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Hybrid Cyanobacterial-Tobacco Rubisco Supports Autotrophic Growth and Procarboxysomal Aggregation - PubMed Skip to main page content
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. 2020 Feb;182(2):807-818.
doi: 10.1104/pp.19.01193. Epub 2019 Nov 19.

Hybrid Cyanobacterial-Tobacco Rubisco Supports Autotrophic Growth and Procarboxysomal Aggregation

Douglas J Orr  1 , Dawn Worrall  2 , Myat T Lin  3 , Elizabete Carmo-Silva  2 , Maureen R Hanson  3 , Martin A J Parry  2
Affiliations

Hybrid Cyanobacterial-Tobacco Rubisco Supports Autotrophic Growth and Procarboxysomal Aggregation

Douglas J Orr et al. Plant Physiol. 2020 Feb.

Abstract

Much of the research aimed at improving photosynthesis and crop productivity attempts to overcome shortcomings of the primary CO2-fixing enzyme Rubisco. Cyanobacteria utilize a CO2-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-CO2 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 CO2 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.

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Figures

Figure 1.
Figure 1.
Replacement of the Rubisco LSU gene (rbcL) in tobacco chloroplasts with SerbcL with or without the ccmM35 gene. A, Gene arrangements of wild-type (WT), SeL, and SeLM35 tobacco lines along with the locations of the EcoRV and KpnI restriction sites used on the DNA blot. The binding site for the digoxigenin (DIG)-labeled DNA probe is shown in green bars. Seeds were obtained from two independent SeL lines and one SeLM35 line. B, DNA-blot analysis of wild-type, SeL, and SeLM35 samples digested with EcoRV and KpnI. All samples produced the expected band on the DNA blot.
Figure 2.
Figure 2.
Tobacco plants expressing cyanobacterial Rubisco LSUs and CcmM35 contain a procarboxysome compartment in the chloroplast. Immunolocalization is shown for Se proteins in the chloroplasts of transplastomic tobacco lines expressing the Rubisco LSU and CcmM35 (SeLM35) or the LSU alone (SeL). Electron micrographs show ultrathin sections of mesophyll cells probed with the indicated primary antibody and a secondary antibody conjugated to 10-nm gold particles. Additional images are presented in Supplemental Figures S3 and S4. Bars show size as indicated.
Figure 3.
Figure 3.
Protein composition of wild-type (WT) tobacco and transplastomic lines expressing β-cyanobacterial carboxysome components. A and B, Polypeptides in leaf extracts prepared from plants of each line were separated by denaturing SDS-PAGE (A) and nondenaturing native PAGE (B) and either stained with Coomassie Blue (top gels) or used for immunoblotting with antibodies against cyanobacterial Rubisco LSU (SeLSU) and CcmM35 and against the tobacco Rubisco small subunit (NtSSU; bottom gels). Images showing blotting of PAGE gels are slices from blots (Supplemental Fig. S5) and show the indicated size regions where the respective antibodies detect proteins of interest. For SDS-PAGE and native PAGE, 10 and 20 µg of total soluble protein was loaded per lane, respectively. Lanes marked M indicate protein markers containing proteins of a range of sizes as indicated at left of each gel. C, SDS-PAGE and native PAGE gels immunoblotted with antibody against NtSSU, loaded with 20 and 40 µg of total soluble protein, respectively.
Figure 4.
Figure 4.
Rubisco and total soluble protein. Rubisco total activity (A), activation state (B), and content (C), and total soluble protein (D), are shown for wild-type (WT) tobacco and transplastomic lines expressing β-cyanobacterial carboxysome components from Se: Rubisco LSU (L), Rubisco SSU (S), and CcmM35 (M35). Values represent means ± se (n = 3–4 biological replicates). Letters denote significant differences (P < 0.05) as determined by Tukey’s honestly significant difference mean-separation test following ANOVA (P values are as indicated).
Figure 5.
Figure 5.
Response of net CO2 assimilation (A) to intercellular CO2 concentrations (Ci). Rates are expressed on an area basis (A) and on a Rubisco active site basis (B) for leaves of wild-type (WT) tobacco and transplastomic lines expressing β-cyanobacterial carboxysome components from Se: Rubisco LSU (L), Rubisco SSU (S), and CcmM35 (M35). Values represent means ± se (n = 3–4 biological replicates).
Figure 6.
Figure 6.
Plant development and growth traits. Shown are a photograph of 33-d-old plants grown in parallel in 4,000 µL L−1 CO2 (A), plant height (B), and leaf area (C) development during the growth cycle of wild-type (WT) tobacco and transplastomic lines expressing β-cyanobacterial carboxysome components from Se: Rubisco LSU (L), Rubisco SSU (S), and CcmM35 (M35). Values represent means ± se (n = 3–5 biological replicates). DAS, Days after sowing.
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