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Full Research Article

Drought Stress Responses in Grass Pea (Lathyrus sativus L.): A Physio-biochemical and Molecular Approach

M
Mala Kumari1,*
0000-0002-9045-772X
R
Rajeev Kumar2
P
Pankaj Kumar Mishra3
A
Anand Kumar4
R
Ranju Kumari1,*
D
Dharamsheela Thakur5
J
Jyoti Kumari1
1Department of Genetics and Plant Breeding, Bihar Agricultural University, Sabour Bhagalpur-813 210, Bihar, India.
2Division of Plant Physiology and Biochemistry, ICAR-Indian Institute of Sugarcane Research, Lucknow-226 002, Uttar Pradesh, India.
3Division of Agriculture, Ramakrishna Mission Vivekananda Educational and Research Institute, Ranchi-834 008, Jharkhand, India.
4Department of Genetics and Plant Breeding, Faculty of Agricultural Sciences, GLA University, Mathura-281 406, Uttar Pradesh, India.
5Department of Molecular Biology and Genetic Engineering, Bihar Agricultural University, Sabour Bhagalpur-813 210, Bihar, India.

  • Submitted03-11-2025|

  • Accepted13-05-2026|

  • First Online 04-06-2026|

  • doi 10.18805/LR-5598

ABSTRACT

Background: Grass pea is a valuable legume crop due to its high protein content and adaptability to harsh environment. Drought is one of the most significant abiotic stresses affecting plant growth, development and productivity. Drought stress has a major impact on the number of physiological and biochemical attributes. Understanding physio-biochemical and molecular responses under drought is crucial for identifying tolerant genotypes and improving stress-resilient cultivars.

Methods: The present study was conducted at Research Cum Instructional Farm, IGKV Raipur, Chhattisgarh, India during rabi season under rainfed condition. A total of 20 Lathyrus genotypes were evaluated for drought tolerance. The experiments were conducted in randomized block design (RBD) with three replications under non-stress (control) and drought stress conditions. Drought was imposed at the flowering stage by withholding irrigation until 80% soil available water was depleted, measured gravimetrically. Standard agronomic practices were followed. Physiological parameters such as leaf area, relative water content (RWC) and chlorophyll content were measured. Biochemical analysis includes different enzymatic activity like SOD, CAT, PPO, POD and ODAP. The molecular analysis was performed using ISSR markers.

Result: Physiological, biochemical and molecular traits is affected by drought stress. Reduction of physiological parameters is observed in stressed plant compared to control. Genotype RLK-83 and RLK-169 exhibit better drought tolerance and lower ODAP content. RLK-78 and RLK-169 showed the highest similarity, while RLK-150 and RLK-310 were the most genetically distinct.

INTRODUCTION

Grass pea (Lathyrus sativus L.) is an annual edible legume crop holds immense significance as a high-protein crop that can be exploited to ensure nutritional security, especially in regions prone to changing environmental conditions (Gupta et al., 2024).  It can also use as a valuable forage due to its high nutritional quality and adaptability (Yildiztekin and Polat, 2026). Ethiopia, Eritrea, India, Bangladesh and Nepal are the major grass pea grower, robustness nature of the crop against drought and flooding and ability to thrive with minimal inputs are the prime reason to cultivate this crop (Edwards et al., 2023). The nutritional value of grass pea extends beyond its high protein content, as it is the sole known source of L-homoarginine (Solovyeva et al., 2020). However, presence of an antinutritional factor β-N-oxalyl-L-α, β-diamino propionic acid (β-ODAP or BOAA), restricts the consumption and acreage area of the crop. Amino acid deficiency of methionine and cysteine causes nutritional deficiency which leads to enhances the β-ODAP, induced neurotoxicity and causes neurolathyrism (Gupta et al., 2024; Yazici et al., 2020) . Thus forth, it becomes a prime concern to reduce β-ODAP content in lathyrus seeds to ensure its contribution in providing food and nutritional security, particularly in developing region of the globe (Lambein et al., 2019). Moreover, β-ODAP levels in grass pea plants and seeds exhibited variability across different locations which influenced by genotype, environmental factors and their complex interactions (Jiao et al., 2011). In context of this some studies have been reported the positive association between high levels of β-ODAP biosynthesis and water stress in grass pea (Basaran et al., 2016; Verma et al., 2022).
       
Drought is one of the most significant abiotic stresses affecting plant growth, development and productivity (Kumar et al., 2023). Drought stress has a major impact on the number of physiological and biochemical attributes such as reduction in the water potential, leaf relative water content and chlorophyll content (Yadav et al., 2013).  Unlike other legumes, the grass pea possesses unique morphological and physiological traits, including thin leaves, winged stem margins, a comprehensive root system and efficient water use efficiency to minimize the stress impact (Kong et al., 2022). This distinctive morpho-physiological character of grass pea contributes in imparting drought tolerance which is emphasizing the crop resilience potential (Lambein et al., 2019).
       
To get the better insight of drought induced responses biochemical markers and associated molecular study are such an important tool to assess the crop response in real time. Furthermore, biochemical mechanism associated with these markers and molecular expression study pattern will accelerate the breeding efforts in an uninterrupted pattern and it could be a sustainable approach to manage the crop (Kumar et al., 2023). Alteration in metabolism under drought stress has been observed and indicates the ROS mediated shift in metabolism from primary to secondary to scavenge the ROS. To maintain a balance between ROS generation and its removal plants developed a number of mechanisms to neutralize these deleterious effects. Endogenous defense weaponry of plants including enzymatic and non-enzymatic antioxidant system get activated upon ROS exposure (Du et al., 2020). Among the enzymatic antioxidants, superoxide dismutase (SOD), polyphenol oxidase (PPO), peroxidases (POD) and catalase (CAT) has key role in ROS scavenging and these are the biochemical marker for drought tolerance in plants (Sharma et al., 2022).
       
