2002 ). In the present analysis, CAT activity was sharply enhanced (Fig. 8d) with a corresponding sharp decline in H 2 O 2 content (Fig. 6c) in salt tolerant genotypes in comparison to the sensitive genotypes. This suggests that the salt tolerant genotypes are more efficient in scavenging H 2 O 2 than the sensitive genotypes to protect against oxidative stress. Likewise, higher levels of CAT activity were associated with increased tolerance in soybean genotypes under conditions of salt stress (Arshi et al. 2012 ). Further, Kaur et al. ( 2023a ) also reported that the salt tolerant genotypes of chickpeas had elevated CAT activity than the sensitive genotypes. The removal of H 2 O 2 from chloroplasts is mediated by another important antioxidant enzyme, GPX (Asada, 2006 ). Salt stress increases GPX activity in plants adapted or tolerant to higher salt concentration (Rahnama and Ebrahimzadeh, 2004 ). In the present study, it was evident that higher salt concentration (150 mM NaCl) was led to correspondingly higher GPX activity in tolerant genotypes than in sensitive genotypes (Fig. 8c). Similar finding was made by Desouky et al. ( 2023 ), which reported a higher GR activity in the tolerant genotypes of faba bean in comparison to the sensitive genotypes. Overall, the analysis revealed that the activity of SOD, CAT, APX, GPX, and GR antioxidant enzymes was assessed as higher in horsegram salt-tolerant genotypes in comparison with salt-sensitive genotypes. Hu et al. ( 2012 ) also reported that the salt-tolerant genotypes had higher antioxidant enzyme activity and had better protection against the oxidative stress than the salt-sensitive genotypes. Differential histochemical staining Recently, various histochemical staining methods in plants have been employed by several investigators for the in-situ visualization of selected plant metabolites (Sharma et al. 2022 ; Sharma et al. 2014 ). In the present study, the trypan blue staining for vitalization of cell viability corroborated the results of percent ion leakage. Ion leakage is considered as an important indicator of cell death (Sharma et al. 2011 ), thus the tolerant genotypes stained lesser than the susceptible genotypes under the present investigation (Fig. 10). Whereas, NBT staining for visualising superoxide (O 2 ⋅ − ) and DAB staining for visualising H 2 O 2 corroborated the results of H 2 O 2 quantification suggesting a stronger oxidative burst in salt-sensitive genotypes in comparison with salt-tolerant genotypes (Fig. 11, 12). A similar increase in superoxide (O 2 ⋅ − ) and H 2 O 2 under salt stress in chilli and maize crops were recorded by Aktas (Aktas et al. 2012 ) and Yang (Yang et al. 2015 ). The present investigation suggests that the tolerant genotypes have a stronger overall biochemical defence machinery than sensitive genotypes in mitigating the negative effects of salt stress. Hence, assessing germplasm variability for salt stress tolerance and identification of tolerant genotypes can be helpful in crop improvement through biotechnological interventions in horsegram. Conclusion and future prospects Salt stress represents a major restriction for plant growth and productivity, especially under saline environments. It causes severe adverse effects on a plants growth and physiology, with an array of associated biochemical changes. In any crop improvement programme against salt stress, it is imperative to explore the variability in the crop germplasm for salt stress tolerance. In the present study, 25 collected genotypes of horsegram with diverse characteristics were screened to identify and shortlist two salt stress tolerant genotypes i.e. , DH-22 and DH-29 and two sensitive genotypes i. e. , DH-11 and DH-12. The current study has provided an insight into salt stress physiology in horsegram, which emphasizes its promise as a hardy crop for saline agriculture. In order to look for the associated biochemical reasons for the salt stress tolerance, a thorough biochemical analysis of the selected genotypes were carried out at varying salt concentrations. The tolerant genotypes had higher growth and improved biomass accumulation under salt stress. The tolerant genotypes had lesser ROS accumulation, MDA content, and cell death, but a higher RWC, free proline accumulation, chlorophyll content, and phenols accumulation than the susceptible genotypes. The tolerant genotypes may be effectively used in plant breeding programmes to establish salt-resistant varieties offering higher yields under saline environments. Further, an attempt might also be made in future to precisely identify the genes intricate in salt stress tolerance through the transcriptomic investigation of the contrasting genotypes in relations of salt stress tolerance. It will facilitate the better understanding of the molecular pathways and regulatory networks connected to salt tolerance in horsegram. Further, it will help in targeting the genes involved in conferring salt stress tolerance to develop salt stress tolerance transgenics using biotechnological interventions. Hence, the present analysis provides a baseline information for the available tolerance to salt stress in horsegram germplasm with the overall aim to achieve global food security and agricultural sustainability. Declarations Conflict of interest The authors declare that they have no conflict of interest. Ethics approval and consent to participate Not applicable Consent for publication Not applicable. Author Contribution Conceptualization, N.S., A.S. and R.K..; Data Curation, N.S. and V.M.; Writing-Original Draft Preparation, N.S.; Writing-Review and Editing, N.S., V.M., A.S. and R.K.; Figure and Table, N.S. and V.M.; Supervision, A.S. and R.K. Acknowledgement Corresponding authors acknowledge the financial grant received from the Council of Scientific and Industrial Research (CSIR-HRDG), New Delhi, India, through an extramural research grant vide project number 38(1477)/19/EMR-II. 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Categorisation of horsegram genotypes on the basis of MFV under 100 mM NaCl stress Genotype MFV (RL) MFV (SL) MFV (GI) Mean MFV Tolerance level DH-2 0.043 0.325 0.666 0.345 SS DH-3 0.156 0.709 0.914 0.593 MST DH-4 0.098 0.000 0.763 0.287 SS DH-7 0.632 0.949 0.709 0.763 ST DH-9 0.772 0.788 0.874 0.811 ST DH-10 1.000 0.634 0.773 0.802 ST DH-11 0.238 0.528 0.000 0.255 HSS DH-12 0.164 0.490 0.017 0.224 HSS DH-13 0.589 0.387 0.782 0.586 MST DH-15 0.567 0.539 0.739 0.615 MST DH-16 0.235 0.440 0.742 0.472 MST DH-17 0.300 0.480 0.730 0.503 MST DH-18 0.845 0.021 0.730 0.532 MST DH-19 0.321 0.096 0.739 0.385 MST DH-21 0.410 0.597 0.763 0.590 MST DH-22 0.869 0.695 1.000 0.854 HST DH-23 0.295 0.755 0.685 0.578 MST DH-24 0.614 0.474 0.348 0.479 MST DH-29 0.883 1 0.900 0.928 HST DH-30 0.525 0.743 0.055 0.441 MST DH-32 0.000 0.353 0.763 0.372 SS DH-33 0.446 0.530 0.811 0.596 MST DH-35 0.073 0.660 0.914 0.549 MST DH-37 0.247 0.896 0.580 0.574 MST DH-38 0.381 0.793 0.730 0.635 MST Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5114057","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":376728151,"identity":"bd3090b0-ca83-41e3-a286-26c369e3241e","order_by":0,"name":"Narayan Singh","email":"","orcid":"","institution":"DAV University","correspondingAuthor":false,"prefix":"","firstName":"Narayan","middleName":"","lastName":"Singh","suffix":""},{"id":376728152,"identity":"daf84679-b91c-4320-a1fc-1988a1971920","order_by":1,"name":"Vasudha Maurya","email":"","orcid":"","institution":"DAV University","correspondingAuthor":false,"prefix":"","firstName":"Vasudha","middleName":"","lastName":"Maurya","suffix":""},{"id":376728153,"identity":"8fbf07c6-90ba-4678-83c0-003f8ea31eb5","order_by":2,"name":"Ashutosh Sharma","email":"","orcid":"","institution":"DAV University","correspondingAuthor":false,"prefix":"","firstName":"Ashutosh","middleName":"","lastName":"Sharma","suffix":""},{"id":376728154,"identity":"07966d13-c517-4583-a2bf-6d1b758eff6d","order_by":3,"name":"Rahul Kumar","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0UlEQVRIiWNgGAWjYFACHgaGBwYHEhjYG4AcAwsitSQAtfDwHABpkSBWCwNQi0QCiEeEFvP2s8ckEgru5NlLPr+64UeBBAN/e3cCXi0yZ/LSJBIMnhXzSOeU3ewBOkzizNkNeLVIMOSYAbUcTuyRzkm7wQPUYiCRS0AL/xuoFskzaTf/EKVFAmaLBPux28TZIvHG2AKopZjnTA7bbRkDCR7CfuHPMbzx4c/hPPb2489uvvljI8ff3otfCxLgMQCTxCoHAfYHpKgeBaNgFIyCEQQABV5GQdxxuP0AAAAASUVORK5CYII=","orcid":"","institution":"DAV University","correspondingAuthor":true,"prefix":"","firstName":"Rahul","middleName":"","lastName":"Kumar","suffix":""}],"badges":[],"createdAt":"2024-09-19 05:31:00","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5114057/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5114057/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":69414680,"identity":"b860ba97-3e8b-44b9-b02e-cdf58db6b305","added_by":"auto","created_at":"2024-11-20 06:52:57","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":538189,"visible":true,"origin":"","legend":"\u003cp\u003eCollected seeds of different horsegram genotypes used in the present study.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5114057/v1/f1f3734818a2f0c5bb75305f.png"},{"id":69413373,"identity":"b9baae3c-cad4-4743-99b9-865a51c89469","added_by":"auto","created_at":"2024-11-20 06:36:57","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":77266,"visible":true,"origin":"","legend":"\u003cp\u003eStress tolerance index of different parameters of horsegram genotypes grown under salt stress. Fig. 1a, 1b, and 1c represent the stress tolerance index of germination index, root length, and shoot length, respectively, of horsegram genotypes grown under salt stress (100 mM NaCl) at 10 DAS.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5114057/v1/2c543c52888c12065b3a7362.png"},{"id":69412975,"identity":"743efd36-e445-4ec2-91e5-522b1097d172","added_by":"auto","created_at":"2024-11-20 06:28:57","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":296162,"visible":true,"origin":"","legend":"\u003cp\u003ePhenotype comparison of salt stress-tolerant and sensitive genotypes at different salt treatments (0, 50, 100, and 150 mM NaCl stress). At 100 and 150 mM NaCl salt, stress-tolerant and sensitive genotypes show remarkable differences in plant growth.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5114057/v1/44d25747015ad15c8d262faa.png"},{"id":69414208,"identity":"159dc997-ab1b-4c39-a621-eafee2f2da3b","added_by":"auto","created_at":"2024-11-20 06:44:57","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":70212,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of different treatments of salt (0, 50, 100, 150 mM NaCl) on shoot length (a), root length (b), fresh weight (c), and dry weight (d). Bars with different letters (a, b, c, d, p, q, r, and s) are significantly different from each other (HSD, p≤0.05). a, b, c, and d are used to compare different treatments within a genotype, whereas p, q, r, and s are used to compare different genotypes within a treatment.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5114057/v1/6b25dbee78d1464f3250586f.png"},{"id":69413374,"identity":"392cc790-7546-47d3-bd50-510d4e1cef86","added_by":"auto","created_at":"2024-11-20 06:36:57","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":90261,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of different treatments of salt (0, 50, 100, 150 mM NaCl) on relative water content (a), chl a+b (b), ratio of chl a/b (c), and carotenoid content (d). Bars with different letters (a, b, c, d, p, q, r, and s) are significantly different from each other (HSD, p≤0.05). a, b, c, and d are used to compare different treatments within a genotype, whereas p, q, r, and s used to compare different genotypes within a treatment.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-5114057/v1/cf829b3963c272e0556300b2.png"},{"id":69412966,"identity":"23c383d2-bc4e-4373-8528-2ba2ab2d5af8","added_by":"auto","created_at":"2024-11-20 06:28:57","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":87493,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of various salt (0, 50, 100, 150 mM NaCl) treatments on proline content (a), MDA content (b), hydrogen peroxide (H2O2) content (c), and percent (%) ion leakage (d). Significant differences between treatments within a genotype are indicated by distinct letters (a, b, c, d), while differences between genotypes within a treatment are denoted by the letters p, q, r and s (HSD, p≤0.05).