Characterization of a Nicotiana tabacum phytochelatin synthase 1 and its response to cadmium stress

Phytochelatin synthase (PCS) is a critical enzyme involved in heavy metal detoxification in organisms. In this study, we aim to comprehensively investigate the molecular and functional characteristics of the <i>PCS1</i> gene from <i>Nicotiana tabacum</i> by examining its enzymatic activity, tissue-specific expression pattern, Cd-induced expression, as well as the impact on Cd tolerance and accumulation. The results demonstrated that the amino acid sequence of NtPCS1 shared a high similarity in its N-terminal region with PCS from other species. The enzymatic activity of NtPCS1 was found to be enhanced in the order Ag²⁺ > Cd²⁺ > Cu²⁺ > Pb²⁺ > Hg²⁺ > Fe²⁺ > Zn²⁺. In addition, RT-PCR data indicated that <i>NtPCS1</i> gene is constitutively expressed, with the highest expression observed in flowers, and that its transcript levels are up-regulated by CdCl₂. When tobacco overexpressing <i>NtPCS1</i> (PCS1 lines) were grown under CdCl₂ stress, they produced more phytochelatins (PCs) than WT plants, but this did not result in increased Cd accumulation. However, in a root growth assay, the PCS1 lines exhibited hypersensitivity to Cd. The overexpression of <i>NtPCS1</i> itself does not appear to be the primary cause of this heightened sensitivity to Cd, as the <i>Arabidopsis thaliana Atpcs1</i> mutant overexpressing <i>NtPCS1</i> actually exhibited enhanced tolerance to Cd. Furthermore, the addition of exogenous glutathione (GSH) progressively reduced the Cd hypersensitivity of the PCS1 lines, with the hypersensitivity even being completely eliminated. Surprisingly, the application of exogenous GSH led to a remarkably enhanced Cd accumulation in the PCS1 lines. This study enriches our understanding of the molecular function of the <i>NtPCS1</i> gene and suggests a promising avenue for Cd tolerance through the heterologous expression of <i>PCS</i> genes in different species.