Current IssueSpecial IssueEditorial BoardOnline First ArticlesArchive
Current IssueSpecial IssueEditorial BoardOnline First ArticlesArchive

Full Research Article

Physiological and Biochemical Characterization of Waterlogging Tolerance in Pigeon pea (Cajanus cajan L.) Genotypes

T
T.N. Tiwari1,*
D
D. Datta1
K
Kalpana Tewari1
V
Vijay Luxmi1
S
S. Ghritlahre1
1ICAR-Indian Institute of Pulses Research, Kalyanpur, Kanpur-208 024, Uttar Pradesh, India.

  • Submitted25-03-2026|

  • Accepted02-09-2026|

  • First Online 16-10-2026|

  • doi 10.18805/LR-5661

ABSTRACT

Background: Waterlogging is a major abiotic constraint limiting pigeon pea productivity, particularly in regions receiving high rainfall during early growth stages or in crop lands with poor drainage. Exposure to prolonged soil saturation during the early vegetative stage disrupts normal physiological processes, resulting in reduced plant growth, impaired metabolism, and yield losses. Deciphering of the adaptive responses associated with waterlogging stress is crucial for devising crop breeding and selection strategy for developing waterlogging-resilient pigeon pea cultivars.

Methods: Thirty pigeon pea genotypes, comprised test genotypes as well as tolerant and susceptible checks, were evaluated under controlled waterlogging conditions during two consecutive growing seasons. Waterlogging stress was imposed for 24, 48 and 72 hours at 30 days after sowing while a parallel set of plants was maintained under normal conditions. Morphological responses, survival percentage, adventitious root formation and aerenchyma development were recorded. Physiological parameters viz. photosynthetic rate, stomatal conductance, transpiration rate, chlorophyll content, nitrogen balance index, flavonols content, anthocyanin content and NDVI were recorded using standard analytical procedures. The biochemical parameters namely, antioxidant activity of enzymes and soluble sugar concentration were also estimated using standard protocol to assess biochemical responses associated with waterlogging stress.

Result: Considerable variation in waterlogging tolerance was observed among the evaluated genotypes. IPAC-79, ICPL-87091, ICPL-20241, ICP-2376, IPAC-212, IPAWL-22-7 and IPAWL-22-8 consistently exhibited superior tolerance by maintaining higher survival, improved photosynthetic performance, greater chlorophyll retention and enhanced antioxidant activity under stress. These genotypes also developed abundant adventitious roots and well-defined aerenchyma tissues, facilitating adaptation to oxygen-deficient conditions. Moderately tolerant genotypes displayed intermediate responses, whereas susceptible genotypes exhibited severe chlorosis, wilting, poor survival and substantial reductions in physiological and biochemical traits in response to prolonged waterlogging stress.

INTRODUCTION

Pigeon pea (Cajanus cajan L.) is one of the most important pulses crops cultivated in tropical and subtropical regions because of its nutritional value, and adaptability to diverse agro-ecological conditions. In addition, it has the ability to improve soil fertility and arability through root exudates, deeper roots network and biological nitrogen fixation. India accounts for the largest share of global pigeon pea production; however, average productivity remains considerably lower than (India-Stat 2020-21) the crop’s genetic potential, owing to several biotic and abiotic constraints (Sultana et al., 2012, 2013).
       
Among the major environmental stresses affecting pigeon pea cultivation, temporary waterlogging represents a serious challenge in the regions that receive high rainfall and lack adequate drainage which results in prolonged flooding. Pigeon pea is predominantly grown during the rainy season, hence plants frequently encounter water logging or water saturated soil conditions during initial vegetative growth stages. Oxygen is depleted in flooded soils which restricts root respiration, impairs nutrient uptake, disrupts water relations and limits normal plant growth. The severity of injury depends on the duration as well as intensity of flooding. Waterlogging often results in partial crop damage and sometimes lead to crop failure due to complete plant mortality.
       
Under waterlogged conditions, diffusion of oxygen in the soil pores declines dramatically, creating hypoxic or anoxic environments around the root system. Consequently, aerobic respiration is inhibited and plants are forced to rely on less efficient anaerobic metabolic pathways (Sairam et al., 2009). The situation aggravates upon extended oxygen deficiency which induces plants to generate excess of reactive oxygen species that lead to membrane damage, chlorophyll degradation, impaired photosynthesis and accelerated senescence. These physiological disturbances collectively reduce plant growth and productivity.
       
The plants possessing inherent tolerance to flooding have evolved a host of adaptive mechanisms that improve survival under oxygen-deficient environments. These adaptations include anatomical, physiological and biochemical processes viz. alteration in plant anatomy-morphology such as formation of adventitious roots, development of aerenchyma tissues, collectively they facilitate internal oxygen transport. Physiological and biochemical adaptations namely, maintenance of photosynthetic capacity and efficient regulation of cellular metabolism enables the plants to recover from waterlogging stress. Similarly, positive adaptations in response to activation of antioxidant defence systems, accumulation of compatible solutes and soluble carbohydrates enhances the resilience of plants in response to waterlogging. The effectiveness of these responses differs substantially among genotypes and largely determines their capacity to withstand waterlogging.

Although genetic variation for waterlogging tolerance has previously been reported in pigeon pea, a comprehensive characterization with integration of morphological, physiological and biochemical traits were scarce. Identifying reliable indicators of tolerance would facilitate efficient screening of breeding materials and accelerated development of waterlogging-resilient cultivars.
       
Therefore, the present investigation was undertaken to study the differential response of thirty pigeon pea genotypes under waterlogging stress for a defined period. Particular emphasis was placed on evaluating morphological adaptations, photosynthetic performance, biochemical defence mechanisms and anatomical modifications associated with tolerance. The information generated from this study is expected to provide valuable insights into the mechanisms governing waterlogging tolerance and identify useful traits for future pigeon pea improvement programmes.

MATERIALS AND METHODS

Plant materials and experimental design
 
The study was conducted during the kharif seasons of 2021-22 and 2022-23 at the Research Farm of ICAR-Indian Institute of Pulses Research (ICAR-IIPR), Kalyanpur, Kanpur, India. Thirty pigeon pea genotypes, representing diverse responses to waterlogging, were selected for evaluation. The experimental material included two standard checks, namely IPAC-79 (waterlogging-tolerant) and ICPL-7035 (waterlogging-susceptible), together with twenty-eight additional breeding lines and cultivars.
       
The experiment was laid out in a completely randomized design (CRD) with three replications. Plants were grown in plastic pots (15 kg capacity) filled with a thoroughly mixed medium consisting of field soil and vermicompost. Five seeds were initially sown in each pot, and after seedling establishment, the population was thinned to three healthy plants per pot to ensure uniform plant density.
 
Waterlogging treatment
 
Waterlogging stress was imposed at 30 days after sowing (DAS) by placing the pots inside concrete tanks and maintaining a water depth of approximately 30 cm (12 inches) above the soil surface. Independent sets of plants were subjected to flooding for 24, 48 and 72 hours, while another set of pots was maintained under normal irrigation and served as the control. Uniform water depth was maintained throughout each treatment period to ensure consistent stress conditions across all genotypes.
 
Morphological evaluation
 
Immediately after completion of each waterlogging treatment, plants were evaluated for visible injury symptoms using a modified 0-3 morphological rating scale, adapted from published protocols (Takele and McDavid, 1995; Zaidi et al., 2007).
• Score 0: Healthy plants without visible symptoms.
• Score 1: Yellowing of leaves.
• Score 2: Yellowing accompanied by temporary wilting.
• Score 3: Yellowing followed by temporary and permanent wilting.
       
Survival percentage was recorded after plant recovery. Morphological observations also included assessment of leaf senescence, stem wilting, plant recovery after stress removal and overall plant vigour.
 
Root anatomical studies
 
Root systems were carefully excavated after the waterlogging treatment to examine adaptive anatomical responses. Particular attention was given to.
• Development and abundance of adventitious roots.
• Length of adventitious roots.
• Formation of aerenchyma tissues within the root cortex.
       
These anatomical characteristics were compared among tolerant, moderately tolerant and susceptible genotypes to identify structural adaptations associated with flooding tolerance.
 
Measurement of physiological traits
 
Physiological responses were quantified using standard non-destructive and gas-exchange techniques.
       
Leaf chlorophyll content, nitrogen balance index (NBI), flavonol index, and anthocyanin index were measured using a Dualex Force-A leaf clip sensor (Force-A, France).
       
The Normalized Difference Vegetation Index (NDVI) was determined using a Green Seeker® optical sensor (Trimble Agriculture) by positioning the sensor approximately 30 cm above the crop canopy under clear- sky conditions.
Gas-exchange measurements, including:
• Net photosynthetic rate.
• Stomatal conductance.
• Transpiration rate.
were recorded at the pod initiation stage using a LI-6400 Portable Photosynthesis System (LI-COR Biosciences, USA) between 11:00 AM and 2:00 PM under natural field conditions.
 