Molecular marker is a proven effective approach to identifying drought tolerance in grass pea cultivars, IISR markers are such a representative that can be utilized in selection and development of climate resilient varieties (Younis et al., 2020; Das et al., 2021).  IISR markers has both AFLP and RAPD characteristics, which enables it to get precise and efficient assessments without requiring prior knowledge of primer sequences, making them extremely useful in evolutionary biology, genome mapping, gene tagging and studies of genetic diversity and phylogeny (Lalrinmawii et al., 2023).
       
Keeping all these facts into consideration present study highlights grass pea’s potential as a resilient crop for drought-prone areas. Physio-biochemical analysis reveals plant responses to drought stress, while genetic diversity using ISSR markers informs breeding programs. Grass pea’s adaptability can contribute to agriculture and plant stress research, offering valuable insights for developing drought-resistant varieties.

MATERIALS AND METHODS

Experimental setup
 
The present study was conducted at Research Cum Instructional Farm, IGKV Raipur, Chhattisgarh, India during rabi season 2021-22 and 2022-23, under rainfed condition. A total of 20 Lathyrus genotypes were evaluated for drought tolerance (Table 1). Drought was imposed at flowering stage through water withholding, while a similar set of crops were raised with adequate amount of available water to check the performance of the crop. Drought stress was imposed through depletion of 80% soil available water and it was measured gravimetrically. The experiments were conducted in randomized block design (RBD) with three replications under non-stress (control) and drought stress conditions. All the genotypes undertaken were raised in a plot of   2 m length keeping row to row distance was 30 cm and 10 cm between plants. Standard agronomic package and practices (Give N, P and K requirement of the crop) were followed as per the recommendation for the crop in the region. 

Table 1: Genotypes accessions of grass pea for drought screening.


 
Analysis of physiological activity
 
Leaf area
 
The length of the midrib is measured thereafter widest width of leaf and width towards tip and base is measured. All the measured width is averaged and multiplied with length.
 
Relative water content (RWC)
 
The leaf relative water content was measured following the method of Barrs and Weatherley (1962), using the following formula:

   
Estimation of chlorophyll
 
The chlorophyll a, chlorophyll b and total chlorophyll content in the leaf sample was estimated as per Arnon (1949). Content of chlorophyll a, chlorophyll b and total chlorophyll were estimated through following formulae:
 
Chl a = 12.9 (A663) - 2.69 (A645) × V /1000 × W
 
Chl b = 22.9 (A645) - 4.68 (A663) × V/ 1000 × W
 
Total chl = Chl a + Chl b
 
Analysis of biochemical activity
 
Enzymatic analysis estimation of 20 genotypes in control and drought condition in each replication was recorded. The leaf samples were collected, processed and analyzed for different enzymatic (SOD, CAT, PPO and POD) activity. The measurement of SOD activity was based on the method given by Beauchamp and Fridovich (1971). Catalase activity was determined by monitoring the disappearance of H2O2 at 240 nm (ε = 40 mM-1 cm-1) as per the method by (Aebi et al., 1984). Polyphenol oxidase activity was estimated as per method described by Sarvesh and Reddy (1988). Peroxidase activity was measured the method reported earlier (Castillo et al., 1984).  ODAP (β -N-oxalyl-L- α, β-diamino propionic acid) content in dry seeds was estimated by using the OPT suggested by Briggs et al. (1983).
 
Molecular studies
 
Molecular diversity among selected genotypes was assessed using ISSR markers following DNA extraction by the modified CTAB method (Jonathan, 1990) and genetic relationships were analyzed using UPGMA clustering with Jaccard’s coefficient in NTSYSpc-2.02e. Eleven ISSR markers with known sequences were taken for the study, out of which nine ISSR primer shows amplification patterns were identified namely, UBC-809, UBC-811, UBC-823, UBC-834, UBC-835, UBC-840, UBC-842 UBC-885 and ISSR1 for polymorphism study. Furthermore, theses markers were used for PCR amplification on all of the selected genotypes.

RESULTS AND DISCUSSION

Effect of drought stress on leaf area, relative water content (RWC) and chlorophyll content 
 
Leaf area
 
Across all genotypes under the study, reduction in leaf area has been observed under drought conditions relative to control grown counterparts. The decrease in leaf area in drought grown lathyrus genotypes indicating the negative impacts of drought stress on leaf growth, consequently growth and development of the crop get hampered. However, genotypes, such as RLK-29 and RLK-30, displayed a significant reduction in leaf area under drought stress which indicates their sensitivity against drought (Fig 1A). Moreover, relatively higher leaf area in RLK-83 and RLK-169 suggesting the tolerance behavior of these genotypes. The minimum reduction in leaf area during drought stress has been observed in RLK-81 and RLK-150 and can be recommended as drought-tolerant that could be useful for breeding programs targeting drought resilience. The substantial loss of leaf area in these genotypes suggests a compromised photosynthetic capacity, as a smaller leaf area reduces the plant’s ability to capture sunlight and produce energy. This sensitivity highlights the need for improvement in such genotypes to enhance their adaptability to drought conditions. Jiang et al., (2013) reported that 20% PEG stressed grass pea seedling for five days shows inward curled leaf margin. Jafarinasab et al., (2022) worked on nine local grass pea genotypes (Baft_1, Baft_2, Bardsir, Dehbakri, Kuhbanan, Rabor, Sirjan, Shiraz and Torbat Heydarieh) collected from different climatic zone of Iran. He found that the leaf area is decreased while soluble sugars and proline content is increased under terminal drought condition (Water withholding at flowering stage). Reduction in leaf area helps in decreases transpirational loss while increased sugar and proline help in osmotic adjustment.