\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-5114057/v1/c2fd3e08bd24a51f30c2fce1.png"},{"id":69412968,"identity":"dbdafa8d-7346-42fb-be58-bf20a4ff0c27","added_by":"auto","created_at":"2024-11-20 06:28:57","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":69125,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of various salt treatments (0, 50, 100, 150 mM NaCl) on total phenolic content (a) and free protein content (b). Significant differences between treatments within a genotype are indicated by distinct letters (a, b, c, d), while differences between genotypes within a treatment are denoted by letters (p, q, r, s) (HSD, p≤0.05).\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-5114057/v1/c63b4962b637b903bd682e20.png"},{"id":69414213,"identity":"6a02b625-ea21-4780-9e2f-732597f507c2","added_by":"auto","created_at":"2024-11-20 06:45:05","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":136896,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of various salt treatments (0, 50, 100, 150 mM NaCl) on the specific activity of superoxide dismutase (a), ascorbate peroxidase (b), guaiacol peroxidase (c), catalase (d), and glutathione reductase. Significant differences between treatments within a genotype are indicated by distinct letters (a, b, c, d), while differences between genotypes within a treatment are denoted by letters (p, q, r, s) (HSD, p≤0.05).\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-5114057/v1/aa2866e9b66daaa22637cce7.png"},{"id":69412969,"identity":"1fb5fc8d-b9e9-4c45-9691-c7581cf73289","added_by":"auto","created_at":"2024-11-20 06:28:57","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":141630,"visible":true,"origin":"","legend":"\u003cp\u003eCorrelation plot depicting Pearson’s correlation analysis between oxidative stress markers and oxidative defence molecules (including growth-related, enzymatic, and non-enzymatic factors) is presented. The size of the circles corresponds to the absolute value of correlation coefficients, while their colour indicates positive or negative correlation values. The color scale ranges from intense blue to intense red, representing correlations from -1 to +1, respectively. RL (root length), SL (shoot length), FW (fresh weight), DW (dry weight), RWC (relative water content), chl a+b (total chlorophyll), chl a/b (chlorophyll a/b ratio), carotenoid, H2O2, % IL (percent ion leakage), TPC (total protein content), proline, MDA (malondialdehyde content), protein, CAT (catalase), SOD (superoxide dismutase), APX (ascorbate peroxidase), GR (glutathione reductase), GPX (guaiacol peroxidase).\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-5114057/v1/5d90cd462a8a85ff319f9087.png"},{"id":69412970,"identity":"17fa47bf-51c3-45b1-b357-8c43f260ddd7","added_by":"auto","created_at":"2024-11-20 06:28:57","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":334636,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of different salt stress on cell viability visualized by using trypan blue staining (a), superoxide anion accumulation stained with NBT (Nitroblue tetrazolium) (b), and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e accumulation stained with DAB (Diaminobenzidine) (c) of horsegram leaves under different salt treatments.\u0026nbsp;\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-5114057/v1/ab9c1cde36c3d96fd9d64686.png"},{"id":69414685,"identity":"46e0566d-4cfb-4cfb-ac87-c65d9df60fe6","added_by":"auto","created_at":"2024-11-20 06:53:04","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3132884,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5114057/v1/8aec521b-962f-426f-b6a0-1ba1cc4a3233.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Exploring Salt Stress Tolerance in Horsegram (Macrotyloma uniflorum): Insights from Growth, Physiology and Biochemical Approaches","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSalt stress is among the top abiotic stresses, posing a serious limitation to global crop productivity (Singh et al. \u003cspan citationid=\"CR95\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). As per recent estimates, the global salt-affected area has grown to up to 1,128\u0026nbsp;million hectares (Mandal et al. \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Salt stress leads to osmotic stress due to ionic imbalances, thereby leading to nutritional imbalances and oxidative bursts (Deinlein et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). The osmotic stress is the major constraint imposed by salt stress, which hampers the process of water uptake (Flowers et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Ion toxicity and nutritional imbalances in plants follow the osmotic imbalance in plants (Munns and Tester \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Plants tolerate salt stress through mechanisms such as limiting the uptake of harmful ions like Na\u0026thinsp;+\u0026thinsp;and Cl-, sequestering them in vacuoles, excluding ions, translocating toxic ions from shoots to roots, scavenging reactive oxygen species (ROS), maintaining adequate K\u0026thinsp;+\u0026thinsp;ion levels, and enhancing their antioxidant defense system (Liang et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). However, the degree of tolerance varies among the genotypes (Munns and Tester \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2008\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eLegumes, in particular, demonstrate a higher tolerance to abiotic stress and are often cultivated by marginal farmers in low-soil fertility or rainfed conditions, contributing significantly to global food security due to their adaptability (Zhu et al. \u003cspan citationid=\"CR108\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Bohra et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The high protein and essential mineral content in legumes makes them vital for managing protein-energy metabolism (PEM) issues. (Foyer et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Considine et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), and are considered important in achieving global food and nutrition security. Legumes improve soil health by fixing atmospheric nitrogen, thereby contributing to the sustainability of cropping systems centered around them. (Foyer et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Orphan legumes, being better adapted to extreme soil and climatic conditions than major legume crops, offer a valuable source of tolerance-related genes for combating various abiotic stresses (Cullis and Kunert, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eHorsegram [\u003cem\u003eMacrotyloma uniflorum\u003c/em\u003e (Lam.) Verdc.], an underutilized legume belonging to the family Fabaceae, is native to South Asia, Australia, the West Indies, and Africa (Chahota et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Its significant contribution to the diet of the marginal Indian population has earned it the alternative name, \u0026lsquo;poor man's food\u0026rsquo; (Kadam and Salunkhe \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e1985\u003c/span\u003e). Horsegram is rich in antioxidants and has high calcium, iron, phosphorus, and vitamin content. It is an important medicinal legume used to treat a wide range of human diseases, including common colds and fevers, renal calculi, leucorrhoea, artery disease, constipation, and many other nutraceutical values (Kaundal et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Kaundal and Kumar \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Besides its use as a pulse, it is also grown entirely as a forage crop in several Indian states. Horsegram germplasm shows considerable variability in its morpho-agronomical characteristics in addition to the biotic and abiotic stress tolerance (Neelam et al. \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Sudhagar et al. \u003cspan citationid=\"CR98\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Past studies have explored its genetic characterization through the use of molecular markers (Chahota et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Kaldate et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Kumar et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMitigating the harmful effects of salinity on crops requires identifying salt-tolerant genotypes in the germplasm and incorporating them into crop improvement strategies. Conventional selection and breeding techniques have recently been applied to study salinity tolerance in legume crops (Atieno et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Kumar et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). However, the limited understanding of salinity tolerance mechanisms and the lack of effective screening methods hinder the progress in breeding for salinity tolerance (Wu et al. \u003cspan citationid=\"CR103\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Moreover, selections based on physiological characteristics can enhance salt tolerance in crops more effectively than selections solely based on agronomic traits (Yousef et al. \u003cspan citationid=\"CR105\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Since the biochemical characterization of salinity stress tolerance was lacking in horsegram, it was necessary to evaluate these parameters in this crop, and hence, the current study aimed to assess various horsegram genotypes for their salt tolerance in order to identify both salt-stress-resistant and susceptible genotypes. Keeping this in mind, our investigation examined the morphological, physiological, and biochemical alterations in salt stress-tolerant and susceptible horsegram genotypes. After the identification of two salt stress-tolerant and two susceptible lines, they were subjected to salt stress of varying concentrations (0, 50, 100, and 150 mM), and a comparison was made at the morphological, physiological, and biochemical levels among these genotypes. This is the first study involving a direct comparison of salt stress-tolerant and susceptible genotypes in terms of growth, physiological, and biochemical changes, including the activities of antioxidant enzymes, in salt stress-tolerant and susceptible horsegram genotypes. Results from this research may be instrumental in developing biotechnological approaches to boost salt tolerance in horsegram and related legumes in an environmentally sustainable way.\u003c/p\u003e"},{"header":"Material and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCollection and screening of horsegram genotypes for salt stress tolerance\u003c/h2\u003e \u003cp\u003eHorsegram genotypes were collected from various regions of India, including Himachal Pradesh, Punjab, and Jammu and Kashmir (Fig.\u0026nbsp;1). The seeds were first surface-sterilized with a 1% (v/v) sodium hypochlorite (NaOCl) solution and then thoroughly rinsed with sterile dH\u003csub\u003e2\u003c/sub\u003eO. Following this, the seeds were soaked in dH\u003csub\u003e2\u003c/sub\u003eO water for 3\u0026ndash;4 hours and placed on Petri dishes lined with Whatman No. 1 filter paper, which had been saturated with 100 mM NaCl. To prevent the evaporation of the salt solution or dH\u003csub\u003e2\u003c/sub\u003eO, the Petri dishes were sealed with parafilm. These dishes were then incubated in a seed germinator under controlled conditions (temperature of 25\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C, a 16-hour photoperiod, and 80\u0026ndash;90% relative humidity). After 10 days, seedling growth was investigated by measuring the root length (RL) and shoot length (SL). Each trial was repeated three times to ensure the accuracy and reliability of the findings.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMeasurement of morpho-physiological traits\u003c/h3\u003e\n\u003cp\u003eAll collected genotypes were evaluated for their tolerance to salt stress by exposing them to 100 mM NaCl and assessing growth parameters such as root length, shoot length, and the number of germinated seeds at the seedling stage (10 days old). The seedlings were then harvested, rinsed with dH\u003csub\u003e2\u003c/sub\u003eO, and gently blotted on tissue paper to eliminate excess moisture prior to measurement. RL and SL were recorded using a thread for precise measurement.