Chloroplast transformation reveals that tonbacco ycf5 is involved in photosynthesis

Hypothetical chloroplast reading frame 5 (ycf5) is encoded by the chloroplast genome and potentially specifies a protein of 313 amino acids in tobacco. A northern blot analysis showed that ycf5 transcripts accumulated at substantial levels in the chloroplasts, but not in the proplastids of nonphotosynthetic cultured cells. In an attempt to determine the function of ycf5, we constructed mutant alleles for the targeted deletion of ycf5. The mutant allele was introduced into the tobacco chloroplast genome by biolistic chloroplast transformation to replace the corresponding wild-typeycf5 alleles by homologous recombination. Homoplasmic ycf5-knockout tobacco plants displayed a pale-green phenotype. An analysis of the transient increase in chlorophyll fluorescence suggested that the electron flow around photosystem II (PSII) was completely impaired in the ycf5-deficient transformants. These findings indicate that ycf5 is indeed a functional gene, and that its gene product is involved in the generation of functional PSII units. This is the first report of the isolation and characterization of ycf5-knockout mutants in higher plants.