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

Plant J. 2014 Jul;79(1):1-12. doi: 10.1111/tpj.12536. Epub 2014 Jun 9.

Abstract

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.

Keywords: CO2 concentration mechanism; Nicotiana benthamiana; bacterial microcompartment; chloroplast engineering; photosynthesis; synthetic biology; β-carboxysome.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Arabidopsis / genetics
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism*
  • Carbon Cycle
  • Carbon Dioxide / metabolism
  • Chloroplast Proteins / genetics
  • Chloroplast Proteins / metabolism*
  • Chloroplasts / metabolism
  • Chloroplasts / ultrastructure
  • Feasibility Studies
  • Gene Expression
  • Genes, Reporter
  • Immunohistochemistry
  • Mesophyll Cells
  • Microscopy, Electron, Transmission
  • Microscopy, Fluorescence
  • Organelles / metabolism
  • Organelles / ultrastructure*
  • Plant Leaves
  • Plants, Genetically Modified
  • Protein Sorting Signals / genetics
  • Protein Transport
  • Synechococcus / genetics*
  • Synechococcus / metabolism
  • Tobacco / genetics
  • Tobacco / metabolism
  • Tobacco / ultrastructure*

Substances

  • Bacterial Proteins
  • Chloroplast Proteins
  • Protein Sorting Signals
  • chloroplast transit peptides
  • Carbon Dioxide