Collaborative Research: From phylogeny to biomolecules: a cross-scale approach to understand the making of a unique carbon-concentrating mechanism in hornworts

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Nature’s carbon fixing enzyme, Rubisco, is notoriously inefficient - often limiting the growth rate of photosynthetic organisms, including crop species. Some organisms have found a way around this problem by packaging Rubisco into compartments, called pyrenoids, which concentrate substrate CO2 around Rubisco active sites. Hornworts are the only land plants that have a pyrenoid. This project seeks to identify the hornwort pyrenoid components, characterize the hornwort carbon-fixing enzyme (i.e. Rubisco) housed within the pyrenoids, and determine how the various pyrenoid components interact with each other. The results of this study will provide evolutionary, biochemical, and mechanistic insights into the hornwort pyrenoid, and make a first step towards gathering the requisite understanding to transplant hornwort pyrenoids into crop plants to boost carbon fixation. This project will also train diverse undergraduates in a range of computational and experimental techniques, including plant tissue culture and transformation and bioinformatics. In addition, a three-part video series will be produced to provide a fun and informative introduction to hornwort diversity, evolution and pyrenoids, and distributed on YouTube and other online platforms. The importance of hornwort and Rubisco research will be further promoted through established podcast series.Photosynthesis is the source of energy for almost every life on earth, and is often limited by the carbon fixing enzyme, Rubisco. Some organisms have evolved ingenious ways to circumvent these limitations, such as pyrenoids, which are a kind of membrane-less organelle that liquid-liquid phase separates Rubisco from the stroma. Pyrenoids actively concentrate substrate CO2 around Rubisco active sites, thereby greatly enhancing the efficiency of carbon fixation. Hornworts are the only land plants with a pyrenoid, and these have translational potential for improving C3 crop efficiency. The fact that hornwort pyrenoids have been gained and lost over the course of evolution offers a unique phylogenetic replication for comparative studies. Putative pyrenoid components will be identified using comparative genomics, RNA-seq, and proteomics, while leveraging repeated pyrenoid-present/absent transitions. The subcellular localization of putative pyrenoid components and Rubisco subunits will be validated using in vivo localization approaches, and Rubisco populations structurally characterized. A constructionist biology approach will identify and characterize interactions between Rubisco and recombinantly-produced putative pyrenoid components in vitro, and will be used to assess the potential for Rubisco phase separation.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.