Biochemical analysis
 
Leaf samples collected immediately after stress treatment were analysed for antioxidant enzyme activities and soluble sugar concentration.
       
Peroxidase activity was estimated following the guaiacol oxidation method described by Castillo et al., (1984). Superoxide dismutase (SOD) activity was determined according to the procedure of Dhindsa et al., (1981), based on inhibition of nitro blue tetrazolium reduction.
       
Total soluble sugars were quantified using the phenol-sulphuric acid method described by DuBois et al., (1956). Ethanol extracts prepared from fresh leaf tissue were reacted with phenol and concentrated sulphuric acid, incubated at room temperature, and absorbance was recorded at 490 nm using a spectrophotometer.
 
Meteorological observations
 
Weather parameters prevailing during the experimental period, including maximum and minimum temperature, rainfall and relative humidity, were recorded from the institute’s meteorological observatory. These observations were used to characterize the environmental conditions under which the experiments were conducted.
 
Statistical analysis
 
Data collected from morphological, physiological and biochemical observations were averaged over replications and subjected to appropriate statistical analyses to compare genotype performance under waterlogging stress. Mean values were used to classify genotypes into tolerant, moderately tolerant, moderately susceptible and susceptible groups based on their overall response to flooding.

RESULTS AND DISCUSSION

Morphological responses to water logging stress
 
Considerable genotypic variation was observed in the response of pigeon pea plants to waterlogging, indicating substantial diversity in their ability to withstand oxygen-deficient soil conditions. The severity of visible injury increased progressively with longer flooding duration; however, the extent of damage differed markedly among genotypes.
       
The tolerant check IPAC-79, together with ICPL-87091, ICPL-20241, ICP-2376, IPAC-212, IPAWL-22-7 and IPAWL-22-8, maintained normal plant appearance even after 72 h of waterlogging. These genotypes exhibited minimal leaf chlorosis, sustained vegetative growth and rapid recovery after drainage, resulting in the highest survival percentages. Their performance demonstrates the presence of effective adaptive mechanisms that enable maintenance of normal physiological activity during transient flooding (Table 1 and Fig 1).

Table 1: Scoring of morphological responses of pigeon pea genotypes exposed to waterlogging stress.



Fig 1: Morphological performance of pigeon pea genotypes after exposure for 72 h to water logging stress.


       
A second group comprising ICP-5028, MA-6 and IPAC-202 displayed moderate level of tolerance. Although mild chlorosis developed after prolonged flooding, these genotypes recovered successfully following stress removal and maintained satisfactory survival. The relatively less injury suggests that the said genotypes have partial tolerance mechanisms capable of alleviating the adverse effects of temporary oxygen deficiency.
       
Moderately susceptible genotypes, including MA-3, NDA-1, IPA-15F, Bahar, IPA-203, IPAC-211, IPAWL-22-6 and IPAWL-22-9, exhibited progressive yellowing, temporary wilting and noticeable reductions in plant vigour as the duration of flooding increased. Although some plants recovered after drainage, overall survival was considerably lower than that of tolerant genotypes.
       
The greatest injury occurred in ICPL-7035, ICPL-87051, ICPL-149, RCM/DD-17, WB-2019/25, WB-2019/26, WB-2019/27, IPAWL-22-1, IPAWL-22-2, IPAWL-22-3, IPAWL-22-4 and IPAWL-22-5. These susceptible genotypes developed severe chlorosis followed by temporary and permanent wilting, extensive leaf senescence and poor recovery after flooding. Consequently, plant mortality was substantially greater in this group as compared to the tolerant group.
       
The progressive decline in survival in tolerant and susceptible genotypes clearly demonstrated that the ability to maintain the cellular function under oxygen deprivation differed genetically among the tested pigeon pea germplasm. The differential survival rates under transient waterlogged conditions have been observed in pigeon pea and other food legume crops. 
 
Adventitious root development
 
Formation of adventitious roots represented one of the earliest adaptive responses to waterlogging. Tolerant genotypes rapidly produced numerous adventitious roots around the stem base shortly after flooding. These roots were not only more abundant but also considerably longer than those observed in susceptible materials (Table 2 and Fig 2).

Table 2: Adventitious root formation in pigeon pea genotypes exposed to water logging stress.



Fig 2: Adventitious roots development in pigeon pea genotypes under water logging stress.


       
The development of waterlogging-induced secondary root structures provides an alternative pathway for oxygen transport and nutrient uptake when the primary root system experiences hypoxia, which is why waterlogging tolerant accessions were able to maintain better plant growth throughout the waterlogged stress period.
       
The production and growth of adventitious roots were differential among the tested genotypes wherein greatest number and length were noted in tolerant genotypes followed by moderately tolerant genotypes. In contrast, susceptible genotypes either failed to develop adventitious roots or produced only a few short roots that contributed little to plant recovery.
       
These observations indicate that rapid induction of adventitious root development is an important morphological marker associated with waterlogging tolerance in pigeon pea and may serve as an effective criterion for screening breeding populations.
 
Aerenchyma formation
 
Microscopic examination of root tissues showed pronounced differences in cortical anatomy among contrasting genotypes. Roots of tolerant genotypes developed extensive aerenchyma tissues following waterlogging (Armstrong, 1979). These enlarged intercellular air spaces with improved internal oxygen diffusion from aerial organs of the plant to submerged roots were key to sustain root respiration under hypoxic conditions (Fig 3).

Fig 3: Aerenchyma development in waterlogging-tolerant genotypes IPAWL22-8.


       
In contrast, susceptible genotypes exhibited little or absence of aerenchyma. The lack of this anatomical adaptation likely restricted oxygen transport within the root system, accelerating tissue injury and reducing plant survival.
       
The simultaneous occurrence of abundant adventitious roots and well-developed aerenchyma demonstrates that tolerant pigeon pea genotypes employ coordinated structural adaptations to minimize the detrimental effects of prolonged soil flooding.
 
Relationship between morphological adaptation and waterlogging tolerance
 
The present investigation demonstrates that tolerance to waterlogging is governed by a combination of complementary adaptive traits rather than by a single characteristic. Genotypes exhibiting high survival consistently combined several advantageous responses, including maintenance of healthy foliage, delayed senescence, prolific adventitious rooting, extensive aerenchyma formation and rapid recovery after drainage (Singh et al., 1986).
       
In waterlogged soils, the soil pores are filled with water which induces hypoxic condition, thereby the level of oxygen rapidly declines and soil becomes oxygen deficient due to highly restricted gaseous diffusion through water as compared to diffusion through air (Orchard and Jessop, 1984, Sairam et al., 2009 and Chaudhary et al., 2011). Under these conditions, susceptible plants experience impaired aerobic respiration, reduced ATP production and disruption of normal root metabolism. The resulting energy deficiency adversely affects nutrient acquisition, photosynthesis and overall plant growth (Marschner, 1995).
       
Conversely, tolerant genotypes appear capable of maintaining oxygen supply to submerged tissues through anatomical modifications that facilitate internal gas movement. The formation of adventitious roots and aerenchyma enhances root aeration and improves survival during temporary flooding (Table 3). 

Table 3: Plant survival in pigeon pea genotypes after exposer of 72 hours of waterlogging stress.


       
Similar adaptive mechanisms have been widely documented in maize, wheat, green gram, soybean and pigeon pea, confirming their central role in waterlogging tolerance (Pourabdal et al., 2008; Jerald Anthony et al., 2024; Thomson et al., 1992; Huang et al., 1994; Saikia et al., 2021, Pires et al., 2002; Shimamura et al., 2003, 2010 and Tiwari et al., 2024). Overall, the observed morphological and anatomical responses clearly differentiate tolerant and susceptible pigeon pea genotypes and provide practical selection criteria for breeding programmes aimed at improving adaptation to waterlogged environments.
 
Physiological and biochemical responses to waterlogging stress
 
Gas exchange characteristics
 
Waterlogging markedly influenced the parameters of gaseous exchange in all the tested pigeon pea genotypes. However, genotypic differences were observed in the magnitude of reduction, which corresponded with the level of waterlogging tolerance (Sharma et al., 2022 and Zhang et al., 2023). The waterlogging tolerant genotypes consistently maintained significantly higher photosynthetic rate, stomatal conductance and transpiration than susceptible genotypes throughout the stress period. The superior physiological performance of IPAC-79, ICPL-87091, ICPL-20241, ICP-2376, IPAC-212, IPAWL-22-7 and IPAWL-22-8 demonstrates their ability to sustain carbon assimilation despite prolonged root-zone hypoxia (Table 4).

Table 4: Effect of waterlogging stress on Photosynthetic Efficiency of pigeon pea genotypes.