\u003c/p\u003e\n\u003ch3\u003eGermination index\u003c/h3\u003e\n\u003cp\u003eThe sprouting seeds were counted for ten days. Using the collected data, the germination index (GI) was determined using the formula:\u003c/p\u003e \u003cp\u003eGermination index = \u0026sum;G\u003csub\u003et\u003c/sub\u003e/T\u003c/p\u003e \u003cp\u003eWhere G\u003csub\u003et\u003c/sub\u003e represents the total number of seeds that have germinated by the T\u003csup\u003eth\u003c/sup\u003e day, T is the number of days since sowing (Li et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eSalt tolerance index\u003c/h3\u003e\n\u003cp\u003eTo evaluate the differences in tolerance levels among the horsegram genotypes, a tolerance index was estimated for the traits, \u003cem\u003ei.e\u003c/em\u003e., RL, SL, and GI, studied in the current investigation using the formula given below:\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:Salt\\:tolerance\\:index=\\frac{{V}_{in}}{{V}_{ic}}$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eHere, STIi represents the tolerance index (TI) of trait i, while V\u003csub\u003ein\u003c/sub\u003e and V\u003csub\u003eic\u003c/sub\u003e correspond to the values of trait i in the stressed treatment and control, respectively. The STI is specific to each trait of each genotype (Li et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eMembership function value (MFV)\u003c/h3\u003e\n\u003cp\u003eThe tolerance index was evaluated using the Membership Function Value (MFV). In fuzzy set theory, the membership function extends the concept of the indicator function from classical sets, representing the degree of truth (Zadeh, \u003cspan citationid=\"CR106\" class=\"CitationRef\"\u003e1965\u003c/span\u003e).\u003cdiv id=\"Equb\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equb\" name=\"EquationSource\"\u003e\n$$\\:{U}_{ij}=\\frac{({U}_{i}-{U}_{jmin})}{{(U}_{jmax}-{U}_{jmin})}$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eHere, Uij is the membership function value of trait i for genotype j in terms of stress tolerance. U\u003csub\u003ejmax\u003c/sub\u003e and U\u003csub\u003ejmin\u003c/sub\u003e represent the highest and lowest STI values of trait i observed across all genotypes, respectively. The average MFV of all traits (U\u003csub\u003ei\u003c/sub\u003e) for each genotype in terms of stress tolerance was calculated. Subsequently, the average MFV for all genotypes was estimated separately.\u003c/p\u003e \u003cp\u003eBased on a previously described method, the stress tolerance levels of genotypes were categorised into five groups (Chen et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), therefore the mean value (Ū) and the standard deviation (SD) of MFV as follows: (1) Ui \u0026ge; Ū + 1.64SD, highly stress-tolerant (HST); (2) Ū + 1SD\u0026thinsp;\u0026le;\u0026thinsp;Ui \u0026le; Ū + 1.64SD, stress-tolerant (ST); (3) Ū \u0026minus;\u0026thinsp;1SD\u0026thinsp;\u0026le;\u0026thinsp;Ui \u0026lt; Ū + 1SD, moderately stress-tolerant (MST); (4) Ū \u0026minus;\u0026thinsp;1.64SD\u0026thinsp;\u0026le;\u0026thinsp;Ui \u0026lt; Ū \u0026minus;\u0026thinsp;1SD, stress-sensitive (SS); (5) Ui \u0026lt; Ū \u0026minus;\u0026thinsp;1.64SD, highly stress-sensitive (HSS).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eSelection of contrasting genotypes\u003c/h2\u003e \u003cp\u003eConsidering the salt tolerance index and MFV analysis, two salt-tolerant and two salt-sensitive genotypes were identified and used for subsequent experimentation at 0, 50, 100 and 150 mM NaCl stresses.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMeasurement of root, shoot length, dry and fresh weight\u003c/h3\u003e\n\u003cp\u003eTo analyse the root and shoot length, fresh weight (FW) of two salt-tolerant and sensitive genotypes, they were measured at 0, 50, 100, and 150 mM NaCl salt concentrations after harvesting 10-day-old seedlings. The samples, once fresh, were placed in an oven at 80\u0026deg;C for 72 hours, and their dry weight (DW) was measured.\u003c/p\u003e\n\u003ch3\u003eRelative water content\u003c/h3\u003e\n\u003cp\u003eTo evaluate the relative water content (RWC), the fresh weight (FW) of the seedlings was measured immediately after harvesting. The harvested seedlings were then submerged in dH\u003csub\u003e2\u003c/sub\u003eO in the closed petri plates. After 24 hours, the turgid weight (TW) of the seedlings was measured. Subsequently, the seedlings were placed in a hot air oven set at 80\u0026deg;C for 72 hours to ensure thorough drying. After drying, the dry weight (DW) of the seedlings was measured using a precise weighing machine. Finally, RWC of the 10-day-old seedlings was calculated using the method described by Barrs and Weatherley (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e1962\u003c/span\u003e).\u003cdiv id=\"Equc\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equc\" name=\"EquationSource\"\u003e\n$$\\:\\text{R}\\text{e}\\text{l}\\text{a}\\text{t}\\text{i}\\text{v}\\text{e}\\:\\text{w}\\text{a}\\text{t}\\text{e}\\text{r}\\:\\text{c}\\text{o}\\text{n}\\text{t}\\text{e}\\text{n}\\text{t}=\\frac{\\text{F}\\text{W}-\\text{D}\\text{W}}{\\text{T}\\text{W}-\\text{F}\\text{W}}\\times\\:100$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eTotal chlorophyll, carotenoid and chl a/b ratio\u003c/h2\u003e \u003cp\u003eTo quantify chlorophyll and carotenoids content, the Lichtenthaler and Wellburm method was used (Lichtenthaler and Wellburm 1983). The 100 mg of freshly harvested leaves of horsegram seedlings were homogenized in liquid nitrogen, followed by the addition of 1.5 ml of 80% acetone (v/v), and centrifuged at 12,000 rpm for 10 minutes. The supernatant was collected, and the absorbance was taken at 645, 662, and 670 nm, and the 80% acetone was used as a blank. The total chlorophyll, chlorophyll a\u0026thinsp;+\u0026thinsp;b (Chl a\u0026thinsp;+\u0026thinsp;b), chlorophyll a and b ratio (Chl a/b), and carotenoids content were determined by the following formulas:\u003c/p\u003e \u003cp\u003e \u003col style=\"list-style-type:lower-roman;\"\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eChl a (\u0026micro;g/gFW) = [11.75 (A\u003csub\u003e662\u003c/sub\u003e) \u0026ndash; 2.35 (A\u003csub\u003e645\u003c/sub\u003e)] \u0026times;20\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eChl b (\u0026micro;g/gFW) = [18.61 (A\u003csub\u003e645\u003c/sub\u003e) \u0026ndash; 3.96 (A\u003csub\u003e662\u003c/sub\u003e)] \u0026times;20\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eCarotenoids (\u0026micro;g/gFW) = [(1000 \u0026times; A\u003csub\u003e470\u003c/sub\u003e\u0026thinsp;\u0026minus;\u0026thinsp;2.27 \u0026times; chl a \u0026minus;\u0026thinsp;81.4 \u0026times; chl b/227] \u0026times;20\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eChl a\u0026thinsp;+\u0026thinsp;b (\u0026micro;g/gFW)\u0026thinsp;=\u0026thinsp;Chl a\u0026thinsp;+\u0026thinsp;Chl b\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eChl a/b\u0026thinsp;=\u0026thinsp;Chl a / Chl b\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003c/ol\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eBiochemical estimation\u003c/h2\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003ch2\u003eFree proline content\u003c/h2\u003e \u003cp\u003eProline content in horsegram seedlings was measured following the Bates et al. (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1973\u003c/span\u003e) method. A 1-gram sample of 10-day-old seedlings was homogenized in 3% (w/v) sulfosalicylic acid (3.6 ml) and centrifuged at 10,000 rpm for 15 minutes. To 1.6 ml of the supernatant, 4 ml of acid ninhydrin (prepared by dissolving 1.25 g of acid ninhydrin in 30 ml of glacial acetic acid and 20 ml of 6M phosphoric acid) and 4 ml of glacial acetic acid were added and the mixture was heated in a water bath at 100\u0026deg;C for 1 hour. The reaction was then stopped on ice and the red color was extracted with 8 ml of toluene. The absorbance of the toluene layer was measured at 520 nm, and proline concentration was assessed using a calibration curve.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eLipid peroxidation\u003c/h2\u003e \u003cp\u003eLipid peroxidation was assessed using the thiobarbituric acid (TBA) reaction with malondialdehyde (MDA), a polyunsaturated fatty acid end product (Hodges et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). One gram of 10-day-old horsegram seedlings was homogenized in 80% acetone (v/v), followed by centrifugation at 10,000 rpm for 10 minutes. The 1 ml supernatant was collected and divided into two test tubes: One with 1 ml of 20% (w/v) TCA (trichloroacetic acid) and the other with 20% (w/v) TCA in 0.65% (w/v) TBA. After heating both tubes in boiling water for one hour and cooling them rapidly, they were centrifuged at 10,000 rpm for 10 minutes to collect the supernatant. Absorbance readings were taken at 600, 532 and 440 nm. The MDA content was evaluated using the following formulae:\u003c/p\u003e \u003cp\u003e \u003col style=\"list-style-type:lower-roman;\"\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003e[(Abs 532\u003csub\u003e(+TBA)\u003c/sub\u003e) - (Abs 600\u003csub\u003e(+TBA)\u003c/sub\u003e) - (Abs 532\u003csub\u003e(-TBA)\u003c/sub\u003e -Abs 600\u003csub\u003e(-TBA)\u003c/sub\u003e)]\u0026thinsp;=\u0026thinsp;A\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003e(Abs 440\u003csub\u003e(+TBA)\u003c/sub\u003e - Abs 600\u003csub\u003e(+TBA)\u003c/sub\u003e) 0.0571\u0026thinsp;=\u0026thinsp;B\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eMDA equivalents (nmol/ml) = (A-B/157000)10\u003csup\u003e6\u003c/sup\u003e\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003c/ol\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eHydrogen peroxide content\u003c/h2\u003e \u003cp\u003eThe H₂O₂ content was measured using the procedure described by Velikova et al. (\u003cspan citationid=\"CR101\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). Freshly collected 10-day-old seedlings were homogenized in 2 ml of 0.1% (w/v) TCA and centrifuged at 10,000 rpm for 10 minutes. A 0.4 ml sample of the supernatant was mixed with 0.4 ml of 10 mM phosphate buffer (pH 7.0), and 0.8 ml of potassium iodide was added. The absorbance of the mixture was recorded at 390 nm, and the concentration of H₂O₂ was determined from a calibration curve.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003ePercent ion leakage\u003c/h2\u003e \u003cp\u003eTo evaluate the percent (%) ion leakage (IL), freshly harvested 10-days-old seedlings were placed in test tubes where ddH\u003csub\u003e2\u003c/sub\u003eO (15 ml) was filled, followed by shaking for 4 hours in a shaking incubator at 30\u0026deg;C. The electrical conductivity of the original medium was measured initially (EC1) as described by Ahmad et al. (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). After that, all the test tubes were autoclaved at 121\u0026deg;C for 20 minutes and then cooled. The electrical conductivity of every test tube was assessed again (EC2), and the % IL was calculated by following formula:\u003c/p\u003e \u003cp\u003e\u003cimg src=\"data:image/png;base64,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\" width=\"156\" height=\"48\"\u003e\u003c/p\u003e\u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eTotal phenolic content\u003c/h2\u003e \u003cp\u003eTo evaluate total phenol content, plant extract of 10-days-old seedlings was treated with 0.5 mL of Folin and Ciocalteu reagent and 2 ml of 20% (w/v) sodium carbonate as described by Singleton's (Singleton et al. \u003cspan citationid=\"CR97\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). After 1 hour of incubation in the dark, absorbance readings at 650 nm were taken. The total phenol content was calculated using a calibration curve of gallic acid.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eProtein extraction and quantification\u003c/h2\u003e \u003cp\u003eProtein extraction from 10-day-old horsegram seedlings was carried out following Vyas et al. (\u003cspan citationid=\"CR102\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Fresh seedlings (1 gram) were homogenized in a pre-chilled mortar with 50 mM extraction buffer containing 1 mM phenylmethylsulphonyl fluoride, 1 mM dithiothreitol, 2 mM EDTA, 10% (w/v) polyvinyl polypyrrolidone, and 0.5% (v/v) Triton X-100. The homogenate was centrifuged at 10,000 rpm for 10 minutes, and the supernatant was used for assessing protein content and antioxidant enzyme activity. Total protein was quantified using the Bradford method (1976) with BSA as the reference standard.