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. 2023 Feb 20;35(2):795-807.
doi: 10.1093/plcell/koac348.

Producing fast and active Rubisco in tobacco to enhance photosynthesis

Taiyu Chen  1   2 , Saba Riaz  3 , Philip Davey  4 , Ziyu Zhao  3 , Yaqi Sun  2 , Gregory F Dykes  2 , Fei Zhou  1 , James Hartwell  2 , Tracy Lawson  4 , Peter J Nixon  3 , Yongjun Lin  1 , Lu-Ning Liu  2   5
Affiliations

Producing fast and active Rubisco in tobacco to enhance photosynthesis

Taiyu Chen et al. Plant Cell. .

Abstract

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) performs most of the carbon fixation on Earth. However, plant Rubisco is an intrinsically inefficient enzyme given its low carboxylation rate, representing a major limitation to photosynthesis. Replacing endogenous plant Rubisco with a faster Rubisco is anticipated to enhance crop photosynthesis and productivity. However, the requirement of chaperones for Rubisco expression and assembly has obstructed the efficient production of functional foreign Rubisco in chloroplasts. Here, we report the engineering of a Form 1A Rubisco from the proteobacterium Halothiobacillus neapolitanus in Escherichia coli and tobacco (Nicotiana tabacum) chloroplasts without any cognate chaperones. The native tobacco gene encoding Rubisco large subunit was genetically replaced with H. neapolitanus Rubisco (HnRubisco) large and small subunit genes. We show that HnRubisco subunits can form functional L8S8 hexadecamers in tobacco chloroplasts at high efficiency, accounting for ∼40% of the wild-type tobacco Rubisco content. The chloroplast-expressed HnRubisco displayed a ∼2-fold greater carboxylation rate and supported a similar autotrophic growth rate of transgenic plants to that of wild-type in air supplemented with 1% CO2. This study represents a step toward the engineering of a fast and highly active Rubisco in chloroplasts to improve crop photosynthesis and growth.

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Conflict of interest statement

Conflict of interest statement. None declared.

Figures

Figure 1
Figure 1
Heterologous assembly of HnRubisco does not require extra chaperones in E. coli. A, Genetic arrangement of the CbbL/S operon in the pAM2991 vector for E. coli expression. B, Native-PAGE (top) and immunoblot (bottom) analysis indicate the formation of CbbL8S8 complexes. From left to right: Rubisco CbbL8S8 complexes purified from WT tobacco leaves, empty vector (EV), total soluble protein of pHnCbbL/S, and HnRuibscoEco purified from pHnCbbL/S. C, Carbon fixation activity of HnRubiscoEco purified from pHnCbbL/S at different CO2 concentrations, fitted with the Michaelis–Menten equation. The kcatC and KC values were 8.85 ± 0.5 s−1 and 182.4 ± 26.9 μM, respectively. Data are presented as mean ± standard deviation (SD, n = 3, Table 1). D, Quantification of the Rubisco active sites as a function of CABP concentration (0, 2.5, 5, and 10 pmol) based on a previously reported procedure (Davidi et al., 2020). The inhibition of CABP is described by a linear model within a certain concentration range (R2 = 0.99). The X-intercept indicates the concentration of Rubisco active sites, and the Y-intercept gives the carboxylation rate without CABP inhibition (Vmax). The specific activity per active site was calculated by dividing Vmax by the number of active sites. Under these conditions, HnRubiscoEco catalyzes 6.84 reactions per second (Table 2).
Figure 2
Figure 2
Engineering HnRubisco in tobacco. A, Gene organization of the HncbbLS locus in the TobHnLS construct, which was transformed into wild-type (WT) tobacco chloroplasts to replace the endogenous NtrbcL gene. T1, AtTpet D; T2, AtTpsb A; IEE, intercistronic expression elements; SD, Shine–Dalgarno sequence. B, DNA gel blot of total genomic DNA of WT and TobHnLS transgenic plants digested by Spe I using the probe indicated in A. A fragment length polymorphism was detected between the transgenic lines and WT. The shifting of the fragment length polymorphism confirmed the complete segregation of the HncbbLS operon into the tobacco chloroplast genome, resulting in homoplasmy. The sizes of DNA markers are indicated in kbp. C, SDS-PAGE (top) and immunoblot analysis (bottom) of total soluble proteins (S) and insoluble proteins (P) indicate the successful expression and solubility of NtRbcL/HnCbbL in both WT and TobHnLS transgenic plants. CbbL/RbcL are ∼50 kDa in size, according to immunoblot analysis using an anti-RbcL antibody. The analysis was performed based on equal protein loading. D, Native-PAGE (top) and immunoblot analysis (bottom) of total soluble proteins confirm that the expressed HnCbbL and HnCbbS form Rubisco CbbL8S8 complexes (∼520 kDa).
Figure 3
Figure 3
Characterization of Rubisco isolated from the leaves of wild-type (NtRubisco) and transgenic plants (HnRubiscoTob). A, SDS-PAGE (top) and immunoblot analysis using α-RbcL and α-6X-Histidine tag antibodies (bottom) of purified Rubisco examining demonstrating the assembly of CbbL and CbbS. No RbcS was detected in the isolated HnRubiscoTob, indicating that NtRbcS and HnCbbLS are structurally incompatible and cannot form a hybrid Rubisco complex. B, Native-PAGE (top) and immunoblot analysis (bottom) confirm that purified HnRubiscoTob is in the CbbL8S8 form. C, Negative-stain EM of purified HnRubiscoTob from the leaves of transgenic plants. HnRubiscoTob shows a typical “dot-ring” Rubisco structure, with an average diameter of 10.7 ± 0.7 nm (n = 92). Scale bar: 50 nm (left), 5 nm (right). D, Selected reference-free 2D class averages of chloroplast-expressed HnRubiscoTob from cryo-EM images in RELION. Scale bar: 5 nm. E, Rubisco activity assays as a function of CO2 concentration reveal a faster catalytic velocity in HnRubiscoTob than in NtRubisco. The kinetic parameters of NtRubisco and HnRubiscoTob were as follows: kcatC and KC of NtRubisco were 3.62 ± 0.1 s−1 and 22.8 ± 2.8 μM, respectively, and kcatC and KC of HnRubiscoTob were 10.0 ± 0.4 s−1 and 166.1 ± 18.3 μM, respectively (n = 3, Table 1). Data were fitted with the Michaelis–Menten equation and are presented as mean ± SD of three independent assays.
Figure 4
Figure 4
HnRubisco supports autotrophic growth of tobacco plants in air with 1% CO2. A, Phenotypes of the transgenic plants and WT grown at 25°C in air with or without 1% (v/v) CO2 on the 33rd day after sowing. The germinated seeds of WT and transgenic plants were sown in the same pot (12 cm × 12 cm) and grown in either ambient air or 1% CO2. With 1% CO2, the transgenic seeds germinated and grew as well as WT. In ambient air, however, the transgenic seeds stopped growing after germination and had completely died 33 days after sowing. See also Supplemental Figure 3. Scale bar: 2 cm. B, HnRubisco-supported growth of TobHnLS tobacco in air supplemented with 1% CO2 at 53 days after sowing, compared with WT. C–E, leaf number (C), plant height (D) and leaf gas-exchange measurements (E) of WT and TobHnLS transgenic plants grown in air with 1% CO2. Leaf gas-exchange analysis of net CO2 assimilation rates (Pn) as a function of intercellular CO2 pressure (Ci) at 25°C and 1,200 μmol photons·m−2·s−1 light density. The measurements were conducted at 42 days after sowing. Data are presented as mean ± SD of three independent transgenic lines.
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