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

Abstract

SUMMARY 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 potential compartmentalization of Rubisco or other proteins.

Streamlined Construction of the Cyanobacterial CO2-Fixing Organelle via Protein Domain Fusions for Use in Plant Synthetic Biology

“…Previous attempts to engineer BMCs have focused on associating heterologous proteins to shells using EPs. For example, through the addition of two different EPs to pyruvate decarboxylase and alcohol dehydrogenase, Lawrence et al (2014) were able to repurpose a propanediol utilization compartment shell for ethanol production; Lin et al (2014b) showed that the encapsulation peptide from CcmN targets yellow fluorescent protein into carboxysome-like structures formed in mutant wild tobacco (Nicotiana benthamiana) plants. In contrast, the approach reported here focuses on assembling a multifunctional BMC core: CcmC nucleates Rubisco, supplies CA activity, and recruits the shell.…”

“…Recent efforts have led to important advances in expressing the b-carboxysome shell and cyanobacterial form 1B Rubisco in chloroplasts (Lin et al, 2014a(Lin et al, , 2014b, which represent the first steps in assembling carboxysomes in plants. More broadly, these results bode well for the prospect of engineering novel types of metabolic nanoreactors based on BMC architectures in heterologous systems (Frank et al, 2013), including cyanobacteria and other microbial platforms for renewable chemical and biofuel production.…”

RNA Recognition Motif-Containing Protein ORRM4 Broadly Affects Mitochondrial RNA Editing and Impacts Plant Development and Flowering

“…Then we mixed the culture expressing GFP N to another culture expressing GFP C in equal volume to a final optical density of 0.5 each and incubated the mixtures in the dark at 28°C for about three hours. Afterward, these mixtures were used to infiltrate leaves from 4-to 6-week-old Nicotiana benthamiana plants grown at 10 h of light per day as described in Lin et al (2014). Within 2 to 4 d after agroinfiltration, the leaf tissues were examined with a confocal microscope (Zeiss LSM 710) through a LD LCI Plan-Apochromat 253/0.8 Imm Korr DIC M27 objective.…”

Light Modulates the Biosynthesis and Organization of Cyanobacterial Carbon Fixation Machinery through Photosynthetic Electron Flow

“…For example, introducing b-carboxysomes into higher plants that use the ancestral C 3 pathway of photosynthesis could potentially enhance photosynthetic carbon fixation and crop production (Lin et al, 2014a(Lin et al, , 2014b. However, engineering of functional carboxysomes requires extensive understanding about the principles underlying the formation of b-carboxysomes and the physiological integration of b-carboxysomes into the cellular metabolism.…”