Oxidative Stress Physiology of Cotton Seedlings under Drought, Linking Water Status, Pigments, Osmolytes, and Antioxidant Enzymes

Drought disrupts cotton seedling physiology by lowering tissue water status, constraining photosynthesis, and intensifying oxidative stress. We evaluated ten upland cotton genotypes in a greenhouse polythene-bag system under graded deficit (100, 75, and 50% field capacity) at the second true-leaf stage. Traits spanned growth and biomass, water status (relative water content, excised-leaf water loss), pigments (chlorophylls, carotenoids), biochemical/osmotic markers (total soluble protein, proline, phenolics, flavonoids), antioxidant enzymes (superoxide dismutase, catalase, ascorbate peroxidase, peroxidase), and oxidative injury (hydrogen peroxide, malondialdehyde). Two-factor factorial ANOVA at P ≤ 0.05 showed significant main effects of genotype and treatment for most traits, with frequent genotype × treatment interactions; Tukey’s test resolved group differences. Correlation analysis revealed hydration and pigments moving together, while enzymatic and non-enzymatic antioxidants clustered and inversely tracked oxidative markers. Water deficit reduced shoot and root growth and chlorophylls, lowered relative water content, and increased excised-leaf water loss. Oxidative load rose, whereas proline, soluble protein, phenolics, flavonoids, and antioxidant enzymes were induced responses that stabilize membranes, support osmotic balance, and detoxify reactive oxygen species. Tolerant genotypes maintained higher relative water content and pigments while restraining hydrogen peroxide and malondialdehyde, incurring smaller growth penalties. Principal component analysis resolved an injury axis dominated by leakage and oxidative markers and a defense axis integrating water status, pigments, osmolytes, and antioxidants, cleanly separating resilient from susceptible ideotypes. These results define a practical seedling-stage selection bundle, high relative water content, low excised-leaf water loss, chlorophyll retention with stable carotenoids, elevated osmolyte and phenolic pools, strong antioxidant activities, and reduced oxidative injury, providing a physiological basis for early screening and breeding.