       
Moderately tolerant genotypes exhibited an intermediate decline in gas exchange, indicating partial disruption of photosynthetic activity. In contrast, susceptible genotypes showed a pronounced decline in all three parameters following 48 and 72 h of waterlogging. Reduced stomatal conductance restricted carbon dioxide diffusion into the leaf, leading to reduced photosynthetic efficiency along with diminished transpiration. These responses collectively reflect impaired water and nutrient transport resulting from oxygen deprivation in the root system.
       
Flood-induced hypoxia disrupts root respiration and ATP production, thereby reducing hydraulic conductivity and limiting the supply of water to aerial tissues. Progressive stomatal closure conserves water but simultaneously restricts CO2‚ uptake, and thereby reduces the rate of carbon fixation through photosynthesis. In addition to stomatal limitations, the structure of chloroplast and associated photosynthetic enzymes are also damaged under sustained waterlogging. It further contributes to decline in photosynthetic performance. Enhanced maintenance of gaseous exchange was observed in tolerant genotypes which indicated greater metabolic stability under flooding stress (Tian et al., 2019).
 
Chlorophyll content and nitrogen balance index
 
Significant differences among genotypes were also observed for chlorophyll concentration and nitrogen balance index (NBI). Waterlogging-tolerant genotypes retained substantially higher chlorophyll levels than susceptible materials, suggesting reduced degradation of photosynthetic pigments during stress (Bansal and Srivastava, 2015). Maintenance of chlorophyll enables continued light harvesting and supports sustained photosynthetic activity, thereby improving plant survival under oxygen-deficient conditions (Table 5).

Table 5: Biochemical attributes as influenced by waterlogging stress.


       
The nitrogen balance index followed a similar trend. Higher NBI values recorded in tolerant genotypes indicate more efficient nitrogen acquisition and assimilation despite restricted root activity. Since leaf nitrogen is closely associated with chlorophyll biosynthesis and Rubisco abundance, maintenance of nitrogen status contributes directly to improved photosynthetic performance during flooding (Bansal and Srivastava, 2017). In contrast, the susceptible genotypes exhibited substantial reduction in chlorophyll as well as NBI, which is reflective of enhanced senescence and impaired metabolic activity.  
 
Vegetation index and photosynthetic integrity
 
The Normalized Difference Vegetation Index (NDVI) remained consistently higher in tolerant genotypes throughout the experiment. Greater NDVI values indicate better preservation of green leaf area and canopy vigour, reflecting sustained physiological activity during waterlogging. Because NDVI integrates canopy greenness and biomass production, it provides a rapid and reliable indicator for identifying tolerant genotypes under field conditions (Table 5).
       
The combined maintenance of chlorophyll content, nitrogen balance and NDVI demonstrates that tolerant pigeon pea genotypes experience considerably less impairment of the photosynthetic apparatus than susceptible lines. The recovery and growth post-waterlogging conditions are positively influenced by higher NDVI, hence, restoration of such physiological characteristics allows continued biomass accumulation and results in improved plant growth recovery after flooding. 
 
Antioxidant defence system
 
Waterlogging induced oxidative stress in all evaluated genotypes, as reflected by changes in antioxidant enzyme activity. However, differential activity of superoxide dismutase (SOD) and peroxidase enzymes was observed among the tolerant and susceptible genotypes, wherein the response of tolerant genotypes was substantially greater than that of susceptible genotypes (Table 6).

Table 6: Effect of water logging stress on anti-oxidant enzymes and soluble sugar in pigeon pea genotypes.


       
The enhanced activity of SOD and peroxidase enzymes indicates a more efficient scavenging of reactive oxygen species (ROS scavenging system). Superoxide dismutase catalyses the conversion of superoxide radicals into hydrogen peroxide, whereas peroxidase subsequently detoxifies hydrogen peroxide into harmless products. Together, these enzymes minimize oxidative damage to cellular membranes, proteins and nucleic acids, thereby preserving cellular integrity during flooding stress (Sairam et al., 2009, Sharma et al., 2024).
       
In susceptible genotypes, comparatively lower antioxidant activity suggests inadequate detoxification of ROS, leading to increased oxidative injury, chlorophyll degradation and membrane instability. The present findings therefore confirm that activation of antioxidant defence constitutes an important biochemical mechanism that contributes to enhanced waterlogging tolerance in pigeon pea.
 
Soluble sugar accumulation
 
The soluble sugar concentration in the leaf tissue was enhanced in response to waterlogging in all genotypes, although the increase was considerably greater in tolerant materials (Table 6). Accumulation of soluble carbohydrates represents an adaptive metabolic response that supports energy production when aerobic respiration is restricted (Mishegyan et al., 2025).
       
Under hypoxic conditions, plants shift from oxidative phosphorylation to anaerobic fermentation, making readily available carbohydrates essential for sustaining ATP generation. Higher soluble sugar concentrations observed in tolerant genotypes therefore provide an important energy reserve that facilitates continued cellular metabolism during oxygen deficiency. In addition, soluble sugars contribute to osmotic adjustment and stabilization of cellular structures, further enhancing waterlogging stress tolerance.

Flavonols and anthocyanins
 
Genotype-dependent reactions to flooding were seen in secondary metabolites. Throughout the stress period, tolerant genotypes maintained a relatively high level of anthocyanin and flavonol indices compared to susceptible genotypes (Table 5).
       
Anthocyanins have potent antioxidant qualities to shield photosynthetic tissues by scavenging reactive oxygen species produced during environmental stress. It limits excessive light absorption and also reduces photo-oxidative damage (Li and Ahammed, 2023). In a similar way, flavonols serve as non-enzymatic antioxidants that can prevent oxidative damage to proteins and membranes (Beegum et al., 2023.)
       
The increased accumulation of these metabolites in tolerant genotypes of pigeon pea suggested that both enzymatic as well as non-enzymatic antioxidant systems operate together to minimize oxidative stress during waterlogging. Such coordinated protective responses are likely to improve physiological stability and higher survival following prolonged flooding.
 
Integrated mechanism of waterlogging tolerance
 
The present investigation indicated that waterlogging tolerance in pigeon pea results from the coordinated interaction of multiple adaptive mechanisms rather than a consequence from the expression of a single trait. Tolerant genotypes simultaneously maintained higher photosynthetic efficiency, greater chlorophyll stability, superior nitrogen status, stronger antioxidant defence, enhanced soluble sugar accumulation and elevated concentrations of protective secondary metabolites. These physiological and biochemical characteristics complemented the structural adaptations, inclusive of stress induced adventitious root formation and development of aerenchyma as described earlier.
       
Collectively, these responses enabled tolerant genotypes to maintain metabolic homeostasis under hypoxic conditions, minimise oxidative damage and fast recovery after withdrawal of waterlogging condition. The integration of anatomical, physiological and biochemical traits, therefore, provides a comprehensive framework for understanding waterlogging stress tolerance in pigeonpea and offers valuable selection criteria for future breeding programmes aimed at developing climate-resilient cultivars.
 
Practical implications
 
The present study provides several traits that can be incorporated into routine screening protocols for breeding programmes targeting waterlogging tolerance. Although survival percentage remains an important selection criterion, combining it with physiological measurements such as chlorophyll content, nitrogen balance index, photosynthetic rate and antioxidant enzyme activity would improve selection accuracy.
       
The tolerant genotypes identified in this investigation represent valuable donor parents for developing improved pigeonpea cultivars adapted to regions experiencing intermittent flooding. Their use in hybridization programmes could accelerate the development of varieties possessing enhanced resilience without compromising productivity.
 
Future research perspectives
 
Response to waterlogging is dependent on the soil and weather parameters as well. Therefore, future studies should validate the identified tolerant genotypes under multi-location field environments representing different soil types and rainfall patterns. Further deciphering of the waterlogging tolerance mechanism will be strengthened by integrating the physiological screening with molecular studies such as proteomic and transcriptomic analyses to facilitate identification of genes regulating waterlogging tolerance. Moreover, studies related to the molecular breeding perspective, genomic approaches such as genome-wide association studies (GWAS), quantitative trait locus (QTL) mapping and marker-assisted selection would facilitate identification of genes regulating waterlogging tolerance. Therefore, comprehensive investigations involving metabolomics, proteomics and root system architecture analysis would further improve understanding of the molecular networks controlling adaptation to flooding stress. In nut shell, an integrated approach involving morpho-physiological, edaphological, genetical, biochemical, plant breeding and genomic science will accelerate the development of climate-resilient pigeon pea cultivars capable of maintaining stable productivity under increasingly erratic rainfall conditions associated with climate change.

