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 transforming the chloroplast genome of tobacco (Nicotiana tabacum). Codon 335 was altered to encode valine instead of leucine. The resulting mutant plants could not grow without atmospheric CO(2) enrichment. In 0.3% (v/v) CO(2), the mutant and wild-type plants produced similar amounts of Rubisco but the extent of carbamylation was nearly twice as great in the mutants. The mutant enzyme's substrate-saturated CO(2)-fixing rate and its ability to distinguish between CO(2) 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 the purified mutant enzyme were the same as those inferred from measurements of photosynthetic gas exchange with leaves of mutant plants. The Michaelis constants for CO(2), O(2), and ribulose-1,5-bisphosphate were reduced and the mutation enhanced oxygenase activity at limiting O(2) concentrations. Consistent with the reduced CO(2) fixation rate at saturating CO(2), the mutant plants grew slower than the wild type but they eventually flowered and reproduced apparently normally. The mutation and its associated phenotype were inherited maternally. The chloroplast-transformation strategy surmounts previous obstacles to mutagenesis of higher-plant Rubisco and allows the consequences for leaf photosynthesis to be assessed.