The two largest chloroplast genome-encoded open reading frames of higher plants are essential genes

The chloroplast genomes of most higher plants contain two giant open readin g frames designated ycf1 and ycf2. In tobacco, ycf1 potentially specifies a protein of 1901 amino acids. The putative gene product of the ycf2 reading frame is a protein of 2280 amino acids. In an attempt to determine the fun ctions of ycf1 and ycf2, we have constructed several mutant alleles for tar geted disruption and/or deletion of these two reading frames. The mutant al leles were introduced into the tobacco plastid genome by biolistic chloropl ast transformation to replace the corresponding wild-type alleles by homolo gous recombination. Chloroplast transformants were obtained for all constru cts and tested for their homoplastomic state. We report here that all trans formed lines remained heteroplastomic even after repeated cycles of regener ation under high selective pressure. A balanced selection was observed in t he presence of the antibiotic spectinomycin, resulting in maintenance of a fairly constant ratio of wild-type versus transformed genome copies. Upon r emoval of the antibiotic and therewith release of the selective pressure, s orting out towards the wild-type plastid genome occurred in all transplasto mic lines. These findings suggest that ycf1 and ycf2 are functional genes a nd encode products that are essential for cell survival. The two reading fr ames are thus the first higher plant chloroplast genes identified as being indispensable.