Physiological and biochemical mechanisms associated with salinity tolerance in sorghum (Sorghum bicolor L.) under drip irrigation

Introduction

Salinity is one of the most serious environmental constraints affecting agricultural productivity worldwide. The continuous use of saline groundwater for irrigation has increased the extent of salt-affected soils, particularly in arid and semi-arid regions. Excess salts in the root zone decrease water availability, disrupt nutrient uptake and induce oxidative stress, ultimately reducing crop growth and productivity. Plants exposed to salinity activate several adaptive mechanisms including osmotic adjustment, antioxidant defense and ionic regulation. Accumulation of compatible solutes such as proline, phenols and soluble sugars helps maintain cell turgor and protect cellular structures. Similarly, antioxidant enzymes such as superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) detoxify reactive oxygen species generated under stress conditions. Sorghum is considered moderately tolerant to salinity; however, varietal differences exist in physiological adaptation and stress tolerance. Therefore, understanding the physiological and biochemical responses of sorghum genotypes under saline irrigation is essential for developing effective management strategies and selecting tolerant varieties. The present study was undertaken to investigate the physiological and biochemical mechanisms associated with salinity tolerance in sorghum under drip irrigation.

A fertilizer dose of N, P 2 O 5 , K₂O @ 100:60:40 kg ha⁻¹ was applied through urea, single superphosphate and muriate of potash respectively. Saline irrigation water was prepared by diluting seawater (38 dS m⁻¹) with fresh water (0.6 dS m⁻¹) and treatments were imposed from 20 DAS through a drip irrigation system scheduled at 100% pan evaporation (Epan) at three-day intervals.

Physiological and Biochemical Analysis

Total chlorophyll content was estimated by the DMSO extraction method using spectrophotometric readings at 645 and 663 nm (Hiscox and Israelstam, 1979) [14] . Relative water content (RWC) was determined following the method of Barrs and Weatherley (1962) [7] , while membrane injury was estimated based on electrolyte leakage according to Towill and Mazur (1975) [35] . Proline content was determined by the acid ninhydrin method (Bates et al., 1973) [8] . Plant samples were wet-digested using a nitric acid-perchloric acid mixture (9:4) and Na⁺ and K⁺ concentrations were determined using standard procedures described by Jackson (1973) [15] and Tandon (1999) [34] . Ionic ratios (Na⁺/K⁺) were calculated from the measured ion concentrations. Data were subjected to analysis of variance and treatment means were compared at the 5% level of significance.

Results and Discussion

Influence of Saline Water Irrigation on Grain and Stover Yield of Sorghum Varieties

Varieties, saline water irrigations and their interactions had marked influence on grain and stover yields. Among the sorghum varieties, variety NTJ-5 recorded significantly the highest grain yield (4822 kg ha⁻¹) and stover yield (8718 kg ha⁻¹), while SVT 68 recorded the lowest grain (3489 kg ha⁻¹) and stover (7971 kg ha⁻¹) yields. This superior performance of NTJ 5(V 1 ) was due to its better genetic potential, higher sink capacity, efficient photosynthate translocation, longer ear head length and heavier ear head weight, contributing to higher grain filling and productivity. Similar results were also reported by Sahar et al. (2024) [30] , Jayalakshmi et al. (2024) [16] . Among salinity levels, Best Available Water (BAW) produced the highest grain (4716 kg ha⁻¹) and stover (8906 kg ha⁻¹) yields, remaining on par with 3 and 6 dS m⁻¹, whereas 12 dS m⁻¹ recorded the lowest grain (3167 kg ha⁻¹) and stover (7074 kg ha⁻¹) yields. Irrigation with BAW caused less osmotic stress on the crop and assisted in improving growth and yield and reduction in grain yield under elevated salinity stress was due to restricted water and nutrient uptake, reduced ear head weight, poor grain filling. Severe salinity adversely affects reproductive development, lowering productivity. These findings are in accordance with Chowdary et al. (2016) [10] , Mageed et al. (2018) [21] .The interaction of variety NTJ-5 under BAW irrigation recorded the highest grain (5667 kg ha⁻¹) and stover (9667 kg ha⁻¹) yields, while variety SVT-68 and 12 dS m⁻¹ salinity level recorded the lowest grain (2870 kg ha⁻¹) and stover (6574 kg ha⁻¹) yields.