Fig 1: (A) Leaf area (cm2), (B) Relative water content (RWC %) and (C) Total chlorophyll content in grass pea within the leaves of 20 genotypes under both control and drought stress conditions.


       
Shamsaee et al., (2025) also found the leaf area index reduction up to 20.32% shows in grass pea under drought stress compared to irrigated condition over two years of treatment (Shamsaee et al., 2025). Many plants show morphological and physiological alterations, due to drought stress like decrease in leaf area, wilting, stomata closure etc. (Bangar et al., 2019).
 
Relative water content
 
Despite the drought tolerant behavior reduction in relative water content has been recorded in all the genotypes grown under drought regime as compared to non-stress counterparts, demonstrating the water stress and its impacts on water retention capacity of leaves. Genotypes RLK-169 and RLK-83 shown relatively higher RWC under drought, suggesting its better water retention capacity and it could be a potential candidate for drought tolerance (Fig 1B). Moreover, RLK-30 and RLK-40 exhibited a significant reduction in RWC indicates its sensitive behavior against drought stress. Similar finding was reported by Talukdar (2013) and Verma et al., (2022). Aloui et al., (2023) also confirmed the reduction in Relative leaf water content (RLWC) in lathyrus decreased by 15% under heat stress and 27% under combined heat and drought stress conditions. Similarly, Chettri et al., (2021) also reported the reduction of RWC under combined drought and salinity stress condition. Shamsaee et al., (2025) revealed that an average 5.5% of RWC is decreased under drought condition compared to normal condition in, while it enhances up to 9.25% by Rhizobium inoculation.
 
Chlorophyll content
 
Chlorophyll content in response to drought condition imposed through different water regime displayed the differential pattern. More specifically chlorophyll content (Chl a and Chl b) was higher in normally grown grass pea genotypes relative to drought stressed grown crop. Amongst the genotypes, RLK-310 (2.6 mg/g), RLK-296 (2.5 mg/g), RLK-24 (2.5 mg/g), RLK-78 (2.4 mg/g) and RLK-24 (2.4 mg/g) displayed higher chlorophyll content under drought conditions (Fig 1C). The higher chlorophyll content noticed in these genotypes suggesting their drought tolerance behavior relative to other genotypes in our study. Notably, the chlorophyll content among the 20 genotypes under drought conditions ranged from 2.1 mg/g (RLK-29, RLK-150) to 2.6 mg/g (RLK-310), with an overall mean of 2.32 mg/g. Sen et al., (2025), studied on five grass pea genotypes (GM-04, GM-02, GM-03, BARI Khesari-2 and BINA Khesari-2) to assess the drought tolerance under three water regimes: control, 60% and 40% field capacity. Under control and moderate field capacity chlorophyll reduction is less compared to stressed condition (40% field capacity).
       
Chlorophyll-a and chlorophyll-b as well as carotenoids showed significant decreases in response to increasing stress level reported by (Kiani et al., 2020). Chettri et al., (2021) documented that the combined effect of drought and salinity stress reduced the chlorophyll content in grass pea. Monteoliva et al., (2021) also find the similar result. Leaf chlorophyll concentration dropped up to 58% under combined drought and heat stress while 37% reduction under individual heat stress compared to controlled condition reported by Aloui et al., (2023). Shamsaee et al., (2025) found that the total chlorophyll content in lathyrus crops decreased by up to 20.3% under drought stress. Our findings align with these studies, indicating a consistent pattern of chlorophyll degradation under stress conditions.
 
Effect of drought stress on antioxidant enzyme activities and ODAP content
 
Effect of drought stress on antioxidant enzymes activities
 
Antioxidant enzymes such as SOD, CAT, PPO and POD activity were assayed for all the 20 genotypes under study in both drought stressed and control grown Lathyrus crop. The activities of these enzymes in Lathyrus leaves significantly enhanced in drought stressed crop relative to normally grown plants (Fig 2). Elevated levels of reactive oxygen species (ROS) induce the activation of antioxidant enzymes in plants, such as ascorbate peroxidase (APX), catalase (CAT), peroxidase (POD) and superoxide dismutase (SOD). These enzymes are vital in preserving cell membrane integrity and preventing lipid peroxidation by efficiently scavenging ROS (Zeng et al., 2023). Genotype RLK-296 shown remarkably higher SOD activity amongst the all genotypes during drought stress. While, RLK-991, RLK-513 and RLK-24 displayed moderate SOD activity. Moreover, the rest of the genotypes revealed more or less comparable SOD activity in drought stressed crop (Fig 2A).

Fig 2: Illustrates alterations in SOD (A), CAT (B), PPO (C) and POD (D) activities in grass pea within the leaves of 20 germplasm under both control and drought stress conditions.


       
The highest catalase activity recorded in RLK-296, while RLK-991, RLK-513 and RLK-24 are categorized as moderate in terms of their catalase activity (Fig 2B). The remaining genotypes displayed the relatively less variation in catalase activity. Exposure to drought stress led to remarkable enhancement in polyphenol oxidase activity in genotypes of grass pea under study (Fig 2C). Moreover, RLK-991, RLK-581 and RLK-24 shows higher activity over other genotypes, while PPO activity in remaining accessions does not display significant changes. The significant increase in peroxidase activity was recorded in drought stress grown crop relative to non-stressed crop (Fig 2D). Remarkably, RLK-40, RLK-51, RLK-310 and RLK-81 displayed the elite behavior in terms of higher POD activity. Moreover, RLK-991, RLK-150, RLK-513, RLK-310, RLK-345 and RLK-24 exhibited moderate increase in POD activity over its normally grown counterparts. It is widely accepted that active oxygen species are responsible for various stress-induced damages to macromolecules and ultimately to cellular structures.
       
