James Barrett (UoY) & Dr Gustaf Degen - University of Sheffield

James Barrett (UoY)

Title: Convergent evolution in the eukaryotic CO₂-fixing organelle

Abstract: Most algae contain a CO₂-fixing organelle called the pyrenoid, which is home to ~50% of global carbon fixation through the enzyme Rubisco. It has recently emerged that pyrenoids are biomolecular condensates; held together by intrinsically disordered “Linker” proteins which bind to Rubisco. Pyrenoid-containing species span vast evolutionary timescales, yet Linker protein sequences are not conserved across them. Here, we developed a bioinformatic approach to identify analogous Linker proteins across species. Our biochemical, structural and physiological characterisation in species of the green lineage demonstrate convergent evolution of these Linker protein function through differing sequence and structural means. By mapping the conservation of these sequences to phylogenetic chronograms, we can tentatively suggest an evolutionary timepoint for green pyrenoids.

Dr Gustaf Degen

Title: Disequilibrium between chloroplast proton motive force and ATP levels in Arabidopsis

Abstract: Current dogma holds that CO₂ fixation by photosynthesis requires additional ATP production via PGR5-dependent cyclic electron transfer (PGR5-CET) to raise the ATP/NADPH ratio produced by linear electron transfer (LET). Yet, to date no direct measurements of chloroplastic ATP in pgr5 mutant exists.  To investigate in vivo ATP dynamics, I used a fluorescent ATP biosensor localised to
the chloroplast stroma combined with advanced illumination-imaging. This approach revealed that while proton motive force (pmf) in the Arabidopsis pgr5CAS mutant is only 50-75% of that of the wild-type, ATP levels are unchanged. These data demonstrate that disequilibrium exists between pmf and ATP concentration in vivo,  indicating that PGR5-CET is not required for ATP augmentation. The severe reduction in CO 2 fixation in the pgr5CAS mutant suggests that PGR5-CET is required for alkalisation of the stroma by pumping additional H + into the thylakoid lumen. This creates a more favourable pH environment for CO 2 -fixing reactions, which exhibit
maximum in vitro activity above the physiological stromal pH, whilst also acidifying the thylakoid lumen to allow for triggering of photoprotective mechanisms. These results reframe our understanding of the physiological bottlenecks of CO 2 -fixation
and reveal possible new avenues of improving photosynthesis by engineering the pH-optimum of Calvin cycle enzymes and by manipulating the pH of the chloroplast stroma.