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eAntioxidant enzymes activity assay\u003c/h2\u003e \u003cp\u003eTo evaluate the superoxide dismutase (SOD) activity, a 1.0 ml reaction mixture was prepared by adding 50 mM phosphate buffer (pH 7.8), 0.1 mM EDTA, 13 mM NBT (nitro blue tetrazolium chloride), 2.0 mM riboflavin, and 16 \u0026micro;l enzyme extract following the Vyas et al. (\u003cspan citationid=\"CR102\" class=\"CitationRef\"\u003e2007\u003c/span\u003e) method. Later, riboflavin was added, and the tubes were exposed to white fluorescent light with an intensity of about 400 \u0026micro;M/m\u003csup\u003e2\u003c/sup\u003e/min. After 15 minutes, the lights were turned off and the tubes were covered with a thick black cloth. The absorbance was subsequently measured at 560 nm. The SOD activity was expressed as the amount of enzyme needed to inhibit NBT photoreduction by 50%.\u003c/p\u003e \u003cp\u003eTo assess ascorbate peroxidase (APX) enzyme activity, a reaction mixture of 1 ml was prepared, containing 50 mM sodium phosphate buffer (pH 7.0), 0.1 mM EDTA, 0.5 mM ascorbic acid, 1 mM H₂O₂, and 10 \u0026micro;l of enzyme extract according to the method by Nakano and Asada (\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e1981\u003c/span\u003e). The decrease in absorbance at 290 nm was monitored every 10 seconds for 1 minute, and the oxidized ascorbate was quantified using an extinction coefficient of 2.6 mM/cm.\u003c/p\u003e \u003cp\u003eTo assess guaiacol peroxidase (GPX) activity, a 1.0 ml reaction mixture was prepared with 50 mM potassium phosphate buffer (pH 7.0), 10 mM H₂O₂, 9.0 mM guaiacol, and 33 \u0026micro;l of enzyme extract according to Fernandez-Gracia et al. (2004). The absorbance change at 436 nm was recorded every 10 seconds over a 1-minute period at 25\u0026deg;C, and GPX activity was determined using an extinction coefficient of 26.6 mM/cm. One unit of GPX activity was defined as the enzyme's ability to oxidize 1.0 \u0026micro;M of guaiacol per minute per gram of fresh tissue.\u003c/p\u003e \u003cp\u003eTo measure catalase (CAT) activity, a 1.0 ml reaction mixture was set up with 50 mM potassium phosphate buffer (pH 7.0), 10 mM H₂O₂, and 20 \u0026micro;l of enzyme extract, according to the method described by Aebi (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1984\u003c/span\u003e). The reaction, conducted at 25\u0026deg;C, was monitored by measuring the decrease in H₂O₂ absorbance at 240 nm every 6 seconds for 1 minute. One unit of catalase activity was defined as the enzyme's capacity to degrade 1.0 micromole of H₂O₂ per minute per gram of fresh weight.\u003c/p\u003e \u003cp\u003eTo analyze the glutathione reductase (GR) activity, a 1 ml reaction mixture was prepared by adding 50 mM potassium phosphate buffer (pH 7.8), 1 mM GSSH, 2 mM EDTA, 0.1 mM NADPH, and 30 \u0026micro;l of enzyme extract following the Jahnke et al. (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e1991\u003c/span\u003e) method. The oxidation of NADPH was initiated at 25\u0026deg;C, followed by measuring the decrease in absorbance at 340 nm for 1 minute at 6-second intervals. At pH 7.8, the one unit of GR activity was defined as the enzyme catalyzing the reduction of one micromole of GSSG per minute per gram of fresh weight.\u003c/p\u003e \u003cp\u003eThe activities of various antioxidant enzymes were expressed in terms of specific activity, calculated using the following formula:\u003cdiv id=\"Equd\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equd\" name=\"EquationSource\"\u003e\n$$\\:\\text{S}\\text{p}\\text{e}\\text{c}\\text{i}\\text{f}\\text{i}\\text{c}\\:\\text{a}\\text{c}\\text{t}\\text{i}\\text{v}\\text{i}\\text{t}\\text{y}\\:=\\:\\frac{\\text{u}\\text{n}\\text{i}\\text{t}\\:\\text{a}\\text{c}\\text{t}\\text{i}\\text{v}\\text{i}\\text{t}\\text{y}}{\\text{t}\\text{o}\\text{t}\\text{a}\\text{l}\\:\\text{p}\\text{r}\\text{o}\\text{t}\\text{e}\\text{i}\\text{n}\\:\\text{c}\\text{o}\\text{n}\\text{t}\\text{e}\\text{n}\\text{t}}$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eHistochemical detection of superoxide anion, hydrogen peroxide, cell viability\u003c/h2\u003e \u003cp\u003eLeaves from 10-day-old seedlings of both salt-sensitive and tolerant horsegram genotypes were utilized for histochemical assays to detect superoxide anion, hydrogen peroxide, and cell viability. The detection of superoxide anion (O₂\u0026sdot;⁻) and hydrogen peroxide was carried out using methods described by Thordal et al. (\u003cspan citationid=\"CR99\" class=\"CitationRef\"\u003e1997\u003c/span\u003e) and Frahry and Schopfer (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2001\u003c/span\u003e), respectively, while cell viability was assessed using trypan blue as per D\u0026iacute;az-Tielas et al. (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). The leaves were stained overnight at room temperature in solutions of NBT (nitro blue tetrazolium chloride), DAB (3,3'-diaminobenzidine), and trypan blue. Following staining, the leaves were transferred to ethanol and boiled to eliminate chlorophyll, enabling clearer visualization of blue or dark brown staining. The stained leaves were then placed on a glass plate and photographed with a white background.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll experiments were conducted in triplicate and results were presented as means\u0026thinsp;\u0026plusmn;\u0026thinsp;SE (standard error). Statistical analysis was performed using two-way ANOVA in SPSS statistics 22.0 software for Windows. Tukey's HSD test was used for pairwise comparisons between treatments and between genotypes at 5% level of significance (p\u0026thinsp;\u0026le;\u0026thinsp;0.05).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003eAssessment of morpho-physiological traits\u003c/h2\u003e \u003cp\u003eThe collected 25 genotypes of horsegram germplasm showed variation in the morphology of seeds (\u003cem\u003ei.e.\u003c/em\u003e, shape, size and colour), as presented in Fig.\u0026nbsp;1. The seeds of these horsegram genotypes exhibited colour like dark-brown, peruvian-brown or orange-brown and reddish-brown.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eAssessment of stress tolerance index of horsegram genotypes at 100 mM NaCl salt stress\u003c/h2\u003e \u003cp\u003eResults showed that GI of genotypes DH-22, DH-3, DH-35, DH-29, DH-9 and DH-33 exhibited slightly higher value of STI as compared to other genotypes when subjected to 100 mM NaCl stress, \u003cem\u003ei.e.\u003c/em\u003e, 1, 0.95, 0.95, 0.94, 0.93, 0.90 respectively while DH-11, DH-12, and DH-30 exhibited lowest STI \u003cem\u003ei.e.\u003c/em\u003e, 0.47, 0.48, 0.50 respectively (Fig.\u0026nbsp;2a), amongst all genotypes. This suggests that the GI of DH-22, DH-3, DH-35, DH-29, DH-9, and DH-33 was more resistant to salt stress, while DH-11, DH-12, and DH-30 were more sensitive.\u003c/p\u003e \u003cp\u003eIt was found that genotypes DH-10, DH-29, DH-22, DH-18 exhibited the highest STI value of root length as compared to other genotypes at 100 mM NaCl salt stress (1.00, 0.93, 0.92, 0.91, respectively), while DH-11, DH-12, DH-35, DH-4 exhibited the least STI (0.32, 0.35, 0.37, 0.39) among all genotypes under consideration (Fig.\u0026nbsp;2b), suggesting that root length of DH-10, DH-29, DH-22, DH-18 was tolerant and DH-11, DH-12, DH-35, DH-4 was sensitive towards salt stress.\u003c/p\u003e \u003cp\u003eIt was observed that genotypes DH-29, DH-7, DH-22, DH-38 exhibited the highest STI value of shoot length as compared to other genotypes at 100 mM NaCl salt stress (0.86, 0.82, 0.79, 0.71, respectively), while DH-4, DH-12, DH-11 exhibited the least STI (0.12, 0.14, 0.19) among all genotypes under consideration (Fig.\u0026nbsp;2c), suggesting that shoot length of DH-29, DH-7, DH-22, DH-38 was tolerant and DH-4, DH-12, DH-11 was sensitive towards salt stress.\u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003eAssessment of MFV and categorisation of genotypes\u003c/h2\u003e \u003cp\u003eAssessment of stress tolerance was done on the basis of MFV. Based on the tolerance index, MFV for each trait of each genotype and mean MFV were calculated separately for salt stress. Mean MFV reflects the tolerance level. The higher the mean MFV, the greater will be the tolerance level of the genotype. Results of table 1 indicated that the highest mean MFV under salt-stressed environments was found in DH-29, DH-22, DH-9, and DH-10 (0.928, 0.854, 0.811, and 0.802, respectively), whereas the lowest mean MFV was found in DH-12, DH-11, DH-4, and DH-2 (0.224, 0.255, 0.287, and 0.345, respectively).\u003c/p\u003e \u003cp\u003eFive categories of horsegram genotypes were identified based on their ability to tolerate 100 mM NaCl stress \u003cem\u003ei.e.\u003c/em\u003e, (1) U\u003csub\u003ei\u003c/sub\u003e \u0026ge; Ū + 1.64SD, highly stress-tolerant (HST); (2) Ū + 1SD\u0026thinsp;\u0026le;\u0026thinsp;U\u003csub\u003ei\u003c/sub\u003e \u0026le; Ū + 1.64SD, stress-tolerant (ST); (3) Ū \u0026minus;\u0026thinsp;1SD\u0026thinsp;\u0026le;\u0026thinsp;U\u003csub\u003ei\u003c/sub\u003e \u0026lt; Ū + 1SD, moderately stress-tolerant (MST); (4) Ū \u0026minus;\u0026thinsp;1.64SD\u0026thinsp;\u0026le;\u0026thinsp;U\u003csub\u003ei\u003c/sub\u003e \u0026lt; Ū \u0026minus;\u0026thinsp;1SD, stress-sensitive (SS); (5) U\u003csub\u003ei\u003c/sub\u003e \u0026lt; Ū \u0026minus;\u0026thinsp;1.64SD, highly stress-sensitive (HSS).\u003c/p\u003e \u003cp\u003eAs depicted in table 1, under 100 mM NaCl stress, 2 horsegram genotypes \u003cem\u003ei.e.\u003c/em\u003e, DH-22, DH-29, were HSS, 3 genotypes \u003cem\u003ei.e.\u003c/em\u003e, DH-7, DH-9, DH-10 were ST, 15 genotypes \u003cem\u003ei.e.\u003c/em\u003e, DH-3, DH-13, DH-15, DH-16, DH-17, DH-18, DH-19, DH-21, DH-23, DH-24, DH-30, DH-33, DH-35, DH-37, DH-38 were MST, 3 genotypes \u003cem\u003ei.e.\u003c/em\u003e, DH-2, DH-4, DH-32 were SS, and 2 genotypes i.e. DH-11 and DH-12 were HSS.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003eSelection of contrasting genotypes\u003c/h2\u003e \u003cp\u003eBased on the salt tolerance index and MFV analysis, two highly salt stress-tolerant genotypes (DH-22, DH-29), and two highly sensitive genotypes (DH-11, DH-12), were selected for the evaluation of morpho-physiological and biochemical traits.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section3\"\u003e \u003ch2\u003eGrowth characteristics\u003c/h2\u003e \u003cdiv id=\"Sec28\" class=\"Section4\"\u003e \u003ch2\u003eRoot shoot length\u003c/h2\u003e \u003cp\u003eIncreasing concentrations of salt led to a higher reduction in plant growth (in terms of root/shoot length) in the sensitive genotypes (DH-11 and DH-12) than the tolerant genotypes (DH-22 and DH-29) in 10-day-old seedlings (Fig.\u0026nbsp;3). The shoot length of tolerant genotypes DH-22 and DH-29 decreased by 16% and 13%, respectively, under the 150 mM NaCl salt treatment. In contrast, the shoot length of sensitive genotypes DH-11 and DH-12 showed reductions of 35% and 23%, respectively, at the same salt concentration. Under 50 mM NaCl salt treatment, the tolerant genotypes DH-22 and DH-29 showed reductions in shoot length of 6% and 2.5%, respectively. Meanwhile, the sensitive genotypes DH-11 and DH-12 exhibited reductions of 11% and 14%, respectively (Fig.\u0026nbsp;4a). At 150 mM NaCl salt treatment, the root length of tolerant genotypes DH-22 and DH-29 decreased by 31% and 29%, respectively. In contrast, sensitive genotypes DH-11 and DH-12 experienced more severe reductions, with decreases of 53% and 57%, respectively. Additionally, at 50 mM NaCl salt treatment, root length significantly decreased by 36% and 37% in the sensitive genotypes (DH-11 and DH-12), Therefore, in the tolerant genotypes, root length remained relatively less unaffected, as shown in Fig.\u0026nbsp;4b.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec29\" class=\"Section2\"\u003e \u003ch2\u003eEffect of salt stress on fresh weight (FW), dry weight (DW) (gm)\u003c/h2\u003e \u003cp\u003eIt was observed that in all genotypes, both FW and DW consistently decreased as salt concentrations increased. Under normal conditions, tolerant genotypes exhibited higher FW and DW compared to sensitive genotypes. Specifically, when exposed to 150 mM NaCl salt stress, the FW of tolerant genotypes decreased by 13% (DH-22) and 15% (DH-29), while the FW of sensitive genotypes decreased by a larger margin, 30% (DH-11) and 40% (DH-12) (Fig.\u0026nbsp;4c). However, under low salt treatment (50 mM NaCl), the FW of tolerant genotypes decreased by 2% (DH-22) and 3% (DH-29), while the FW of sensitive genotypes decreased by 13% (DH-11) and 22% (DH-12). When subjected to high salt treatment (150 mM NaCl), the DW decreased by 15% and 14% in tolerant genotypes DH-22 and DH-29, respectively; a significantly higher decline of 44% and 37% was observed in sensitive genotypes DH-11 and DH-12, respectively (Fig.