Hybrid Cyanobacterial-Tobacco Rubisco Supports Autotrophic Growth and Procarboxysomal Aggregation

Much of the research aimed at improving photosynthesis and crop productivity attempts to overcome shortcomings of the primary CO 2 -fixing enzyme Rubisco. Cyanobacteria utilize a CO 2 -concentrating mechanism (CCM), which encapsulates Rubisco with poor specificity but a relatively fast catalytic rate within a carboxysome microcompartment. Alongside the active transport of bicarbonate into the cell and localization of carbonic anhydrase within the carboxysome shell with Rubisco, cyanobacteria are able to overcome the limitations of Rubisco via localization within a high-CO 2 environment. As part of ongoing efforts to engineer a β-cyanobacterial CCM into land plants, we investigated the potential for Rubisco large subunits (LSU) from the β-cyanobacterium Synechococcus elongatus (Se) to form aggregated Rubisco complexes with the carboxysome linker protein CcmM35 within tobacco ( Nicotiana tabacum ) chloroplasts. Transplastomic plants were produced that lacked cognate Se Rubisco small subunits (SSU) and expressed the Se LSU in place of tobacco LSU, with and without CcmM35. Plants were able to form a hybrid enzyme utilizing tobacco SSU and the Se LSU, allowing slow autotrophic growth in high CO 2 CcmM35 was able to form large Rubisco aggregates with the Se LSU, and these incorporated small amounts of native tobacco SSU. Plants lacking the Se SSU showed delayed growth, poor photosynthetic capacity, and significantly reduced Rubisco activity compared with both wild-type tobacco and lines expressing the Se SSU. These results demonstrate the ability of the Se LSU and CcmM35 to form large aggregates without the cognate Se SSU in planta, harboring active Rubisco that enables plant growth, albeit at a much slower pace than plants expressing the cognate Se SSU.