Cadmium detoxification mechanisms in Populus alba: ecophysiological perspectives and the role of thiol-peptides

Due to its exceptional toxicity to all biological systems, cadmium (Cd) is a heavy metal (HM) frequently released into the environment by both natural and man-made sources and is particularly pervasive in soils competing with the plant for nutrient uptake. In contrast to organic pollutants, HMs are non-biodegradable and tend to accumulate in living organisms. For these reasons HMs and specifically Cd are dangerous even at low concentrations as they can result in micronutrient deficiencies. This impactful problem of HM pollution in environment is a significant risk to public health worldwide. Populus alba has been identified as a promising phytoremediator due to its ability to withstand various HMs, employing different tolerance and detoxification mechanisms, including the production of phytochelatins (PCn), which are cysteine-rich oligopeptides synthesized from glutathione (GSH) by the enzyme phytochelatin synthase (PCS). The principal mechanism of intracellular HMs detoxification involves the formation of PCn-HM complexes, which are then sequestered in the vacuole for detoxification. To investigate the thiol-peptides role in Populus alba HM detoxification, we developed an HPLC-MS/MS method for quantifying GSH and PCn in plant matrices under CTRL and Cd2+-stressed conditions. The first experimental setup provided the evaluation of biometric parameters, fluorescence and photosynthetic efficiency, and thiol-peptide quantifications in Populus alba plants exposed to high Cd2+ stressed conditions (50 and 100 μM CdSO4) in a woody plant solid medium with vitamins (pH 5.85) for two months. To assess short-term responses of Populus alba, a second set of experiments was conducted: one-month-old plants were transferred to Gamborg’s B5 liquid medium (pH 5.85) containing various concentrations of CdSO4 (5, 10, 20, 36 μM) for exposure durations of 24-48-72 hours. The plants' shoots and roots were sampled and analysed to assess GSH and PCn levels at different exposure times and Cd2+ concentrations. Concurrently, the growth medium was analysed to track the potential expulsion of GSH and PCn- Cd2+ complexes from the roots. Preliminary results indicated an increase in both intracellular and extracellular GSH and PCn concentrations, which correlated with longer treatment durations and higher Cd²⁺ concentrations. At the same time, to investigate whether thiol-peptide release observed in complex plants also occurs in simpler cellular organisms, we used immortalized Arabidopsis thaliana cells as a model. Cells were exposed to control conditions and varying concentrations of Cd²⁺ (10, 20, and 36 µM) for 48 and 72 hours. Analytical evaluation of GSH and PCn demonstrated that eukaryotic cells effectively employ defence mechanisms against HM stress. Notably, at 20 µM Cd²⁺ after 48 hours, significant thiol-peptide production was observed both intracellularly and extracellularly. To investigate the release of thiol-peptides into the extracellular environment an inhibition study of hypothetical plasmalemma membrane PCn/ PCn- Cd2+ transporters was conducted. This study highlights a remarkable ability of Populus alba to tolerate Cd stress, through the investigation of its detoxification mechanisms. Notably, the focus on PCn revealed their dual role: not only they chelate Cd at the intracellular level, but they also contribute to the formation of an extracellular barrier, further protecting the plant from metal toxicity.