\u0026nbsp;4d).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePhysiological traits\u003c/h3\u003e\n\u003cdiv id=\"Sec31\" class=\"Section2\"\u003e \u003ch2\u003eRelative water content\u003c/h2\u003e \u003cp\u003eIn this investigation, it was observed that a decrease in relative water content (RWC) in both genotypes with increased salt concentration. The tolerant genotypes maintained a higher RWC compared to the sensitive genotypes. Specifically, in the salt-sensitive genotypes DH-11 and DH-12, the RWC decreased by 3% and 4%, respectively, when treated with 50 mM NaCl. However, in the tolerant genotypes DH-22 and DH-29, the RWC only decreased by 1.6% and 1%, respectively, under the same treatment conditions. When exposed to 150 mM NaCl salt treatment, RWC decreased by 9% and 11% in sensitive genotypes DH-11 and DH-12, respectively, therefore, a lower decrease of 6% and 2% was observed in tolerant genotypes DH-22 and DH-29, respectively (Fig.\u0026nbsp;5a).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec32\" class=\"Section2\"\u003e \u003ch2\u003eTotal Chlorophyll (a\u0026thinsp;+\u0026thinsp;b) content, ratio of a/b and carotenoid content\u003c/h2\u003e \u003cp\u003eThe results indicated a consistent decreasing pattern in chlorophyll a\u0026thinsp;+\u0026thinsp;b and carotenoid content across different genotypes and salt stress treatments. Salt-tolerant genotypes DH-22 and DH-29 maintained higher chlorophyll a\u0026thinsp;+\u0026thinsp;b and carotenoid content as compared to salt-sensitive genotypes DH-11 and DH-12 under all salt stress concentrations (Fig.\u0026nbsp;5b, d). As the NaCl concentration increased from 50 mM to 150 mM, there was a noticeable reduction in chl a\u0026thinsp;+\u0026thinsp;b for all genotypes, indicating salt-induced chlorophyll degradation. In sensitive genotypes, DH-11 and DH-12, total chlorophyll content decreased by 74% and 73%, respectively, while in tolerant genotypes DH-22 and DH-29, it decreased by 62% and 59%, respectively, at the 150 mM NaCl salt treatment. Initially, under control conditions, the ratio of chl a/b showed no significantly variation between the genotypes. However, as salt concentration increased, the ratio of chl a/b decreased in tolerant genotypes and increased in sensitive genotypes (Fig.\u0026nbsp;5c). The ratio of chl a/b increased by 18% and 15% in sensitive genotypes DH-11 and DH-12, respectively, while in tolerant genotypes DH-22 and DH-29, it decreased by 18% and 9%, respectively, at the 150 mM NaCl salt treatment. Tolerant genotypes DH-22 and DH-29 exhibited a decrease in carotenoid content of 26% and 17% at 50 mM NaCl, 39% and 42% at 100 mM NaCl and 60% and 54% at 150 mM NaCl salt stress, respectively, therefore, sensitive genotypes DH-11 and DH-12 experienced reductions of 36% and 27% at 50 mM NaCl, 61% and 56% at 100 mM NaCl and 73% and 70% at 150 mM NaCl salt stress, respectively (Fig.\u0026nbsp;5d).\u003c/p\u003e \u003cdiv id=\"Sec33\" class=\"Section3\"\u003e \u003ch2\u003eBiochemical traits\u003c/h2\u003e \u003c/div\u003e \u003cdiv id=\"Sec34\" class=\"Section3\"\u003e \u003ch2\u003eFree proline content\u003c/h2\u003e \u003cp\u003eIt was observed that the salt-tolerant genotypes DH-22 and DH-29 consistently exhibited higher levels of free proline accumulation compared to the salt-sensitive genotypes DH-11 and DH-12 across all salt stress concentrations. As the concentration of NaCl increased from 50 mM to 150 mM, there was a clear trend of increasing proline accumulation in salt-tolerant genotypes, followed by sensitive genotypes. Under 100 mM NaCl salt stress, free proline accumulation increased by 40% in the tolerant genotypes DH-22 and DH-29, and by 40% and 37% in the salt-sensitive genotypes DH-11 and DH-12, respectively. In the sensitive genotypes DH-11 and DH-12, proline accumulation increased by 45% and 49%, respectively, under 150 mM NaCl salt treatment. However, in tolerant genotypes DH-22 and DH-29, it increased by 53% and 53%, respectively, under 150 mM NaCl salt stress (Fig.\u0026nbsp;6a).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e\n\u003ch3\u003eLipid peroxidation (MDA Content) and cell viability\u003c/h3\u003e\n\u003cp\u003eGenotypes DH-22 and DH-29, which are salt-tolerant, showed reduced MDA content relative to the salt-sensitive DH-11 and DH-12. This suggests a more effective antioxidant defence system or lower susceptibility to oxidative damage. The MDA content showed a significant increasing trend with higher salt concentrations, indicating a dose-dependent response to salt stress. With rising salt concentrations, salt-sensitive genotypes (DH-11 and DH-12) was experienced more significant oxidative stress than the tolerant genotypes. Specifically, in the sensitive genotypes DH-11 and DH-12, MDA content increased by 29% and 22% at 50 mM NaCl, 65% and 86% under 100 mM NaCl and 136% and 128% at 150 mM NaCl salt stress, respectively. Conversely, it was found out that the tolerant genotypes DH-22 and DH-29 showed increases of 15% and 16% at 50 mM NaCl, 66% and 54% at 100 mM NaCl and 91% and 81% at 150 mM NaCl salt stress, respectively (Fig.\u0026nbsp;6b).\u003c/p\u003e \u003cp\u003eFurthermore, Cell viability in horsegram leaves, visualized with trypan blue staining, was depicted in Fig.\u0026nbsp;10. The salt-tolerant and sensitive seedlings grown in salt-stress conditions showed different staining patterns with trypan blue, reflecting varying levels of cell viability. Tolerant genotypes DH-22 and DH-29 displayed a light staining pattern with increased salt concentration, while sensitive genotypes DH-11 and DH-12 exhibited a dark staining pattern. The findings revealed that sensitive genotypes were more prone to oxidative stress due to salt stress compared to the tolerant genotypes.\u003c/p\u003e\n\u003ch3\u003eHydrogen peroxide content\u003c/h3\u003e\n\u003cp\u003eIn the present investigation, Salt-tolerant genotypes (DH-22 and DH-29) showed significantly lower hydrogen peroxide content as compared to salt-sensitive genotypes (DH-11 and DH-12) under both normal and salt stress conditions (Fig.\u0026nbsp;6c). In the salt-tolerant genotypes, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e content increased by 0.12% (DH-22) and 0.46% (DH-29). In contrast, in the sensitive genotypes, it increased by 0.62% (DH-11) and 0.07% (DH-12) when subjected to 50 mM NaCl salt stress. However, at 150 mM NaCl salt stress, the increase was 0.84% (DH-22) and 0.09% (DH-29) for the salt-tolerant genotypes, while in the sensitive genotypes, it increased by 0.66% (DH-11) and 0.78% (DH-12). At salt stress of 150 mM NaCl, the increase in DH-22 was 0.84%, in DH-29 was 0.09%, in DH-11 was 0.66% and in DH-12 was 0.78%. The NBT and DAB staining indicated a lower accumulation of O2\u003csup\u003e\u0026minus;\u003c/sup\u003e and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e in tolerant genotypes as compared to the sensitive genotypes (Fig.\u0026nbsp;11, 12). This suggests that the tolerant genotypes had lower accumulation of ROS and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e as compared to the sensitive genotypes.\u003c/p\u003e \u003cdiv id=\"Sec37\" class=\"Section2\"\u003e \u003ch2\u003ePercent ion leakage\u003c/h2\u003e \u003cp\u003eOur investigation showed that the percent (%) ion leakage increased significantly with rising salt concentrations in both tolerant and sensitive genotypes. Tolerant genotypes DH-22 and DH-29 generally demonstrate lower % ion leakage as compared to sensitive genotypes DH-11 and DH-12 (Fig.\u0026nbsp;6d). The lowest % ion leakage was observed at 50 mM NaCl salt stress in salt-tolerant genotypes DH-22 (28%) and DH-29 (32%) as opposed to sensitive genotypes DH-11 (47%) and DH-12 (43%). The highest % ion leakage occurred at 150 mM NaCl salt treatment in sensitive genotypes DH-11 (81%) and DH-12 (78%) as compared to tolerant genotypes DH-22 (65%) and DH-29 (69%). Additionally, It was observed through trypan blue staining that the tolerant genotypes exhibited reduced cell death relative to the sensitive genotypes (Fig.\u0026nbsp;10).\u003c/p\u003e \u003cdiv id=\"Sec38\" class=\"Section3\"\u003e \u003ch2\u003eTotal phenolic content\u003c/h2\u003e \u003cp\u003eThe increase in salt concentrations led to a significant rise in Total Phenolic Content (TPC) in both sensitive and tolerant genotypes. In tolerant genotypes DH-22 and DH-29, the TPC levels increased by 27% and 38% respectively, while in sensitive genotypes DH-11 and DH-12, it was increased by 19% and 16% respectively at 100 mM NaCl. This suggested that the TPC content in tolerant genotypes has increased significantly within the treatment as well as between the genotypes. At 150 mM NaCl salt stress, TPC content increased by 34% (DH-22) and 36% (DH-29) as compared to 31% (DH-11) and 29% (DH-12) in sensitive genotypes (Fig.\u0026nbsp;7a). The findings showed that tolerant genotypes had higher TPC levels than sensitive genotypes, implying that they might have boosted their antioxidant defense mechanisms in response to salt stress by elevating TPC.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec39\" class=\"Section2\"\u003e \u003ch2\u003eTotal protein content\u003c/h2\u003e \u003cp\u003eThe total protein content in salt-tolerant genotypes DH-22 and DH-29 was significantly higher than in salt-sensitive genotypes DH-11 and DH-12 under control conditions, as shown in Fig.\u0026nbsp;7b. As salt stress increased, the protein content decreased in both the salt-tolerant and sensitive genotypes. In the tolerant genotypes, the total protein content decreased by 11% in DH-22 and 7% in DH-29, while in the sensitive genotypes, the greater reduction was observed, with reductions of 21% in DH-11 and 12% in DH-12 at the 100 mM NaCl salt treatment. Furthermore, at the 150 mM NaCl salt treatment, the total protein content decreased by 13% in DH-22 and 20% in DH-29 among the tolerant genotypes, while it decreased by 28% in DH-11 and 14% in DH-12 among the sensitive genotypes. Tolerant genotypes maintain higher protein content as compared to sensitive genotypes as salt concentrations increase.\u003c/p\u003e \u003cdiv id=\"Sec40\" class=\"Section3\"\u003e \u003ch2\u003eActivity of antioxidant enzymes\u003c/h2\u003e \u003cp\u003e \u003cb\u003eSuperoxide dismutase\u003c/b\u003e \u003c/p\u003e \u003cp\u003eSuperoxide dismutase (SOD) is a crucial antioxidant enzyme involved in scavenging superoxide radicals, offering insights into the plant's response to oxidative stress. It was observed that the specific activity of SOD was significantly higher in tolerant genotypes DH-22 and DH-29 under control conditions and increased significantly with rising salt concentrations in both tolerant and sensitive genotypes (Fig.\u0026nbsp;8a). In tolerant genotypes, the specific activity of SOD increased by 60% (DH-22) and 84% (DH-29), while in sensitive genotypes, it increased by 116% (DH-11) and 105% (DH-12) at 150 mM NaCl salt treatment. At 50 mM NaCl salt treatment, the specific activity of SOD increased by 15% and 19% in tolerant genotypes DH-22 and DH-29, respectively, but in sensitive genotypes DH-11 and DH-12, it increased by 34% and 18%, respectively. The higher SOD activity in tolerant genotypes indicates a more efficient ROS scavenging system, contributing to their ability to survive oxidative stress in salt stress conditions.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAscorbate peroxidase\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe specific activity of APX did not increase significantly at 50 mM NaCl. However, at 100 mM NaCl, the specific activity of APX increased significantly in both salt-sensitive genotypes (DH-11, DH-12) and salt-tolerant genotypes (DH-22, DH-29. In the tolerant genotypes, the specific activity of APX increased by 317% (DH-22), and 322% (DH-29) and in sensitive genotypes, it increased by 281% (DH-11), 231% (DH-12) under 100 mM NaCl salt treatment. Furthermore, under 150 mM NaCl salt treatment, specific activity increased by 587% (DH-22), 622% (DH-29) in tolerant genotypes, and 427% (DH-11), 370% (DH-12) in sensitive genotypes. In particular, under conditions of elevated salt stress, genotypes that were tolerant showed higher APX activity in comparison to sensitive genotypes (Fig.\u0026nbsp;8b). This shows that to efficiently scavenge hydrogen peroxide and lessen salt-induced oxidative stress, the tolerant genotypes may strengthen their antioxidant defense mechanisms by upregulating APX activity.