In-situ high-resolution 3D imaging combined with proteomics and metabolomics reveals enlargement of subcellular architecture and enhancement of photosynthesis pathways in nuclear-irradiated Chlorella pyrenoidosa

Chlorella pyrenoidosa is a microalga that holds enormous potential as a source of renewable energy. Nuclear radiation is commonly used to modify natural microalgal phenotypes to enhance specified functions. However, the mechanisms underlying such modifications are poorly understood. Here, we use a multidisciplinary approach to analyze wild-type Chlorella pyrenoidosa and a nuclear-irradiated mutant known for its higher growth rate and photosynthetic efficiency. A combination of focused ion beam scanning electron microscopy (FIB-SEM), cryo-FIB milling, and cryo-electron tomography (cryo-ET) achieve visualization of the microalgae and their organelles in unprecedented detail. Compared to the wild-type cell, the nuclear-irradiated mutant and its subcellular organelles, including the chloroplast, significantly increase in volume by 1.2-fold. Moreover, proteomics and metabolomics data indicate rubisco overexpression by 1.07-fold in the mutant, consistent with enhancement of carbon fixation in photosynthesis and the observed enlargement of subcellular morphology. Furthermore, cryo-ET reveals enlarged membrane width in the thylakoid of the mutant, suggesting an upregulation role for chlorophyll synthesis and chloroplast construction. Collectively, our studies reveal the detailed architectures of C. pyrenoidosa wild-type and a mutant with higher growth rate and photosynthetic efficiency, providing a basis for understanding how subcellular organelles function collaboratively to support macroscopic traits.