Univrsity of Tehran Press Iranian Journal of Field Crop Science 2008-4811 57 1 2026 03 21 Biochemical, Physiological Changes and Expression of Some Drought Tolerance Genes in Lentil (Lens culinaris) Biochemical, Physiological Changes and Expression of Some Drought Tolerance Genes in Lentil (Lens culinaris) 97 118 105732 10.22059/ijfcs.2025.390766.655137 FA Saeed Mohammadi Department of Agronomy & Plant Breeding, College of Agriculture and Natural Resources, University of Tehran, Karaj, Iran Mohammad Reza Bihamta Department of Agronomy & Plant Breeding, College of Agriculture and Natural Resources, University of Tehran, Karaj, Iran Ali Reza Abbasi Department of Agronomy & Plant Breeding, College of Agriculture and Natural Resources, University of Tehran, Karaj, Iran Sajad Rashidi-Monfared Agricultural biotechnology Department, Faculty of Agriculture, Tarbiat modares university, Tehran, Iran Abdolrahman Rasoulnia Department of Agronomy & Plant Breeding, College of Agriculture and Natural Resources, University of Tehran, Karaj, Iran Pouya Najibnejad Department of Agronomy & Plant Breeding, College of Agriculture and Natural Resources, University of Tehran, Karaj, Iran Journal Article 2025 07 21 Introduction. Drought stress represents one of the most formidable abiotic challenges to global agricultural productivity, particularly in arid and semi-arid regions where water scarcity is a pervasive issue. The escalating frequency, intensity, and duration of drought events, exacerbated by climate change, pose a significant threat to food security and sustainable agricultural practices worldwide. Lentil (Lens culinaris), a diploid legume, stands as a cornerstone of global food systems, ranking as the sixth most important pulse crop by production volume. Its nutritional density, providing substantial plant protein, complex carbohydrates, dietary fiber, and essential micronutrients, underscores its critical role in human diets. In Iran, where rainfed lentil cultivation is prevalent and water scarcity is a national concern, understanding and mitigating the adverse effects of drought on this vital crop is paramount. This study was designed to elucidate the multifaceted responses of lentil to varying degrees of drought stress, focusing on two distinct genotypes: One previously identified as drought-sensitive (FLIP2002-55) and another as drought-tolerant (FLIP2002-57). Materials and Methods. The experimental design was employed a factorial arrangement based on a randomized complete block design with three replications, conducted under controlled greenhouse conditions. Drought stress was incrementally applied at three distinct levels: Control (90% field capacity), moderate stress (60% field capacity), and severe stress (30% field capacity). Subsequently, comprehensive measurements were taken on a wide array of morphological, physiological, and biochemical traits, alongside an investigation into the relative expression of key drought-responsive genes using quantitative real-time PCR (qRT-PCR). Results and Discussion. Morphological assessments revealed significant reductions in plant height, shoot and root fresh and dry weights, and leaf area index across both genotypes as drought intensity increased. Physiological analyses demonstrated a significant decline in relative leaf water content (RWC) with escalating drought stress in both genotypes, although the tolerant genotype generally maintained higher RWC values, indicative of better osmotic adjustment and cell wall elasticity. Electrolyte leakage, a direct measure of membrane damage, significantly increased in both genotypes under drought, with a more pronounced increase observed in the sensitive genotype. This highlights the superior membrane stability of the tolerant genotype under stress. Biochemical investigations revealed a consistent pattern of stress-induced changes. Increasing drought intensity led to a significant decrease in chlorophyll and total leaf protein content, consistent with oxidative damage to photosynthetic machinery and protein degradation. Conversely, the concentrations of carotenoids, proline, total carbohydrates, malondialdehyde (MDA), and hydrogen peroxide (H2O2) significantly increased. Furthermore, the activities of key antioxidant enzymes, including catalase (CAT), ascorbate peroxidase (APX), and guaiacol peroxidase (GPX), were evaluated. While the overall trend showed increased antioxidant enzyme activity under moderate stress, particularly in the tolerant genotype, severe stress sometimes led to a decline, possibly due to enzyme denaturation or overwhelming oxidative load. The tolerant genotype consistently demonstrated a more robust and sustained antioxidant defense system compared to the sensitive genotype, which is critical for detoxifying ROS and mitigating oxidative damage. At the molecular