Tokarz et al., (2020) observed that the activity of POD and CAT in root and shoot of grass pea seedling under drought and salinity stress. He found that the activity of CAT increase in root and shoot of PEG media and decreased in shoot in NaCl media. The POD activity increased in shoot in both media while decreased in root in PEG media and increased in NaCl media. The basis is NaCl (salinity)and PEG (drought) execute different form of osmotic and ionic stress which leads to tissue specific antioxidant enzyme regulation.
       
The increase in antioxidant enzyme activities like SOD, CAT, POD is more in lathyrus compared to pea under 20% PEG stressed media (Jiang et al., 2013). Because lathyrus is inherently drought tolerant and have more effective ROS-scavenging defense system.
       
The activity of antioxidant enzyme under drought stress is also reported in different crops including soybean (Wang et al., 2019), faba bean (Desoky et al., 2021), pigeon pea (Radadiya et al., 2016), tomato (Raja et al., 2020), wheat (Abid et al., 2018) etc.
 
ODAP
 
The ODAP (β-N-Oxalyl-L-α, β-diaminopropionic acid) content (%) increases under drought conditions across all genotypes, suggesting that drought stress enhances ODAP accumulation in grass pea (Fig 3). Basaran et al., (2016) documented a positive correlation between elevated β-ODAP biosynthesis and water stress in grass pea. Moreover, RLK-30 and RLK-24 displayed highest ODAP content in both stressed and non-stress plants indicating the natural accumulation of ODAP in Lathyrus. RLK-83 and RLK-169 shown relatively lower ODAP content, even under drought conditions, making them preferable for breeding programs targeting low-toxin varieties. Aloui et al., (2023) reported a significant rise in ODAP content under drought stress and combined drought-heat stress, with increases of 33% and 83%, respectively, compared to normal conditions.

Fig 3: Illustrates alterations in ODAP content in grass pea within the leaves of 20 germplasm under both control and drought stress conditions.


       
Verma et al., (2022) also observed the positive correlation of drought stress and β-ODAP content in grass pea. She used transcriptome profiling of two genotype, a somaclone ratan and its parent pusa-24. Differential gene expression analysis showed the up regulation of β-ODAP biosynthetic gene under stress condition.
       
Tokarz et al., (2020) found that the ODAP accumulation is increase under PEG stress in lathyrus seedling. He found that the ODAP content in shoot is higher in PEG media compared to NaCl whereas, its content is more in root in NaCl media compared to PEG media. This is because differential accumulation of ODAP, NaCl stress encourages root-localized accumulation whereas PEG-induced drought stress selectively boosts ODAP synthesis and transport to shoots. These findings suggest that environmental stressors play a crucial role in modulating β-ODAP levels, potentially impacting the safety and resilience of grass pea crops.
 
Molecular diversity analysis of genotypes
 
Similarity matrices were obtained by using NTSYS (Numerical Taxonomy System Biostatistics) computer programme. Cluster analysis was performed using UPGMA (unweighed pair group method with arithmetic averages) method that displayed the similarity coefficient 0.50 to 0.95 among the genotypes.
       
At the mean similarity coefficient of 0.72, analyzed genotypes classified into 5 clusters in dendrogram (Fig 4). Cluster I consists of eight genotypes namely, RLK-29, RLK-580, RLK-581, RLK-30, RLK-9, RLK-513, RLK-345 and RLK-962; whereas, RLK-51 and RLK-81 are in cluster II. However, cluster III consists of four genotypes namely, RLK-78, RLK-169, RLK-46 and RLK-991. Cluster IV and cluster V representing RLK-150 RLK-310 respectively. RLK-78 and RLK-169 are most similar with Similarity coefficient 0.95 and RLK-150 and RLK-310 are least similar with Similarity coefficient 0.71 (Fig 4). Further research by Younis et al., (2020) demonstrated that ISSR markers effectively identify genetic traits associated with drought tolerance, facilitating the selection and development of more resilient cultivars. For grass pea breeding programme ISSR marker is mostly utilized for analyzing diversity and evolutionary relationship among the species (Das et al., 2021).

Fig 4: Dendrogram depicts the genetic relationships among the sixteen different lines, established through analysis of ISSR markers.

CONCLUSION

Drought stress significantly affects physiological traits and ODAP content in grass pea genotypes, Genotype RLK-83 and RLK-169 exhibit better drought tolerance and lower ODAP content. These genotypes are promising candidate for breeding program targeting low toxin and enhanced drought tolerance. RLK296 possess a strong antioxidant defense system, reducing oxidative damage caused by drought induced reactive oxygen species. Such genotype are promising candidates for breeding programs aimed at developing drought-tolerant grass pea cultivars. Genetic diversity analysis using ISSR markers revealed significant polymorphism among grass pea genotypes, with similarity coefficients ranging from 0.50 to 0.95. UPGMA clustering divided the genotypes into five clusters, indicating genetic variation. RLK-78 and RLK-169 showed the highest similarity (0.95), while RLK-150 and RLK-310 were the most genetically distinct (0.71). This diversity provides valuable insights for selecting diverse parents in breeding programs to enhance drought tolerance and other desired traits.

ACKNOWLEDGEMENT

The authors are sincerely thankful to Indira Gandhi Krishi Vishwavidyalaya, Raipur, Chhattisgarh for providing financial support and necessary facilities in carrying out the present investigation. Authors are also thankful to Publication Unit of BAU Sabour for providing communication number (BAU Communication No. 1821/240813) for submission of article for publication.
 
Disclaimers
 
The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.

CONFLICT OF INTEREST

All authors declare that they have no conflict of interest.