CONCLUSION

Among the evaluated materials, IPAC-79, ICPL-87091, ICPL-20241, ICP-2376, IPAC-212, IPAWL-22-7 and IPAWL-22-8 consistently exhibited superior performance under waterlogged conditions. The results further indicate that traits viz. adventitious root development, aerenchyma formation, chlorophyll retention, photosynthetic stability, antioxidant capacity and accumulation of soluble sugars can serve as reliable physiological markers for the identification of waterlogging-tolerant genotypes. Incorporation of these morphological/physiological/biochemical marker traits into breeding programmes may substantially improve the efficiency of selecting waterlogging tolerant breeding and parental lines for developing climate-resilient pigeonpea cultivars adapted to flood-prone environments.

CONFLICT OF INTEREST

All authors declare that they have no conflicts of interest.

REFERENCES


  1. Armstrong W. (1979). Aeration in higher plants. Advances in Botanical Research. 7: 225-332.

  2. Bansal, Ruchi and Srivastava J.P. (2017). Effect of water logging on root anatomy and nitrogen distribution in pigeon pea [Cajanus cajan (L.) Mill.sp.]. Indian Journal of Plant Physiology. 22(1): 130-134.

  3. Bansal, Ruchi and Srivastava, J.P. (2015). Effect of water logging on photosynthetic and biochemical parameters in pigeon pea. Russian Journal of Plant Physiology. 62(3): 322-332.

  4. Beegum, S., Troung, V., Bheemanahalli, R., Brand, D., Reddy, V. and Reddy, K.R. (2023). Developing functional relationships between water logging and cotton growth and physiology- towards water logging. Front. Plant Sci. Sec. Plant Abiotic Stress. 31(14): 1174682. https://doi.org/10.3389.

  5. Castillo, F.I., Penel, I. and Greppin, H. (1984). Peroxidase release induced by ozone in sedum album leaves. Plant Physiology.  74(4): 846-851.

  6. Chaudhary, A.K., Sultana, R., Pratap, A., Nadarajan, N. and Jha, U.C. (2011). Breeding for abiotic stresses in pigeon pea. Journal of Food Legumes. 24(3): 165-174.

  7. Dhindsa, R.A., Plumb-Dhindsa, P. and Thorpe, T.A. (1981). Leaf senescence: Correlated with increased permeability and lipid peroxidation and decreased levels of superoxide dismutase and catalase. Journal of Experimental Botany. 32(1): 93-101.

  8. DuBois, M., Gilles, K., Hamilton, J., Rebers, P. and Smith, F. (1956). Colorimetric method for determination of sugars and related substances. Analytical Chemistry. 28(3): 350-356.

  9. Huang, B., Johnson, J.W., Nesmith, D.S. and Bridges, D.C. (1994). Root and shoot growth of wheat genotypes in response to hypoxia and subsequent resumption of aeration. Crop Sci. 34(6): 1538-1544.

  10. India Stat. (2021). Land Use Data Classification/Agriculture Statistics https://www.indiastat.com/data/agriculture/land-use-classification/data-ye.

  11. Jerald Anthony, C., Esteban, Nenita, B. and Baldo (2024). Effect and recovery of maize (Zea mays linn) to water logging imposed to early seedling stage. Agricultural Science Digest. 44(3): 414-420. doi: 10.18805/ag.DF-500.

  12. Li, Z. and Ahammad, G.J. (2023). Plant stress response and adaptation via anthocyanins: A review. Plant Stress. 10: 100230.

  13. Marschner, H. (1995). Mineral Nutrition of Higher Plants. Second edition. London, Academic Press Limited. pp. 889.

  14. Mishegyan, A., Mentreddy, S.R., Duong, L. and Basu, C. (2025). Soluble sugar accumulation and differential gene- expression linked to water logging tolerance in turmeric (Curcuma longa). Plant-Environment Interaction. https://doi.org/10.10 80/17429145.2025.2558701.

  15. Orchard, P.W. and Jessop, R.S. (1984). The response of sorghum and sunflower to short term waterlogging. I. Effects of stage of development and duration of waterlogging on growth and yield. Plant Soil. 81: 119-132.

  16. Pires, J.L.F., Soprano, E. and Cassol, B. (2002). Morph physiological changes of soybean in flooded soils. Pesquisa Agropecuaria Brasileira. 37(1): 41-50.

  17. Pourabdal, L., Heidary, R. and Farboodnia, T. (2008). Effects of Different Flooding periods on Some Histochemicals of Zea mays L. Seedlings. Plant Sci. Res. 1(1): 8-12.

  18. Saikia, B., Prakash, K. and Ranjan, D. (2021). Effect of Simulated waterlogging condition imposed at early vegetative growth on final yield in green gram (vigna radiata). Legume Research. 44(10): 1226-1232. doi: 10.18805/LR-4238.

  19. Sairam, R.K., Umutha, D.K., Ezhilmathi, K., Chinnusamy, V. and Meena, R.C. (2009). Water logging induced oxidative stress and antioxidant enzyme activities in pigeon pea. Biologia Plantarum. 53(3): 493-504.

  20. Sharma, S., Bhatt, U., Shah, G. and Soni, V. (2024). Assessment of water logging induced changes in enzymatic antioxidants and carbohydrate metabolism in peanut genotypes. Biochemistry and Biophysics Reports. 39: 101794.

  21. Sharma, S., Bhatt, U., Sharma, J., Kalaji, H.M., Mojski, J. and Soni, V. (2022). Ultrastructure adaptability and alleviation mechanisms of photosynthetic apparatus in plants under water logging: A review. Photosynthetica. 60(3): 430-444.

  22. Shimamura, S., Mochizuki, T., Nada, Y. and Fukuyama, M. (2003). Formation and function of secondary aerenchyma in hypocotyl, roots and nodules of soybean (Glycine max) under flooded conditions. Plant Soil. 251: 351-359.

  23. Shimamura, S., Yamamoto, R., Nakamura, T., Shimada, S. and Komatsu, S. (2010). Stem hypertrophic lenticels and secondary aerenchyma enable oxygen transport to roots of soybean in flooded soil. Ann. Bot. 106(2): 277-284.

  24. Singh, K., Sharma, S.P., Singh, T.K. and Singh, Y. (1986). Effect of waterlogging on growth, yield and nutrient concentration of black gram and green gram under subtropical condition of Varanasi. Ann. Agr. Res. 7: 169-177.

  25. Sultana, R., Vales, M., Saxena, K.B., Rathore, A., Rao, S. and Kumar, R. (2013). Waterlogging tolerance in pigeon pea [Cajanus cajan (L.) Millsp.]: Genotypic variability and identification of tolerant genotypes. The J. Agric. Sci. 151(5): 659-671.

  26. Sultana, R., Vales, M.I., Saxena, K.B., Rathore, A., Rao, S., Rao, S.K., Mula, M.G. and Kumar, R.V. (2012). Waterlogging tolerance in pigeon pea [Cajanus cajan (L.) Millsp]: genotypic variability and identification of tolerant genotypes. J. Agril. Sci. pp. 1-14. ISSN1469-5146.

  27. Takele, A. and Mcdavid, C.R. (1995). The response of pigeon pea cultivars to short durations of waterlogging. African Crop Sci. J. 3(1): 51-58.

  28. Thomson, C.J., Colmer, T.D., Watkin, E.L.J. and Greenway, H. (1992). Tolerance of wheat (Triticum aestivum cvs. Gamenya and Kite) and triticale (Triticosecale cv. Muir) to waterlogging. New Phytol. 120: 335-344.

  29. Tian, L., Li, J., Bi, W., Zuo, S., Li, l., Li, W. and Sun, L. (2019). Effect of water logging stress at different growth stages on the photosynthetic characteristics and grain yield of spring maize (Zea mays L.) under field conditions. Agriculture Water Management. 218: 250-258.

  30. Tiwari, T.N., Datta, D., Tiwari, K. and Vijay Luxmi (2024). Physiological characterization of pigeon pea genotypes [Cajanus cajan (L.) Millsp.] for water logging tolerance. Legume Research-An International Journal. 49(3): 397-405. doi: 10.18805/LR-5342.

  31. Zaidi, P.H., Maniselvan, P., Yadav, P., Singh, A.K., Sultana, R., Dureja, P., Singh, R.P. and Srinivasan, G. (2007). Stress-adaptive changes in tropical maize (Zea mays L.) under excessive soil moisture stress. Maydica. 52: 159-171.

  32. Zhang, B., Maoxiangsun, Liu, W., Lian, M., Yang, S., Peng, F., Yuansong, X. (2023). Water logging resistance and evaluation of Physiological mechanism of three peach (Prunuspersica) rootstocks. Protoplasma. 260: 1375- 1388. https://do i.Org/10.1007/s00709-023-01850-W.Epu b2023Apr3.
Published In
Legume Research
Article Metrics
Metrics Icon
21
Views
0
Citations
Reviewed By
Share Article
Download Article
In this Article
    Contact us
    Follow us

    Full Research Article

    Physiological and Biochemical Characterization of Waterlogging Tolerance in Pigeon pea (Cajanus cajan L.) Genotypes

    T
    T.N. Tiwari1,*
    D
    D. Datta1
    K
    Kalpana Tewari1
    V
    Vijay Luxmi1
    S
    S. Ghritlahre1
    1ICAR-Indian Institute of Pulses Research, Kalyanpur, Kanpur-208 024, Uttar Pradesh, India.

