The dependency of red Rubisco on its cognate activase for enhancing plant photosynthesis and growth

Abstract

Plant photosynthesis and growth are often limited by the activity of the CO₂-fixing enzyme Rubisco. The broad kinetic diversity of Rubisco in nature is accompanied by differences in the composition and compatibility of the ancillary proteins needed for its folding, assembly, and metabolic regulation. Variations in the protein folding needs of catalytically efficient red algae Rubisco prevent their production in plants. Here, we show this impediment does not extend to Rubisco from Rhodobacter sphaeroides (RsRubisco)-a red-type Rubisco able to assemble in plant chloroplasts. In transplastomic tobRsLS lines expressing a codon optimized Rs-rbcLS operon, the messenger RNA (mRNA) abundance was ∼25% of rbcL transcript and RsRubisco ∼40% the Rubisco content in WT tobacco. To mitigate the low activation status of RsRubisco in tobRsLS (∼23% sites active under ambient CO₂), the metabolic repair protein RsRca (Rs-activase) was introduced via nuclear transformation. RsRca production in the tobRsLS::X progeny matched endogenous tobacco Rca levels (∼1 µmol protomer·m²) and enhanced RsRubisco activation to 75% under elevated CO₂ (1%, vol/vol) growth. Accordingly, the rate of photosynthesis and growth in the tobRsLS::X lines were improved >twofold relative to tobRsLS. Other tobacco lines producing RsRubisco containing alternate diatom and red algae S-subunits were nonviable as CO₂-fixation rates (k_cat^c) were reduced >95% and CO₂/O₂ specificity impaired 30-50%. We show differences in hybrid and WT RsRubisco biogenesis in tobacco correlated with assembly in Escherichia coli advocating use of this bacterium to preevaluate the kinetic and chloroplast compatibility of engineered RsRubisco, an isoform amenable to directed evolution.

Keywords: Rubisco activase; carbon fixation; chloroplast transformation; photosynthesis.

Fig. 1.

RsRubisco expression in tobacco chloroplasts. (A) Transformation of the tobRr plastome with plasmid pLEVRsLS produced tobRsLS lines where the rbcM gene coding R. rubrum L₂ Rubisco (located in place of the native rbcL in WT tobacco) was replaced with the RsRubisco operon (RsrbcL-RsS) and aadA selectable marker gene. Dashed lines and numbering relative to tobacco plastome (GenBank accession no. Z00044) indicate plastome sequence in pLEVRsLS used to facilitate homologous recombination. The tobacco rbcL promoter/5′UTR (P) and first 42 nucleotides of native rbcL sequence (the 5UTR probe) are conserved in each tobacco genotype. The aadA probe DNA region is shown. accD, plastome genes; T, tobacco rbcL 3′UTR; T, psbA 3′UTR; t, rps16 3′UTR; atpB. (B) Coomassie stain and immunoblot native-PAGE analyses confirming the production of RsL₈S₈ Rubisco complexes in tobRsLS. The varying area of soluble leaf protein analyzed is indicated in italics. (C) Phenotype of a T₁ tobRsLS plant (line #1) and WT tobacco grown at 25 °C in air containing 1% (vol/vol) CO₂. Arrows indicate leaves sampled for RNA and protein analyses in B, D, and E. (D) Blots of 3 µg of total leaf RNA hybridized with the 5UTR-probe showing the WT rbcL mRNA levels are four- to fivefold higher than the Rs-rbcLS and Rs-rbcLS-aadA polycistronic mRNAs (the latter detected by the aadA probe) produced in tobRsLS lines #1 and #2. (E) SDS/PAGE–immunoblot analysis of the total and soluble leaf protein fractions showing the RsRubisco produced in the tobRsLS lines is fully soluble and does not contain tobacco S-subunits.

Fig. 2.

