Integrated assessment of photosynthesis, water use efficiency and yield in cotton under differential irrigation regimes

Introduction

Cotton (Gossypium hirsutum L.) is one of the most widely cultivated fiber crops globally, serving as a vital component of the textile industry and providing significant income to farmers in tropical and subtropical regions (Khan et al., 2019) [6] . In India, cotton occupies nearly 12 million hectares, with a substantial proportion grown in semi-arid zones where rainfall is erratic and irrigation resources are limited. Despite its moderate drought tolerance, cotton's growth, reproductive development and final yield are highly dependent on adequate water availability during critical phenological stages, particularly flowering and boll formation (Pettigrew, 2004; Sarwar et al., 2015) [9,12] . The increasing frequency of drought events, declining groundwater tables and climate variability are exacerbating water scarcity in many cotton-growing regions (Abdelraheem et al., 2019) [1] . These challenges underscore the need for strategies that enhance water productivity while maintaining yield stability. Deficit irrigation, when carefully timed, has shown potential in improving water use efficiency (WUE) in cotton without causing severe yield losses (Reddy et al., 2015; Zhang et al., 2010) [10,14] . However, crop response to water deficit is highly genotype-dependent and stage-specific, making it essential to dissect physiological responses across genotypes and growth stages. Physiological traits such as photosynthetic rate, stomatal conductance, transpiration and leaf water potential offer valuable insights into plant water relations and have been proposed as functional indicators of drought tolerance (Flexas et al., 2004; Chaves et al., 2009) [4,3] . Additionally, traits like light interception and WUE are important determinants of crop performance under water-limited environments (Gaju et al., 2011) [5] . Integrating these physiological indicators with yield traits can aid in identifying resilient genotypes and optimizing irrigation schedules. Despite these advances, comprehensive field studies evaluating cotton responses to moisture deficit using integrated physiological and agronomic parameters remain limited. Therefore, the present study aimed to (i) assess the effects of irrigation levels and stress imposition at different growth stages on physiological traits and yield in two cotton genotypes, (ii) identify physiological markers associated with water use efficiency and drought tolerance and (iii) explore genotype-specific responses to water deficit in a semi-arid agroecological context.

Material and Methods Experimental Site and Climate

The field experiment was conducted during the kharif season at the Agriculture Research Station, Hebballi Farm, UAS, Dharwad. The site falls under a semi-arid agroclimatic zone with an average annual rainfall of approximately 659.06 mm. The soil at the site was classified as clayey, neutral pH and good drainage.

Experimental Design and Treatments

The study was laid out in a split-plot design with three replications. The main plot factor comprised four irrigation levels, based on field capacity (FC): M 1 -100% FC (Wellirrigated control), M 2 -75% FC, M 3 -50% FC and M 4 -Rainfed (No irrigation after establishment). The sub-plot factor included two cotton genotypes: V 1 -GSHV-97/162 and V 2 -LRA-5166. Water was applied via surface irrigation. Soil moisture content was monitored using the gravimetric method to maintain field capacity thresholds in M 1 -M 3 . In each main plot, three irrigation withdrawal stages were imposed as sub-subplot treatments to induce growth-stage-specific water stress: S 1 -No irrigation from 10 to 40 days after sowing (vegetative stage), S 2 -No irrigation from 50 to 80 DAS (flowering stage) and S 3 -No irrigation from 85 to 120 DAS (boll development stage)

Crop Management

Standard agronomic practices for cotton cultivation in the region were followed. Seeds were sown at a spacing of 90 cm × 60 cm. Fertilizer was applied at the recommended rate of 120:60:60 kg N: P₂O₅:K₂O ha⁻¹. All plots received uniform weed and pest management.

