In situ 3D comparison of Chlorella pyrenoidosa with nuclear-irradiated mutagenic strains by using focused ion beam milling and cryo-electron tomography

Microalgae are highly efficient photosynthetic organisms that hold enormous potential as sources of renewable energy. In particular, Chlorella pyrenoidosa displays a rapid growth rate, high tolerance to light, and high lipid content, making it especially valuable for applications such as flue gas CO2 fixation, biofuel production, and nutritional extracts. In order to unveil its full potential, it is necessary to characterize its subcellular architecture. Here, we achieved three-dimensional (3D) visualization of the architectures of C. pyrenoidosa cells, by combining focused ion beam scanning electron microscopy (FIB/SEM), cryo-FIB milling, and cryo-electron tomography (cryo-ET). These high-resolution images bring to light intricate features of intact organelles, including thylakoid membranes, pyrenoid, starch granules, mitochondria, nucleus, lipid droplets and vacuoles, as well as the fine architectures within the chloroplast, including the concave-convex pyrenoid, plastoglobules, thylakoid tips, and convergence zones. Significantly, comparative analysis of wild-type and nuclear-irradiated mutagenic strains determined that cell volume and surface area of mutant cells have increased substantially to 2.2-fold and 1.7-fold, respectively, consistent with up-regulation of the enzyme Rubisco and enhanced photosynthetic metabolic processes. Moreover, quantitative analysis established that the thylakoid membrane width in mutant cells increased to 1.3-fold, while the membrane gap decreased to 0.8-fold, possibly contributing to the higher biomass growth rate of mutant cells. Our work reveals the first 3D subcellular architectures of C. pyrenoidosa cell and provides a structural framework for unlocking the higher growth rate in microalgae relevant to a wide range of industrial applications. ### Competing Interest Statement The authors have declared no competing interest.