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Link to original content: https://pubmed.ncbi.nlm.nih.gov/25231869
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. 2014 Sep 25;513(7519):547-50.
doi: 10.1038/nature13776. Epub 2014 Sep 17.

A faster Rubisco with potential to increase photosynthesis in crops

Myat T Lin  1 , Alessandro Occhialini  2 , P John Andralojc  3 , Martin A J Parry  3 , Maureen R Hanson  4
Affiliations

A faster Rubisco with potential to increase photosynthesis in crops

Myat T Lin et al. Nature. .

Abstract

In photosynthetic organisms, D-ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is the major enzyme assimilating atmospheric CO2 into the biosphere. Owing to the wasteful oxygenase activity and slow turnover of Rubisco, the enzyme is among the most important targets for improving the photosynthetic efficiency of vascular plants. It has been anticipated that introducing the CO2-concentrating mechanism (CCM) from cyanobacteria into plants could enhance crop yield. However, the complex nature of Rubisco's assembly has made manipulation of the enzyme extremely challenging, and attempts to replace it in plants with the enzymes from cyanobacteria and red algae have not been successful. Here we report two transplastomic tobacco lines with functional Rubisco from the cyanobacterium Synechococcus elongatus PCC7942 (Se7942). We knocked out the native tobacco gene encoding the large subunit of Rubisco by inserting the large and small subunit genes of the Se7942 enzyme, in combination with either the corresponding Se7942 assembly chaperone, RbcX, or an internal carboxysomal protein, CcmM35, which incorporates three small subunit-like domains. Se7942 Rubisco and CcmM35 formed macromolecular complexes within the chloroplast stroma, mirroring an early step in the biogenesis of cyanobacterial β-carboxysomes. Both transformed lines were photosynthetically competent, supporting autotrophic growth, and their respective forms of Rubisco had higher rates of CO2 fixation per unit of enzyme than the tobacco control. These transplastomic tobacco lines represent an important step towards improved photosynthesis in plants and will be valuable hosts for future addition of the remaining components of the cyanobacterial CCM, such as inorganic carbon transporters and the β-carboxysome shell proteins.