    • Submitted25-03-2026|

    • Accepted02-09-2026|

    • First Online 16-10-2026|

    • doi 10.18805/LR-5661

    ABSTRACT

    Background: Waterlogging is a major abiotic constraint limiting pigeon pea productivity, particularly in regions receiving high rainfall during early growth stages or in crop lands with poor drainage. Exposure to prolonged soil saturation during the early vegetative stage disrupts normal physiological processes, resulting in reduced plant growth, impaired metabolism, and yield losses. Deciphering of the adaptive responses associated with waterlogging stress is crucial for devising crop breeding and selection strategy for developing waterlogging-resilient pigeon pea cultivars.

    Methods: Thirty pigeon pea genotypes, comprised test genotypes as well as tolerant and susceptible checks, were evaluated under controlled waterlogging conditions during two consecutive growing seasons. Waterlogging stress was imposed for 24, 48 and 72 hours at 30 days after sowing while a parallel set of plants was maintained under normal conditions. Morphological responses, survival percentage, adventitious root formation and aerenchyma development were recorded. Physiological parameters viz. photosynthetic rate, stomatal conductance, transpiration rate, chlorophyll content, nitrogen balance index, flavonols content, anthocyanin content and NDVI were recorded using standard analytical procedures. The biochemical parameters namely, antioxidant activity of enzymes and soluble sugar concentration were also estimated using standard protocol to assess biochemical responses associated with waterlogging stress.

    Result: Considerable variation in waterlogging tolerance was observed among the evaluated genotypes. IPAC-79, ICPL-87091, ICPL-20241, ICP-2376, IPAC-212, IPAWL-22-7 and IPAWL-22-8 consistently exhibited superior tolerance by maintaining higher survival, improved photosynthetic performance, greater chlorophyll retention and enhanced antioxidant activity under stress. These genotypes also developed abundant adventitious roots and well-defined aerenchyma tissues, facilitating adaptation to oxygen-deficient conditions. Moderately tolerant genotypes displayed intermediate responses, whereas susceptible genotypes exhibited severe chlorosis, wilting, poor survival and substantial reductions in physiological and biochemical traits in response to prolonged waterlogging stress.

    INTRODUCTION

    Pigeon pea (Cajanus cajan L.) is one of the most important pulses crops cultivated in tropical and subtropical regions because of its nutritional value, and adaptability to diverse agro-ecological conditions. In addition, it has the ability to improve soil fertility and arability through root exudates, deeper roots network and biological nitrogen fixation. India accounts for the largest share of global pigeon pea production; however, average productivity remains considerably lower than (India-Stat 2020-21) the crop’s genetic potential, owing to several biotic and abiotic constraints (Sultana et al., 2012, 2013).
           
    Among the major environmental stresses affecting pigeon pea cultivation, temporary waterlogging represents a serious challenge in the regions that receive high rainfall and lack adequate drainage which results in prolonged flooding. Pigeon pea is predominantly grown during the rainy season, hence plants frequently encounter water logging or water saturated soil conditions during initial vegetative growth stages. Oxygen is depleted in flooded soils which restricts root respiration, impairs nutrient uptake, disrupts water relations and limits normal plant growth. The severity of injury depends on the duration as well as intensity of flooding. Waterlogging often results in partial crop damage and sometimes lead to crop failure due to complete plant mortality.
           
    Under waterlogged conditions, diffusion of oxygen in the soil pores declines dramatically, creating hypoxic or anoxic environments around the root system. Consequently, aerobic respiration is inhibited and plants are forced to rely on less efficient anaerobic metabolic pathways (Sairam et al., 2009). The situation aggravates upon extended oxygen deficiency which induces plants to generate excess of reactive oxygen species that lead to membrane damage, chlorophyll degradation, impaired photosynthesis and accelerated senescence. These physiological disturbances collectively reduce plant growth and productivity.
           
    The plants possessing inherent tolerance to flooding have evolved a host of adaptive mechanisms that improve survival under oxygen-deficient environments. These adaptations include anatomical, physiological and biochemical processes viz. alteration in plant anatomy-morphology such as formation of adventitious roots, development of aerenchyma tissues, collectively they facilitate internal oxygen transport. Physiological and biochemical adaptations namely, maintenance of photosynthetic capacity and efficient regulation of cellular metabolism enables the plants to recover from waterlogging stress. Similarly, positive adaptations in response to activation of antioxidant defence systems, accumulation of compatible solutes and soluble carbohydrates enhances the resilience of plants in response to waterlogging. The effectiveness of these responses differs substantially among genotypes and largely determines their capacity to withstand waterlogging.

    Although genetic variation for waterlogging tolerance has previously been reported in pigeon pea, a comprehensive characterization with integration of morphological, physiological and biochemical traits were scarce. Identifying reliable indicators of tolerance would facilitate efficient screening of breeding materials and accelerated development of waterlogging-resilient cultivars.
           
    Therefore, the present investigation was undertaken to study the differential response of thirty pigeon pea genotypes under waterlogging stress for a defined period. Particular emphasis was placed on evaluating morphological adaptations, photosynthetic performance, biochemical defence mechanisms and anatomical modifications associated with tolerance. The information generated from this study is expected to provide valuable insights into the mechanisms governing waterlogging tolerance and identify useful traits for future pigeon pea improvement programmes.

    MATERIALS AND METHODS

    Plant materials and experimental design
     
    The study was conducted during the kharif seasons of 2021-22 and 2022-23 at the Research Farm of ICAR-Indian Institute of Pulses Research (ICAR-IIPR), Kalyanpur, Kanpur, India. Thirty pigeon pea genotypes, representing diverse responses to waterlogging, were selected for evaluation. The experimental material included two standard checks, namely IPAC-79 (waterlogging-tolerant) and ICPL-7035 (waterlogging-susceptible), together with twenty-eight additional breeding lines and cultivars.
           
    The experiment was laid out in a completely randomized design (CRD) with three replications. Plants were grown in plastic pots (15 kg capacity) filled with a thoroughly mixed medium consisting of field soil and vermicompost. Five seeds were initially sown in each pot, and after seedling establishment, the population was thinned to three healthy plants per pot to ensure uniform plant density.
     
    Waterlogging treatment
     
    Waterlogging stress was imposed at 30 days after sowing (DAS) by placing the pots inside concrete tanks and maintaining a water depth of approximately 30 cm (12 inches) above the soil surface. Independent sets of plants were subjected to flooding for 24, 48 and 72 hours, while another set of pots was maintained under normal irrigation and served as the control. Uniform water depth was maintained throughout each treatment period to ensure consistent stress conditions across all genotypes.
     
    Morphological evaluation
     
    Immediately after completion of each waterlogging treatment, plants were evaluated for visible injury symptoms using a modified 0-3 morphological rating scale, adapted from published protocols (Takele and McDavid, 1995; Zaidi et al., 2007).
    • Score 0: Healthy plants without visible symptoms.
    • Score 1: Yellowing of leaves.
    • Score 2: Yellowing accompanied by temporary wilting.
    • Score 3: Yellowing followed by temporary and permanent wilting.
           
    Survival percentage was recorded after plant recovery. Morphological observations also included assessment of leaf senescence, stem wilting, plant recovery after stress removal and overall plant vigour.
     
    Root anatomical studies
     
    Root systems were carefully excavated after the waterlogging treatment to examine adaptive anatomical responses. Particular attention was given to.
    • Development and abundance of adventitious roots.
    • Length of adventitious roots.
    • Formation of aerenchyma tissues within the root cortex.
           
    These anatomical characteristics were compared among tolerant, moderately tolerant and susceptible genotypes to identify structural adaptations associated with flooding tolerance.
     
    Measurement of physiological traits
     
    Physiological responses were quantified using standard non-destructive and gas-exchange techniques.
           
    Leaf chlorophyll content, nitrogen balance index (NBI), flavonol index, and anthocyanin index were measured using a Dualex Force-A leaf clip sensor (Force-A, France).
           
    The Normalized Difference Vegetation Index (NDVI) was determined using a Green Seeker® optical sensor (Trimble Agriculture) by positioning the sensor approximately 30 cm above the crop canopy under clear- sky conditions.
    Gas-exchange measurements, including:
    • Net photosynthetic rate.
    • Stomatal conductance.
    • Transpiration rate.
    were recorded at the pod initiation stage using a LI-6400 Portable Photosynthesis System (LI-COR Biosciences, USA) between 11:00 AM and 2:00 PM under natural field conditions.
     
