Modifying Plant Photosynthesis and Growth via Simultaneous Chloroplast Transformation of Rubisco Large and Small Subunits.

Engineering improved Rubisco to enhance photosynthesis is challenged by the alternate locations of the chloroplast rbcL gene and nuclear RbcS genes. Here we develop a RNAi-RbcS tobacco (Nicotiana tabacum) master-line, tobRrΔS, for producing homogenous plant Rubisco by rbcL-rbcS operon chloroplast transformation. Four genotypes encoding alternative rbcS genes and adjoining 5'-intergenic sequences revealed that Rubisco production was highest (50% of wild type) in the lines incorporating a rbcS gene whose codon use and 5'UTR matched rbcL. Additional tobacco genotypes produced here incorporated differing potato (Solanum tuberosum) rbcL-rbcS operons that either encoded one of three mesophyll small subunits (pS1, pS2, pS3) or the potato trichome pST-subunit. The pS3-subunit impaired potato Rubisco production by ~15% relative to the lines producing pS1, pS2 or pST. However, the βA-βB loop Asn-55-His and Lys-57-Ser substitutions in the pS3-subunit improved carboxylation rates by 13% and carboxylation efficiency (CE) by 17% relative to potato Rubisco incorporating pS1 or pS2-subunits. Tobacco photosynthesis and growth were most impaired in lines producing potato Rubisco incorporating the pST-subunit, which reduced CE and CO2/O2 specificity 40% and 15% respectively. Returning the rbcS gene to the plant plastome provides an effective bioengineering chassis to introduce and evaluate novel homogeneous Rubisco complexes in a whole plant context.