\u003c/p\u003e \u003cp\u003e \u003cb\u003eGuaiacol peroxidase\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe specific activity of GPX was significantly higher in salt-tolerant genotypes DH-22 and DH-29 compared to salt-sensitive genotypes DH-11 and DH-12. This elevated activity was noted as salt concentrations increased. At 150 mM NaCl salt treatment, the activity of GPX increased by 145% (DH-11), 187% (DH-12) in salt-sensitive genotypes and 98% (DH-22), 120% (DH-29) in salt-tolerant genotypes. Additionally, at 50 mM NaCl salt treatment, GPX activity increased by 20% (DH-22), 23% (DH-29) in tolerant genotypes and 53% (DH-11), 72% (DH-12) in sensitive genotypes. The analysis shows that the maximum GPX activity was increased in the sensitive genotypes compared to the tolerant genotypes, the tolerant genotypes still maintained higher GPX activity overall (Fig.\u0026nbsp;8c).\u003c/p\u003e \u003cp\u003e \u003cb\u003eCatalase\u003c/b\u003e \u003c/p\u003e \u003cp\u003eHigher CAT specific activity was detected in the salt-tolerant genotypes DH-22 and DH-29 compared to the salt-sensitive genotypes DH-11 and DH-12. It was observed that CAT activity was increased significantly with higher salt concentrations in both salt-tolerant and salt-sensitive genotypes. In salt-tolerant genotypes, CAT activity increased by 388% (DH-22) and 395% (DH-29) at 150 mM salt treatment and also in salt-sensitive genotypes, it increased by 444% (DH-11) and 482% (DH-12). The 50 mM NaCl treatment produced the smallest boost in CAT activity, with an increase of 77% (DH-22), 72% (DH-29) in salt-tolerant genotypes, and a 70% (DH-11), 72% (DH-12) increment in salt-sensitive genotypes. These results indicate that there is no significant change in both categories of genotypes, but salt-tolerant genotypes exhibit higher CAT activity as compared to salt-sensitive genotypes at all salt concentrations, as illustrated in Fig.\u0026nbsp;8d.\u003c/p\u003e \u003cp\u003e \u003cb\u003eGlutathione reductase\u003c/b\u003e \u003c/p\u003e \u003cp\u003eIn plants, Glutathione reductase (GR) is an important enzyme involved in replenishing reduced glutathione, which helps in the antioxidant protective mechanism when plants are under stress from their environment. In our results, the specific activity of GR was significantly increased as salt concentrations rose in both salt-tolerant genotypes (DH-22, DH-29) and sensitive genotypes (DH-11, DH-12) (Fig.\u0026nbsp;8e). However, the activity of GR was higher in the tolerant genotypes compared to the sensitive genotypes in all treatments. For instance, at 50 mM NaCl salt treatment, GR activity was increased by 34% (DH-22), 37% (DH-29) in tolerant genotypes 26% (DH-11) and 30% (DH-12) in sensitive genotypes. At 150 mM NaCl salt treatment, GR activity increased by 89% (DH-22), 108% (DH-29) in tolerant genotypes and 75% (DH-11), 73% (DH-12) in sensitive genotypes. This analysis revealed that tolerant genotypes may elevate their antioxidant protective mechanism by increasing glutathione reductase activity to maintain the reduced state of glutathione and counteract salt-induced oxidative stress.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCorrelation analysis\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe correlation plot in Fig.\u0026nbsp;9 shows the results of Pearson's correlation analysis of oxidative stress markers and oxidative defence molecules. It includes various factors such as growth-related parameters and enzymatic and non-enzymatic components. Root length (RL), shoot length (SL), dry weight (DW), relative water content (RWC), chlorophyll a\u0026thinsp;+\u0026thinsp;b, carotenoid and protein levels were all strongly positively correlated. On the other hand, hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e), total phenolic content (TPC) and guaiacol peroxidase (GPX) show weak positive correlations. Moreover, the chlorophyll a/b ratio, percentage of ion leakage, and malondialdehyde (MDA) levels exhibited clear negative correlations. It was further observed that proline content and all antioxidant enzymes (SOD, CAT, APX, and GR) showed weak negative correlations, excluding GPX.\u003c/p\u003e \u003cp\u003e \u003cb\u003eHistochemical visualization of superoxide anion, H\u003c/b\u003e \u003csub\u003e \u003cb\u003e2\u003c/b\u003e \u003c/sub\u003e \u003cb\u003eO\u003c/b\u003e \u003csub\u003e \u003cb\u003e2\u003c/b\u003e \u003c/sub\u003e \u003cb\u003eaccumulation and cell viability\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo visualize cell death in \u003cem\u003ein-situ\u003c/em\u003e, we used the trypan blue staining method. We observed the highest level of blue color in trypan blue staining (Fig.\u0026nbsp;10) in the salt-sensitive genotypes (DH-11, DH-12) as compared to the salt-tolerant genotypes (DH-22, DH-29) when treated with 150 mM NaCl salt. It was observed that, as the salt treatment decreased, the blue color gradually faded, reaching its maximum in the salt-tolerant genotypes compared to the salt-sensitive genotypes. As a result, the least amount of blue color was reported in the control groups of both sensitive and tolerant genotypes. Thus, it suggests the lack of cell death in these groups.\u003c/p\u003e \u003cp\u003eTo detect superoxide ions and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e accumulation in both sensitive and tolerant genotypes, we used NBT (Nitro-blue Tetrazolium chloride) and DAB (Diaminobenzidine) staining. When exposed to 150 mM NaCl salt treatment, maximum blue spots were observed in NBT staining and a dark brown color in the salt-sensitive genotypes (DH-11, DH-12) compared to the salt-tolerant genotypes (DH-22, DH-29) (Fig.\u0026nbsp;11). In the control group and both sensitive and tolerant genotypes, minimal blue spots and dark brown colour were observed. However, as salt stress increased, there was a gradual rise in blue color spots and dark brown color, indicating an accumulation of superoxide ions and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, with the maximum accumulation found in the salt-sensitive genotypes, in contrast to the salt-tolerant genotypes.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eAddressing salt stress is crucial for overcoming one of the main challenges to crop productivity and global food security. The pulses like horsegram (poor man’s pulse) contribute greatly to ensure protein rich food, specifically for the unprivileged human population. Moreover, there is considerable variability in horsegram germplasm for salt stress tolerance, which can be utilized for crop improvement. Hence, identifying the biochemical changes related to salt stress tolerance and susceptibility is crucial. The current investigation was carried out to identify salt-stress tolerant and susceptible genotypes in horsegram and unravel the biochemical mechanisms underlying tolerance and susceptibility.\u003c/p\u003e \u003cp\u003eDuring long-term evolution, plants have designed sophisticated responses to various environmental stresses. Within the same species, different germplasms exhibit varying responses to identical stresses following their growth under diverse environmental forms or breeding for specific purposes (Li et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Identifying highly salt-tolerant and sensitive genotypes requires the screening of multiple genotypes.\u003c/p\u003e \u003cp\u003eSalinity tolerance, a complex, polygenic, and quantitative trait, is significantly influenced by environmental conditions, making genotype screening for salt tolerance challenging (Atieno et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The 10-days old seedling screening provides preliminary insights into the variation in salt tolerance among different genotypes (Sikder et al. \u003cspan citationid=\"CR94\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Previous studies by Ramadan et al. (\u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), Kaur et al. (\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2023b\u003c/span\u003e), Kaur et al. (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2022b\u003c/span\u003e) and Shaheenuzzamn, (\u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), identified root length (RL) and shoot length (SL) as critical traits under salinity stress, suggesting their use in genotype screening for salt tolerance.\u003c/p\u003e \u003cp\u003eHorsegram, considered moderately salt-tolerant, exhibits variability in response to saline environments across different genotypes (Pantola et al. 2020). Salt stress negatively impacts the normal growth and development of chickpeas, affecting traits like plant length, fresh and dry weight, germination index, and leaf area (Alom et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTo evaluate the effects of salt stress on different horsegram genotypes, RL, SL, and GI were employed in the current study. As a result, through the analysis of phenotypic and physiological traits, a reliable and efficient method was used to identify horsegram genotypes tolerant to salt stress. The initial step of this analysis was to assess the STI values for RL, SL, and GI. STI is a prevalent tool for selecting superior genotypes adapted to stress (Jha et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The higher the value of STI at the seedling stage of plants, the greater the plants' tolerance to salt stress, which can be attributed to greater accumulation of compatible solutes such as proline, an elevation in the K+, Ca2+, and Mg2+, as well as greater retention of K + in photosynthetic tissues, thereby reducing sodium ion uptake and efficient Na + sequestration in vacuoles (Boussora et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Singh et al. \u003cspan citationid=\"CR95\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Rahman et al. \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Further, the MFV was calculated for all the genotypes for all traits for further validation of results obtained from STI. The MFV value acts as an indicator of plant stress tolerance (Gholizadeh et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Greater the MFV, higher will be the tolerance, and vice-versa. Results show that among 25 genotypes under study, DH-22 and DH-29 displayed the highest mean MFV, and DH-11, DH-12 displayed the least MFV value under salt stress. Thus, in the present study involving 25 genotypes, two genotypes, \u003cem\u003eviz\u003c/em\u003e., DH-22 and DH-29, were identified as salt stress-tolerant, while the other two genotypes, \u003cem\u003eviz\u003c/em\u003e., DH-11 and DH-12, were recognized as salt stress-susceptible.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEffect of salt on growth parameters in tolerant vis-à-vis sensitive genotypes\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe salt sensitive genotypes exhibited higher reduction in shoot and root length, in contrast to the tolerant genotypes at seedling stage under 100 mM NaCl salt stress conditions. (Fig.\u0026nbsp;4a, b). Assessing growth-related parameters is a key method for evaluating salt tolerance in various plant genotypes, at seedling stage of 10 days. Other studies by researchers were also used 10-days seedling for screening of tolerant and sensitive genotypes of different crops (Mi et al. \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Alom et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Khatun et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Mano et al. \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e1996\u003c/span\u003e). Generally, the salt stress affected seedlings exhibit slow growth or reduced mobilization of food, thereby delaying the cell division and damaging the growing hypocotyls (Müller et al. \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). This leads to a metabolic reprogramming so that most of the metabolites are utilized for protection against salt-induced damage rather than promoting growth (Negrão et al. \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). In some similar analysis involving pulses, it was analysed that the salt tolerant genotypes of Pisum sativum and Cajanus cajan revealed a lesser decrease in root-shoot length, fresh weight, and dry weight when compared with the salt sensitive ones. (Khan et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Joshi et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In the present study also, the tolerant genotypes displayed a lesser reduction in dry and fresh weight, when compared to sensitive genotypes (Fig.