    Biochemical analysis
     
    Leaf samples collected immediately after stress treatment were analysed for antioxidant enzyme activities and soluble sugar concentration.
           
    Peroxidase activity was estimated following the guaiacol oxidation method described by Castillo et al., (1984). Superoxide dismutase (SOD) activity was determined according to the procedure of Dhindsa et al., (1981), based on inhibition of nitro blue tetrazolium reduction.
           
    Total soluble sugars were quantified using the phenol-sulphuric acid method described by DuBois et al., (1956). Ethanol extracts prepared from fresh leaf tissue were reacted with phenol and concentrated sulphuric acid, incubated at room temperature, and absorbance was recorded at 490 nm using a spectrophotometer.
     
    Meteorological observations
     
    Weather parameters prevailing during the experimental period, including maximum and minimum temperature, rainfall and relative humidity, were recorded from the institute’s meteorological observatory. These observations were used to characterize the environmental conditions under which the experiments were conducted.
     
    Statistical analysis
     
    Data collected from morphological, physiological and biochemical observations were averaged over replications and subjected to appropriate statistical analyses to compare genotype performance under waterlogging stress. Mean values were used to classify genotypes into tolerant, moderately tolerant, moderately susceptible and susceptible groups based on their overall response to flooding.

    RESULTS AND DISCUSSION

    Morphological responses to water logging stress
     
    Considerable genotypic variation was observed in the response of pigeon pea plants to waterlogging, indicating substantial diversity in their ability to withstand oxygen-deficient soil conditions. The severity of visible injury increased progressively with longer flooding duration; however, the extent of damage differed markedly among genotypes.
           
    The tolerant check IPAC-79, together with ICPL-87091, ICPL-20241, ICP-2376, IPAC-212, IPAWL-22-7 and IPAWL-22-8, maintained normal plant appearance even after 72 h of waterlogging. These genotypes exhibited minimal leaf chlorosis, sustained vegetative growth and rapid recovery after drainage, resulting in the highest survival percentages. Their performance demonstrates the presence of effective adaptive mechanisms that enable maintenance of normal physiological activity during transient flooding (Table 1 and Fig 1).

    Table 1: Scoring of morphological responses of pigeon pea genotypes exposed to waterlogging stress.



    Fig 1: Morphological performance of pigeon pea genotypes after exposure for 72 h to water logging stress.


           
    A second group comprising ICP-5028, MA-6 and IPAC-202 displayed moderate level of tolerance. Although mild chlorosis developed after prolonged flooding, these genotypes recovered successfully following stress removal and maintained satisfactory survival. The relatively less injury suggests that the said genotypes have partial tolerance mechanisms capable of alleviating the adverse effects of temporary oxygen deficiency.
           
    Moderately susceptible genotypes, including MA-3, NDA-1, IPA-15F, Bahar, IPA-203, IPAC-211, IPAWL-22-6 and IPAWL-22-9, exhibited progressive yellowing, temporary wilting and noticeable reductions in plant vigour as the duration of flooding increased. Although some plants recovered after drainage, overall survival was considerably lower than that of tolerant genotypes.
           
    The greatest injury occurred in ICPL-7035, ICPL-87051, ICPL-149, RCM/DD-17, WB-2019/25, WB-2019/26, WB-2019/27, IPAWL-22-1, IPAWL-22-2, IPAWL-22-3, IPAWL-22-4 and IPAWL-22-5. These susceptible genotypes developed severe chlorosis followed by temporary and permanent wilting, extensive leaf senescence and poor recovery after flooding. Consequently, plant mortality was substantially greater in this group as compared to the tolerant group.
           
    The progressive decline in survival in tolerant and susceptible genotypes clearly demonstrated that the ability to maintain the cellular function under oxygen deprivation differed genetically among the tested pigeon pea germplasm. The differential survival rates under transient waterlogged conditions have been observed in pigeon pea and other food legume crops. 
     
    Adventitious root development
     
    Formation of adventitious roots represented one of the earliest adaptive responses to waterlogging. Tolerant genotypes rapidly produced numerous adventitious roots around the stem base shortly after flooding. These roots were not only more abundant but also considerably longer than those observed in susceptible materials (Table 2 and Fig 2).

    Table 2: Adventitious root formation in pigeon pea genotypes exposed to water logging stress.



    Fig 2: Adventitious roots development in pigeon pea genotypes under water logging stress.


           
    The development of waterlogging-induced secondary root structures provides an alternative pathway for oxygen transport and nutrient uptake when the primary root system experiences hypoxia, which is why waterlogging tolerant accessions were able to maintain better plant growth throughout the waterlogged stress period.
           
    The production and growth of adventitious roots were differential among the tested genotypes wherein greatest number and length were noted in tolerant genotypes followed by moderately tolerant genotypes. In contrast, susceptible genotypes either failed to develop adventitious roots or produced only a few short roots that contributed little to plant recovery.
           
    These observations indicate that rapid induction of adventitious root development is an important morphological marker associated with waterlogging tolerance in pigeon pea and may serve as an effective criterion for screening breeding populations.
     
    Aerenchyma formation
     
    Microscopic examination of root tissues showed pronounced differences in cortical anatomy among contrasting genotypes. Roots of tolerant genotypes developed extensive aerenchyma tissues following waterlogging (Armstrong, 1979). These enlarged intercellular air spaces with improved internal oxygen diffusion from aerial organs of the plant to submerged roots were key to sustain root respiration under hypoxic conditions (Fig 3).

    Fig 3: Aerenchyma development in waterlogging-tolerant genotypes IPAWL22-8.


           
    In contrast, susceptible genotypes exhibited little or absence of aerenchyma. The lack of this anatomical adaptation likely restricted oxygen transport within the root system, accelerating tissue injury and reducing plant survival.
           
    The simultaneous occurrence of abundant adventitious roots and well-developed aerenchyma demonstrates that tolerant pigeon pea genotypes employ coordinated structural adaptations to minimize the detrimental effects of prolonged soil flooding.
     
    Relationship between morphological adaptation and waterlogging tolerance
     
    The present investigation demonstrates that tolerance to waterlogging is governed by a combination of complementary adaptive traits rather than by a single characteristic. Genotypes exhibiting high survival consistently combined several advantageous responses, including maintenance of healthy foliage, delayed senescence, prolific adventitious rooting, extensive aerenchyma formation and rapid recovery after drainage (Singh et al., 1986).
           
    In waterlogged soils, the soil pores are filled with water which induces hypoxic condition, thereby the level of oxygen rapidly declines and soil becomes oxygen deficient due to highly restricted gaseous diffusion through water as compared to diffusion through air (Orchard and Jessop, 1984, Sairam et al., 2009 and Chaudhary et al., 2011). Under these conditions, susceptible plants experience impaired aerobic respiration, reduced ATP production and disruption of normal root metabolism. The resulting energy deficiency adversely affects nutrient acquisition, photosynthesis and overall plant growth (Marschner, 1995).
           
    Conversely, tolerant genotypes appear capable of maintaining oxygen supply to submerged tissues through anatomical modifications that facilitate internal gas movement. The formation of adventitious roots and aerenchyma enhances root aeration and improves survival during temporary flooding (Table 3). 

    Table 3: Plant survival in pigeon pea genotypes after exposer of 72 hours of waterlogging stress.


           
    Similar adaptive mechanisms have been widely documented in maize, wheat, green gram, soybean and pigeon pea, confirming their central role in waterlogging tolerance (Pourabdal et al., 2008; Jerald Anthony et al., 2024; Thomson et al., 1992; Huang et al., 1994; Saikia et al., 2021, Pires et al., 2002; Shimamura et al., 2003, 2010 and Tiwari et al., 2024). Overall, the observed morphological and anatomical responses clearly differentiate tolerant and susceptible pigeon pea genotypes and provide practical selection criteria for breeding programmes aimed at improving adaptation to waterlogged environments.
     
    Physiological and biochemical responses to waterlogging stress
     
    Gas exchange characteristics
     
    Waterlogging markedly influenced the parameters of gaseous exchange in all the tested pigeon pea genotypes. However, genotypic differences were observed in the magnitude of reduction, which corresponded with the level of waterlogging tolerance (Sharma et al., 2022 and Zhang et al., 2023). The waterlogging tolerant genotypes consistently maintained significantly higher photosynthetic rate, stomatal conductance and transpiration than susceptible genotypes throughout the stress period. The superior physiological performance of IPAC-79, ICPL-87091, ICPL-20241, ICP-2376, IPAC-212, IPAWL-22-7 and IPAWL-22-8 demonstrates their ability to sustain carbon assimilation despite prolonged root-zone hypoxia (Table 4).

    Table 4: Effect of waterlogging stress on Photosynthetic Efficiency of pigeon pea genotypes.