Relative Water Content (RWC)

Among the varieties, SVT 68 (V 3 ) recorded the highest RWC (82.9%), which was statistically on par with NTJ 5 (V 1 ), whereas SVT 55 (V 2 ) recorded the lowest RWC (72.0%). Among the salinity levels, BAW (S1) recorded the highest RWC (86.8%), which was statistically comparable with 3 dS m⁻¹ (S 2 ) and 6 dS m⁻¹ (S 3 ) but significantly superior to the higher salinity levels. The lowest RWC (61.4%) was observed under 12 dS m⁻¹ (S 5 ). The reduction in RWC with increasing salinity may be attributed to loss of cell turgor due to osmotic stress. Accumulation of solutes in plant cells facilitates osmotic adjustment, helping maintain cell volume and reduce water loss under saline conditions (Fahmy and Esmat, 2014) [12] . The interaction effect was significant, with the highest RWC (92.2%) recorded in SVT 68 (V 3 ) under BAW (S1) and the lowest (52.4%) in SVT 55 (V 2 ) under 12 dS m⁻¹ (S 5 ). Similar findings were reported by Sarker and Oba (2020) [32] and Pattanagul and Thitisaksakul (2008) [27] . RWC is an important indicator of leaf survival and metabolic activity and is widely used to distinguish salttolerant and sensitive genotypes under salinity stress (Sinclair and Ludlow, 1986) [33] . Data showed significant variation in RWC at maturity among varieties, salinity levels, and their interaction. SVT 68 (V 3 ) recorded the highest RWC (72.3%), which was statistically comparable with NTJ 5 (V 1 ), whereas SVT 55 (V 2 ) recorded the lowest (62.4%). These genotypic differences in RWC reflect the interaction between genetic characteristics and environmental factors governing plant water retention (Dehnavi et al., 2024) [11] . Among the salinity treatments, BAW (S 1 ) recorded the highest RWC (76.9%), which was statistically on par with 3 dS m⁻¹ (S 2 ) and 6 dS m⁻¹ (S 3 ), whereas the lowest RWC (50.1%) was observed under 12 dS m⁻¹ (S 5 ). The continuous decline in RWC with increasing salinity indicates reduced water uptake and loss of cellular turgor due to osmotic stress (Zhang et al., 2022) [37] . The interaction effect was also significant, with the highest RWC (78.4%) recorded in SVT 68 (V 3 ) under BAW (S 1 ) and the lowest (40.0%) in SVT 55 (V 2 ) under 12 dS m⁻¹ (S 5 ). These findings agree with Ashraf et al. (2023) [6] . The relatively higher RWC in tolerant varieties may be attributed to greater osmotic adjustment, which lowers cellular osmotic potential, enhances root water uptake, and maintains cell turgidity under salt stress (Sarker and Oba, 2020) [32] .