Physiological Observations

Physiological traits were recorded at 60, 90 and 120 DAS. Photosynthetic rate (µmol CO₂ m⁻² s⁻¹), stomatal conductance (mol H₂O m⁻² s⁻¹) and transpiration rate (mmol H₂O m⁻² s⁻¹) were measured using a portable Infrared Gas Analyzer (IRGA, LI-6400, LI-COR Inc., USA). Water use efficiency (WUE) was computed as the ratio of photosynthesis to transpiration. Leaf water potential (bars) was measured using a pressure chamber (Scholander-type) at 90 and 120 DAS. Percent light absorption was measured at 90 and 120 DAS using a line quantum sensor (Model LI-191R, LI-COR Inc.), with values calculated from light intercepted by the canopy. Yield and Yield Components viz., Seed cotton yield (kg ha⁻¹) from the net plot area, Number of bolls per plant, based on five randomly selected plants per plot and Mean boll weight (g), based on 20 bolls sampled at random were recorded at harvesting.

Statistical Analysis

Data were analyzed using analysis of variance (ANOVA) following the split-plot design structure. Treatment means were compared using the least significant difference (LSD) test at 5% significance level. Homogeneity of variance was verified prior to analysis. A correlation matrix and heatmap were constructed using Python 3.11 (libraries: pandas, seaborn, matplotlib) to visualize the interdependence among yield and physiological traits across irrigation treatments.

Results and Discussion

Effect of Irrigation Levels on Yield Performance

The influence of irrigation levels on cotton yield was profound and statistically significant (Table 7). Under full irrigation (M 1 ), genotype V 1 (GSHV-97/162) achieved the highest average yield (1602.33 kg/ha), whereas under rainfed conditions (M 4 ), the yield declined sharply to 1406.00 kg/ha, reflecting a ~12.3% yield penalty. A similar but slightly more pronounced decline was observed for genotype V 2 (LRA-5166), from 1603.77 kg/ha (M 1 ) to 1405.77 kg/ha (M 4 ). These yield reductions were directly associated with decreasing soil moisture levels and the corresponding physiological impairments, consistent with earlier studies that emphasize the vulnerability of cotton to moisture stress, particularly during critical reproductive stages (Pettigrew, 2004; Sarwar et al., 2015) [9,12] .

Photosynthesis and Gas Exchange Dynamics

Photosynthetic rate (Pn) was highly responsive to irrigation and varied across developmental stages (Table 1). At 90 DAS, cotton plants under M1 maintained high photosynthetic rates in both genotypes (V 1: 26.60 µmol/m²/s; V 2: 30.83 µmol/m²/s). These values dropped significantly under water-deficit conditions (M 4 ), especially in V 2 , which decreased to 23.92 µmol/m²/s. Reduced photosynthetic activity under water stress is attributed to stomatal limitations, decline in chlorophyll content and impaired mesophyll conductance, corroborated by earlier work on drought responses in cotton (Flexas et al., 2004; Abdelraheem et al., 2019) [4,1] . This decline in photosynthesis coincided with a reduction in stomatal conductance (gs), as shown in Table 2. Under M 1 , V 2 maintained a gs of 0.052 µmol/m²/s at 90 DAS, which dropped to 0.049 under M 4 . This suggests partial stomatal closure to conserve water, but at the cost of limiting CO₂ influx. Transpiration rates followed a parallel trend (Table 3), with M 1 conditions supporting high evapotranspiration (6.74 µmol CO₂/m²/s in V 2 at 90 DAS), while M 4 saw a reduction to 4.02 µmol CO₂/m²/s. These reductions in Pn, gs and E under drought stress demonstrate the complex interplay between gas exchange parameters, which collectively affect dry matter production and yield (Chaves et al., 2009) [3] .

Water Use Efficiency (WUE)

Water use efficiency, defined as the ratio of carbon gained to water lost, improved modestly under moderate stress (M 2 ), but declined under severe stress (M 4 ), especially at 120 DAS (Table 4). Genotype V1 maintained higher WUE than V 2 across most stages and treatments. At 90 DAS, WUE reached 0.41 µmol CO₂/mmol H₂O in V 1 under M 2 , but fell to 0.34 under M 4 . These results align with prior findings that moderate stress may enhance WUE by optimizing stomatal function, but excessive water deficit suppresses both photosynthesis and transpiration, thus compromising efficiency (Zhang et al., 2010; Liu et al., 2013) [14,7] .