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Figures

Extended Data Figure 1
Extended Data Figure 1. Rubisco and CcmM35 content of SeLSM35 tobacco leaves
The stated concentrations of purified Se Rubisco (a) and CcmM35 (b) proteins were used as standards. a, Immunoblot using an antibody against cyanobacterial LSU (top) and the standard curve used to estimate the amount of cyanobacterial Rubisco in uk1-uk3 samples extracted from SeLSM35 tobacco leaves (bottom). b, Immunoblot using an antibody against CcmM (top) and the standard curve used to estimate the amount of CcmM35 in uk4-uk6 samples extracted from SeLSM35 tobacco leaves (bottom). The band intensities in the two standard curves were obtained with ImageJ software (NIH, USA) and the standard curves with Microsoft Excel. c, The absolute and relative amounts (mean ± standard deviation) of CcmM35 and cyanobacterial Rubisco in SeLSM35 tobacco line from two separate measurements. Each Rubisco holoenzyme is assumed to be composed of 8 LSU and an unknown quantity of SSU.
Extended Data Figure 2
Extended Data Figure 2. Electron micrographs of ultrathin sections of leaf mesophyll cells from the chloroplast transformant SeLSM35 showing big compartments (black arrows) containing cyanobacterial Rubisco and CcmM35 in the chloroplast stroma
Leaf tissues were prepared by high pressure freeze fixation (HPF) in combination with immunogold labeling using an antibody against CcmM. A secondary antibody conjugated with 10 nm gold particles was used for the labelling. Scale bars = 500 nm.
Extended Data Figure 3
Extended Data Figure 3. Rubisco-specific 14CO2 fixation by crude leaf homogenates from tobacco lines expressing cyanobacterial Rubisco (SeLSX and SeLSM35) and wild type tobacco (wt)
a, Carboxylase activity assayed with [+] and without [−] RuBP. b, Carboxylase activity assayed with [+] and without [−] the inhibitor CABP. The rates of carboxylase activity (mols fixed. mol act sites−1. s−1) are the means ± standard deviation of the 2,4 & 10 min data obtained in assays at 125 μM steady-state [14CO2] (i.e. 10 mM NaH14CO3, at pH 8.0).
Figure 1
Figure 1. Replacement of the tobacco chloroplast rbcL with cyanobacterial genes
a, Gene arrangements of the rbcL locus in the wild-type, SeLSX and SeLSM35 tobacco lines. Endogenous chloroplast DNA elements are shown in grey and the newly introduced segments in black. The intergenic regions IG1, IG2, IG3 and IG4 include TpetD(At)-IEE-SD, TpsbA(At)-IEE-SD, Trps16(At)-IEE-SD and TpsbA(At)-IEE-SD18 respectively. The selectable marker operon (SMO) includes LoxP-PpsbA-aadA-Trps16-LoxP. The probe recognizes the rbcL promoter (PrbcL) region. The NheI and NdeI sites used in the DNA blot along with the lengths of the expected DNA fragments detected by the probe are indicated. b, DNA blot analysis of wild-type, SeLSX and SeLSM35 lines digested with NdeI and NheI. c, Analyses of RT-PCR products of 6 genes. Nt-rbcL is the only tobacco gene; all other genes are the transgenes introduced into the tobacco chloroplast genome. x = rbcX, M = ccmM35.
Figure 1
Figure 1. Replacement of the tobacco chloroplast rbcL with cyanobacterial genes
a, Gene arrangements of the rbcL locus in the wild-type, SeLSX and SeLSM35 tobacco lines. Endogenous chloroplast DNA elements are shown in grey and the newly introduced segments in black. The intergenic regions IG1, IG2, IG3 and IG4 include TpetD(At)-IEE-SD, TpsbA(At)-IEE-SD, Trps16(At)-IEE-SD and TpsbA(At)-IEE-SD18 respectively. The selectable marker operon (SMO) includes LoxP-PpsbA-aadA-Trps16-LoxP. The probe recognizes the rbcL promoter (PrbcL) region. The NheI and NdeI sites used in the DNA blot along with the lengths of the expected DNA fragments detected by the probe are indicated. b, DNA blot analysis of wild-type, SeLSX and SeLSM35 lines digested with NdeI and NheI. c, Analyses of RT-PCR products of 6 genes. Nt-rbcL is the only tobacco gene; all other genes are the transgenes introduced into the tobacco chloroplast genome. x = rbcX, M = ccmM35.
Figure 2
Figure 2. Cyanobacterial proteins in chloroplasts
a, Coomassie-stained gel and immunoblot of 14 μg of total leaf protein from wild-type (wt), SeLSX and SeLSM35 tobacco lines. Immunoblots were probed with the antibodies indicated. Molecular weights (MW; kDa) of standard proteins are shown. (*) MW of tobacco SSU; (°) MW of cyanobacterial SSU. b-d, Electron micrographs of leaf sections showing Rubisco’s localization in the stroma of mesophyll chloroplasts of (b) wild-type, (c) SeLSX and (d) SeLSM35 tobacco lines. Leaf tissues were prepared by high pressure freeze fixation (HPF) in combination with immunogold labeling using an anti-tobacco Rubisco antibody (b) or an anti-cyanobacterial Rubisco antibody (c,d) and a secondary antibody conjugated with 10 nm gold particles, which are indicated with either black circles or arrows. Scale bars = 500 nm (for the top panels in b,d), 200 nm (for c and the bottom panels in b,d).
Figure 2
Figure 2. Cyanobacterial proteins in chloroplasts
a, Coomassie-stained gel and immunoblot of 14 μg of total leaf protein from wild-type (wt), SeLSX and SeLSM35 tobacco lines. Immunoblots were probed with the antibodies indicated. Molecular weights (MW; kDa) of standard proteins are shown. (*) MW of tobacco SSU; (°) MW of cyanobacterial SSU. b-d, Electron micrographs of leaf sections showing Rubisco’s localization in the stroma of mesophyll chloroplasts of (b) wild-type, (c) SeLSX and (d) SeLSM35 tobacco lines. Leaf tissues were prepared by high pressure freeze fixation (HPF) in combination with immunogold labeling using an anti-tobacco Rubisco antibody (b) or an anti-cyanobacterial Rubisco antibody (c,d) and a secondary antibody conjugated with 10 nm gold particles, which are indicated with either black circles or arrows. Scale bars = 500 nm (for the top panels in b,d), 200 nm (for c and the bottom panels in b,d).
Figure 3
Figure 3. Phenotype of the wild-type and transplastomic tobacco lines
Plant were grown at atmospheric CO2 level about 9000 ppm. Pictures showing 6-week old (a) wild-type (b) SeLSX, and (c) SeLSM35; and 10-week old (d) SeLSX and (e) SeLSM35 tobacco lines grown in the same conditions. Scale bars = 5 cm.
Figure 4
Figure 4. Carboxylase activities at different [14CO2]
CO2 fixation by crude leaf homogenates from tobacco lines expressing cyanobacterial Rubisco (SeLSX and SeLSM35) and wild-type tobacco (wt). The rates of carboxylase activity (mol CO2 fixed. mol active sites−1.s−1) at each point of the curves are the means ± standard deviation of the 2,4 & 6 min data obtained in two independent assays at different [CO2] (125 μM; 250 μM; 640 μM).
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