level, qRT-PCR was employed to assess the relative expression of five key genes implicated in drought tolerance pathways: Beta Amylase (BA), Dehydration-Responsive Element-Binding protein (DREBIC), ABA-WDS induced protein (ABAWDS), High Chlorophyll Fluorescence 136 (HCF136), and myo-inositol monophosphatase (MIMP). In the tolerant genotype, severe drought stress significantly upregulated the expression of ABAWDS and HCF136 genes by approximately 2.5-fold and 2-fold, respectively, compared to control conditions. ABAWDS is associated with abscisic acid (ABA) signaling, a crucial hormone in drought response, mediating stomatal closure and root growth adjustments. The increased expression of HCF136, a gene involved in photosystem II stability, suggests an adaptive mechanism to maintain photosynthetic efficiency under stress. Conversely, the expression of Beta Amylase, DREBIC, and MIMP genes significantly decreased in the tolerant genotype under severe drought. While Beta Amylase is involved in starch degradation for sugar accumulation, its downregulation might indicate a shift in carbohydrate metabolism or a genotype-specific response. Similarly, the reduced expression of DREBIC, a transcription factor typically associated with stress gene activation, and MIMP, involved in myo-inositol metabolism, in the tolerant genotype under severe stress warrants further investigation to fully understand their nuanced roles in this specific context. In the sensitive genotype, HCF136 showed a modest increase (approximately 1.5-fold), while the other genes (ABAWDS, DREBIC, BA, and MIMP) exhibited a general downregulation or minimal change, indicating a less effective molecular response to drought compared to the tolerant genotype. Conclusion. These findings collectively underscore the intricate interplay of morphological, physiological, biochemical, and molecular mechanisms contributing to drought tolerance in lentil. The tolerant genotype's superior performance across multiple parameters – including better maintenance of water status, reduced membrane damage, enhanced antioxidant defense, and specific gene expression patterns – highlights its adaptive capacity. The differential gene expression profiles, particularly the upregulation of ABAWDS and HCF136 in the tolerant genotype, provide valuable molecular markers for drought resilience. This comprehensive analysis not only deepens our understanding of drought tolerance mechanisms in Lens culinaris but also offers critical insights for future lentil breeding programs. By leveraging these identified traits and genetic markers, it is possible to develop and select new drought-tolerant lentil varieties, thereby enhancing crop productivity and ensuring food security in regions increasingly threatened by water scarcity. Further research should focus on validating these findings in field conditions and exploring the functional genomics of these candidate genes to accelerate the development of climate-resilient lentil cultivars. Introduction. Drought stress represents one of the most formidable abiotic challenges to global agricultural productivity, particularly in arid and semi-arid regions where water scarcity is a pervasive issue. The escalating frequency, intensity, and duration of drought events, exacerbated by climate change, pose a significant threat to food security and sustainable agricultural practices worldwide. Lentil (Lens culinaris), a diploid legume, stands as a cornerstone of global food systems, ranking as the sixth most important pulse crop by production volume. Its nutritional density, providing substantial plant protein, complex carbohydrates, dietary fiber, and essential micronutrients, underscores its critical role in human diets. In Iran, where rainfed lentil cultivation is prevalent and water scarcity is a national concern, understanding and mitigating the adverse effects of drought on this vital crop is paramount. This study was designed to elucidate the multifaceted responses of lentil to varying degrees of drought stress, focusing on two distinct genotypes: One previously identified as drought-sensitive (FLIP2002-55) and another as drought-tolerant (FLIP2002-57). Materials and Methods. The experimental design was employed a factorial arrangement based on a randomized complete block design with three replications, conducted under controlled greenhouse conditions. Drought stress was incrementally applied at three distinct levels: Control (90% field capacity), moderate stress (60% field capacity), and severe stress (30% field capacity). Subsequently, comprehensive measurements were taken on a wide array of morphological, physiological, and biochemical traits, alongside an investigation into the relative expression of key drought-responsive genes using quantitative real-time PCR (qRT-PCR). Results and Discussion. Morphological