Autophagy-like induction by the overexpression of the chloroplast protease subunit CrClpP5 increases nickel tolerance in Chlamydomonas reinhardtii

Heavy metals are toxic to living organisms even at trace levels and persist in the environment due to their non-degradability. Understanding the mechanisms of heavy metal response and adaptation is therefore crucial. As a key stress defense system, the chloroplast Clp protease complex plays a central role in degrading damaged proteins within chloroplasts. This study characterized the molecular responses of the Clp complex to nickel (Ni) and cadmium (Cd) stress in Chlamydomonas reinhardtii, revealing significant upregulation of CrClpP5 specifically under Ni stress. Subsequently, a CrClpP5-overexpressing transgenic strain (OE-CrClpP5) was generated to investigate its role in Ni stress regulation. Phenotypic analysis demonstrated that OE-CrClpP5 cells exhibited significantly higher Ni tolerance than wild-type (WT) controls, evidenced by enhanced chlorophyll retention, increased biomass accumulation, and elevated antioxidant enzyme activities. Ultrastructural examination revealed the presence of autophagosome-like structures in unstressed OE-CrClpP5 cells. Furthermore, the up-regulated expression pattern of autophagy-related genes in OE-CrClpP5 confirmed the activation of autophagy, indicating that chloroplast CrClpP5 overexpression activates cytoplasmic ATG pathway associated with autophagy formation. These findings elucidate a novel mechanism of chloroplast-protease-mediated autophagy activation for heavy metal stress mitigation and provide a theoretical foundation for developing microalgae-based bioremediation technologies for heavy metal pollution.