INVESTIGATING THE MOLECULAR BASIS OF THE HORNWORT PYRENOID
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Pyrenoid-based CO2-concentrating mechanisms (pCCMs) enhance photosynthetic efficiency by elevating CO2 levels around the carbon-fixing enzyme Rubisco. While pCCMs have been studied extensively in green algae such as Chlamydomonas reinhardtii, their application to crop improvement has been limited by the evolutionary distance between algae and land plants. Hornworts, the only extant land plants known to possess a pyrenoid, offer a promising alternative model for translational research. My dissertation focuses on the hornwort Anthoceros agrestis and presents the first comprehensive investigation into the spatial organization and molecular basis of its pCCM. I found that the hornwort pyrenoid exhibits hallmark features of algal pyrenoids, yet also has key differences in its structure and material properties. Using a combination of phylogenomic analysis, subcellular localization studies, and proteomics, I identified key pCCM components—such as CAH3, BST, and LCIB—previously characterized in Chlamydomonas, suggesting a conserved molecular framework that may be ancestral to land plants. Building on this, I developed the first spatial model of a land plant pCCM, laying a foundation for future functional studies. In parallel, I uncovered a novel mechanism of Rubisco condensation in A. agrestis involving a previously uncharacterized Rubisco small subunit (RbcS) with an extended C-terminal domain. This RbcS, here called RbcS-STAR, acts as a Rubisco linker protein and is both necessary and sufficient to induce pyrenoid formation. Expression of RbcS-STAR in a pyrenoid-lacking hornwort species (Anthoceros fusiformis) and in Arabidopsis resulted in the formation of proto-pyrenoids, demonstrating that it can drive Rubisco phase separation in diverse plant contexts. Structural and biochemical analyses revealed how RbcS-STAR mediates Rubisco condensation, providing an alternative to previously described mechanisms. Together, this work not only advances our understanding of the evolution and diversity of pCCMs but also introduces RbcS-STAR as a powerful tool for engineering synthetic pyrenoids in crop species. My findings highlight hornworts as a valuable model system for overcoming current barriers in translational photosynthesis research.