Persistent and reversible labile Cu(I) heterogeneity reflects stress response during chronic copper exposure in microalgae.

Phenotypic heterogeneity has emerged as a critical yet insufficiently explored factor influencing organismic responses to environmental stress. Here, we investigated the long-term dynamics and biological implications of cell-to-cell heterogeneity in labile Cu(I) bioaccumulation in the model microalga Chlamydomonas reinhardtii under 4 months of Cu exposure. Flow cytometric analysis revealed a sustained subpopulation of cells containing elevated labile Cu(I) levels, with the Cu(I) positive fraction stabilizing at approximately 13% under high Cu conditions. This finding suggested that the observed heterogeneity was not transient but might represent a potentially persistent trait. This heterogeneity increased with external Cu levels in medium, indicating strong environmental modulation. Notably, total cellular Cu content did not strictly correlate with growth inhibition, whereas the proportion of Cu(I) positive cells closely followed reductions in cell density. Quantitative correlation and regression analyses further demonstrated that the proportion of Cu(I) positive cells showed a stronger association with growth inhibition than total cellular Cu accumulation. Confocal imaging further revealed the heterogeneous mitochondrial morphology, linking intracellular Cu(I) imbalance to organelle dysfunction. Upon transfer to stress-free conditions, Cu(I) positive cells could rapidly convert to a low-Cu(I) state, accompanied by decreased intracellular Cu content but largely restored viability and redox status, although photosynthetic efficiency remained partially impaired. These findings revealed that Cu(I)-associated heterogeneity was persistent, environmentally responsive, and associated with physiological changes in microalgae. This persistent heterogeneity may represent a type of stress response phenotype and provides a potential biomarker for evaluating metal toxicity in phytoplankton.