Expressing RsRca in tobRsLS improved growth. (A) Genetic detail of the nucleus transforming plasmid pBinTP-cbbX. LB/RB, Left/Right T-DNA borders; nptII, gene coding kanamycin resistance; P_CaMV, cauliflower mosaic virus 35S promoter; P_MS/T_MS, mannopine synthase promoter/terminator; P_nos/T_nos, nopaline synthase promoter/terminator; TP, tobacco S-subunit transit peptide. (B) Phenotype of the plants at the indicated ages postcotyledon emergence (pce) grown at 25 °C in air with 1% (vol/vol) CO₂ under ∼400 ± 100 µmol photons.m⁻²·s⁻¹. (C) Comparative growth measured as a function of plant height of WT tobacco (squares), tobRsLS#1 (circles), and RsRca-expressing tobRsLS::X lines (triangles; line #7 white, #8 black), n = 5 ± SD for each genotype. Age of plants analyzed by leaf gas exchange in Fig. 3 is shaded gray. Comparison of the Rubisco (D), tobacco Rubisco activase (NtRca) (E), and RsRca contents (F) in the youngest nearly fully expanded leaves (14 ± 1 cm in diameter) from five (±SD) plants at 30 ± 3 cm in height of each genotype. See SI Appendix, Fig. S3 for example quantification of NtRca and RsRca. Letters show the ranking of the means following a one-way ANOVA and post hoc Tukey test (different letters indicate statistical differences at the 5% level, P < 0.05).

Fig. 3.

RsRca and high CO₂ is needed to activate RsRubisco and stimulate leaf photosynthesis. (A) Leaf gas exchange measurements of CO₂-assimilation rates (A) at 25 °C under varying intercellular CO₂ pressures (C_i) made at 1,200 µmol photons·m⁻²·s⁻¹ illumination (plants analyzed indicated in Fig. 2C). Despite sharing comparable levels of RsRubisco (8.2 ± 0.7 µmol active site·m⁻²; see Fig. 2D), the photosynthetic rates in the tobRsLS::X lines (open symbols) were ∼twofold higher than the tobRsLS lines but lower than that modeled (see SI Appendix, Supplementary Information Text) using the RsRubisco kinetics in Table 1. (B) The effect of growth CO₂ and RsRca expression on the in vivo activation status of RsRubisco in plants acclimated overnight to air (ambient 0.04% (vol/vol) CO₂, open bars) or in the high CO₂ (1% [vol/vol]) growth chamber (filled bars). The activation status of R. sphaeroides Rubisco was stimulated by high CO₂ growth, and RsRca coexpression while in wild type the activation status of tobacco Rubisco declined with increasing CO₂. n = 3 and 5 (±SD) for air and high CO₂ samples, respectively. Solid lines in A represent the modeled A-C_i response accounting for the low activation status of RsRubisco (as indicated) in the tobRsLS and tobRsLS::X leaves during gas exchange. Different letters in B indicate statistical differences (P < 0.05) following a one-way ANOVA and post hoc Tukey test.

Fig. 4.

Comparing WT and hybrid RsRubisco production in E. coli and tobacco chloroplasts. (A) RsRubisco operons comprising an Rs-rbcL gene and differing rbcS genes coding either G. sulphuraria (GsS), G. monilis (GmS), P. tricornutum (PtS), or tobacco (NtS) S-subunits (see SI Appendix, Fig. S4 for sequence and structure information) were cloned into pLEV4 and transformed into the tobRr plastome (Fig. 1A) or cloned into pET28a(+) and expressed in E. coli BL21(DE3). (B) Differences in the assembly of the WT and hybrid RsRubisco isoforms in E. coli (n = 3 ± SD) and tobacco chloroplasts (n = 3 or 4 ± SD) was analyzed by native-PAGE and quantified by [¹⁴C]-CABP binding (Rubisco amount loaded per lane shown in brackets). The relative levels of each RsRubisco produced in E. coli (filled bars) matched that made in tobacco chloroplasts (open bars). Hybrid RsRubisco comprising NtS-subunits were not detected (nd). The measured CO₂-fixation rates (k_cat^c) and CO₂/O₂ specificity (S_C/O) for each Rubisco is shown in italics. Further analysis of the transformed samples is shown in SI Appendix, Fig. S5.