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 <i>Rhodobacter sphaeroides</i> (<i>Rs</i>Rubisco)-<i>a</i> red-type Rubisco able to assemble in plant chloroplasts. In transplastomic tob<i>Rs</i>LS lines expressing a codon optimized <i>Rs</i>-<i>rbcLS</i> operon, the messenger RNA (mRNA) abundance was ∼25% of <i>rbcL</i> transcript and <i>Rs</i>Rubisco ∼40% the Rubisco content in WT tobacco. To mitigate the low activation status of <i>Rs</i>Rubisco in tob<i>Rs</i>LS (∼23% sites active under ambient CO₂), the metabolic repair protein <i>Rs</i>Rca (<i>Rs</i>-activase) was introduced via nuclear transformation. <i>Rs</i>Rca production in the tob<i>Rs</i>LS::X progeny matched endogenous tobacco Rca levels (∼1 µmol protomer·m²) and enhanced <i>Rs</i>Rubisco activation to 75% under elevated CO₂ (1%, vol/vol) growth. Accordingly, the rate of photosynthesis and growth in the tob<i>Rs</i>LS::X lines were improved >twofold relative to tob<i>Rs</i>LS. Other tobacco lines producing <i>Rs</i>Rubisco containing alternate diatom and red algae S-subunits were nonviable as CO₂-fixation rates (<i>k</i>_cat^c) were reduced >95% and CO₂/O₂ specificity impaired 30-50%. We show differences in hybrid and WT <i>Rs</i>Rubisco biogenesis in tobacco correlated with assembly in <i>Escherichia coli</i> advocating use of this bacterium to preevaluate the kinetic and chloroplast compatibility of engineered <i>Rs</i>Rubisco, an isoform amenable to directed evolution.