assessments revealed significant reductions in plant height, shoot and root fresh and dry weights, and leaf area index across both genotypes as drought intensity increased. Physiological analyses demonstrated a significant decline in relative leaf water content (RWC) with escalating drought stress in both genotypes, although the tolerant genotype generally maintained higher RWC values, indicative of better osmotic adjustment and cell wall elasticity. Electrolyte leakage, a direct measure of membrane damage, significantly increased in both genotypes under drought, with a more pronounced increase observed in the sensitive genotype. This highlights the superior membrane stability of the tolerant genotype under stress. Biochemical investigations revealed a consistent pattern of stress-induced changes. Increasing drought intensity led to a significant decrease in chlorophyll and total leaf protein content, consistent with oxidative damage to photosynthetic machinery and protein degradation. Conversely, the concentrations of carotenoids, proline, total carbohydrates, malondialdehyde (MDA), and hydrogen peroxide (H2O2) significantly increased. Furthermore, the activities of key antioxidant enzymes, including catalase (CAT), ascorbate peroxidase (APX), and guaiacol peroxidase (GPX), were evaluated. While the overall trend showed increased antioxidant enzyme activity under moderate stress, particularly in the tolerant genotype, severe stress sometimes led to a decline, possibly due to enzyme denaturation or overwhelming oxidative load. The tolerant genotype consistently demonstrated a more robust and sustained antioxidant defense system compared to the sensitive genotype, which is critical for detoxifying ROS and mitigating oxidative damage. At the molecular level, qRT-PCR was employed to assess the relative expression of five key genes implicated in drought tolerance pathways: Beta Amylase (BA), Dehydration-Responsive Element-Binding protein (DREBIC), ABA-WDS induced protein (ABAWDS), High Chlorophyll Fluorescence 136 (HCF136), and myo-inositol monophosphatase (MIMP). In the tolerant genotype, severe drought stress significantly upregulated the expression of ABAWDS and HCF136 genes by approximately 2.5-fold and 2-fold, respectively, compared to control conditions. ABAWDS is associated with abscisic acid (ABA) signaling, a crucial hormone in drought response, mediating stomatal closure and root growth adjustments. The increased expression of HCF136, a gene involved in photosystem II stability, suggests an adaptive mechanism to maintain photosynthetic efficiency under stress. Conversely, the expression of Beta Amylase, DREBIC, and MIMP genes significantly decreased in the tolerant genotype under severe drought. While Beta Amylase is involved in starch degradation for sugar accumulation, its downregulation might indicate a shift in carbohydrate metabolism or a genotype-specific response. Similarly, the reduced expression of DREBIC, a transcription factor typically associated with stress gene activation, and MIMP, involved in myo-inositol metabolism, in the tolerant genotype under severe stress warrants further investigation to fully understand their nuanced roles in this specific context. In the sensitive genotype, HCF136 showed a modest increase (approximately 1.5-fold), while the other genes (ABAWDS, DREBIC, BA, and MIMP) exhibited a general downregulation or minimal change, indicating a less effective molecular response to drought compared to the tolerant genotype. Conclusion. These findings collectively underscore the intricate interplay of morphological, physiological, biochemical, and molecular mechanisms contributing to drought tolerance in lentil. The tolerant genotype's superior performance across multiple parameters – including better maintenance of water status, reduced membrane damage, enhanced antioxidant defense, and specific gene expression patterns – highlights its adaptive capacity. The differential gene expression profiles, particularly the upregulation of ABAWDS and HCF136 in the tolerant genotype, provide valuable molecular markers for drought resilience. This comprehensive analysis not only deepens our understanding of drought tolerance mechanisms in Lens culinaris but also offers critical insights for future lentil breeding programs. By leveraging these identified traits and genetic markers, it is possible to develop and select new drought-tolerant lentil varieties, thereby enhancing crop productivity and ensuring food security in regions increasingly threatened by water scarcity. Further research should focus on validating these findings in field conditions and exploring the functional genomics of these candidate genes to accelerate the development of climate-resilient lentil cultivars.

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