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

Erasing Rubisco small subunit nuclear synthesis in tobacco provides a new plant chassis for investigating novel Rubisco complexes in a whole plant context via chloroplast transformation of both its large and small subunits. Engineering improved Rubisco for the enhancement of photosynthesis is challenged by the alternate locations of the chloroplastrbcLgene and nuclearRbcSgenes. Here we develop an RNAi-RbcStobacco (Nicotiana tabacum) master-line, tobRr Delta S, for producing homogenous plant Rubisco byrbcL-rbcS operon chloroplast transformation. Four genotypes encoding alternativerbcSgenes and adjoining 5 '-intergenic sequences revealed that Rubisco production was highest (50% of the wild type) in the lines incorporating arbcSgene whose codon use and 5 ' untranslated-region matchedrbcL. Additional tobacco genotypes produced here incorporated differing potato (Solanum tuberosum)rbcL-rbcSoperons that either encoded one of three mesophyll small subunits (pS1, pS2, and pS3) or the potato trichome pS(T)-subunit. The pS3-subunit caused impairment of potato Rubisco production by similar to 15% relative to the lines producing pS1, pS2, or pS(T). However, the beta A-beta B 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 pS(T)-subunit, which reduced CE and CO2/O(2)specificity 40% and 15%, respectively. Returning therbcSgene to the plant plastome provides an effective bioengineering chassis for introduction and evaluation of novel homogeneous Rubisco complexes in a whole plant context.