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

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.

Laura H. Gunn, Elena Martin Avila, Rosemary Birch, and Spencer M. Whitney

PNAS October 13, 2020 117 (41) 25890-25896.

Significance

The specialized assembly requirements of Rubisco hamper its bioengineering in plants, especially in regard to transforming in “red” Rubiscos from algae with better CO2-fixing properties that could enhance crop photosynthesis and growth. We show this assembly incompatibility does not extend to the “red” type Rubisco from Rhodobacter sphaeroides. Despite evolving from a different phylogenetic lineage to plant Rubisco, the assembly requirements of RsRubisco are readily met in chloroplasts as well as Escherichia coli. Coexpressing its cognate Rubisco activase enhanced RsRubisco activity and improved plant photosynthesis and growth twofold. RsRubisco provides a protein scaffold for red Rubisco bioengineering in E. coli and plants—requiring future optimisation of chloroplast RsRubisco expression and catalytic repair.

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 (kcatc) 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.

 

See https://www.pnas.org/content/117/41/25890

 

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.

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