A digital PCR-based platform for rapid assessment of chloroplast stress adaptation in microalgal metabolic engineering.

Microalgae rapidly adjust their chloroplast physiology in response to environmental stress, and these adaptive responses are closely associated with cellular fitness and metabolic performance. However, conventional assessments of stress adaptation primarily rely on growth characteristics, pigment accumulation, or physiological measurements, which often require extended cultivation periods and may not capture early molecular responses. In this study, we introduce a digital PCR (dPCR)-based platform for rapid assessment of chloroplast stress adaptation in microalgae. The platform quantifies the chloroplast-to-nuclear genome copy number ratio (C/N ratio) using multiplex dPCR and utilizes this metric as a molecular indicator of chloroplast acclimation. As a proof-of-concept, the assay was applied to the halotolerant microalga Dunaliella salina cultivated under different salinity stress conditions. Distinct temporal changes in the C/N ratio were observed across salinity treatments, indicating dynamic chloroplast genome remodeling during stress adaptation. The assay enabled sensitive detection of chloroplast responses at early cultivation stages, prior to the appearance of clear phenotypic differences. These findings demonstrate that chloroplast-to-nuclear genome quantification by dPCR provides a rapid and reproducible approach for monitoring chloroplast stress adaptation in microalgae. The proposed platform offers a practical molecular tool for strain evaluation, cultivation optimization, and stress-response studies, and may support future applications in microalgal biotechnology and industrial production systems.