\u0026nbsp;4c, d).\u003c/p\u003e \u003cp\u003e \u003cb\u003eComparative study on physiological traits\u003c/b\u003e \u003c/p\u003e \u003cp\u003eRWC is an important physiological trait which can be employed as an important indicator to discriminate the salt tolerance ability among genotypes (Sánchez-Rodríguez et al. \u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). The present analysis revealed that RWC declined in both genotypes, yet the tolerant genotypes preserved higher RWC levels compared to the sensitive ones with increasing salt concentrations. Likewise, in pea and chickpea, sensitive genotypes showed a greater decrease in RWC than tolerant genotypes (Khan et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Kaur et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eChlorophyll, a pervasive natural green pigment, is found in the leaves and other parts of plants and is necessary for photosynthesis. (Humphrey \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Zahra et al. \u003cspan citationid=\"CR107\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). When chlorophyll absorbs a photon of light (quantum), it rapidly migrates through the pigments to the reaction center, where photochemical conversion of light energy to chemical energy occurs (Najar et al. \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In the present investigation, the total chlorophyll (chl a + b) and carotenoid content decreased significantly with the increased salt concentration; The tolerant genotypes of horsegram, however, sustained higher chl a + b and carotenoid content than their sensitive genotypes (Fig.\u0026nbsp;5c, d). Moreover, an increase in salt concentrations led to a rise in the Chl a/b ratio in sensitive genotypes, while it dropped in tolerant genotypes. (Fig.\u0026nbsp;5c). The decrease in the chl a/b ratio could be linked to the greater extent of chl b degradation occurring in the sensitive genotypes. Further, in a study conducted on mungbean, it was observed that chl and carotenoid content decreased sharply in sensitive genotypes than the tolerant ones, under salt stress (Alharby et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Sehrawat et al. \u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Here, it is important to mention that the decreases in chlorophyll contents due to higher chlorophyllase activity associated with decline in plant growth under salt stress (Khan et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). The decrease in chlorophyll observed in this analysis was associated with reduced growth.\u003c/p\u003e \u003cp\u003e \u003cb\u003eComparative evaluation of biochemical parameters\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe present revealed that salt stress led to higher levels of MDA, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, and percent ion leakage, although the extent of these increases varied between genotypes. Plants often experience an oxidative burst, involving a significant increase in ROS levels under stress (Kaur et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Salt stress induces the formation of ROS, including superoxide radicals (O\u003csub\u003e2\u003c/sub\u003e⋅\u003csup\u003e−\u003c/sup\u003e) hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e), and hydroxyl radicals (OH⋅) (ElSayed et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). These ROS lead to increased membrane lipid peroxidation, resulting in membrane decay and therefore, an enhanced ion leakage. Moreover, Damage caused by ROS extends to important macromolecules such as lipids, nucleic acids, proteins and photosynthetic pigments (Rezayian et al. \u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In the present analysis, the tolerant genotypes displayed the lesser increase in MDA content, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and percent ion leakage, than the susceptible genotypes with increase salt concentration (Fig.\u0026nbsp;6b, c, d). Further, mungbean sensitive genotypes demonstrated significantly higher levels of MDA content and ROS compared to the tolerant genotypes (Rohman et al. \u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In another experiment on barley and rice, it was observed that % ion leakage increased sharply in sensitive genotypes than the tolerant ones under salt stress (Mahlooji et al. \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Singh et al. \u003cspan citationid=\"CR96\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eGenerally, plants accumulate phenols and osmoprotectant like proline under abiotic stress including salt stress. Free proline and phenols have multiple functions in plant which include regulation of osmotic pressure, protection of membrane integrity, stabilization of enzymes/proteins, maintenance of appropriate NADP\u003csup\u003e+\u003c/sup\u003e, NADPH ratios, scavenger of ROS, and free radicals (Mohamed and Aly \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Misra and Saxena \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Tiwari et al. \u003cspan citationid=\"CR100\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). In the present analysis the tolerant genotypes accumulated higher proline (Fig.\u0026nbsp;6a) and phenolics (Fig.\u0026nbsp;7a) in comparison to sensitive genotypes. The synthesis of phenolic compounds through the phenylpropanoid pathway is enhanced by salinity through the stimulation of the enzyme phenylalanine ammonia lyase (PAL) (Gao et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2008\u003c/span\u003e), and thus leads to a higher TPC (Lim et al. \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Falcinelli et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Kiani et al. proposed that proline and total phenolic content accumulation helps in enhancing stress tolerance under salt stress (Kiani et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). It is reported that salt tolerant genotypes of chickpea and soyabean accumulated higher proline and phenolic content rather than the sensitive genotypes (Kaur et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2022a\u003c/span\u003e; El-Esawi et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAs polymers of amino acids, proteins are essential for various functions in plant structure and various plant functions including plant defense (Jain et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In the present investigation, the protein content decreases with increase in salt concentration in both salt-sensitive and tolerant genotypes (Fig.\u0026nbsp;7b), but in the tolerant genotype’s protein content exhibited greater stability than than sensitive ones. In the previous studies in common bean, a similar reduction in the protein content was reported under stress. However, the salt tolerant genotypes displayed a lesser reduction in protein content than the sensitive genotypes (Azimychetabi and Sabokdast \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Farhangi-Abriz and Torabian, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003eComparative analysis of antioxidant enzymes’ activities\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo reduce the ROS-triggered oxidative damage under various abiotic stress, plants possess a wide array of antioxidants. Besides the non-enzymatic antioxidants in the plant cells like phenolics, plant cells possess enzymatic antioxidants like SOD, APX, GR, and CAT \u003cem\u003eetc\u003c/em\u003e (Sharma et al. \u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In the present investigation, the activities of CAT, APX, GPX, GR, and SOD were analyzed under salt stress.\u003c/p\u003e \u003cp\u003eSuperoxide dismutase (SOD) is an important enzyme which catalyzes the conversion of O\u003csub\u003e2\u003c/sub\u003e⋅\u003csup\u003e−\u003c/sup\u003e into H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. Its activity generally increases under the salt stress conditions (Rasool et al. \u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Khoshbakht et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In the present investigation, the activity of SOD significantly increased under the salt stress; however, the pattern of increase differed among the salt sensitive and tolerant genotypes. In the present analysis, the tolerant genotypes showed a higher rise in SOD activity than the sensitive. Similarly, the activity of SOD in the salt-tolerant genotypes of \u003cem\u003eVigna radiata\u003c/em\u003e was recorded to be higher than the sensitive genotypes (Rohman et al. \u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). This enhanced activity of SOD under stress seems to provide tolerant genotypes with better protection against salinity including oxidative damage of cell membranes, as judged by the lower rates of lipid peroxidation (Fig.\u0026nbsp;8a).\u003c/p\u003e \u003cp\u003eAPX and GR are two key enzymes of the ascorbate-glutathione interaction pathway (Noctor and Foyer \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). APX converts ascorbate into dehydroascorbate, which is employed as a crucial substrate for H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e detoxification (Anjum et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Pang et al. 2010). Both APX and GR are involved in the oxidation and reduction of ascorbate, glutathione and NADPH in the ascorbate-glutathione cycle (Kunert and Foyer, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In the present study, The rise in APX and GR activities under salt stress was greater in the tolerant genotypes compared to the sensitive ones. A corresponding trend was observed in some other investigations on salt stress. Chakraborty et al. (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) and Kaur et al. (\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2023a\u003c/span\u003e) reported that the tolerant genotypes of peanut and chickpea had higher activity of APX and GR than the sensitive genotypes facing salt stress (Figs.\u0026nbsp;8 and 18). The higher APX and GR activity may be able to elevate NADP\u003csup\u003e+\u003c/sup\u003e concentrations to gain electrons emerging from the electron transport chain of photosynthesis, consequently reducing ROS generation (Reddy et al. \u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Catalase scavenges H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e by directly converting it to H\u003csub\u003e2\u003c/sub\u003eO and O\u003csub\u003e2\u003c/sub\u003e in the peroxisomes as well as glyoxysome (Mittler, \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2002\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn the present analysis, CAT activity was sharply enhanced (Fig.\u0026nbsp;8d) with a corresponding sharp decline in H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e content (Fig.\u0026nbsp;6c) in salt tolerant genotypes in comparison to the sensitive genotypes. This suggests that the salt tolerant genotypes are more efficient in scavenging H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e than the sensitive genotypes to protect against oxidative stress. Likewise, higher levels of CAT activity were associated with increased tolerance in soybean genotypes under conditions of salt stress (Arshi et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Further, Kaur et al. (\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2023a\u003c/span\u003e) also reported that the salt tolerant genotypes of chickpeas had elevated CAT activity than the sensitive genotypes.\u003c/p\u003e \u003cp\u003eThe removal of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e from chloroplasts is mediated by another important antioxidant enzyme, GPX (Asada, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Salt stress increases GPX activity in plants adapted or tolerant to higher salt concentration (Rahnama and Ebrahimzadeh, \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). In the present study, it was evident that higher salt concentration (150 mM NaCl) was led to correspondingly higher GPX activity in tolerant genotypes than in sensitive genotypes (Fig.\u0026nbsp;8c). Similar finding was made by Desouky et al. (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), which reported a higher GR activity in the tolerant genotypes of faba bean in comparison to the sensitive genotypes.