Membrane Injury

Among the varieties, NTJ 5 (V 1 ) recorded the lowest membrane injury (41.3%), which was statistically comparable with SVT 68 (V 3 ), while SVT 55 (V 2 ) recorded the highest (56.0%). The lower membrane injury in NTJ 5 and SVT 68 indicates better membrane stability under salt stress (Adilakshmi, 2016) [2] . Membrane injury increased progressively with salinity, with the lowest value (34.2%) under BAW (S 1 ), statistically at par with 3 dS m⁻¹ (S 2 ), and the highest (48.3%) under 12 dS m⁻¹ (S 5 ). This increase has been associated with enhanced H₂O₂ accumulation and electrolyte leakage under salt stress (Nawaz et al., 2010; Kukreja et al., 2006; Rani, 2004) [26,19,29] . The interaction effect was significant, with the lowest membrane injury (27.1%) in NTJ 5 (V 1 ) under BAW (S 1 ) and the highest (53.3%) in SVT 55 (V 2 ) under 12 dS m⁻¹ (S 5 ) (Munira et al., 2015) [25] . Similarly, at maturity stage significant effects of varieties, salinity levels and their interaction on membrane injury was recorded. NTJ 5 (V 1 ) recorded the lowest membrane injury (36.8%), statistically on par with SVT 68 (V 3 ), whereas SVT 55 (V 2 ) recorded the highest (47.3%). Membrane injury increased from 35.3% under BAW (S 1 ) to 59.9% under 12 dS m⁻¹ (S 5 ). Higher electrolyte leakage under salinity reflects membrane damage caused by increased cell permeability and lipid peroxidation (Makarana et [22,13,20] . The interaction effect was significant, with the lowest membrane injury (31.2%) in NTJ 5 (V 1 ) under BAW (S 1 ) and the highest (67.2%) in SVT 55 (V 2 ) under 12 dS m⁻¹ (S 5 ), in agreement with Sarker and Oba (2020) [32] .

Chlorophyll Content

Total chlorophyll content at flowering was not significantly influenced by sorghum varieties. Numerically, SVT 68 (V 3 ) recorded the highest chlorophyll content (2.2 mg g⁻¹), while NTJ 5 (V 1 ) and SVT 55 (V 2 ) recorded the lowest (2.0 mg g⁻¹). Salinity levels significantly affected total chlorophyll content. The highest chlorophyll content (2.4 mg g⁻¹) was recorded under BAW (S 1 ), which was statistically comparable with 3 dS m⁻¹ (S 2 ) and significantly superior to the higher salinity levels, whereas the lowest value (1.9 mg g⁻¹) was observed under 12 dS m⁻¹ (S 5 ). The decline in chlorophyll content with increasing salinity may be attributed to chlorophyll oxidation by free radicals and disruption of pigment protein complexes by salt ions (Vahid et al., 2011; Kato and Shimizu, 1985) [36,17] . The interaction effect between varieties and salinity levels at flowering was non-significant. At maturity, total chlorophyll content also did not differ significantly among varieties. However, SVT 68 (V 3 ) recorded the highest numerical value (1.7 mg g⁻¹), while SVT 55 (V 2 ) recorded the lowest (1.5 mg g⁻¹). Salinity significantly influenced chlorophyll content at maturity, with the highest value (2.0 mg g⁻¹) recorded under BAW (S 1 ), statistically on par with 3 dS m⁻¹ (S 2 ) and the lowest (1.2 mg g⁻¹) under 12 dS m⁻¹ (S ). The reduction in chlorophyll under saline conditions may be due to disruption of chloroplast membrane integrity and oxidative degradation of chlorophyll pigments caused by excessive reactive oxygen species (Ahmad et al., 2022) [3] . The interaction effect at maturity was significant. The highest chlorophyll content (2.1 mg g⁻¹) was recorded in SVT 68 (V 3 ) under BAW (S 1 ), whereas the lowest (1.1 mg g⁻¹) was observed in SVT 55 (V 2 ) under 12 dS m⁻¹ (S 5 ).

