Structural and functional characterisation of two red plastid pyrenoids

Algal pyrenoids are thought to have evolved multiple times over the course of natural history. Despite this, many algal lineages remain understudied in the context of their carbon concentrating mechanisms. I aim to bridge that gap in knowledge by studying the structure and function of proteins involved in formation of red plastid pyrenoids from two diverse algae – the diatom Thalassiosira pseudonana and rhodophyte Porphyridium purpureum.

In vitro investigation of shell proteins forming a diffusion barrier in the Thalassiosira pseudonana pyrenoid has revealed extensive concentration dependent self-assembly. My work optimised expression and purification methods and tested several approaches to investigate homo- and hetero-oligomerisation.

In Porphyridium purpureum, bioinformatics and proteomics have revealed putative proteins involved in the CO2 concentrating mechanisms, including a Rubisco linker protein named PERL1, which could be involved in biogenesis of the phase separated condensate. Imaging revealed the putative linker to be localised to pyrenoid, and in vitro assays demonstrated that PERL1 binds Rubisco in detergent sensitive manner. PERL1 also appears to homotypically phase separate and recruits Rubisco into the condensate. A cryo-electron microscopy structure shows the interface of the interaction while cryo-electron tomography analysis shows that the pyrenoid is bisected by a complex network of thylakoids and lacks an obvious diffusion barrier that could prevent CO2 leakage.

My overall findings present the importance of research into a diverse systems not only for better understanding of the evolution of CO2 concentrating mechanisms, but also as a way of discovering new pyrenoid assembly principles which could be utilised in future plant engineering.