           
    Moderately tolerant genotypes exhibited an intermediate decline in gas exchange, indicating partial disruption of photosynthetic activity. In contrast, susceptible genotypes showed a pronounced decline in all three parameters following 48 and 72 h of waterlogging. Reduced stomatal conductance restricted carbon dioxide diffusion into the leaf, leading to reduced photosynthetic efficiency along with diminished transpiration. These responses collectively reflect impaired water and nutrient transport resulting from oxygen deprivation in the root system.
           
    Flood-induced hypoxia disrupts root respiration and ATP production, thereby reducing hydraulic conductivity and limiting the supply of water to aerial tissues. Progressive stomatal closure conserves water but simultaneously restricts CO2‚ uptake, and thereby reduces the rate of carbon fixation through photosynthesis. In addition to stomatal limitations, the structure of chloroplast and associated photosynthetic enzymes are also damaged under sustained waterlogging. It further contributes to decline in photosynthetic performance. Enhanced maintenance of gaseous exchange was observed in tolerant genotypes which indicated greater metabolic stability under flooding stress (Tian et al., 2019).
     
    Chlorophyll content and nitrogen balance index
     
    Significant differences among genotypes were also observed for chlorophyll concentration and nitrogen balance index (NBI). Waterlogging-tolerant genotypes retained substantially higher chlorophyll levels than susceptible materials, suggesting reduced degradation of photosynthetic pigments during stress (Bansal and Srivastava, 2015). Maintenance of chlorophyll enables continued light harvesting and supports sustained photosynthetic activity, thereby improving plant survival under oxygen-deficient conditions (Table 5).

    Table 5: Biochemical attributes as influenced by waterlogging stress.


           
    The nitrogen balance index followed a similar trend. Higher NBI values recorded in tolerant genotypes indicate more efficient nitrogen acquisition and assimilation despite restricted root activity. Since leaf nitrogen is closely associated with chlorophyll biosynthesis and Rubisco abundance, maintenance of nitrogen status contributes directly to improved photosynthetic performance during flooding (Bansal and Srivastava, 2017). In contrast, the susceptible genotypes exhibited substantial reduction in chlorophyll as well as NBI, which is reflective of enhanced senescence and impaired metabolic activity.  
     
    Vegetation index and photosynthetic integrity
     
    The Normalized Difference Vegetation Index (NDVI) remained consistently higher in tolerant genotypes throughout the experiment. Greater NDVI values indicate better preservation of green leaf area and canopy vigour, reflecting sustained physiological activity during waterlogging. Because NDVI integrates canopy greenness and biomass production, it provides a rapid and reliable indicator for identifying tolerant genotypes under field conditions (Table 5).
           
    The combined maintenance of chlorophyll content, nitrogen balance and NDVI demonstrates that tolerant pigeon pea genotypes experience considerably less impairment of the photosynthetic apparatus than susceptible lines. The recovery and growth post-waterlogging conditions are positively influenced by higher NDVI, hence, restoration of such physiological characteristics allows continued biomass accumulation and results in improved plant growth recovery after flooding. 
     
    Antioxidant defence system
     
    Waterlogging induced oxidative stress in all evaluated genotypes, as reflected by changes in antioxidant enzyme activity. However, differential activity of superoxide dismutase (SOD) and peroxidase enzymes was observed among the tolerant and susceptible genotypes, wherein the response of tolerant genotypes was substantially greater than that of susceptible genotypes (Table 6).

    Table 6: Effect of water logging stress on anti-oxidant enzymes and soluble sugar in pigeon pea genotypes.


           
    The enhanced activity of SOD and peroxidase enzymes indicates a more efficient scavenging of reactive oxygen species (ROS scavenging system). Superoxide dismutase catalyses the conversion of superoxide radicals into hydrogen peroxide, whereas peroxidase subsequently detoxifies hydrogen peroxide into harmless products. Together, these enzymes minimize oxidative damage to cellular membranes, proteins and nucleic acids, thereby preserving cellular integrity during flooding stress (Sairam et al., 2009, Sharma et al., 2024).
           
    In susceptible genotypes, comparatively lower antioxidant activity suggests inadequate detoxification of ROS, leading to increased oxidative injury, chlorophyll degradation and membrane instability. The present findings therefore confirm that activation of antioxidant defence constitutes an important biochemical mechanism that contributes to enhanced waterlogging tolerance in pigeon pea.
     
    Soluble sugar accumulation
     
    The soluble sugar concentration in the leaf tissue was enhanced in response to waterlogging in all genotypes, although the increase was considerably greater in tolerant materials (Table 6). Accumulation of soluble carbohydrates represents an adaptive metabolic response that supports energy production when aerobic respiration is restricted (Mishegyan et al., 2025).
           
    Under hypoxic conditions, plants shift from oxidative phosphorylation to anaerobic fermentation, making readily available carbohydrates essential for sustaining ATP generation. Higher soluble sugar concentrations observed in tolerant genotypes therefore provide an important energy reserve that facilitates continued cellular metabolism during oxygen deficiency. In addition, soluble sugars contribute to osmotic adjustment and stabilization of cellular structures, further enhancing waterlogging stress tolerance.

    Flavonols and anthocyanins
     
    Genotype-dependent reactions to flooding were seen in secondary metabolites. Throughout the stress period, tolerant genotypes maintained a relatively high level of anthocyanin and flavonol indices compared to susceptible genotypes (Table 5).
           
    Anthocyanins have potent antioxidant qualities to shield photosynthetic tissues by scavenging reactive oxygen species produced during environmental stress. It limits excessive light absorption and also reduces photo-oxidative damage (Li and Ahammed, 2023). In a similar way, flavonols serve as non-enzymatic antioxidants that can prevent oxidative damage to proteins and membranes (Beegum et al., 2023.)
           
    The increased accumulation of these metabolites in tolerant genotypes of pigeon pea suggested that both enzymatic as well as non-enzymatic antioxidant systems operate together to minimize oxidative stress during waterlogging. Such coordinated protective responses are likely to improve physiological stability and higher survival following prolonged flooding.
     
    Integrated mechanism of waterlogging tolerance
     
    The present investigation indicated that waterlogging tolerance in pigeon pea results from the coordinated interaction of multiple adaptive mechanisms rather than a consequence from the expression of a single trait. Tolerant genotypes simultaneously maintained higher photosynthetic efficiency, greater chlorophyll stability, superior nitrogen status, stronger antioxidant defence, enhanced soluble sugar accumulation and elevated concentrations of protective secondary metabolites. These physiological and biochemical characteristics complemented the structural adaptations, inclusive of stress induced adventitious root formation and development of aerenchyma as described earlier.
           
    Collectively, these responses enabled tolerant genotypes to maintain metabolic homeostasis under hypoxic conditions, minimise oxidative damage and fast recovery after withdrawal of waterlogging condition. The integration of anatomical, physiological and biochemical traits, therefore, provides a comprehensive framework for understanding waterlogging stress tolerance in pigeonpea and offers valuable selection criteria for future breeding programmes aimed at developing climate-resilient cultivars.
     
    Practical implications
     
    The present study provides several traits that can be incorporated into routine screening protocols for breeding programmes targeting waterlogging tolerance. Although survival percentage remains an important selection criterion, combining it with physiological measurements such as chlorophyll content, nitrogen balance index, photosynthetic rate and antioxidant enzyme activity would improve selection accuracy.
           
    The tolerant genotypes identified in this investigation represent valuable donor parents for developing improved pigeonpea cultivars adapted to regions experiencing intermittent flooding. Their use in hybridization programmes could accelerate the development of varieties possessing enhanced resilience without compromising productivity.
     
    Future research perspectives
     
    Response to waterlogging is dependent on the soil and weather parameters as well. Therefore, future studies should validate the identified tolerant genotypes under multi-location field environments representing different soil types and rainfall patterns. Further deciphering of the waterlogging tolerance mechanism will be strengthened by integrating the physiological screening with molecular studies such as proteomic and transcriptomic analyses to facilitate identification of genes regulating waterlogging tolerance. Moreover, studies related to the molecular breeding perspective, genomic approaches such as genome-wide association studies (GWAS), quantitative trait locus (QTL) mapping and marker-assisted selection would facilitate identification of genes regulating waterlogging tolerance. Therefore, comprehensive investigations involving metabolomics, proteomics and root system architecture analysis would further improve understanding of the molecular networks controlling adaptation to flooding stress. In nut shell, an integrated approach involving morpho-physiological, edaphological, genetical, biochemical, plant breeding and genomic science will accelerate the development of climate-resilient pigeon pea cultivars capable of maintaining stable productivity under increasingly erratic rainfall conditions associated with climate change.

