Characterisation of Chlamydomonas reinhardtii bestrophin-like proteins expressed in C3 land plants

Many photosynthetic species have evolved CO₂-concentrating mechanisms (CCMs) to improve the efficiency of CO₂ assimilation by Rubisco. C3 plants, which include major crops like wheat and rice, lack a CCM and thus, engineering a heterologous CCM into important C3 crops has become a key strategic ambition to enhance yield potential. The green alga Chlamydomonas reinhardtii has the best characterised algal CCM and has become the blueprint for engineering an algal CCM into C3 plants. The Chlamydomonas CCM is based on a Rubisco-dense microcompartment called a pyrenoid, which is supplied with concentrated CO₂ via inorganic carbon transport and carbonic anhydrase activity. Structurally, the pyrenoid is traversed by tubular extensions of the thylakoid membrane, known as the pyrenoid tubules, which are thought to be the site of CO₂ release into the pyrenoid. In this thesis, I assess four bestrophin-like proteins from the Chlamydomonas CCM for their compatibility and function in model C3 plants, to inform efforts to reconstitute a pyrenoid based CCM in C3 plants. In the third chapter, I characterise the first three bestrophin-like proteins (BST1-3), which are thought to be involved in the transport of bicarbonate into the thylakoid lumen. In the current model, bicarbonate then diffuses along the thylakoid lumen and into the pyrenoid tubules. Once in the pyrenoid tubules, bicarbonate is converted to CO₂, which can then diffuse through the tubule membrane into the Rubisco matrix where it is fixed. When I expressed BST1-3 proteins in the C3 plants Arabidopsis thaliana (Arabidopsis) and Nicotiana benthamiana (N. benthamiana), I found they were successfully localised to the thylakoid membrane and assembled into complexes. Further work in Arabidopsis suggested that BST1 and BST2 may function as bicarbonate channels in Arabidopsis but additional validation is required. I also present preliminary evidence that BST1-3 form heterooligomeric channels. Finally, I explored the interaction of BST1-3 with the carbonic anhydrase proteins LCIB and LCIC and found that the previously reported interactions for these proteins are likely mediated by the C-termini of BST1-3. In my fourth chapter, I characterise the fourth bestrophin-like protein, BST4 (also known as Rubisco-binding membrane protein 1), which is hypothesised to function as a linker protein to bring the pyrenoid tubules and the Rubisco matrix together. A heterologous Rubisco condensate in Arabidopsis chloroplasts has already been achieved, and structurally, one of the next steps is to incorporate thylakoid membranes to supply CO₂. I tested whether BST4 could promote the incorporation of thylakoid membrane into this Rubisco condensate in Arabidopsis and found that although BST4 co-localised with the condensate, it was insufficient for the incorporation of thylakoid membrane. Finally, I explored the channel function of BST4 and found that like BST1-3, BST4 localises to the stroma lamellae thylakoid membranes and assembles as a complex. I found that BST4 had adverse effects on plant growth but only when fused to mNeon and co-expressed with the Chlamydomonas Rubisco small subunit or when the C-terminal was mutated or truncated, suggesting BST4 may be subject to C-terminal regulation. However, when BST4 was expressed without a tag in Arabidopsis I found no impact on photosynthetic processes.