Investigation into the dynamic nature of the pyrenoid of Chlamydomonas reinhardtii

Our rising global population requires innovation to increase crop yields and provide food security.One promising solution is to improve the performance of Rubisco, an important enzyme which fixes CO2 into organic matter.In our current atmosphere, O2 competes with CO2 at the active site of Rubisco, leading to the energetically wasteful process of photorespiration.To overcome this problem, single-celled photosynthetic eukaryotes have evolved a biophysical CO2-concentrating mechanism (CCM) to concentrate CO2 around Rubisco.Critical to CCM function is the assembly of Rubisco into a liquid-liquid phase separated (LLPS) organelle called the pyrenoid.Rubisco is packaged with its linker protein Essential Pyrenoid Component 1 (EPYC1) to form the pyrenoid matrix, which is surrounded by a starch sheath.This study aims to investigate the mobility of pyrenoid components including Rubisco under different growth conditions, using Fluorescence Recovery After Photobleaching (FRAP).A dual-tagged Rubisco-starch line was created to simultaneously observe Rubisco and starch movement during cell division, using time-lapse confocal microscopy.The modification of the starch sheath during pyrenoid division was further investigated using a bioinformatics approach, revealing potential candidates involved in pyrenoid starch degradation and synthesis.It is hypothesised that HCO3 -delivery to the pyrenoid matrix requires conversion from CO2 by low-CO2-inducible proteins LCIB/C, and transport across thylakoids by Bestrophin-like proteins (BST1-3).The role of these complexes in recycling escaping CO2 is explored.Preliminary results allow further investigation into the dynamic nature of the pyrenoid, which will help guide current efforts to engineer a pyrenoid into higher plants.unending enthusiasm and encouragement throughout the project.Despite the isolation that 2020 brought, Luke continued to make me feel part of a team and provided fantastic advice and support in unprecedented times.I am extremely grateful for the skills he taught me, and for giving me this opportunity.I want to thank the post-docs in the lab, whose guidance went beyond science.Thank you to Dr. Charlotte Walker for her patient teaching and her words of wisdom: "it's a marathon, not a sprint!"Thank you also to Dr. Gary Yates who taught me the value of absorbing 'espresso shots' of relevant information, and for being equally as excited at getting some great microscopy images.Thank you both for your pep-talks, they were a source of great encouragement.Thank you also to Dr. Irina Grouneva for always pointing me in the right direction in the lab, and to Dr. Philipp Girr for sharing your interesting plans