\u003c/p\u003e \u003cp\u003eOverall, the analysis revealed that the activity of SOD, CAT, APX, GPX, and GR antioxidant enzymes was assessed as higher in horsegram salt-tolerant genotypes in comparison with salt-sensitive genotypes. Hu et al. (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) also reported that the salt-tolerant genotypes had higher antioxidant enzyme activity and had better protection against the oxidative stress than the salt-sensitive genotypes.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDifferential histochemical staining\u003c/b\u003e \u003c/p\u003e \u003cp\u003eRecently, various histochemical staining methods in plants have been employed by several investigators for the \u003cem\u003ein-situ\u003c/em\u003e visualization of selected plant metabolites (Sharma et al. \u003cspan citationid=\"CR93\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Sharma et al. \u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). In the present study, the trypan blue staining for vitalization of cell viability corroborated the results of percent ion leakage. Ion leakage is considered as an important indicator of cell death (Sharma et al. \u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), thus the tolerant genotypes stained lesser than the susceptible genotypes under the present investigation (Fig.\u0026nbsp;10). Whereas, NBT staining for visualising superoxide (O\u003csub\u003e2\u003c/sub\u003e⋅\u003csup\u003e−\u003c/sup\u003e) and DAB staining for visualising H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e corroborated the results of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e quantification suggesting a stronger oxidative burst in salt-sensitive genotypes in comparison with salt-tolerant genotypes (Fig.\u0026nbsp;11, 12). A similar increase in superoxide (O\u003csub\u003e2\u003c/sub\u003e⋅\u003csup\u003e−\u003c/sup\u003e) and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e under salt stress in chilli and maize crops were recorded by Aktas (Aktas et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) and Yang (Yang et al. \u003cspan citationid=\"CR104\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe present investigation suggests that the tolerant genotypes have a stronger overall biochemical defence machinery than sensitive genotypes in mitigating the negative effects of salt stress. Hence, assessing germplasm variability for salt stress tolerance and identification of tolerant genotypes can be helpful in crop improvement through biotechnological interventions in horsegram.\u003c/p\u003e "},{"header":"Conclusion and future prospects","content":"\u003cp\u003eSalt stress represents a major restriction for plant growth and productivity, especially under saline environments. It causes severe adverse effects on a plants growth and physiology, with an array of associated biochemical changes. In any crop improvement programme against salt stress, it is imperative to explore the variability in the crop germplasm for salt stress tolerance. In the present study, 25 collected genotypes of horsegram with diverse characteristics were screened to identify and shortlist two salt stress tolerant genotypes \u003cem\u003ei.e.\u003c/em\u003e, DH-22 and DH-29 and two sensitive genotypes i.\u003cem\u003ee.\u003c/em\u003e, DH-11 and DH-12. The current study has provided an insight into salt stress physiology in horsegram, which emphasizes its promise as a hardy crop for saline agriculture. In order to look for the associated biochemical reasons for the salt stress tolerance, a thorough biochemical analysis of the selected genotypes were carried out at varying salt concentrations. The tolerant genotypes had higher growth and improved biomass accumulation under salt stress. The tolerant genotypes had lesser ROS accumulation, MDA content, and cell death, but a higher RWC, free proline accumulation, chlorophyll content, and phenols accumulation than the susceptible genotypes. The tolerant genotypes may be effectively used in plant breeding programmes to establish salt-resistant varieties offering higher yields under saline environments.\u003c/p\u003e\u003cp\u003eFurther, an attempt might also be made in future to precisely identify the genes intricate in salt stress tolerance through the transcriptomic investigation of the contrasting genotypes in relations of salt stress tolerance. It will facilitate the better understanding of the molecular pathways and regulatory networks connected to salt tolerance in horsegram. Further, it will help in targeting the genes involved in conferring salt stress tolerance to develop salt stress tolerance transgenics using biotechnological interventions. Hence, the present analysis provides a baseline information for the available tolerance to salt stress in horsegram germplasm with the overall aim to achieve global food security and agricultural sustainability.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eConceptualization, N.S., A.S. and R.K..; Data Curation, N.S. and V.M.; Writing-Original Draft Preparation, N.S.; Writing-Review and Editing, N.S., V.M., A.S. and R.K.; Figure and Table, N.S. and V.M.; Supervision, A.S. and R.K.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eCorresponding authors acknowledge the financial grant received from the Council of Scientific and Industrial Research (CSIR-HRDG), New Delhi, India, through an extramural research grant vide project number 38(1477)/19/EMR-II. Corresponding authors also acknowledge the infrastructural and financial support provided by DAV University, Jalandhar vide sanction order DAVU/REGR/2023/278(a).\u003c/p\u003e\u003ch2\u003eData availability statement\u003c/h2\u003e \u003cp\u003eData sharing is not applicable as no new data were generated or analysed during this study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAebi H (1984) Catalase in Vitro. Methods Enzymol 105:121\u0026ndash;126. https://doi.org/10.1016/S0076-6879(84)05016-3\u003c/li\u003e\n\u003cli\u003eAhmad I, Akhtar MJ, Asghar HN et al (2016) Differential effects of plant growth-promoting rhizobacteria on maize growth and cadmium uptake. 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Plant Physiol 137:1189\u0026ndash;1196. https://doi.org/10.1104/pp.104.058891\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table 1","content":"\u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height:150%;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:150%;font-family:\"Times New Roman\";'\u003eTable 1. Categorisation of horsegram genotypes on the basis of MFV under 100 mM NaCl stress\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003ctable style=\"border-collapse: collapse;border: none;width: 602px;\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 75.8pt;border: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003eGenotype\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 74.75pt;border-top: 1pt solid windowtext;border-right: 1pt solid windowtext;border-bottom: 1pt solid windowtext;border-image: initial;border-left: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003eMFV (RL)\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 74.75pt;border-top: 1pt solid windowtext;border-right: 1pt solid windowtext;border-bottom: 1pt solid windowtext;border-image: initial;border-left: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003eMFV (SL)\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 74.75pt;border-top: 1pt solid windowtext;border-right: 1pt solid windowtext;border-bottom: 1pt solid windowtext;border-image: initial;border-left: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003eMFV (GI)\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 74.8pt;border-top: 1pt solid windowtext;border-right: 1pt solid windowtext;border-bottom: 1pt solid windowtext;border-image: initial;border-left: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003eMean MFV\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 75.95pt;border-top: 1pt solid windowtext;border-right: 1pt solid windowtext;border-bottom: 1pt solid windowtext;border-image: initial;border-left: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: normal;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003eTolerance level\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 75.8pt;border-right: 1pt solid windowtext;border-bottom: 1pt solid windowtext;border-left: 1pt solid windowtext;border-image: initial;border-top: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 150%;'\u003e\u003cspan style='font-size:16px;line-height:150%;font-family: \"Times New Roman\",serif;'\u003eDH-2\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 74.75pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 150%;'\u003e\u003cspan style='font-size:16px;line-height:150%;font-family: \"Times New Roman\",serif;'\u003e0.043\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 74.75pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 150%;'\u003e\u003cspan style='font-size:16px;line-height:150%;font-family: \"Times New Roman\",serif;'\u003e0.325\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 74.75pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 150%;'\u003e\u003cspan style='font-size:16px;line-height:150%;font-family: \"Times New Roman\",serif;'\u003e0.666\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 74.8pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 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74.75pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 150%;'\u003e\u003cspan style='font-size:16px;line-height:150%;font-family: \"Times New Roman\",serif;'\u003e0.685\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 74.8pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:justify;line-height: 150%;'\u003e\u003cspan style='font-size:16px;line-height:150%;font-family: \"Times New Roman\",serif;'\u003e0.578\u003c/span\u003e\u003c/p\u003e\n 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Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"antioxidant enzymes, proline, lipid peroxidation, histochemical staining, NaCl stress","lastPublishedDoi":"10.21203/rs.3.rs-5114057/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5114057/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSoil salinity is one of the significant challenges affecting seed germination, growth, physiology, and crop productivity worldwide. Horsegram is known for its higher tolerance to stress conditions than other legumes and therefore is a potential pulse and fodder crop in salt-affected areas. During this study, 25 horsegram genotypes were screened under a controlled environment to identify salt-tolerant and sensitive genotypes based on the salt tolerance index and membership function value of 10-day-old seedlings. DH-22 and DH-29 were identified as salt-tolerant, while DH-11 and DH-12 were identified as salt-sensitive genotypes. These genotypes were further analyzed under 0, 50, 100, and 150 mM NaCl treatments to examine various growth, physiological, and biochemical parameters. The analysis revealed that the tolerant genotypes exhibited higher root and shoot length, dry and fresh weight, relative water content, chlorophyll and carotenoids content, free proline and phenolic content, and enhanced activity of antioxidant enzymes such as catalase, ascorbate peroxidase, glutathione reductase, superoxide dismutase, and guaiacol peroxidase. Further, lipid peroxidation, hydrogen peroxide content, and percent ion leakage decreased in the tolerant genotypes than in the sensitive genotypes. Additionally, the tolerant genotypes displayed less cell death and lower accumulation of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and superoxide ions in histochemical staining, which may play a vital role in cellular protection during salt stress tolerance.\u003c/p\u003e","manuscriptTitle":"Exploring Salt Stress Tolerance in Horsegram (Macrotyloma uniflorum): Insights from Growth, Physiology and Biochemical Approaches","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-20 06:28:52","doi":"10.21203/rs.3.rs-5114057/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"0f761270-9775-43ce-b88b-f7f42f927f7a","owner":[],"postedDate":"November 20th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-12-16T08:53:08+00:00","versionOfRecord":[],"versionCreatedAt":"2024-11-20 06:28:52","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5114057","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5114057","identity":"rs-5114057","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}