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. Here, we show this impediment does not extend to Rubisco from Rhodobacter sphaeroides (RsRubisco)-a redtype Rubisco able to assemble in plant chloroplasts. In transplastomic tobRsLS lines expressing a codon optimized Rs-rbcLS operon, themessenger RNA (mRNA) abundance was similar to 25% of rbcL transcript and RsRubisco similar to 40% the Rubisco content in WT tobacco. To mitigate the low activation status of RsRubisco in tobRsLS (similar to 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 (similar to 1 mu mol protomer.m(2)) 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/O-2 specificity impaired 30-50%. We show differences in hybrid andWT 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.