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    Drought Stress Responses in Grass Pea (Lathyrus sativus L.): A Physio-biochemical and Molecular Approach

    M
    Mala Kumari1,*
    0000-0002-9045-772X
    R
    Rajeev Kumar2
    P
    Pankaj Kumar Mishra3
    A
    Anand Kumar4
    R
    Ranju Kumari1,*
    D
    Dharamsheela Thakur5
    J
    Jyoti Kumari1
    1Department of Genetics and Plant Breeding, Bihar Agricultural University, Sabour Bhagalpur-813 210, Bihar, India.
    2Division of Plant Physiology and Biochemistry, ICAR-Indian Institute of Sugarcane Research, Lucknow-226 002, Uttar Pradesh, India.
    3Division of Agriculture, Ramakrishna Mission Vivekananda Educational and Research Institute, Ranchi-834 008, Jharkhand, India.
    4Department of Genetics and Plant Breeding, Faculty of Agricultural Sciences, GLA University, Mathura-281 406, Uttar Pradesh, India.
    5Department of Molecular Biology and Genetic Engineering, Bihar Agricultural University, Sabour Bhagalpur-813 210, Bihar, India.

    • Submitted03-11-2025|

    • Accepted13-05-2026|

    • First Online 04-06-2026|

    • doi 10.18805/LR-5598

    ABSTRACT

    Background: Grass pea is a valuable legume crop due to its high protein content and adaptability to harsh environment. Drought is one of the most significant abiotic stresses affecting plant growth, development and productivity. Drought stress has a major impact on the number of physiological and biochemical attributes. Understanding physio-biochemical and molecular responses under drought is crucial for identifying tolerant genotypes and improving stress-resilient cultivars.

    Methods: The present study was conducted at Research Cum Instructional Farm, IGKV Raipur, Chhattisgarh, India during rabi season under rainfed condition. A total of 20 Lathyrus genotypes were evaluated for drought tolerance. The experiments were conducted in randomized block design (RBD) with three replications under non-stress (control) and drought stress conditions. Drought was imposed at the flowering stage by withholding irrigation until 80% soil available water was depleted, measured gravimetrically. Standard agronomic practices were followed. Physiological parameters such as leaf area, relative water content (RWC) and chlorophyll content were measured. Biochemical analysis includes different enzymatic activity like SOD, CAT, PPO, POD and ODAP. The molecular analysis was performed using ISSR markers.

    Result: Physiological, biochemical and molecular traits is affected by drought stress. Reduction of physiological parameters is observed in stressed plant compared to control. Genotype RLK-83 and RLK-169 exhibit better drought tolerance and lower ODAP content. RLK-78 and RLK-169 showed the highest similarity, while RLK-150 and RLK-310 were the most genetically distinct.

    INTRODUCTION

    Grass pea (Lathyrus sativus L.) is an annual edible legume crop holds immense significance as a high-protein crop that can be exploited to ensure nutritional security, especially in regions prone to changing environmental conditions (Gupta et al., 2024).  It can also use as a valuable forage due to its high nutritional quality and adaptability (Yildiztekin and Polat, 2026). Ethiopia, Eritrea, India, Bangladesh and Nepal are the major grass pea grower, robustness nature of the crop against drought and flooding and ability to thrive with minimal inputs are the prime reason to cultivate this crop (Edwards et al., 2023). The nutritional value of grass pea extends beyond its high protein content, as it is the sole known source of L-homoarginine (Solovyeva et al., 2020). However, presence of an antinutritional factor β-N-oxalyl-L-α, β-diamino propionic acid (β-ODAP or BOAA), restricts the consumption and acreage area of the crop. Amino acid deficiency of methionine and cysteine causes nutritional deficiency which leads to enhances the β-ODAP, induced neurotoxicity and causes neurolathyrism (Gupta et al., 2024; Yazici et al., 2020) . Thus forth, it becomes a prime concern to reduce β-ODAP content in lathyrus seeds to ensure its contribution in providing food and nutritional security, particularly in developing region of the globe (Lambein et al., 2019). Moreover, β-ODAP levels in grass pea plants and seeds exhibited variability across different locations which influenced by genotype, environmental factors and their complex interactions (Jiao et al., 2011). In context of this some studies have been reported the positive association between high levels of β-ODAP biosynthesis and water stress in grass pea (Basaran et al., 2016; Verma et al., 2022).
           
    Drought is one of the most significant abiotic stresses affecting plant growth, development and productivity (Kumar et al., 2023). Drought stress has a major impact on the number of physiological and biochemical attributes such as reduction in the water potential, leaf relative water content and chlorophyll content (Yadav et al., 2013).  Unlike other legumes, the grass pea possesses unique morphological and physiological traits, including thin leaves, winged stem margins, a comprehensive root system and efficient water use efficiency to minimize the stress impact (Kong et al., 2022). This distinctive morpho-physiological character of grass pea contributes in imparting drought tolerance which is emphasizing the crop resilience potential (Lambein et al., 2019).
           
    To get the better insight of drought induced responses biochemical markers and associated molecular study are such an important tool to assess the crop response in real time. Furthermore, biochemical mechanism associated with these markers and molecular expression study pattern will accelerate the breeding efforts in an uninterrupted pattern and it could be a sustainable approach to manage the crop (Kumar et al., 2023). Alteration in metabolism under drought stress has been observed and indicates the ROS mediated shift in metabolism from primary to secondary to scavenge the ROS. To maintain a balance between ROS generation and its removal plants developed a number of mechanisms to neutralize these deleterious effects. Endogenous defense weaponry of plants including enzymatic and non-enzymatic antioxidant system get activated upon ROS exposure (Du et al., 2020). Among the enzymatic antioxidants, superoxide dismutase (SOD), polyphenol oxidase (PPO), peroxidases (POD) and catalase (CAT) has key role in ROS scavenging and these are the biochemical marker for drought tolerance in plants (Sharma et al., 2022).
           
