β‐Carboxysomal proteins assemble into highly organized structures in Nicotiana chloroplasts

• Published in: The Plant journal : for cell and molecular biology Vol. 79; no. 1; pp. 1 - 12
• Main Authors: Lin, Myat T, Occhialini, Alessandro, Andralojc, P. John, Devonshire, Jean, Hines, Kevin M, Parry, Martin A. J, Hanson, Maureen R
• Format: Journal Article
• Language: English
• Published: England Blackwell Science 01-07-2014
• Subjects: agroinfiltration; Arabidopsis - genetics; bacterial microcompartment; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; C3 plants; carbon; Carbon Cycle; carbon dioxide; Carbon Dioxide - metabolism; chemical reactions; chloroplast engineering; Chloroplast Proteins - genetics; Chloroplast Proteins - metabolism; chloroplasts; Chloroplasts - metabolism; Chloroplasts - ultrastructure; CO2 concentration mechanism; Cyanobacterium; Feasibility Studies; Gene Expression; Genes, Reporter; Immunohistochemistry; Mesophyll Cells; Microscopy, Electron, Transmission; Microscopy, Fluorescence; Nicotiana - genetics; Nicotiana - metabolism; Nicotiana - ultrastructure; Nicotiana benthamiana; Organelles - metabolism; Organelles - ultrastructure; oxygen; photorespiration; photosynthesis; Plant Leaves; Plants, Genetically Modified; prokaryotic cells; Protein Sorting Signals - genetics; Protein Transport; proteins; ribulose-bisphosphate carboxylase; Synechococcus - genetics; Synechococcus - metabolism; Synechococcus elongatus; synthetic biology; transgenic plants; β‐carboxysome; synthetic biology; Nicotiana benthamiana; bacterial microcompartment; photosynthesis; chloroplast engineering; β-carboxysome; CO2 concentration mechanism
• ISSN: 0960-7412; 1365-313X
• DOI: 10.1111/tpj.12536

The photosynthetic efficiency of C3 plants suffers from the reaction of ribulose 1,5‐bisphosphate carboxylase/oxygenase (Rubisco) with O₂ instead of CO₂, leading to the costly process of photorespiration. Increasing the concentration of CO₂ around Rubisco is a strategy used by photosynthetic prokaryotes such as cyanobacteria for more efficient incorporation of inorganic carbon. Engineering the cyanobacterial CO₂‐concentrating mechanism, the carboxysome, into chloroplasts is an approach to enhance photosynthesis or to compartmentalize other biochemical reactions to confer new capabilities on transgenic plants. We have chosen to explore the possibility of producing β‐carboxysomes from Synechococcus elongatus PCC7942, a model freshwater cyanobacterium. Using the agroinfiltration technique, we have transiently expressed multiple β‐carboxysomal proteins (CcmK2, CcmM, CcmL, CcmO and CcmN) in Nicotiana benthamiana with fusions that target these proteins into chloroplasts, and that provide fluorescent labels for visualizing the resultant structures. By confocal and electron microscopic analysis, we have observed that the shell proteins of the β‐carboxysome are able to assemble in plant chloroplasts into highly organized assemblies resembling empty microcompartments. We demonstrate that a foreign protein can be targeted with a 17‐amino‐acid CcmN peptide to the shell proteins inside chloroplasts. Our experiments establish the feasibility of introducing carboxysomes into chloroplasts for the potential compartmentalization of Rubisco or other proteins.