Proline Accumulation

Among the varieties, SVT 68 (V 3 ) recorded the highest proline content at flowering (327.9 µg g⁻¹), which was statistically on par with NTJ 5 (V 1 ) and superior to SVT 55 (V 2 ), which recorded the lowest value (264.5 µg g⁻¹). Similar results were reported by Makarana et al. (2019) [22] and Adilakshmi (2016) [2] . Proline content increased progressively with salinity, from 183.9 µg g⁻¹ under BAW (S 1 ), statistically comparable with 3 dS m⁻¹ (S 2 ), to 469.3 µg g⁻¹ under 12 dS m⁻¹ (S 5 ). Proline accumulation under salinity promotes osmotic adjustment, facilitates water uptake, maintains cell turgidity, and protects cellular functions under stress (Abdelaziz et al., 2014; Sairam et al., 2002; Misra and Gupta, 2005) [1,31,24] . The interaction effect was significant, with the lowest proline content (178.8 µg g⁻¹) in SVT 55 (V 2 ) under BAW (S 1 ) and the highest (470.7 µg g⁻¹) in SVT 68 (V3) under 12 dS m⁻¹ (S 5 ) (Bavei et al., 2011) [9] . At maturity, SVT 68 (V 3 ) recorded the highest proline content (255.1 µg g⁻¹), statistically on par with NTJ 5 (V 1 ), while SVT 55 (V 2 ) recorded the lowest (218.0 µg g⁻¹), corroborating Alaei et al. (2020) [4] . Proline content increased from 154.2 µg g⁻¹ under BAW (S 1 ) to 305.6 µg g⁻¹ under 12 dS m⁻¹ (S 5 ). The increase may be attributed to the role of proline in scavenging reactive oxygen species and maintaining the integrity of cellular macromolecules under stress (Rahdari et al., 2012; Koc et al., 2010) [28,18] , as also reported by Bavei et al. (2011) [9] . The interaction effect was significant, with the lowest proline content (136.6 µg g⁻¹) in SVT 55 (V 2 ) under BAW (S 1 ) and the highest (329.7 µg g⁻¹) in SVT 68 (V 3 ) under 12 dS m⁻¹ (S 5 ).

Influence of Saline Water Irrigation on Na⁺/K⁺ Ratio

At flowering, the Na⁺/K⁺ ratio differed significantly among the sorghum varieties. SVT 55 (V 2 ) recorded the highest ratio (0.54), which was significantly superior to the other varieties, whereas SVT 68 (V 3 ) recorded the lowest ratio (0.45). Salinity significantly increased the Na⁺/K⁺ ratio, with the lowest value (0.39) recorded under BAW (S 1 ), which was statistically comparable with 3 dS m⁻¹ (S 2 ), and the highest (0.59) under 12 dS m⁻¹ (S 5 ). Increasing salinity reduced K⁺ concentration while enhancing Na⁺ accumulation, resulting in a higher Na⁺/K⁺ ratio, as reported by Vahid et al. (2011) [36] . Salt stress also promotes nonspecific ion uptake, leading to greater Na⁺ accumulation in plant tissues (Anower-Rokebul et al., 2013) [5] . The interaction between varieties and salinity levels at flowering was significant. The lowest Na⁺/K⁺ ratio (0.36) was observed in SVT 68 (V 3 ) under BAW (S 1 ), whereas SVT 55 (V 2 ) under 12 dS m⁻¹ (S 5 ) recorded the highest ratio (0.69). At maturity, the Na⁺/K⁺ ratio did not differ significantly among the varieties, although SVT 55 (V 2 ) recorded the highest numerical value (0.79) and SVT 68 (V 3 ) the lowest (0.72). Salinity continued to increase the Na⁺/K⁺ ratio, from 0.64 under BAW (S 1 ), statistically comparable with 3 dS m⁻¹ (S 2 ), to 0.88 under 12 dS m⁻¹ (S 5 ). The increase was associated with greater Na⁺ accumulation and a decline in K⁺ concentration, in agreement with Menezes et al. (2017) [23] . Selective uptake of K⁺ over Na⁺ is considered a major physiological mechanism contributing to salt tolerance in plants The interaction effect between varieties and salinity levels at maturity was not significant.

Conclusion

Significantly the highest grain and stover yields were recorded under variety NTJ 5 and pertaining to interaction variety NTJ 5 and BAW irrigation recorded highest grain and stover yields. Salinity significantly affected physiological and biochemical processes in sorghum.

Increasing irrigation water salinity reduced relative water content, and chlorophyll concentration while increasing membrane injury. Salinity stress induced accumulation of proline and enhanced antioxidant enzyme activities. These responses contributed to osmotic adjustment and protection against oxidative damage. Among the tested varieties, SVT-68 exhibited superior physiological adaptation through better water status, membrane stability, osmolyte accumulation and antioxidant defense. Therefore, SVT-68 may be considered a physiologically tolerant genotype suitable for cultivation under saline irrigation conditions.

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