The ycf 9 (orf 62) gene in the plant chloroplast genome encodes a hydrophobic protein of stromal thylakoid membranes

There are still some open reading frames, orfs, with unknown function in the higher plant chloroplast genome. Of these conserved orfs, designated as ycfs (hypothetical chloroplast open reading frames), one is ycf 9 (orf 62) in the transcription unit with the psbC and psbD genes. The aim of this work was to investigate the function of ycf 9 by insertional inactivation of the gene with a selectable marker cassette, consisting of the aadA coding region connected to the trc promoter and rrnB terminator. This cassette was inserted 19 bp downstream from the start of the coding region of the tobacco ycf 9 gene. Two DNA constructs with the aadA cassette in opposite orientations were precipitated on 1 mu m gold particles and delivered into leaves of Nicotiana tabacum, cultivar Samsun, by the biolistic method. Spectinomycin-resistant plants regenerated following bombardment with only the construct containing the aadA gene in the opposite orientation as ycf 9, In spite of several subsequent regeneration cycles on spectinomycin, the transplastomic plants did not reach homoplasmicity. This suggests that the ycf 9 gene product is essential for chloroplast function. Using a polyclonal antibody raised against the inner part of the gene product, the polypeptide was localized in the stromal thylakoid membranes of chloroplasts.