In recent decades, heavy metal stress has emerged as a significant abiotic factor contributing to orchard contamination. High levels of lead (Pb) and copper (Cu) in the soil result from their frequent use as fungicides and pesticides. Additionally, food contamination has persisted due to over 50 years of insecticide applications such as lead arsenate and copper sulfate. Sour orange (Citrus aurantium L.) is known for its medicinal properties, attributed to its bioactive compounds like phenolics, flavonoids, and essential oils. This study examines the impact of proline, a common compatible osmolyte which plays a crucial role in antioxidant activity, on growth and biochemical characteristics in sour orange plants exposed to lead and copper-contaminated soils. Citrus plants were cultivated in a greenhouse with varying concentrations of copper and lead (500, 800μM), including a combination of Cu+Pb (500, 800μM), and exogenous proline treatment (20mM). The volatile constituents of the essential oils from the peel of sour oranges were analyzed using GC-MS. Furthermore, the photosynthetic machinary was estimated by calculating the assimilation and transpiration rate of CO2 and the water use efficiency using a portable photosynthesis system. Morphological characteristics such as height showed notable

decreases due to photosynthetic disturbances especially at simultaneously high levels of Cu+Pb (800μM). In addition CuSO4 treatment at high levels (800μM) with simultaneously high Pb(NO3)2 concentrations caused significant increased, in lipid peroxidation (MDA), proline and hydrogen peroxide (H2O2) content, while the antioxidant activity was increased. Proline treatment generally increased tolerance to copper and lead, and enhanced the accumulation of phenols and soluble sugars compared to untreated plants. These findings suggest variations in antioxidant responses to oxidative stress induced by copper and lead, potentially linked to the application of exogenous proline. This treatment appears to elevate antioxidants, thereby protecting membrane functions from ROS-induced damage in citrus plants.