Melatonin Enhances Antioxidant Defense and Physiological Stability in Grapevine (<i>Vitis vinifera</i> L.) Cultivars ‘Merlot’ and ‘Erciş’ under UV-B Stress

Climate change-driven environmental stresses, particularly ultraviolet-B (UV-B) radiation, pose severe threats to grapevine (Vitis vinifera L.) productivity and physiological stability. This study investigated the protective role of melatonin in in vitro plantlets of two grapevine cultivars, ‘Merlot’ and ‘Erciş’, subjected to low (≈8.25 μW cm−2, 16 h) and high (≈33 μW cm−2, 4 h) UV-B exposure. Significant cultivar-specific responses were observed (p < 0.001). The ‘Erciş’ cultivar exhibited higher oxidative stress, with malondialdehyde (MDA) levels reaching 24.30 mmol g−1 FW in control plants compared with 14.91 ± 0.25 mmol g−1 FW in ‘Merlot’. Melatonin provided dose-dependent mitigation, reducing MDA to 12.68 in ‘Erciş’ and 8.52 ± 0.13 in ‘Merlot’ at 200 μmol L−1. Antioxidant enzyme activities increased significantly: superoxide dismutase rose from 0.02 ± 0.01 to 0.10 EU g−1 in ‘Erciş’ and to 0.13 EU g−1 in ‘Merlot’, catalase increased up to 0.08 in ‘Erciş’ and 0.16 in ‘Merlot’, while ascorbate peroxidase reached 1.06 ± 0.02 and 1.20 ± 0.03, respectively. Pigment traits also improved, with chlorophyll content increasing to 23.70 μg cm−2 in ‘Merlot’ and 22.66 μg cm−2 in ‘Erciş’, alongside enhanced nitrogen balance index values. Secondary metabolites were elevated, particularly total phenolic content (8.23 GAE 100 g−1 in ‘Erciş’ and 5.99 in ‘Merlot’) and antioxidant capacity (17.24 and 8.15 μmol TE g−1, respectively). Correlation analyses revealed strong positive associations between melatonin and antioxidant enzymes (r = 0.54–0.85), while principal component analysis explained 64.71% of total variance, separating cultivars and treatments clearly. Clustering patterns showed distinct grouping of enzymatic defenses, phenolic compounds, and pigments, reflecting coordinated protective mechanisms. Overall, melatonin application, especially at 200 μmol L−1, significantly enhanced enzymatic and non-enzymatic antioxidant defenses, stabilized photosynthetic pigments, and reduced oxidative damage, with stronger protective efficiency in ‘Merlot’. The research provided valuable insights for developing biotechnological approaches to enhance grape stress tolerance in the context of climate change challenges.