    Molecular marker is a proven effective approach to identifying drought tolerance in grass pea cultivars, IISR markers are such a representative that can be utilized in selection and development of climate resilient varieties (Younis et al., 2020; Das et al., 2021).  IISR markers has both AFLP and RAPD characteristics, which enables it to get precise and efficient assessments without requiring prior knowledge of primer sequences, making them extremely useful in evolutionary biology, genome mapping, gene tagging and studies of genetic diversity and phylogeny (Lalrinmawii et al., 2023).
           
    Keeping all these facts into consideration present study highlights grass pea’s potential as a resilient crop for drought-prone areas. Physio-biochemical analysis reveals plant responses to drought stress, while genetic diversity using ISSR markers informs breeding programs. Grass pea’s adaptability can contribute to agriculture and plant stress research, offering valuable insights for developing drought-resistant varieties.

    MATERIALS AND METHODS

    Experimental setup
     
    The present study was conducted at Research Cum Instructional Farm, IGKV Raipur, Chhattisgarh, India during rabi season 2021-22 and 2022-23, under rainfed condition. A total of 20 Lathyrus genotypes were evaluated for drought tolerance (Table 1). Drought was imposed at flowering stage through water withholding, while a similar set of crops were raised with adequate amount of available water to check the performance of the crop. Drought stress was imposed through depletion of 80% soil available water and it was measured gravimetrically. The experiments were conducted in randomized block design (RBD) with three replications under non-stress (control) and drought stress conditions. All the genotypes undertaken were raised in a plot of   2 m length keeping row to row distance was 30 cm and 10 cm between plants. Standard agronomic package and practices (Give N, P and K requirement of the crop) were followed as per the recommendation for the crop in the region. 

    Table 1: Genotypes accessions of grass pea for drought screening.


     
    Analysis of physiological activity
     
    Leaf area
     
    The length of the midrib is measured thereafter widest width of leaf and width towards tip and base is measured. All the measured width is averaged and multiplied with length.
     
    Relative water content (RWC)
     
    The leaf relative water content was measured following the method of Barrs and Weatherley (1962), using the following formula:

       
    Estimation of chlorophyll
     
    The chlorophyll a, chlorophyll b and total chlorophyll content in the leaf sample was estimated as per Arnon (1949). Content of chlorophyll a, chlorophyll b and total chlorophyll were estimated through following formulae:
     
    Chl a = 12.9 (A663) - 2.69 (A645) × V /1000 × W
     
    Chl b = 22.9 (A645) - 4.68 (A663) × V/ 1000 × W
     
    Total chl = Chl a + Chl b
     
    Analysis of biochemical activity
     
    Enzymatic analysis estimation of 20 genotypes in control and drought condition in each replication was recorded. The leaf samples were collected, processed and analyzed for different enzymatic (SOD, CAT, PPO and POD) activity. The measurement of SOD activity was based on the method given by Beauchamp and Fridovich (1971). Catalase activity was determined by monitoring the disappearance of H2O2 at 240 nm (ε = 40 mM-1 cm-1) as per the method by (Aebi et al., 1984). Polyphenol oxidase activity was estimated as per method described by Sarvesh and Reddy (1988). Peroxidase activity was measured the method reported earlier (Castillo et al., 1984).  ODAP (β -N-oxalyl-L- α, β-diamino propionic acid) content in dry seeds was estimated by using the OPT suggested by Briggs et al. (1983).
     
    Molecular studies
     
    Molecular diversity among selected genotypes was assessed using ISSR markers following DNA extraction by the modified CTAB method (Jonathan, 1990) and genetic relationships were analyzed using UPGMA clustering with Jaccard’s coefficient in NTSYSpc-2.02e. Eleven ISSR markers with known sequences were taken for the study, out of which nine ISSR primer shows amplification patterns were identified namely, UBC-809, UBC-811, UBC-823, UBC-834, UBC-835, UBC-840, UBC-842 UBC-885 and ISSR1 for polymorphism study. Furthermore, theses markers were used for PCR amplification on all of the selected genotypes.

    RESULTS AND DISCUSSION

    Effect of drought stress on leaf area, relative water content (RWC) and chlorophyll content 
     
    Leaf area
     
    Across all genotypes under the study, reduction in leaf area has been observed under drought conditions relative to control grown counterparts. The decrease in leaf area in drought grown lathyrus genotypes indicating the negative impacts of drought stress on leaf growth, consequently growth and development of the crop get hampered. However, genotypes, such as RLK-29 and RLK-30, displayed a significant reduction in leaf area under drought stress which indicates their sensitivity against drought (Fig 1A). Moreover, relatively higher leaf area in RLK-83 and RLK-169 suggesting the tolerance behavior of these genotypes. The minimum reduction in leaf area during drought stress has been observed in RLK-81 and RLK-150 and can be recommended as drought-tolerant that could be useful for breeding programs targeting drought resilience. The substantial loss of leaf area in these genotypes suggests a compromised photosynthetic capacity, as a smaller leaf area reduces the plant’s ability to capture sunlight and produce energy. This sensitivity highlights the need for improvement in such genotypes to enhance their adaptability to drought conditions. Jiang et al., (2013) reported that 20% PEG stressed grass pea seedling for five days shows inward curled leaf margin. Jafarinasab et al., (2022) worked on nine local grass pea genotypes (Baft_1, Baft_2, Bardsir, Dehbakri, Kuhbanan, Rabor, Sirjan, Shiraz and Torbat Heydarieh) collected from different climatic zone of Iran. He found that the leaf area is decreased while soluble sugars and proline content is increased under terminal drought condition (Water withholding at flowering stage). Reduction in leaf area helps in decreases transpirational loss while increased sugar and proline help in osmotic adjustment.

