PKG drives metabolic adaptation and salt stress response mechanisms in Chromochloris zofingiensis

Salt stress impacts crop and microalgae growth via osmotic stress, ion toxicity, and oxidative stress. Microalgae, with their efficient photosynthesis and diverse metabolism, offer promising solutions to resource scarcity and environmental challenges. The nitric oxide (NO)-cGMP-protein kinase G (PKG) signaling cascade is a conserved stress-response pathway that regulates ion homeostasis and antioxidant defense in eukaryotes. Although PKG has been shown to modulate stress adaptation through the GAMYB transcription factor in plants, its role in microalgae remains unknown. To elucidate the function of PKG in salt stress adaptation, this study focuses on a mutant strain of Chromochloris zofingiensis with inactivated cGMP-dependent protein kinase G (PKG) (Cz-pkg), and compares it to the wild-type strain (Chromochloris zofingiensis wild type, Cz-WT), analyzing their differential responses under varying salt concentrations in terms of growth, pigment metabolism, lipid accumulation, and gene expression. We found that PKG inactivation markedly reduced salt tolerance and led to a complete loss of astaxanthin accumulation, while significantly suppressing lipid biosynthesis. Transcriptomic analysis further revealed that PKG regulates key genes involved in the MEP pathway, carotenoid metabolism, and fatty acid synthesis, particularly enhancing antioxidant defense and carbon flux toward astaxanthin and lipid production. These results demonstrate that PKG acts as a central regulator in microalgal adaptation to salt stress by coordinating antioxidant responses and metabolic reprogramming. This study provides the first evidence of PKG's role in microalgal salt tolerance, offering insights for engineering stress-resilient strains and salt-tolerant crops.