    CONCLUSION

    Among the evaluated materials, IPAC-79, ICPL-87091, ICPL-20241, ICP-2376, IPAC-212, IPAWL-22-7 and IPAWL-22-8 consistently exhibited superior performance under waterlogged conditions. The results further indicate that traits viz. adventitious root development, aerenchyma formation, chlorophyll retention, photosynthetic stability, antioxidant capacity and accumulation of soluble sugars can serve as reliable physiological markers for the identification of waterlogging-tolerant genotypes. Incorporation of these morphological/physiological/biochemical marker traits into breeding programmes may substantially improve the efficiency of selecting waterlogging tolerant breeding and parental lines for developing climate-resilient pigeonpea cultivars adapted to flood-prone environments.

    CONFLICT OF INTEREST

    All authors declare that they have no conflicts of interest.

    REFERENCES


    1. Armstrong W. (1979). Aeration in higher plants. Advances in Botanical Research. 7: 225-332.

    2. Bansal, Ruchi and Srivastava J.P. (2017). Effect of water logging on root anatomy and nitrogen distribution in pigeon pea [Cajanus cajan (L.) Mill.sp.]. Indian Journal of Plant Physiology. 22(1): 130-134.

    3. Bansal, Ruchi and Srivastava, J.P. (2015). Effect of water logging on photosynthetic and biochemical parameters in pigeon pea. Russian Journal of Plant Physiology. 62(3): 322-332.

    4. Beegum, S., Troung, V., Bheemanahalli, R., Brand, D., Reddy, V. and Reddy, K.R. (2023). Developing functional relationships between water logging and cotton growth and physiology- towards water logging. Front. Plant Sci. Sec. Plant Abiotic Stress. 31(14): 1174682. https://doi.org/10.3389.

    5. Castillo, F.I., Penel, I. and Greppin, H. (1984). Peroxidase release induced by ozone in sedum album leaves. Plant Physiology.  74(4): 846-851.

    6. Chaudhary, A.K., Sultana, R., Pratap, A., Nadarajan, N. and Jha, U.C. (2011). Breeding for abiotic stresses in pigeon pea. Journal of Food Legumes. 24(3): 165-174.

    7. Dhindsa, R.A., Plumb-Dhindsa, P. and Thorpe, T.A. (1981). Leaf senescence: Correlated with increased permeability and lipid peroxidation and decreased levels of superoxide dismutase and catalase. Journal of Experimental Botany. 32(1): 93-101.

    8. DuBois, M., Gilles, K., Hamilton, J., Rebers, P. and Smith, F. (1956). Colorimetric method for determination of sugars and related substances. Analytical Chemistry. 28(3): 350-356.

    9. Huang, B., Johnson, J.W., Nesmith, D.S. and Bridges, D.C. (1994). Root and shoot growth of wheat genotypes in response to hypoxia and subsequent resumption of aeration. Crop Sci. 34(6): 1538-1544.

    10. India Stat. (2021). Land Use Data Classification/Agriculture Statistics https://www.indiastat.com/data/agriculture/land-use-classification/data-ye.

    11. Jerald Anthony, C., Esteban, Nenita, B. and Baldo (2024). Effect and recovery of maize (Zea mays linn) to water logging imposed to early seedling stage. Agricultural Science Digest. 44(3): 414-420. doi: 10.18805/ag.DF-500.

    12. Li, Z. and Ahammad, G.J. (2023). Plant stress response and adaptation via anthocyanins: A review. Plant Stress. 10: 100230.

    13. Marschner, H. (1995). Mineral Nutrition of Higher Plants. Second edition. London, Academic Press Limited. pp. 889.

    14. Mishegyan, A., Mentreddy, S.R., Duong, L. and Basu, C. (2025). Soluble sugar accumulation and differential gene- expression linked to water logging tolerance in turmeric (Curcuma longa). Plant-Environment Interaction. https://doi.org/10.10 80/17429145.2025.2558701.

    15. Orchard, P.W. and Jessop, R.S. (1984). The response of sorghum and sunflower to short term waterlogging. I. Effects of stage of development and duration of waterlogging on growth and yield. Plant Soil. 81: 119-132.

    16. Pires, J.L.F., Soprano, E. and Cassol, B. (2002). Morph physiological changes of soybean in flooded soils. Pesquisa Agropecuaria Brasileira. 37(1): 41-50.

    17. Pourabdal, L., Heidary, R. and Farboodnia, T. (2008). Effects of Different Flooding periods on Some Histochemicals of Zea mays L. Seedlings. Plant Sci. Res. 1(1): 8-12.

    18. Saikia, B., Prakash, K. and Ranjan, D. (2021). Effect of Simulated waterlogging condition imposed at early vegetative growth on final yield in green gram (vigna radiata). Legume Research. 44(10): 1226-1232. doi: 10.18805/LR-4238.

    19. Sairam, R.K., Umutha, D.K., Ezhilmathi, K., Chinnusamy, V. and Meena, R.C. (2009). Water logging induced oxidative stress and antioxidant enzyme activities in pigeon pea. Biologia Plantarum. 53(3): 493-504.

    20. Sharma, S., Bhatt, U., Shah, G. and Soni, V. (2024). Assessment of water logging induced changes in enzymatic antioxidants and carbohydrate metabolism in peanut genotypes. Biochemistry and Biophysics Reports. 39: 101794.

    21. Sharma, S., Bhatt, U., Sharma, J., Kalaji, H.M., Mojski, J. and Soni, V. (2022). Ultrastructure adaptability and alleviation mechanisms of photosynthetic apparatus in plants under water logging: A review. Photosynthetica. 60(3): 430-444.

    22. Shimamura, S., Mochizuki, T., Nada, Y. and Fukuyama, M. (2003). Formation and function of secondary aerenchyma in hypocotyl, roots and nodules of soybean (Glycine max) under flooded conditions. Plant Soil. 251: 351-359.

    23. Shimamura, S., Yamamoto, R., Nakamura, T., Shimada, S. and Komatsu, S. (2010). Stem hypertrophic lenticels and secondary aerenchyma enable oxygen transport to roots of soybean in flooded soil. Ann. Bot. 106(2): 277-284.

    24. Singh, K., Sharma, S.P., Singh, T.K. and Singh, Y. (1986). Effect of waterlogging on growth, yield and nutrient concentration of black gram and green gram under subtropical condition of Varanasi. Ann. Agr. Res. 7: 169-177.

    25. Sultana, R., Vales, M., Saxena, K.B., Rathore, A., Rao, S. and Kumar, R. (2013). Waterlogging tolerance in pigeon pea [Cajanus cajan (L.) Millsp.]: Genotypic variability and identification of tolerant genotypes. The J. Agric. Sci. 151(5): 659-671.

    26. Sultana, R., Vales, M.I., Saxena, K.B., Rathore, A., Rao, S., Rao, S.K., Mula, M.G. and Kumar, R.V. (2012). Waterlogging tolerance in pigeon pea [Cajanus cajan (L.) Millsp]: genotypic variability and identification of tolerant genotypes. J. Agril. Sci. pp. 1-14. ISSN1469-5146.

    27. Takele, A. and Mcdavid, C.R. (1995). The response of pigeon pea cultivars to short durations of waterlogging. African Crop Sci. J. 3(1): 51-58.

    28. Thomson, C.J., Colmer, T.D., Watkin, E.L.J. and Greenway, H. (1992). Tolerance of wheat (Triticum aestivum cvs. Gamenya and Kite) and triticale (Triticosecale cv. Muir) to waterlogging. New Phytol. 120: 335-344.

    29. Tian, L., Li, J., Bi, W., Zuo, S., Li, l., Li, W. and Sun, L. (2019). Effect of water logging stress at different growth stages on the photosynthetic characteristics and grain yield of spring maize (Zea mays L.) under field conditions. Agriculture Water Management. 218: 250-258.

    30. Tiwari, T.N., Datta, D., Tiwari, K. and Vijay Luxmi (2024). Physiological characterization of pigeon pea genotypes [Cajanus cajan (L.) Millsp.] for water logging tolerance. Legume Research-An International Journal. 49(3): 397-405. doi: 10.18805/LR-5342.

    31. Zaidi, P.H., Maniselvan, P., Yadav, P., Singh, A.K., Sultana, R., Dureja, P., Singh, R.P. and Srinivasan, G. (2007). Stress-adaptive changes in tropical maize (Zea mays L.) under excessive soil moisture stress. Maydica. 52: 159-171.

    32. Zhang, B., Maoxiangsun, Liu, W., Lian, M., Yang, S., Peng, F., Yuansong, X. (2023). Water logging resistance and evaluation of Physiological mechanism of three peach (Prunuspersica) rootstocks. Protoplasma. 260: 1375- 1388. https://do i.Org/10.1007/s00709-023-01850-W.Epu b2023Apr3.
    In this Article
      Contact us
      Follow us
      Published In
      Legume Research

      Editorial Board

      View all (0) Â