    Fig 1: (A) Leaf area (cm2), (B) Relative water content (RWC %) and (C) Total chlorophyll content in grass pea within the leaves of 20 genotypes under both control and drought stress conditions.


           
    Shamsaee et al., (2025) also found the leaf area index reduction up to 20.32% shows in grass pea under drought stress compared to irrigated condition over two years of treatment (Shamsaee et al., 2025). Many plants show morphological and physiological alterations, due to drought stress like decrease in leaf area, wilting, stomata closure etc. (Bangar et al., 2019).
     
    Relative water content
     
    Despite the drought tolerant behavior reduction in relative water content has been recorded in all the genotypes grown under drought regime as compared to non-stress counterparts, demonstrating the water stress and its impacts on water retention capacity of leaves. Genotypes RLK-169 and RLK-83 shown relatively higher RWC under drought, suggesting its better water retention capacity and it could be a potential candidate for drought tolerance (Fig 1B). Moreover, RLK-30 and RLK-40 exhibited a significant reduction in RWC indicates its sensitive behavior against drought stress. Similar finding was reported by Talukdar (2013) and Verma et al., (2022). Aloui et al., (2023) also confirmed the reduction in Relative leaf water content (RLWC) in lathyrus decreased by 15% under heat stress and 27% under combined heat and drought stress conditions. Similarly, Chettri et al., (2021) also reported the reduction of RWC under combined drought and salinity stress condition. Shamsaee et al., (2025) revealed that an average 5.5% of RWC is decreased under drought condition compared to normal condition in, while it enhances up to 9.25% by Rhizobium inoculation.
     
    Chlorophyll content
     
    Chlorophyll content in response to drought condition imposed through different water regime displayed the differential pattern. More specifically chlorophyll content (Chl a and Chl b) was higher in normally grown grass pea genotypes relative to drought stressed grown crop. Amongst the genotypes, RLK-310 (2.6 mg/g), RLK-296 (2.5 mg/g), RLK-24 (2.5 mg/g), RLK-78 (2.4 mg/g) and RLK-24 (2.4 mg/g) displayed higher chlorophyll content under drought conditions (Fig 1C). The higher chlorophyll content noticed in these genotypes suggesting their drought tolerance behavior relative to other genotypes in our study. Notably, the chlorophyll content among the 20 genotypes under drought conditions ranged from 2.1 mg/g (RLK-29, RLK-150) to 2.6 mg/g (RLK-310), with an overall mean of 2.32 mg/g. Sen et al., (2025), studied on five grass pea genotypes (GM-04, GM-02, GM-03, BARI Khesari-2 and BINA Khesari-2) to assess the drought tolerance under three water regimes: control, 60% and 40% field capacity. Under control and moderate field capacity chlorophyll reduction is less compared to stressed condition (40% field capacity).
           
    Chlorophyll-a and chlorophyll-b as well as carotenoids showed significant decreases in response to increasing stress level reported by (Kiani et al., 2020). Chettri et al., (2021) documented that the combined effect of drought and salinity stress reduced the chlorophyll content in grass pea. Monteoliva et al., (2021) also find the similar result. Leaf chlorophyll concentration dropped up to 58% under combined drought and heat stress while 37% reduction under individual heat stress compared to controlled condition reported by Aloui et al., (2023). Shamsaee et al., (2025) found that the total chlorophyll content in lathyrus crops decreased by up to 20.3% under drought stress. Our findings align with these studies, indicating a consistent pattern of chlorophyll degradation under stress conditions.
     
    Effect of drought stress on antioxidant enzyme activities and ODAP content
     
    Effect of drought stress on antioxidant enzymes activities
     
    Antioxidant enzymes such as SOD, CAT, PPO and POD activity were assayed for all the 20 genotypes under study in both drought stressed and control grown Lathyrus crop. The activities of these enzymes in Lathyrus leaves significantly enhanced in drought stressed crop relative to normally grown plants (Fig 2). Elevated levels of reactive oxygen species (ROS) induce the activation of antioxidant enzymes in plants, such as ascorbate peroxidase (APX), catalase (CAT), peroxidase (POD) and superoxide dismutase (SOD). These enzymes are vital in preserving cell membrane integrity and preventing lipid peroxidation by efficiently scavenging ROS (Zeng et al., 2023). Genotype RLK-296 shown remarkably higher SOD activity amongst the all genotypes during drought stress. While, RLK-991, RLK-513 and RLK-24 displayed moderate SOD activity. Moreover, the rest of the genotypes revealed more or less comparable SOD activity in drought stressed crop (Fig 2A).

    Fig 2: Illustrates alterations in SOD (A), CAT (B), PPO (C) and POD (D) activities in grass pea within the leaves of 20 germplasm under both control and drought stress conditions.


           
    The highest catalase activity recorded in RLK-296, while RLK-991, RLK-513 and RLK-24 are categorized as moderate in terms of their catalase activity (Fig 2B). The remaining genotypes displayed the relatively less variation in catalase activity. Exposure to drought stress led to remarkable enhancement in polyphenol oxidase activity in genotypes of grass pea under study (Fig 2C). Moreover, RLK-991, RLK-581 and RLK-24 shows higher activity over other genotypes, while PPO activity in remaining accessions does not display significant changes. The significant increase in peroxidase activity was recorded in drought stress grown crop relative to non-stressed crop (Fig 2D). Remarkably, RLK-40, RLK-51, RLK-310 and RLK-81 displayed the elite behavior in terms of higher POD activity. Moreover, RLK-991, RLK-150, RLK-513, RLK-310, RLK-345 and RLK-24 exhibited moderate increase in POD activity over its normally grown counterparts. It is widely accepted that active oxygen species are responsible for various stress-induced damages to macromolecules and ultimately to cellular structures.
           