Light Absorption and Canopy Behavior

A marked reduction in percent light absorption was observed under reduced irrigation (Table 5). At 120 DAS, V 2 absorbed up to 43.20% of incident light under M 1 , but only 25.20% under M 4 . Reduced light interception under drought conditions may result from a decrease in leaf area index, chlorophyll degradation and premature senescence (Gaju et al., 2011) [5] . Genotype V 1 maintained better light absorption under stress, suggesting superior canopy retention and possibly deeper rooting, which may have facilitated better water uptake.

Water Potential and Plant Water Status

Water potential measurements (Table 6) confirmed significant stress under limited irrigation. At 120 DAS, V 2 recorded a water potential of -23.08 bars under M 4 , indicating severe dehydration. In contrast, M 1 conditions maintained relatively higher water potential values (-18.44 bars in V 2 ), reflecting adequate soil moisture availability. These values are consistent with the physiological thresholds identified by Turner et al. (2001) [13] , who noted that cotton's critical water potential threshold for stomatal closure is around -20 bars.

Yield Components and Their Contribution

Yield decline under drought was also linked to reductions in boll number and boll weight (Table 7). Under M 1 , V 1 produced 17.55 bolls per plant and an average boll weight of 4.15 g. These values dropped significantly under M 4 to 9.55 bolls and 2.83 g boll weight, respectively. The sensitivity of reproductive structures to drought-particularly flower shedding and reduced boll filling-has been documented in numerous cotton studies (Saeed et al., 2014; Nawaz et al., 2020) [11,8] .

Genotypic Response and Stability

Across all measured parameters, V 1 (GSHV-97/162) outperformed V 2 (LRA-5166) under limited water availability. V 1 exhibited better retention of photosynthesis, higher WUE, more stable light absorption and less negative water potential values. These traits contribute to better drought resilience and are desirable in breeding programs for semi-arid regions. Similar genotypic stability under stress conditions has been reported by Ali et al. (2015) [2] , who found that genotypes with higher WUE and leaf retention maintain yield under drought.

Integrated Analysis of Physiological and Agronomic Traits Using Heatmap Visualization

A heatmap was used to visualize the integrated response of cotton to four irrigation regimes (M 1 -M 4 ), based on key physiological traits and yield (Fig. 1). The analysis showed a clear decline in yield, photosynthetic rate, transpiration, light absorption and water potential from full irrigation (M 1 ) to rainfed (M 4 ) conditions. Seed cotton yield dropped from 1603.05 kg/ha (M 1 ) to 1405.88 kg/ha (M 4 ), closely tracking reductions in photosynthesis (from 28.7 to 24.1 µmol m⁻² s⁻¹) and light absorption (from 42.7% to 26.4%).

Water use efficiency (WUE) was highest under moderate stress (M 2 ), indicating partial physiological compensation. Stomatal conductance showed minor variation, suggesting a balance between water conservation and carbon assimilation. Water potential became more negative with increasing stress, confirming drought severity. The heatmap highlighted strong trait interdependence and showed that drought stress impairs photosynthetic efficiency, water transport and ultimately yield. Genotype V 1 generally outperformed V 2 under stress, showing better physiological stability. This visual analysis supports the use of integrated trait-based approaches to guide irrigation management and genotype selection for drought resilience.

Conclusion

The present study demonstrated that deficit irrigation significantly affects the physiological performance and yield of cotton. Among the treatments, moderate water stress (75% field capacity) optimized water use efficiency without causing substantial yield loss, while severe stress (rainfed) led to marked declines in photosynthesis, transpiration, light absorption, and boll development. Genotype GSHV-97/162 (V1) exhibited superior drought tolerance compared to LRA-5166 (V2), maintaining higher gas exchange rates, WUE, and yield under stress. The integrated use of physiological indicators and heatmap analysis proved effective in identifying trait interdependencies and genotype resilience. These findings underscore the importance of genotype selection and growth-stage-specific irrigation management for sustaining cotton productivity in waterlimited environments.

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