Background In recent decades, arsenic (As) toxicity has emerged as a significant challenge in many countries. It not only reduces the growth and performance of plants, but also poses a threat to human health. The synthesis of compatible solutes, particularly proline, is a mechanism plants utilize to cope with stress. Investigating the metabolic pathways of proline would deepen our understanding for future molecular breeding or genetic engineering efforts. Therefore, the aim of this study was to explore the metabolic and catabolic pathways of proline, as well as the morpho-physiological traits of tobacco, under As stress. Results The results revealed a significant decrease in morphological traits and photosynthetic efficiency, chlorophyll content, and total soluble protein content with increasing As concentration. The results also showed that proline content, total soluble carbohydrates, hydrogen peroxide, and malondialdehyde, as well as the activity of two antioxidant enzymes, superoxide dismutase and ascorbate peroxidase, increased with increasing As concentration. At 10 mg As Kg−1 soil, the expression of Δ1-pyrroline-carboxylate synthetase (P5CS) and P5C reductase (P5CR) genes was not different from the control, but their expression increased significantly at 20 and 40 mg As Kg−1 soil. At 10 mg As Kg−1 soil, the expression of proline dehydrogenase (PDH) and P5C dehydrogenase (P5CDH) genes decreased sharply compared to the control but remained unchanged at 20 and 40 mg As Kg−1 soil. At 10 and 20 mg As Kg−1 soil, expression of the ornithine δ-aminotransferase (OAT) gene was unchanged compared to the control, but at 40 mg As Kg−1 soil, it increased sharply. Conclusion The results showed that the accumulation of proline at the lowest (10 mg As Kg−1 soil) tested As concentration was due to a decrease in the expression of proline catabolic genes (PDH and P5CDH), while the genes involved in proline synthesis did not play a role. At 20 mg As Kg−1 soil, proline accumulation was caused by the increased expression of genes (P5CS and P5CR) involved in the glutamate pathway of proline synthesis. Additionally, at the highest concentration of arsenic (40 mg As Kg−1 soil), the OAT gene, which is active in the ornithine pathway, was also involved in proline synthesis, along with the P5CS and P5CR genes.
