Form I Rubiscos from non-green algae are expressed abundantly but not assembled in tobacco chloroplasts

Non-green algae have Rubiscos that are phylogenetically distinct from their counterparts in green algae and higher plants. Some non-green-algal Rubisc os are more specific for CO2, relative to O-2, than higher-plant Rubiscos, sometimes coupled with lower Michaelis constants for CO2. If these Rubiscos could be substituted for the higher-plant enzyme, and if they functioned s uccessfully in the higher-plant chloroplast and were regulated appropriatel y, they would improve the CO2 use and quantum efficiency of higher-plant ph otosynthesis. To assess the feasibility of expressing non-green algal Rubis cos in higher-plant chloroplasts, we inserted the rbcLS operons from the rh odophyte Galdieria sulphuraria and the diatom Phaeodactylum tricornutum int o the inverted repeats of the plastid genome of tobacco, leaving the tobacc o rbcL gene unaltered. Homoplasmic transformants were selected. The transge nes directed the synthesis of abundant amounts of transcripts and both subu nits of the foreign Rubiscos. In some circumstances, leaves of the transfor mants with the P. tricornutum Rubisco contained as much foreign Rubisco pro tein as endogenous tobacco Rubisco (>30% of the soluble leaf protein). Howe ver, the subunits of the foreign Rubiscos were not properly folded and/or a ssembled. All the foreign large subunits and most of the foreign small subu nits were recovered in the insoluble fractions of leaf extracts. Edman sequ encing yielded the expected N-terminal sequences for the foreign small subu nits but the N-termini of the foreign large subunits were blocked. Accumula tion of large amounts of denatured foreign Rubisco in the leaves, particula rly of the P. tricornutum transformants, caused a reduction in the amount o f tobacco Rubisco present, with concomitant reductions in leaf CO2 assimila tion and plant growth.