Directed mutation of the Rubisco large subunit of tobacco influences photorespiration and growth

The gene for the large subunit of Rubisco was specifically mutated by trans forming the chloroplast genome of tobacco (Nicotiana tabacum). Codon 335 wa s altered to encode valine instead of leucine. The resulting mutant plants could not grow without atmospheric CO2 enrichment. In 0.3% (v/v) CO2, the m utant and wildtype plants produced similar amounts of Rubisco but the exten t of carbamylation was nearly twice as great in the mutants. The mutant enz yme's substrate-saturated CO2-fixing rate and its ability to distinguish be tween CO2 and O-2 as substrates were both reduced to 25% of the wild type's values. Estimates of these parameters obtained from kinetic assays with th e purified mutant enzyme were the same as those inferred from measurements of photosynthetic gas exchange with leaves of mutant plants. The Michaelis constants for CO2, O-2, and ribulose-1,5-bisphosphate were reduced and the mutation enhanced oxygenase activity at limiting O-2 concentrations. Consis tent with the reduced CO2 fixation rate at saturating CO2, the mutant plant s grew slower than the wild type but they eventually flowered and reproduce d apparently normally. The mutation and its associated phenotype were inher ited maternally. The chloroplast-transformation strategy surmounts previous obstacles to mutagenesis of higher-plant Rubisco and allows the consequenc es for leaf photosynthesis to be assessed.