    Tokarz et al., (2020) observed that the activity of POD and CAT in root and shoot of grass pea seedling under drought and salinity stress. He found that the activity of CAT increase in root and shoot of PEG media and decreased in shoot in NaCl media. The POD activity increased in shoot in both media while decreased in root in PEG media and increased in NaCl media. The basis is NaCl (salinity)and PEG (drought) execute different form of osmotic and ionic stress which leads to tissue specific antioxidant enzyme regulation.
           
    The increase in antioxidant enzyme activities like SOD, CAT, POD is more in lathyrus compared to pea under 20% PEG stressed media (Jiang et al., 2013). Because lathyrus is inherently drought tolerant and have more effective ROS-scavenging defense system.
           
    The activity of antioxidant enzyme under drought stress is also reported in different crops including soybean (Wang et al., 2019), faba bean (Desoky et al., 2021), pigeon pea (Radadiya et al., 2016), tomato (Raja et al., 2020), wheat (Abid et al., 2018) etc.
     
    ODAP
     
    The ODAP (β-N-Oxalyl-L-α, β-diaminopropionic acid) content (%) increases under drought conditions across all genotypes, suggesting that drought stress enhances ODAP accumulation in grass pea (Fig 3). Basaran et al., (2016) documented a positive correlation between elevated β-ODAP biosynthesis and water stress in grass pea. Moreover, RLK-30 and RLK-24 displayed highest ODAP content in both stressed and non-stress plants indicating the natural accumulation of ODAP in Lathyrus. RLK-83 and RLK-169 shown relatively lower ODAP content, even under drought conditions, making them preferable for breeding programs targeting low-toxin varieties. Aloui et al., (2023) reported a significant rise in ODAP content under drought stress and combined drought-heat stress, with increases of 33% and 83%, respectively, compared to normal conditions.

    Fig 3: Illustrates alterations in ODAP content in grass pea within the leaves of 20 germplasm under both control and drought stress conditions.


           
    Verma et al., (2022) also observed the positive correlation of drought stress and β-ODAP content in grass pea. She used transcriptome profiling of two genotype, a somaclone ratan and its parent pusa-24. Differential gene expression analysis showed the up regulation of β-ODAP biosynthetic gene under stress condition.
           
    Tokarz et al., (2020) found that the ODAP accumulation is increase under PEG stress in lathyrus seedling. He found that the ODAP content in shoot is higher in PEG media compared to NaCl whereas, its content is more in root in NaCl media compared to PEG media. This is because differential accumulation of ODAP, NaCl stress encourages root-localized accumulation whereas PEG-induced drought stress selectively boosts ODAP synthesis and transport to shoots. These findings suggest that environmental stressors play a crucial role in modulating β-ODAP levels, potentially impacting the safety and resilience of grass pea crops.
     
    Molecular diversity analysis of genotypes
     
    Similarity matrices were obtained by using NTSYS (Numerical Taxonomy System Biostatistics) computer programme. Cluster analysis was performed using UPGMA (unweighed pair group method with arithmetic averages) method that displayed the similarity coefficient 0.50 to 0.95 among the genotypes.
           
    At the mean similarity coefficient of 0.72, analyzed genotypes classified into 5 clusters in dendrogram (Fig 4). Cluster I consists of eight genotypes namely, RLK-29, RLK-580, RLK-581, RLK-30, RLK-9, RLK-513, RLK-345 and RLK-962; whereas, RLK-51 and RLK-81 are in cluster II. However, cluster III consists of four genotypes namely, RLK-78, RLK-169, RLK-46 and RLK-991. Cluster IV and cluster V representing RLK-150 RLK-310 respectively. RLK-78 and RLK-169 are most similar with Similarity coefficient 0.95 and RLK-150 and RLK-310 are least similar with Similarity coefficient 0.71 (Fig 4). Further research by Younis et al., (2020) demonstrated that ISSR markers effectively identify genetic traits associated with drought tolerance, facilitating the selection and development of more resilient cultivars. For grass pea breeding programme ISSR marker is mostly utilized for analyzing diversity and evolutionary relationship among the species (Das et al., 2021).

    Fig 4: Dendrogram depicts the genetic relationships among the sixteen different lines, established through analysis of ISSR markers.

    CONCLUSION

    Drought stress significantly affects physiological traits and ODAP content in grass pea genotypes, Genotype RLK-83 and RLK-169 exhibit better drought tolerance and lower ODAP content. These genotypes are promising candidate for breeding program targeting low toxin and enhanced drought tolerance. RLK296 possess a strong antioxidant defense system, reducing oxidative damage caused by drought induced reactive oxygen species. Such genotype are promising candidates for breeding programs aimed at developing drought-tolerant grass pea cultivars. Genetic diversity analysis using ISSR markers revealed significant polymorphism among grass pea genotypes, with similarity coefficients ranging from 0.50 to 0.95. UPGMA clustering divided the genotypes into five clusters, indicating genetic variation. RLK-78 and RLK-169 showed the highest similarity (0.95), while RLK-150 and RLK-310 were the most genetically distinct (0.71). This diversity provides valuable insights for selecting diverse parents in breeding programs to enhance drought tolerance and other desired traits.

    ACKNOWLEDGEMENT

    The authors are sincerely thankful to Indira Gandhi Krishi Vishwavidyalaya, Raipur, Chhattisgarh for providing financial support and necessary facilities in carrying out the present investigation. Authors are also thankful to Publication Unit of BAU Sabour for providing communication number (BAU Communication No. 1821/240813) for submission of article for publication.
     
    Disclaimers
     
    The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.

    CONFLICT OF INTEREST

    All authors declare that they have no conflict of interest.

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